New Patent, WO 2016110874, Artemisinin , IPCA Laboratories Ltd

PATENTS, Uncategorized Comments Off

Jul 182016

New Patent, WO 2016110874, Artemisinin , IPCA Laboratories Ltd

FOR Cancer; Parasitic infection; Plasmodium falciparum infection; Viral infection

WO-2016110874

KUMAR, Ashok; (IN).
SINGH, Dharmendra; (IN).
MAURYA, Ghanshyam; (IN).
WAKCHAURE, Yogesh; (IN)

Dr. Ashok Kumar, President – Research and Development (Chemical) at IPCA LABORATORIES LTD

IPCA LABORATORIES LIMITED [IN/IN]; 48, Kandivli Industrial Estate, Charkop, Kandivali (West), Mumbai 400067 (IN)

Novel process for preparing artemisinin or its derivatives such as dihydroartemisinin, artemether, arteether and artesunate. Also claims novel intermediates of artemesinin such as artemisinic acid or dihydroartemisinic acid. Discloses the use of artemisinin or its derivatives, for treating malaria, cancer, viral and parasitic infections.

In July 2016, Newport Premium™ reported that IPCA was capable of producing commercial quantities of artemether, arteether and artesunate; and holds an inactive US DMF for artemether since February 2009. In July 2016, IPCA’s website lists artemether, arteether and artesunate under its products and also lists artemether and artesunate as having EDMF and WHO certificates. The assignee also has Canada HPFB certificate for artemether.

The Central Drug Research Institute (CDRI) in collaboration with IPCA is developing CDRI-97/78 (1,2,4 trioxane derivative), a synthetic artemisinin substitute for treating drug resistant Plasmodium falciparum infection. In July 2016, CDRI-97/78 was reported to be in phase 1 clinical development. IPCA in collaboration with CDRI was also investigating CDRI-99/411, a synthetic artemisinin substitute for treating malaria; but its development had been presumed to have been discontinued; however, this application’s publication would suggest otherwise.

Writeup

Artemisinin is an active phytoconstituent of Chinese medicinal herb Artemisia annua, useful for the treatment of malaria. Generally, artemisinin/artemisinic acid is obtained by extraction of the plant, Artemisia annua. The plant Artemisia annua was first mentioned in an ancient Chinese medicine book written on silk in the West Han Dynasty at around 200 B.C. The plant’s anti-malarial application was first described in a Chinese pharmacopeia, titled “Chinese Handbook of Prescriptions for Emergency Treatments,” written at around 340 A.D.

Artemisinin being poorly bioavailable limits its effectiveness. Therefore semisynthetic derivatives of artemisinin such as artesunate, dihydroartemisinin, artelinate, artemether, arteether have been developed to improve the bioavailability of Artemisinin.

Artemisinin and its derivatives – dihydroartemisinin, artemether, arteether, and artesunate being a class of antimalarials compounds used for the treatment of uncomplicated, severe complicated/cerebral and multi drug resistant malaria. Additionally, there are research findings that artemisinin and its derivatives show anti-parasite, anti-cancer, and anti-viral activities.

Dihydroartemisinin Artesunate

The content of Artemisinin in the plant Artemisia annua varies significantly according to the climate and region/geographical area where it is cultivated. Further, the extraction methods provide artemisinin or artemisinic acid from the plant in very poor yields and therefore not sufficient to accommodate the ever-growing need for this important drug. Consequently, widespread use of these valuable drugs has been hampered due to the low availability of this natural product. Therefore, research has focused on the syntheses of this valuable drug in a larger scale to meet the increasing global demand and accordingly ample literature is available on the synthesis of artemisinin or its derivatives, but no commercial success being reported / known till date.

Artemisinin can be prepared synthetically from its precursors such as artemisinic acid or dihydroartemisinic acid according to literature methods known to skilled artisans. For example, dihydroartemisinic acid can be converted to artemisinin by a combination of photooxidation and air-oxidation processes as described in U.S. Patent No. 4,992,561.

Amorphadiene is an early starting material for synthesis of Artemisinic acid or dihydroartemisinic acid, which is an important intermediate for producing Artemisinin commercially, and WO2006128126 reported a preparation method as mentioned in scheme- 1.

acid

In accordance with the scheme 1, the amorphadiene is treated with di(cyclohexyl)borane ( δΗι ΒΗ followed by reaction with H2O2 in presence of NaOH to obtain the amorph-4-ene 12-ol which is further oxidized to dihydroartemisinic acid using CrCb/ifcSC^. The formation of amorph-4-ene 12-ol is taking place via epoxidation of the exocyclic double bond. However, the reported yields of this synthesis are very low, making it unviable to produce artemisinic acid at a cheaper cost than natural extraction, for commercial use.

Amorpha -4, 11-diene

A similar method is published in, WO2009088404, for synthesis of dihydroartemisinic acid through preparation of amorph-4-ene-12-ol via epoxide formation, albeit, predominantly at exo position by reacting the amorpha-4,11-diene with H2O2 in presence of porphyrin catalyst (TDCPPMnCl). During reaction, epoxidation also occurred at endo position leading to formation of Amorphadiene- 4,5- epoxide that remain as impurity. The formed exo epoxide (amorphadiene – 11, 12 – epoxide) is further reduced to get amorph- 4-ene 12-ol and then converted to dihydroartemisinic acid and finally converted into artemisinin.

Amorphadiene-11,12-epoxide

This process involves expensive & industry unfriendly reagents. Moreover, desired stereo isomers were obtained only in poor yields, because several purification steps were needed to get desired stereo isomers leading to escalated production/operational costs.

Therefore there remains a need in the art to improve the yield of Dihydroartemisinic acid, which could potentially reduce the cost of production of Artemisinin and/or its derivatives. Consequently it is the need of the hour to provide a synthetic and economically viable process to meet the growing worldwide demand by improving the process for Artemisinin and/or its derivatives to obtain them in substantially higher yields with good purity by plant friendly operations like crystallization/extractions rather than column chromatography/other cost constraint procedures.

Therefore, the object of the invention is to prepare Artemisinic acid of formula-II, Dihydroartemisinic acid of formula-IIa, Artemisinin and its derivatives through Amorphadiene- 4,5- epoxide.

DHAA methyl ester

Scheme 2

Method 4 (From compound of formula IV (R = CI)):

In the 4-neck round bottom flask was charged Diphenyl sulfoxide (23.8 g), NaHC03 (32.96 g) and DMSO (80 ml) at 30^°C. Further a solution of compound of formula IV (R = CI) (10 g) in DMSO (20 ml) was charged to the reaction mass at 30^°C followed by heating and maintaining the temperature for 40 hours at 80^°C till completion. DMSO was distilled out under vacuum. The reaction mass was cooled followed by charging water

(100 ml) and toluene (100 ml) to the reaction mass with stirring for 30 minutes at 28^°C. The layers were separated out and aqueous layer was back extracted with toluene (2 X 100 ml). The organic layer was washed with water (100 ml) and saturated brine solution (100 ml). Solvent was distilled out under vacuum at 50^°C, and the crude mass degassed under vacuum at 50-55^°C. IPA (40 ml) was charged to the mass. Simultaneous addition of hydrazine hydrate (65% in aqueous solution) (3.8 g) and hydrogen peroxide (50% in aqueous solution) (2.5 ml) was done at 30-32^°C over a period of 3.25 hours. After completion, reaction mass was cooled up to 5-10^°C and water (100ml) was added to the reaction mass. The pH of the reaction mass was adjusted to 3.8 with dilute 8% aqueous HC1 (24 ml) at 10^°C. Ethyl acetate (60 ml) was added to the reaction mass at 10^°C and stirred for 15 minutes at 15-20^°C. The layers were separated. Aqueous layer was back extracted with ethyl acetate (2 X 20 ml). The combined organic layer was washed with 10%) sodium metabisulfite solution (50 ml), water (50 ml) and saturated brine solution (50 ml). The organic layer was distilled out under vacuum at 45^°C and the obtained crude mass was degassed at 50-55^°C. To this was added DME (40 ml), Biphenyl (0.9 g) and Li-metal (1.63 g) and the reaction mass was maintained for 10 hours at 80-85^°C till reaction completion. The reaction mass was cooled up to 0-5^°C followed by drop wise addition of water within one hour, and the reaction stirred for two hours at 20-25^°C. Toluene (35 ml) was charged with stirring and layers were separated. The aqueous layer was washed with toluene (35 ml) and the combined toluene layer was washed with water (20 ml). The combined aqueous layer was again washed with toluene (20 ml). The aqueous layer was cooled to 10-15^°C and pH adjusted to 3.5-4 with dilute 16% aqueous HC1. MDC (50 ml) was charged and stirred 30 minutes at 20-25^°C followed by separation of layers. The aqueous layer extracted with MDC (25 ml) and the combined MDC layer was washed with water (50 ml), then with saturated NaCl solution (25 ml). The solvent was distilled out under vacuum at 40-45^°C and the crude mass (Purity: 70-80%>) was degassed at 65-70°C. The crude product (10 g) was dissolved in ethyl acetate (200 ml). 10%> aqueous NaOH (100 ml) was charged to the reaction mass and stirred one hour at 20^°C followed by layer separation. Again 10%> aqueous NaOH (100ml) was added to the organic layer, stirred for 30 minutes and layers were separated out. The pH of the combined NaOH solution wash was adjusted to 4.0 with dilute 16%> aqueous HC1 at 5-10^°C under stirring. Ethyl acetate (850 ml) was charged to aqueous acidic mass, stirred 30 minutes and layers were separated out. The aqueous layer was back extracted with ethyl acetate (2 X 30 ml) and the combined organic layer was washed with water (100 ml) and saturated brine (50 ml). The organic layer was dried over sodium chloride, solvent was distilled out under vacuum and the purified mass was degassed under vacuum at 50-55^°C to obtain Dihydroartemisinic acid (Purity: 90-95%).

b) Methyl ester of Dihydroartemisinic acid:

To a clear solution of Dihydroartemisinic acid (40 g) dissolved in MDC (120 ml) was added thionyl chloride (SOCh) (14.85 ml) at 10±2°C and reaction mass was heated to reflux temperature 40±2°C. After the completion of reaction, solvent was distilled out and excess SOCh was removed under reduced pressure. The resulting concentrated mass of acid chloride was dissolved in MDC (200 ml). In another RBF was taken triethylamine (30.6 ml) and methanol (120 ml). To this solution was added above acid chloride solution at 30±2°C and maintained till completion of reaction. To the reaction mass was added water (400 ml) and organic layer was separated. The aqueous layer was washed with MDC and mixed with main organic layer and the combined organic layer was back washed with water till neutral pH. Then organic layer was concentrated to give methyl ester of Dihydroartemisinic acid as a brown color oily mass.

Weight: 41.88 gm

Yield = 98%

c) Artemisinin:

Methyl ester of dihydroartemisinic acid (67.7 g) was dissolved in methanol (338 ml). To this solution was added Sodium molybdate (29.5 g), 50% hydrogen peroxide (147.3 g) was added at 30±2°C and reaction was maintained for 3-4 hours. After completion of reaction was added water (300 ml) and MDC (300 ml) to the reaction mass. The organic layer was separated and aqueous layer washed with MDC (100 ml). The combined organic layer was concentrated to 475 ml containing hydroperoxide intermediate and directly used for next stage reaction. In another RBF containing MDC (475 ml) was added benzene sulfonic acid (1.27 g) and Indion resin (6.7 g). This heterogeneous solution was saturated with oxygen by passing O2 gas for 10 min at 0±2°C. To this was added previous stage hydroperoxide solution at same temperature with continuous 02 gas purging within 30-40 minutes. The oxygen gas was passed at same temp for 4 hours and temperature raised to 15±2°C with continued passing of oxygen for 5 hours. The

mixture was stirred at 25-30°C for 8-10 hours followed by filtration of resin. The filtrate was washed with water (200 ml X 3) and the combined aqueous layer back washed with MDC (50 ml). The combined organic layer was concentrated to give crude Artemisinin. Weight: 54 gm

Yield= 70.7%

Purification of Artemisinin:

Crude Artemisinin (10 g) was dissolved in ethyl acetate (25 ml) at 45-50°C. The solution was cooled to 30-35°C followed by addition of n-Hexane (100 ml). The material was isolated, stirred for 2 hours, filtered and vacuum dried at 45°C.

Weight: 4 gm

Yield: 40%

THE VIEWS EXPRESSED ARE MY PERSONAL AND IN NO-WAY SUGGEST THE VIEWS OF THE PROFESSIONAL BODY OR THE COMPANY THAT I REPRESENT, amcrasto@gmail.com, +91 9323115463 India

////////New Patent, WO 2016110874, Artemisinin , IPCA Laboratories Ltd, malaria, Cancer, Parasitic infection, Plasmodium falciparum infection, Viral infection, artemether artemisinin, artemotil, artenimol, artesunate,

Galunisertib

Phase 3 drug, Uncategorized Comments Off

May 042016

Galunisertib

A TGF-beta receptor type-1 inhibitor potentially for the treatment of myelodysplastic syndrome (MDS) and solid tumours.

LY-2157299

CAS No.700874-72-2

4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide

6-Quinolinecarboxamide, 4-[5,6-dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-

700874-72-2

Molecular FormulaC₂₂H₁₉N₅O
Average mass369.419 Da

4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide

4-(2-(6-Methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-carboxamide monohydrate

Anal. Calcd for C₂₂H₁₉N₅O·H₂O: C, 68.20; H, 5.46; N, 18.08. Found: C, 68.18; H, 5.34; N, 17.90.

¹H NMR (DMSO-d₆: δ) 1.74 (s, 3H), 2.63 (m, 2H), 2.82 (br s, 2H), 4.30 (t, J = 7.2 Hz, 2H), 6.93 (m, 1H), 7.37 (s, 1H), 7.41 (d, J = 4.4 Hz, 1H), 7.56 (m, 1H), 7.58 (m, 1H), 8.04, (s, 1H), 8.04 (d, J = 4.4 Hz, 1H), 8.12 (dd, J = 8.8, 1.6 Hz, 1H), 8.25 (d, J = 2.0 Hz, 1H), 8.87 (d, J = 4.4 Hz, 1H).

¹³C NMR (DMSO-d₆: δ) 22.56, 23.24, 25.58, 48.01, 109.36, 117.74, 121.26, 122.95, 126.73, 127.16 (2C), 129.01, 131.10, 136.68, 142.98, 147.20, 148.99, 151.08, 151.58, 152.13, 156.37, 167.47.

IR (KBr): 3349, 3162, 3067, 2988, 2851, 1679, 1323, 864, 825 cm^–1.

HRMS (m/z M + 1): Calcd for C₂₂H₁₉N₅O: 370.1653. Found: 370.1662.

GalunisertibAn orally available, small molecule antagonist of the tyrosine kinase transforming growth factor-beta (TGF-b) receptor type 1 (TGFBR1), with potential antineoplastic activity. Upon administration, galunisertib specifically targets and binds to the kinase domain of TGFBR1, thereby preventing the activation of TGF-b-mediated signaling pathways. This may inhibit the proliferation of TGF-b-overexpressing tumor cells. Dysregulation of the TGF-b signaling pathway is seen in a number of cancers and is associated with increased cancer cell proliferation, migration, invasion and tumor progression.

OriginatorEli Lilly
DeveloperEli Lilly; National Cancer Institute (USA); Vanderbilt-Ingram Cancer Center; Weill Cornell Medical College
ClassAntineoplastics; Pyrazoles; Pyridines; Pyrroles; Quinolines; Small molecules
Mechanism of ActionPhosphotransferase inhibitors; Transforming growth factor beta1 inhibitors
- Phase II/IIIMyelodysplastic syndromes
- Phase IIBreast cancer; Glioblastoma; Hepatocellular carcinoma
- Phase I/IIGlioma; Non-small cell lung cancer; Pancreatic cancer
- Phase ICancer; Solid tumours
Most Recent Events
- 26 Apr 2016Eli Lilly plans a pharmacokinetics phase I trial in Healthy volunteers in United Kingdom (PO) (NCT02752919)
- 16 Apr 2016Pharmacodynamics data from a preclinical study in Cancer presented at the 107th Annual Meeting of the American Association for Cancer Research (AACR-2016)
- 06 Apr 2016Eli Lilly and AstraZeneca plan a phase Ib trial for Pancreatic cancer (Second-line therapy or greater, Metastatic disease, Recurrent, Combination therapy) in USA, France, Italy, South Korea and Spain (PO) (NCT02734160)

Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients.

Company	Eli Lilly and Co.
Description	Transforming growth factor (TGF) beta receptor 1 (TGFBR1; ALK5) inhibitor
Molecular Target	Transforming growth factor (TGF) beta receptor 1 (TGFBR1) (ALK5)
Mechanism of Action	Transforming growth factor (TGF) beta 1 inhibitor
Therapeutic Modality	Small molecule

Bristol-Myers Squibb and Lilly Enter Clinical Collaboration Agreement to Evaluate Opdivo (nivolumab) in Combination with Galunisertib in Advanced Solid Tumors

Bristol-Myers Squibb and Lilly

NEW YORK & INDIANAPOLIS–(BUSINESS WIRE)– Bristol-Myers Squibb Company (NYSE:BMY) and Eli Lilly and Company (NYSE:LLY) announced today a clinical trial collaboration to evaluate the safety, tolerability and preliminary efficacy of Bristol-Myers Squibb’s immunotherapy Opdivo (nivolumab) in combination with Lilly’s galunisertib (LY2157299). The Phase 1/2 trial will evaluate the investigational combination of Opdivo and galunisertib as a potential treatment option for patients with advanced (metastatic and/or unresectable) glioblastoma, hepatocellular carcinoma and non-small cell lung cancer.

Opdivo is a human programmed death receptor-1 (PD-1) blocking antibody that binds to the PD-1 receptor expressed on activated T-cells. Galunisertib (pronounced gal ue” ni ser’tib) is a TGF beta R1 kinase inhibitor that in vitro selectively blocks TGF beta signaling. TGF beta promotes tumor growth, suppresses the immune system and increases the ability of tumors to spread in the body. This collaboration will address the hypothesis that co-inhibition of PD-1 and TGF beta negative signals may lead to enhanced anti-tumor immune responses than inhibition of either pathway alone.

“Advanced solid tumors represent a serious unmet medical need among patients with cancer,” said Michael Giordano, senior vice president, Head of Development, Oncology, Bristol-Myers Squibb. “Our clinical collaboration with Lilly underscores Bristol-Myers Squibb’s continued commitment to explore combination regimens from our immuno-oncology portfolio with other mechanisms of action that may accelerate the development of new treatment options for patients.”

“Combination therapies will be key to addressing tumor heterogeneity and the inevitable resistance that is likely to develop to even the most promising new tailored therapies,” said Richard Gaynor, M.D., senior vice president, Product Development and Medical Affairs, Lilly Oncology. “To that end, having multiple cancer pathways and technology platforms will be critical in an era of combinations to ensure sustainability beyond any single asset.”

The study will be conducted by Lilly. Additional details of the collaboration were not disclosed.

About Galunisertib

Galunisertib (pronounced gal ue” ni ser’tib) is Lilly’s TGF beta R1 kinase inhibitor that in vitro selectively blocks TGF beta signaling. TGF beta promotes tumors growth, suppresses the immune system, and increases the ability of tumors to spread in the body.

Immune function is suppressed in cancer patients, and TGF beta worsens immunosuppression by enhancing the activity of immune cells called T regulatory cells. TGF beta also reduces immune proteins, further decreasing immune activity in patients

Galunisertib is currently under investigation as an oral treatment for advanced/metastatic malignancies, including Phase 2 evaluation in hepatocellular carcinoma, myelodysplastic syndromes (MDS), glioblastoma, and pancreatic cancer.

PATENT

WO 2004048382

The disclosed invention also relates to the select compound of Formula II:

Formula II

2-(6-methyl-pyridin-2-yI)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[l,2- bjpyrazole and the phannaceutically acceptable salts thereof.

The compound above is genetically disclosed and claimed in PCT patent application PCT/US02/11884, filed 13 May 2002, which claims priority from U.S. patent application U. S . S .N. 60/293 ,464, filed 24 May 2001 , and incorporated herein by reference. The above compound has been selected for having a surprisingly superior toxicology profile over the compounds specifically disclosed in application cited above.

The following scheme illustrates the preparation of the compound of Formula II.

Scheme II

Cs₂C0₃

The following examples further illustrate the preparation of the compounds of this invention as shown schematically in Schemes I and II. Example 1

Preparation of 7-(2-morpholin-4-yI-ethoxy)-4-(2-pyridin-2-yl-5,6-dihydro-4H- pyrroIo[l,2-b]pyrazol-3-yl)-q inoline

A. Preparation of 4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)- 7-[2-(tetrahydropyran-2-yIoxy)ethoxy]quinoIine

Heat 4-(2-pyridm-2-yl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)-quinolin-7-ol (376 mg, 1.146 mmol), cesium carbonate (826 mg, 2.54 mmol), and 2-(2- bromoethoxy)tetrahydro-2H-pyran (380 μL, 2.52 mmol) in DMF (5 mL) at 120 °C for 4 hours. Quench the reaction with saturated sodium chloride and then extract with chloroform. Dry the organic layer over sodium sulfate and concentrate in vacuo. Purify the reaction mixture on a silica gel column eluting with dichloromethane to 10% methanol in dichloromethane to give the desired subtitled intermediate as a yellow oil (424 mg, 81%). MS ES⁺m/e 457.0 (M+l).

EXAMPLE 2

Preparation of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[l,2- b]pyrazole

A. Preparation of 6-bromo-4-methyI-quinoline

Stir a solution of 4-bromo-phenylamine (1 eq), in 1,4-dioxane and cool to approximately 12 °C. Slowly add sulfuric acid (2 eq) and heat at reflux. Add methyl vinyl ketone (1.5 eq) drop wise into the refluxing solution. Heat the solution for 1 hour after addition is complete. Evaporate the reaction solution to dryness and dissolve in methylene chloride. Adjust the solution to pH 8 with 1 M sodium carbonate and extract three times with water. Chromatograph the residue on SiO (70/30 hexane/ethyl acetate) to obtain the desired subtitled inteπnediate. MS ES+ m e = 158.2 (M+l). B. Preparation of 6-methyl-pyridine-2-carboxylic acid methyl ester

Suspend 6-methyl-pyridine-2-carboxylic acid (10 g, 72.9 mmol) in methylene chloride (200 mL). Cool to 0 °C. Add methanol (10 mL), 4-dimethylaminopyridine (11.6 g, 94.8 mmol), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)

(18.2 g, 94.8 mmol). Stir the mixture at room temperature for 6 hours, wash with water and brine, and dry over sodium sulfate. Filter the mixture and concentrate in vacuo.

Chromatograph the residue on SiO₂ (50% ethyl acetate/hexanes) to obtain the desired subtitled intermediate, 9.66 g (92%), as a colorless liquid. 1H NMR (CDC1₃) 6 7.93-7.88 (m, IH), 7.75-7.7 (m, IH), 7.35-7.3 (m, IH), 4.00 (s, 3H), 2.60 (s, 3H).

C. Preparation of 2-(6-bromo-quinoIin-4-yl)-l-(6-methyl-pyridin-2-yl)-ethanone Dissolve 6-bromo-4-methyl-quinoline (38.5 g, 153 mmol) in 600 mL dry THF.

Cool to -70° C and treat with the dropwise addition of 0.5 M potassium hexamethyldisilazane (KN(SiMe )₂ (400 mL, 200 mmol) over 2 hours while keeping the temperature below -65 °C. Stir the resultant solution at -70°C for 1 hour and add a solution of 6-methylpyridine-2-carboxylic acid methyl ester (27.2, 180 mmol) in 100 mL dry THF dropwise over 15 minutes. During the addition, the mixture will turn from dark red to pea-green and form a precipitate. Stir the mixture at -70°C over 2 hours then allow it to warm to ambient temperature with stirring for 5 hours. Cool the mixture then quench with 12 N HC1 to pH=l . Raise the pH to 9 with solid potassium carbonate. Decant the solution from the solids and extract twice with 200 mL ethyl acetate. Combine the organic extracts, wash with water and dry over potassium carbonate. Stir the solids in 200 mL water and 200 mL ethyl acetate and treat with additional potassium carbonate. Separate the organic portion and dry with the previous ethyl acetate extracts. Concentrate the solution in vacuo to a dark oil. Pass the oil through a 300 mL silica plug with methylene chloride then ethyl acetate. Combine the appropriate fractions and concentrate in vacuo to yield an amber oil. Rinse the oil down the sides of the flask with methylene chloride then dilute with hexane while swirling the flask to yield 38.5 g (73.8 %) of the desired subtitled intermediate as a yellow solid. MS ES+ = 341 (M+l)v D. Preparation of l-[2-(6-bromo-quinolin-4-yI)-l-(6-methyl-pyridin-2-yl)- ethylideneamino]-pyrrolidin-2-one

Stir a mixture of 2-(6-bromo-quinolin-4-yl)-l-(6-methyl-pyridin-2-yl)-ethanone (38.5 g, 113 mmol) and 1-aminopyrrolidinone hydrochloride (20 g, 147 mmol) in 115 mL pyridine at ambient temperature for 10 hours. Add about 50 g 4 A unactivated sieves. Continue stirring an additional 13 h and add 10-15 g silica and filter the mixture through a 50 g silica plug. Elute the silica plug with 3 L ethyl acetate. Combine the filtrates and concentrate in vacuo. Collect the hydrazone precipitate by filtration and suction dry to yield 33.3 g (69.7%) of the desired subtitled intermediate as an off-white solid. MS ES⁺ = 423 (M+l).

E. Preparation of 6-bromo-4-[2-(6-methyl-pyridin-2-yι)-5,6-dihydro-4H- pyrrolo[l,2-b]pyrazol-3-yl]-quinoline

To a mixture of (1.2 eq.) cesium carbonate and l-[2-(6-bromo-qumolin-4-yl)-l- (6-methyl-pyridin-2-yl)-ethylideneamino]-pyrrolidin-2-one (33.3 g, 78.7 mmol) add 300 mL dry N,N-dimethylformamide. Stir the mixture 20 hours at 100°C. The mixture may turn dark during the reaction. Remove the N,N-dimethylformamide in vacuo. Partition the residue between water and methylene chloride. Extract the aqueous portion with additional methylene chloride. Filter the organic solutions through a 300 mL silica plug, eluting with 1.5 L methylene chloride, 1.5 L ethyl acetate and 1.5 L acetone. Combine the appropriate fractions and concentrate in vacuo. Collect the resulting precipitate by filtration to yield 22.7 g (71.2%) of the desired subtitled intermediate as an off-white solid. MS ES⁺ = 405 (M+l).

F. Preparation of 4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2- b]pyrazol-3-yl]-quinoline-6-carboxylic acid methyl ester

Add 6-bromo-4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2- b]pyrazol-3-yl]-quinoline (22.7 g, 45 mmol) to a mixture of sodium acetate (19 g, 230 mmol) and the palladium catalyst [1,1 ‘- bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1:1) (850 mg, 1.04 mmol) in 130 mL methanol. Place the mixture under 50 psi carbon monoxide atmosphere and stir while warming to 90° C over 1 hour and with constant charging with additional carbon monoxide. Allow the mixture to cool over 8 hours, recharge again with carbon monoxide and heat to 90 °C. The pressure may rise to about 75 PSI. The reaction is complete in about an hour when the pressure is stable and tic (1 : 1 toluene/acetone) shows no remaining bromide. Partition the mixture between methylene chloride (600 mL) and water (1 L). Extract the aqueous portion with an additional portion of methylene chloride (400 mL.) Filter the organic solution through a 300 mL silica plug and wash with 500 mL methylene chloride, 1200 mL ethyl acetate and 1500 mL acetone. Discard the acetone portion. Combine appropriate fractions and concentrate to yield 18.8 g (87.4%) of the desired subtitled intermediate as a pink powder. MS ES⁺ = 385 (M+l).

G. Preparation of 2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yι)-5,6- dihydro-4H-pyrrolo[l,2-b]pyrazole

Warm a mixture of 4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[l,2- b]pyrazol-3-yl]-quinolme-6-carboxylic acid methyl ester in 60 mL 7 N ammonia in methanol to 90 °C in a stainless steel pressure vessel for 66 hours. The pressure will rise to about 80 PSI. Maintain the pressure for the duration of the reaction. Cool the vessel and concentrate the brown mixture in vacuo. Purify the residual solid on two 12 g Redi- Pak cartridges coupled in series eluting with acetone. Combine appropriate fractions and concentrate in vacuo. Suspend the resulting nearly white solid in methylene chloride, dilute with hexane, and filter. The collected off-white solid yields 1.104 g (63.8%) of the desired title product. MS ES⁺ = 370 (M+l).

PAPER

http://pubs.acs.org/doi/abs/10.1021/op4003054

Application of Kinetic Modeling and Competitive Solvent Hydrolysis in the Development of a Highly Selective Hydrolysis of a Nitrile to an Amide

Jeffry K. Niemeier*, Roger R. Rothhaar*, Jeffrey T. Vicenzi, and John A. Werner

Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States

Org. Process Res. Dev., 2014, 18 (3), pp 410–416

DOI: 10.1021/op4003054

Publication Date (Web): February 11, 2014

*Telephone: (317) 276-2066. E-mail: niemeier_jeffry_k@lilly.com (J.K.N.)., *Telephone: (317) 433-3769. E-mail: rrothhaar@lilly.com(R.R.R.).

Abstract

A combination of mechanism-guided experimentation and kinetic modeling was used to develop a mild, selective, and robust hydroxide-promoted process for conversion of a nitrile to an amide using a substoichiometric amount of aqueous sodium hydroxide in a mixed water and N-methyl-2-pyrrolidone solvent system. The new process eliminated a major reaction impurity, minimized overhydrolysis of the product amide by selection of a solvent that would be sacrificially hydrolyzed, eliminated genotoxic impurities, and improved the intrinsic safety of the process by eliminating the use of hydrogen peroxide. The process was demonstrated in duplicate on a 90 kg scale, with 89% isolated yield and greater than 99.8% purity.

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REFERENCES

1: Rodón J, Carducci M, Sepulveda-Sánchez JM, Azaro A, Calvo E, Seoane J, Braña I, Sicart E, Gueorguieva I, Cleverly A, Pillay NS, Desaiah D, Estrem ST, Paz-Ares L, Holdhoff M, Blakeley J, Lahn MM, Baselga J. Pharmacokinetic, pharmacodynamic and biomarker evaluation of transforming growth factor-β receptor I kinase inhibitor, galunisertib, in phase 1 study in patients with advanced cancer. Invest New Drugs. 2014 Dec 23. [Epub ahead of print] PubMed PMID: 25529192.

2: Kovacs RJ, Maldonado G, Azaro A, Fernández MS, Romero FL, Sepulveda-Sánchez JM, Corretti M, Carducci M, Dolan M, Gueorguieva I, Cleverly AL, Pillay NS, Baselga J, Lahn MM. Cardiac Safety of TGF-β Receptor I Kinase Inhibitor LY2157299 Monohydrate in Cancer Patients in a First-in-Human Dose Study. Cardiovasc Toxicol. 2014 Dec 9. [Epub ahead of print] PubMed PMID: 25488804.

3: Rodon J, Carducci MA, Sepulveda-Sanchez JM, Azaro A, Calvo E, Seoane J, Brana I, Sicart E, Gueorguieva I, Cleverly AL, Sokalingum Pillay N, Desaiah D, Estrem ST, Paz-Ares L, Holdoff M, Blakeley J, Lahn MM, Baselga J. First-in-Human Dose Study of the Novel Transforming Growth Factor-β Receptor I Kinase Inhibitor LY2157299 Monohydrate in Patients with Advanced Cancer and Glioma. Clin Cancer Res. 2014 Nov 25. pii: clincanres.1380.2014. [Epub ahead of print] PubMed PMID: 25424852.

4: Huang C, Wang H, Pan J, Zhou D, Chen W, Li W, Chen Y, Liu Z. Benzalkonium Chloride Induces Subconjunctival Fibrosis Through the COX-2-Modulated Activation of a TGF-β1/Smad3 Signaling Pathway. Invest Ophthalmol Vis Sci. 2014 Nov 18;55(12):8111-22. doi: 10.1167/iovs.14-14504. PubMed PMID: 25406285.

5: Cong L, Xia ZK, Yang RY. Targeting the TGF-β receptor with kinase inhibitors for scleroderma therapy. Arch Pharm (Weinheim). 2014 Sep;347(9):609-15. doi: 10.1002/ardp.201400116. Epub 2014 Jun 11. PubMed PMID: 24917246.

6: Gueorguieva I, Cleverly AL, Stauber A, Sada Pillay N, Rodon JA, Miles CP, Yingling JM, Lahn MM. Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokinetic/pharmacodynamic model. Br J Clin Pharmacol. 2014 May;77(5):796-807. PubMed PMID: 24868575; PubMed Central PMCID: PMC4004400.

7: Oyanagi J, Kojima N, Sato H, Higashi S, Kikuchi K, Sakai K, Matsumoto K, Miyazaki K. Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor. Exp Cell Res. 2014 Aug 15;326(2):267-79. doi: 10.1016/j.yexcr.2014.04.009. Epub 2014 Apr 26. PubMed PMID: 24780821.

8: Giannelli G, Villa E, Lahn M. Transforming growth factor-β as a therapeutic target in hepatocellular carcinoma. Cancer Res. 2014 Apr 1;74(7):1890-4. doi: 10.1158/0008-5472.CAN-14-0243. Epub 2014 Mar 17. Review. PubMed PMID: 24638984.

9: Dituri F, Mazzocca A, Peidrò FJ, Papappicco P, Fabregat I, De Santis F, Paradiso A, Sabbà C, Giannelli G. Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II. PLoS One. 2013 Jun 27;8(6):e67109. Print 2013. PubMed PMID: 23826206; PubMed Central PMCID: PMC3694933.

10: Bhola NE, Balko JM, Dugger TC, Kuba MG, Sánchez V, Sanders M, Stanford J, Cook RS, Arteaga CL. TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Invest. 2013 Mar 1;123(3):1348-58. doi: 10.1172/JCI65416. Epub 2013 Feb 8. PubMed PMID: 23391723; PubMed Central PMCID: PMC3582135.

11: Bhattachar SN, Perkins EJ, Tan JS, Burns LJ. Effect of gastric pH on the pharmacokinetics of a BCS class II compound in dogs: utilization of an artificial stomach and duodenum dissolution model and GastroPlus,™ simulations to predict absorption. J Pharm Sci. 2011 Nov;100(11):4756-65. doi: 10.1002/jps.22669. Epub 2011 Jun 16. PubMed PMID: 21681753.

12: Bueno L, de Alwis DP, Pitou C, Yingling J, Lahn M, Glatt S, Trocóniz IF. Semi-mechanistic modelling of the tumour growth inhibitory effects of LY2157299, a new type I receptor TGF-beta kinase antagonist, in mice. Eur J Cancer. 2008 Jan;44(1):142-50. Epub 2007 Nov 26. PubMed PMID: 18039567.

References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539082/

http://www.ncbi.nlm.nih.gov/pubmed/26057634

https://clinicaltrials.gov/ct2/show/NCT0242334

Bhattachar, Shobha N.; Journal of Pharmaceutical Sciences 2011, 100(11), 4756-4765

Investigational new drugs (2015), 33(2), 357-70.

//////////TGF-β, TGF-βRI kinase inhibitor, ALK5, galunisertib, LY2157299, cancer, clinical trials, PHASE 3

CC1=CC=CC(=N1)C2=NN3CCCC3=C2C4=C5C=C(C=CC5=NC=C4)C(=O)N

INCB24360 (epacadostat)

phase 2, Uncategorized Comments Off

Apr 182016

Epacadostat

(Z)-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazole-3-carboxamidine

1,2,5-Oxadiazole-3-carboximidamide, 4-[[2-[(aminosulfonyl)amino]ethyl]amino]-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-

1204669-58-8

INCB024360

N-(3-Brom-4-fluorphenyl)-N’-hydroxy-4-{[2-(sulfamoylamino)ethyl]amino}-1,2,5-oxadiazol-3-carboximidamid

UNII 71596A9R13

(Z)-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-(2-(sulfamoylamino)ethylamino)-1,2,5-oxadiazole-3-carboximidamide

1,2,5-Oxadiazole-3-carboximidamide, 4-[[2-[(aminosulfonyl)amino]ethyl]amino]-N’-(3-bromo-4-fluorophenyl)-N-hydroxy-

Molecular Formula, C₁₁H₁₃BrFN₇O₄S

Average mass438.233 Da

cas 1204669-58-8 (or 1204669-37-3)

Synonym:	IDO1 inhibitor INCB024360 indoleamine-2,3-dioxygenase inhibitor INCB024360
Code name:	INCB 024360 INCB024360
Chemical structure:	1,2,5-Oxadiazole-3-carboximidamide, 4-((2-((Aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-, (C(Z))-

Company	Incyte Corp.
Description	Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor
Molecular Target	Indoleamine 2,3-dioxygenase 1 (IDO1)
Mechanism of Action	Indoleamine 2,3-dioxygenase (INDO) inhibitor
Therapeutic Modality	Small molecule

OriginatorIncyte Corporation
DeveloperFred Hutchinson Cancer Research Center; Incyte Corporation; Merck AG
ClassAmides; Antineoplastics; Imides; Oxadiazoles; Small molecules
- Phase IIFallopian tube cancer; Malignant melanoma; Non-small cell lung cancer; Ovarian cancer; Peritoneal cancer; Solid tumours
Most Recent Events
- 15 Jan 2016Phase-II clinical trials in Solid tumours (Combination therapy, Late-stage disease, Second-line therapy or greater) in USA (PO)
- 11 Jan 2016Phase-II clinical trials in Non-small cell lung cancer (Combination therapy, Late-stage disease, Second-line therapy or greater) in USA (PO)
- 11 Jan 2016The US FDA and Health Canada approve IND application and Clinical Trial Application, respectively, for a phase Ib trial in Ovarian cancer (Combination therapy, Recurrent, Second-line therapy or greater)

In 2016, orphan drug designation was assigned to the compound in the US. for the treatment of stage IIB-IV melanoma

EpacadostatAn orally available hydroxyamidine and inhibitor of indoleamine 2,3-dioxygenase (IDO1), with potential immunomodulating and antineoplastic activities. epacadostat targets and binds to IDO1, an enzyme responsible for the oxidation of tryptophan into kynurenine. By inhibiting IDO1 and decreasing kynurenine in tumor cells, epacadostat increases and restores the proliferation and activation of various immune cells, including dendritic cells (DCs), NK cells, and T-lymphocytes, as well as interferon (IFN) production, and a reduction in tumor-associated regulatory T cells (Tregs). Activation of the immune system, which is suppressed in many cancers, may inhibit the growth of IDO1-expressing tumor cells. IDO1 is overexpressed by a variety of tumor cell types and DCsINCB24360 (epacadostat), An Agent For Cancer Immunotherapy

Incyte and Merck Expand Clinical Collaboration to Include Phase 3 Study Investigating the Combination of Epacadostat with Keytruda® (pembrolizumab) as First-line Treatment for Advanced Melanoma

Pivotal study to evaluate Incyte’s IDO1 inhibitor in combination with Merck’s anti-PD-1 therapy in patients with advanced or metastatic melanoma

WILMINGTON, Del. and KENILWORTH, N.J. — October 13, 2015 — Incyte Corporation (Nasdaq: INCY) and Merck (NYSE:MRK), known as MSD outside the United States and Canada, today announced the expansion of the companies’ ongoing clinical collaboration to include a Phase 3 study evaluating the combination of epacadostat, Incyte’s investigational selective IDO1 inhibitor, with Keytruda® (pembrolizumab), Merck’s anti-PD-1 therapy, as first-line treatment for patients with advanced or metastatic melanoma. The Phase 3 study, which is expected to begin in the first half of 2016, will be co-funded by Incyte and Merck.

“We are very pleased to expand our collaboration with Merck and to move the clinical development program for epacadostat in combination with Keytruda into Phase 3,” said Hervé Hoppenot, President and Chief Executive Officer of Incyte. “We believe the combination of these two immunotherapies shows promise and, if successfully developed, may help to improve clinical outcomes for patients with metastatic melanoma.”

“The initiation of this large Phase 3 study with Incyte in the first-line advanced melanoma treatment setting is an important addition to our robust immunotherapy clinical development program for Keytruda,” said Dr. Roger Dansey, senior vice president and therapeutic area head, oncology late-stage development, Merck Research Laboratories. “We continue to explore the benefit that Keytruda brings to patients suffering from advanced melanoma when used alone, and we are pleased to be able to add this important combination study with epacadostat to our Keytruda development program.”

Under the terms of the agreement Incyte and Merck have also agreed, for a period of two years, not to initiate new pivotal studies of an IDO1 inhibitor in combination with a PD-1/PD-L1 antagonist as first-line therapy in advanced or metastatic melanoma with any third party. During this time, the companies will each offer the other the opportunity to collaborate on any new pivotal study involving an IDO1 inhibitor in combination with a PD-1/PD-L1 antagonist for types of melanoma and lines of therapy outside of the current collaboration agreement.

The agreement is between Incyte and certain subsidiaries and Merck through its subsidiaries.

Epacadostat and Keytruda are part of a class of cancer treatments known as immunotherapies that are designed to enhance the body’s own defenses in fighting cancer; the two therapies target distinct regulatory components of the immune system. IDO1 is an immunosuppressive enzyme that has been shown to induce regulatory T cell generation and activation, and allow tumors to escape immune surveillance. Keytruda is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Preclinical evidence suggests that the combination of these two agents may lead to an enhanced anti-tumor immune response compared with either agent alone.

Safety and efficacy data from the ongoing Phase 1/2 study evaluating the combination of epacadostat with Keytruda in patients with advanced malignancies is scheduled to be highlighted as a late-breaking oral presentation (Abstract #142) at the upcoming Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting & Associated Programs, November 4–8, 2015 at the Gaylord National Resort & Convention Center in National Harbor, MD.

Metastatic Melanoma

Melanoma, the most serious form of skin cancer, strikes adults of all ages and accounts for approximately five percent of all new cases of cancer in the United States each year. The number of new cases of melanoma continues to rise by almost three percent each year which translates to 76,000 new cases yearly in the U.S. alone.[i] The 5-year survival rate for late-stage or metastatic disease is 15 percent.[ii]

About Epacadostat (INCB024360)

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that has been shown to induce regulatory T cell generation and activation, and allow tumors to escape immune surveillance. Epacadostat is an orally bioavailable small molecule inhibitor of IDO1 that has nanomolar potency in both biochemical and cellular assays and has demonstrated potent activity in enhancing T lymphocyte, dendritic cell and natural killer cell responses in vitro, with a high degree of selectivity. Epacadostat has shown proof-of-concept clinical data in patients with unresectable or metastatic melanoma in combination with the CTLA-4 inhibitor ipilimumab, and is currently in four proof-of-concept clinical trials with PD-1 and PD-L1 immune checkpoint inhibitors in a variety of cancer histologies.

PATENT

WO 2014066834

https://www.google.com/patents/WO2014066834A1?cl=en

EXAMPLE 1

4-({2-[(Aminosulfonyl)amino]ethyl}amino)- V-(3-bromo-4-fluorophenyl)- V -hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step 1: 4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide

[00184] Malononitrile (320.5 g, 5 mol) was added to water (7 L) preheated to 45 °C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 °C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 °C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 °C. The reaction mixture was cooled to 13 °C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 °C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27 °C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 °C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 °C) to give the desired product (644 g, 90%). LCMS for C3H6N5O2

(M+H)+: m/z = 144.0. ¹³C MR (75 MHz, CD3OD): δ 156.0, 145.9, 141.3. Step 2: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride [00185] 4-Amino-N^,-hydroxy-l ,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 – 45 °C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 °C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 °C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl i-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 °C) to give the desired product (256 g, 53.4%). LCMS for C3H4CIN4O2 (M+H)+: m/z = 162.9. ¹³C NMR (100 MHz, CD3OD): 5 155.8, 143.4, 129.7.

Step 3: 4-Amino-N’-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidamide [00186] 4-Amino-N-hydroxy-l ,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5 °C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 °C. The reaction was cooled to 0 – 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 1 19%) as a crude dark oil.

LCMS for C₆Hi_{2 5}0₃ (M+H)⁺: m/z = 202.3. 1H NMR (400 MHz, DMSO- ): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J = 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J = 5.8 Hz, 2 H), 3.08 (s, 3 H).

Step 4: N’-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide

[00187] 4-Amino-N-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3- carboximidamide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 °C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf = 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C6H12N5O3 (M+H)⁺: m/z = 202.3 ^LH NMR (400 MHz, OMSO-d₆): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J = 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H). Step 5: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride

[00188] At room temperature N’-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3-carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 °C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 – 8 °C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for

C₆HioClN₄03 (M+H)⁺: m/z = 221.0. ^!H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).

Step 6 : N-(3-Bromo-4-fluorophenyl)-N’-hydroxy-4- [(2-methoxyethyl)amino] – 1 ,2,5- oxadiazole-3-carboximidamide [00189] N-Hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4-fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 °C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrF₅0₃ (M+H)⁺: m/z = 374.0, 376.0. 1H NMR (400 MHz, DMSO- t_f): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J = 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).

Step 7: 4-(3-Bromo-4-fluorophenyl)-3-{4- [(2-methoxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one

[00190] A mixture of N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-[(2- methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), 1,1 ‘- carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 °C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 N HC1 (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for

(M+H)⁺: m/z = 400.0, 402.0. 1H NMR (400 MHz, DMSO-c½): δ 7.94 (t, J = 8.2 Hz, 1 H), 7.72 (dd, J = 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J = 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).

Step 8: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one

[00191] 4-(3-Bromo-4-fluoroplienyl)-3-{4-[(2-metlioxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 °C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 °C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 – 5 °C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 °C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for Ci₂HioBrFN₅0₄ (M+H)⁺: m/z = 386.0, 388.0.

1H NMR (400 MHz, DMSO-^): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J = 8.7 Hz, 1 H), 6.33 (t, J = 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).

Step 9 : 2-({4- [4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro- 1 ,2,4-oxadiazol-3-yl] – l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate

[00192] To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]- l,2,5-oxadiazol-3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 °C. After 2 h, the reaction mixture was washed with water (5 L), brine (1 L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for C HnBrFNsOeS a (M+Na)⁺: m/z = 485.9, 487.9. ^!H NMR (400 MHz, DMSO- d₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.75 (t, J = 5.9 Hz, 1 H), 4.36 (t, J = 5.3 Hz, 2 H), 3.58 (dd, J = 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).

Step 10: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- l,2,4-oxadiazol-5(4H)-one

To a solution of 2-({4-[4-(3-bromo-4-f uorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50 °C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for CnHgBrFNsOs a

(M+Na)⁺: m/z = 433.0, 435.0. ^!H NMR (400 MHz, DMSO-J₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J = 5.7 Hz, 1 H), 3.54 (t, J = 5.3 Hz, 2 H), 3.45 (dd, J= 1 1.1 , 5.2 Hz, 2 H).

Step 11: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-

1.2.4- oxadiazol-5(4H)-one hydrochloride

[00194] Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-

1.2.5- oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-l ,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 °C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (-1.5 L), diluted with water (4 L), and the pH adjusted to 9 carefully with solid potassium carbonate (250 g – added in small portions: watch foaming). Di-ieri-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pH was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 THF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 °C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 °C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in 1 ,4-dioxane, 4 L, 16 mol). tert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 41 13 g, 95%). LCMS for

Ci₂H_nBrFN₆0₃ (M+H)⁺: m/z = 384.9, 386.9. 1H NMR (400 MHz, DMSO-^): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.78 (t, J = 6.1 Hz, 1 H), 3.51 (dd, J = 1 1.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).

Step 12: tert-Butyl ({[2-({4-[4-(3-bromo-4-nuorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product # 142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 °C. teri-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 °C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-bvAy\ [chlorosulfonyl]carbamate.

A 22 L flask was charged with 3- {4-[(2-aminoethyl)amino]- 1 ,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 °C with an ice/salt bath, the solution oi tert- Vmtvl i Vi 1 r>rosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 °C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 °C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 °C, split into two portions, and neutralized with 10% cone HC1 (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (-25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off- white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). 1H NMR (400 MHz, DMSO-^): δ 10.90 (s, 1 H), 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J = 8.6 Hz, 1 H), 6.58 (t, J = 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J= 12.1 , 5.9 Hz, 2 H), 1.41 (s, 9 H).

Step 13: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

[00198] To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-bvXyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40- 50 °C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 °C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C₁₂H₁₂BrFN₇0₅S (M+H)⁺: m/z = 463.9, 465.9. 1H NMR (400 MHz, DMSO- ): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J = 5.9 Hz, 1H), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J = 12.7, 6.3 Hz, 2 H), 3.11 (dd, J = 12.3, 6.3 Hz). Step 14: 4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N’- hydroxy-l,2,5-oxadiazole-3-carboximidamide

[00199] To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5- dihydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 N NaOH (4 L) was added at a rate so that the temperature did not exceed 10 °C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HC1 (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2:1 watenethanol, 14.1 L each flask) and dried in a 50 °C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for CnHnBrF yC S (M+H)⁺: m/z = 437.9, 439.9. i NMR (400 MHz, DMSO-J^): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J = 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).

PATENT

WO 2010005958

https://www.google.com/patents/WO2010005958A2?cl=en

EXAMPLES Example 1

4-({2-[(Aminosulfonyl)amino]ethyl}amino)-7V-(3-bromo-4-fluorophenyl)-iV’-hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step A: 4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide

Malononitrile [Aldrich, product # M1407] (320.5 g, 5 mol) was added to water (7 L) preheated to 45 ⁰C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 ⁰C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 ⁰C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 ⁰C. The reaction mixture was cooled to 13 ⁰C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 ⁰C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27⁰C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 ⁰C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 ⁰C) to give the desired product (644 g, 90%). LCMS for C₃H₆N₅O₂ (M+H)+: m/z = 144.0. ¹³C NMR (75 MHz, CD₃OD): δ 156.0, 145.9, 141.3. Step B: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride

4-Amino-N’-hydroxy-l,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 – 45 ⁰C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 ⁰C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 ⁰C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl f-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 ⁰C) to give the desired product (256 g, 53.4%). LCMS for C₃H₄ClN₄O₂(M+H)+: m/z = 162.9. 13c NMR (100 MHz, CD₃OD): δ 155.8, 143.4, 129.7.

Step C: 4-Amino-N’-hydroxy-N-(2-methoxyethyl)- 1 ,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5⁰C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 ⁰C. The reaction was cooled to 0 – 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 119%) as a crude dark oil. LCMS for C₆Hi₂N₅O₃ (M+H)⁺: m/z = 202.3. ¹H NMR (400 MHz, DMSO-J₆): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J= 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J= 5.8 Hz, 2 H), 3.08 (s, 3 H).

Step D: N’-Hydroxy-4-[(2-methoxyethyl)amino]-l ,2,5-oxadiazole-3-carboximidamide

4-Amino-N’-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidaniide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 ⁰C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf= 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z = 202.3 ¹H NMR (400 MHz, DMSO-Gk): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J= 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H).

Step E: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride

Ν. ,Ν O

At room temperature N’-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3- carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 ⁰C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 – 8 ⁰C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for C₆Hi₀ClN₄O₃ (M+H)⁺: m/z = 221.0. ¹H NMR (400 MHz, DMSO-J₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).

Step F: N-(3-Bromo-4-fluorophenyl)-N’-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3 -carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4- fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 nrn^‘n. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 ⁰C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrFN₅O₃ (M+H)⁺: m/z = 374.0, 376.0. ¹H NMR (400 MHz, DMSO-J₆): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J= 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).

Step G: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

A mixture of N-(3-bromo-4-fluorophenyl)-N’-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5- oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), l,r-carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 ⁰C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 Ν HCl (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z = 400.0, 402.0. ¹H NMR (400 MHz, OMSO-d₆): δ 7.94 (t, J= 8.2 Hz, 1 H), 7.72 (dd, J= 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J= 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).

Step H: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-liydroxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyetliyl)amino]-l,2,5-oxadiazol-3-yl}-l,2,4- oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 ⁰C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 ⁰C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 – 5 ⁰C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 ⁰C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z = 386.0, 388.0. ¹H NMR (400 MHz, DMSO-^₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J= 8.7 Hz, 1 H), 6.33 (t, J= 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).

Step I: 2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]-l,2,5- oxadiazol-3-yl}amino)ethyl methanesulfonate

To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 ⁰C. After 2 h, the reaction mixture was washed with water (5 L), brine (I L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for

Ci₃HnBrFN₅O₆SNa (M+Na)⁺: m/z = 485.9, 487.9. ¹H NMR (400 MHz, DMSCW₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J- 5.9 Hz, 1 H), 4.36 (t, J= 5.3 Hz, 2 H), 3.58 (dd, J= 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).

Step J: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one

To a solution of 2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50⁰C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for C₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z =

433.0, 435.0. ¹H NMR (400 MHz, DMSO-*/*): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J= 5.7 Hz, 1 H), 3.54 (t, J= 5.3 Hz, 2 H), 3.45 (dd, J= 11.1, 5.2 Hz, 2 H).

Step K: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one hydrochloride

Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-l,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 ⁰C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (~1.5 L), diluted with water (4 L), and the pΗ adjusted to 9 carefully with solid potassium carbonate (250 g – added in small portions: watch foaming). Di-fe/t-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pΗ was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 TΗF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 ⁰C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 ⁰C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).

In a 22 L flask was charged hydrogen chloride (4 N solution in 1,4-dioxane, 4 L, 16 mol). fert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 4113 g, 95%). LCMS for C₁₂H_nBrFN₆O₃ (M+H)⁺: m/z

= 384.9, 386.9. ¹H NMR (400 MHz, DMSO-J₆): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.78 (t, J= 6.1 Hz, 1 H), 3.51 (dd, J= 11.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).

Step L: tert-Butyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol- 3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product #

142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 ⁰C. tert-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 ⁰C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-butyl [chlorosulfonyljcarbamate.

A 22 L flask was charged with 3-{4-[(2-aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3- bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 ⁰C with an ice/salt bath, the solution of tert-butyl [chlorosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 ⁰C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 ⁰C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 ⁰C, split into two portions, and neutralized with 10% cone HCl (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (~25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off-white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). *Η NMR (400 MHz, OMSO-d₆): δ 10.90 (s, 1 H), 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.6 Hz, 1 H), 6.58 (t, J= 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J = 12.1, 5.9 Hz, 2 H), 1.41 (s, 9 H). Step M: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dmydro-l ,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-butyl ({[2- ({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3- yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40-50 ⁰C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 ⁰C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C]₂H₁₂BrFN₇O₅S (M+H)⁺: m/z = 463.9, 465.9.

¹H NMR (400 MHz, OM$>O-d₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J= 5.9 Hz, IH), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J= 12.7, 6.3 Hz, 2 H), 3.11 (dd, J= 12.3, 6.3 Hz).

Step N: 4-( {2-[(Aminosulfonyl)amino]ethyl} amino)-N-(3-bromo-4-fluorophenyl)-N- hydroxy-l,2,5-oxadiazole-3-carboximidamide

To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 Ν NaOH (4 L) was added at a rate so that the temperature did not exceed 10 ⁰C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HCl (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2: 1 water: ethanol, 14.1 L each flask) and dried in a 50 ⁰C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for C_nH₁₄BrFN₇O₄S (M+H)⁺: m/z = 437.9, 439.9. ¹H NMR (400 MHz, DMSO-J₆): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J= 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).

The final product was an anhydrous crystalline solid. The water content was determined to be less than 0.1% by Karl Fischer titration.

CLIP

INCB24360

Company:Incyte Corp.
Target: IDO1
Disease: Cancer

Incyte’s Andrew P. Combs presented the company’s clinical candidate for cancer immunotherapy. The basic tenet of this burgeoning field is that the human body’s immune system is a tremendous resource for fighting disease; scientists just need to figure out how to unleash it. One target that’s proven to be particularly attractive for this purpose in recent years is indoleamine-2,3-dioxygenase-1, or IDO1 (C&EN, April 6, page 10).

IDO1 plays a role in signaling the immune system to stand down from attacking foreign bodies it might otherwise go after, such as fetuses. Tumors also produce IDO1 to evade the immune system, so molecules that can inhibit this enzyme could bring the full force of the body’s defenses to bear on these deadly invaders.

Incyte’s search for an IDO1 inhibitor began with a high-throughput screen, which led to a proof-of-concept compound. But the compound had poor oral bioavailability. What’s more, the molecule and its analogs underwent glucuronidation during its metabolism: Enzymes tacked on a glucuronic acid group to the structure’s amidoxime, which was key to its activity.

The chemists reasoned they could block this metabolism by sterically hindering that position. Making such molecules proved to be more difficult than they expected. But then they unearthed a Latvian paper from 1993 that gave them the synthetic method they needed to make the series of compounds that would lead to their clinical candidate INCB24360 (epacadostat).

With its furazan core, as well as its amidoxime, bromide, and sulfuric diamide functional groups, INCB24360 is something of an odd duck, Combs acknowledged. “Some of you in the audience may be looking at this and saying, ‘That molecule does not look like something I would bring forward or maybe even make,’ ” he said, noting that the structure breaks many medicinal chemistry rules. “We’re a data-centric company, and we followed the data, not the rules,” Combs told C&EN.

The compound has completed Phase I clinical trials and is now being used in collaborative studies with several other pharmaceutical companies that combine INCB24360 with other cancer immunotherapy agents.

TEAMWORK

Incyte’s team (from left): Andrew Combs, Dilip Modi, Joe Glenn, Brent Douty, Padmaja Polam, Brian Wayland, Rick Sparks, Wenyu Zhu, and Eddy Yue.

Credit: Incyte

WO2007113648A2 *	Mar 26, 2007	Oct 11, 2007	Pfizer Products Inc.	Ctla4 antibody combination therapy
US20070185165 *	Dec 19, 2006	Aug 9, 2007	Combs Andrew P	N-hydroxyamidinoheterocycles as modulators of indoleamine 2,3-dioxygenase
US20100055111 *	Feb 14, 2008	Mar 4, 2010	Med. College Of Georgia Research Institute, Inc.	Indoleamine 2,3-dioxygenase, pd-1/pd-l pathways, and ctla4 pathways in the activation of regulatory t cells
US20120058079 *	Nov 11, 2011	Mar 8, 2012	Incyte Corporation, A Delaware Corporation	1,2,5-Oxadiazoles as Inhibitors of Indoleamine 2,3-Dioxygenase

REFERENCES

1: Vacchelli E, Aranda F, Eggermont A, Sautès-Fridman C, Tartour E, Kennedy EP, Platten M, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology. 2014 Dec 15;3(10):e957994. eCollection 2014 Nov. Review. PubMed PMID: 25941578; PubMed Central PMCID: PMC4292223.

2: Liu X, Shin N, Koblish HK, Yang G, Wang Q, Wang K, Leffet L, Hansbury MJ, Thomas B, Rupar M, Waeltz P, Bowman KJ, Polam P, Sparks RB, Yue EW, Li Y, Wynn R, Fridman JS, Burn TC, Combs AP, Newton RC, Scherle PA. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010 Apr 29;115(17):3520-30. doi: 10.1182/blood-2009-09-246124. Epub 2010 Mar 2. PubMed PMID: 20197554.

3: Koblish HK, Hansbury MJ, Bowman KJ, Yang G, Neilan CL, Haley PJ, Burn TC, Waeltz P, Sparks RB, Yue EW, Combs AP, Scherle PA, Vaddi K, Fridman JS. Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors. Mol Cancer Ther. 2010 Feb;9(2):489-98. doi: 10.1158/1535-7163.MCT-09-0628. Epub 2010 Feb 2. PubMed PMID: 20124451.

//////////1204669-58-8 , INCB024360, INCB24360, epacadostat, PHASE 2, CANCER, orphan drug designation

Fc1ccc(cc1Br)N/C(=N\O)c2nonc2NCCNS(N)(=O)=O

AMG 337

phase 2, Uncategorized Comments Off

Apr 182016

PIC CREDIT.BETHANY HALFORD

Name: AMG-337(AMG337; AMG 337)
Cas 1173699-31-4
Formula: C23H22FN7O3
M.Wt: 463.46
Chemical Name: 6-[(1R)-1-[8-fluoro-6-(1-methylpyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl]-3-(2-methoxyethoxy)-5-methylidene-1,6-naphthyridine

(R)-6-(1-(8-fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one

(R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one

6-{ (lR)-l-[8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)[l,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl}-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“Compound M”),

PHASE 2 CANCER OF ESOPHAGUS

AMG-337 is a potent and highly selective small molecule ATP-competitive MET kinase inhibitor. AMG 337 inhibits MET kinase activity with an IC50 of < 5nM in enzymatic assays.
IC50 value: < 5nM [1]
Target: MET
in vitro: AMG-337 demonstrates exquisite selectivity for MET when profiled against a diverse panel of over 400 protein and lipid kinases in a competitive binding assay. In cellular assays, AMG 337 inhibits HGF-dependent MET phosphorylation with an IC50 of < 10 nM. [1] AMG 337 is a selective inhibitor of Met, which inhibits multiple mechanisms of Met activation. [2]
in vivo: AMG-337 demonstrates robust activity in MET-dependent cancer models. Oral administration of AMG 337 results in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling

AMG 337 is a potent and highly selective small molecule ATP-competitive MET kinase inhibitor that demonstrates robust activity in MET-dependent cancer models. In enzymatic assays, AMG 337 inhibited MET kinase activity with an IC₅₀ less than 5 nM. AMG 337 demonstrated exquisite selectivity for MET when profiled against a diverse panel of over 400 protein and lipid kinases in a competitive binding assay. In cellular assays, AMG 337 inhibited HGF-dependent MET phosphorylation with an IC₅₀ of less than 10 nM [1].

AMG 337 was profiled in cell viability assays using a diverse panel of over 200 cancer cell lines where on treatment with AMG 337 affected the viability of only two gastric cancer cell lines (SNU-5 and Hs746T), both of which harbor amplification of the MET gene. The AMG 337 IC₅₀ in the two sensitive cell lines was less than 50 nM, and greater than 10 µM in all other tested cell lines.

The receptor tyrosine kinase c-Met and its natural ligand, hepatocyte growth factor (HGF), are involved in cell proliferation, migration, and invasion and are essential for normal embryonic development. Deregulation of c-Met/HGF signaling can lead to tumorigenesis and metastasis and has been implicated in a variety of cancers. Several mechanisms lead to deregulation, including overexpression of c-Met and/or HGF, amplification of the MET gene, or activating mutations of c-Met, all of which have been found in human cancers.

AMG 337 is a potent and highly selective inhibitor of wild-type and some mutant forms of MET. In a competitive binding assay conducted on 402 human kinases, AMG 337 bound only to MET. In a cell viability study, the only cell lines that responded to an AMG 337 analog were gastric cancer cells harboring MET gene amplification. None of the other cell lines were sensitive to the AMG 337 analog and none harbored MET gene amplification. In secondary pharmacology assays with transporters, enzymes, ion channels, and receptors, binding to the adenosine transporter was the only activity inhibited.

In vivo, oral administration of AMG 337 resulted in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling. Further studies in an expanded panel of additional cancer cell lines derived from gastric, NSCLC, and esophageal cancer confirmed that the in-vitro anti-proliferative activity of AMG 337 correlated with amplification of MET. In those cell lines, treatment with AMG 337 inhibited downstream PI3K and MAPK signaling pathways, which translated into growth arrest as evidenced by an accumulation of cells in the G1 phase of the cell cycle, a concomitant reduction in DNA synthesis, and the induction of apoptosis [1].

In a small subset of patients with MET-amplified gastrointestinal (GI) tumors, monotherapy with the investigational agent AMG 337 produced a “dramatic” response. Of the 13 patients with MET-amplified gastric and esophageal cancers, eight experienced a response. The overall response rate in this group of patients was 62%. Response was rapid, with time to response being 4 weeks in most cases. Patients achieved tumor shrinkage and symptomatic improvement. One patient achieved a complete response and is still on treatment at 155 weeks; the others achieved partial responses or stable disease. This has led to further trials, including Phase II trials MET amplified gastric/esophageal adenocarcinoma or other solid tumors.

PAPER

Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo Antitumor Activity.

Boezio, A.A., Copeland, K.W., Rex, K., K Albrecht, B., Bauer, D., Bellon, S.F., Boezio, C., Broome, M.A., Choquette, D., Coxon, A., Dussault, I., Hirai, S., Lewis, R., Lin, M.H., Lohman, J., Liu, J., Peterson, E.A., Potashman, M., Shimanovich, R., Teffera, Y., Whittington, D.A., Vaida, K.R., Harmange, J.C.

(2016) J.Med.Chem. 59: 2328-2342

DOI: 10.1021/acs.jmedchem.5b01716

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01716

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

(R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (23)

Step 1: Coupling 9c and 13c in MeCN for 30 min at room temperature resulted in 86% yield. LRMS (ESI): m/z (M + H) 482.2. Step 2: THF for 50 min at room temperature resulted in 48% yield. The racemate was purified by supercritical fluid chromatography (SFC) by repeating 0.75 mL injections of a 30 mg/mL solution onto a Chiralpak AS-H, 2 cm × 15 cm (i.d. × length) column, eluting with 20% i-PrOH and 80% CO₂ at a flow rate of 50 mL/min to provide 120 mg peak 1 (23) with >99% ee and 150 mg of peak 2 (ent-23) with >99% ee.(29) ¹H NMR (400 MHz, Chloroform-d): δ 8.72 (d, J = 2.93 Hz, 1H), 8.31 (d, J = 0.78 Hz, 1H), 8.15 (d, J = 2.84 Hz, 1H), 7.72 (s, 1H), 7.61 (s, 1H), 7.42 (d, J = 7.82 Hz, 1H), 7.09 (dd, J = 0.73, 10.61 Hz, 1H), 7.05 (q, J= 7.00 Hz, 1H), 6.82 (d, J = 7.82 Hz, 1H), 4.26–4.37 (m, 2H), 3.97 (s, 3H), 3.80–3.88 (m, J = 3.80, 5.10 Hz, 2H), 3.49 (s, 3H), 2.15 (d, J = 7.14 Hz, 3H). HRMS (ESI): m/z (M + H) calcd, 464.1859; found, 464.1841. The solid was recrystallized in EtOH followed by the addition of H₂O to form crystalline free base monohydrate form I with a dehydration event at 40–55 °C followed by a melt at 151–153 °C. The solid could also be recrystallized in EtOH under anhydrous conditions to form crystalline anhydrous free base form I with a melting point of 151–153 °C.

PATENT

WO 2009091374

http://www.google.com/patents/WO2009091374A2?cl=en

Example 515

(SV6-(l-f8-fluoro-6-(3-methvIisoxazol-5-vn-|l,2,41triazoIo[4,3-a1pyridin-3-vncthvn-3-(f2- methoxyethoxy)methv.)-l,6-naphthyridin-5(6HVone Synthesized in the same general manner as that previously described for example 509 using General Method N. Chiral separation by preparative SFC (Chiralpak® AD-H (20 x 150 mm, 5Dm), 25% MeOH, 75% CO₂, 0.2% DEA; 100 bar system pressure; 75 mL/min; t_r 4.75min). On the basis of previous crystallographic data and potency recorded for related compound in the same program, the absolute stereochemistry has been assigned to be the S enantiomer. M/Z – 465.2 [M+H], calc 464.16 for C₂₃H₂iFN₆O₄

Example 516 ri?)-6-ri-(8-fluoro-6-(l-methyl-lH-pyrazol-4-vn-H.2.41triazolo[4,3-alpyridin-3-yl)ethyl)- 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one The title compound was synthesized using General Method N. Chiral separation by preparative SFC (Chiralpak® AS-H (20 x 150 mm, 5 Dm), 20% iPrOH, 80% CO₂; 100 bar system pressure, 50 mL/min; t_r 1.67 min). On the basis of previous crystallographic data and potency recorded for related compound in the same program, the absolute stereochemistry has been assigned to be the R enantiomer. M/Z = 464.2 [M+H], calc 463.18 for C₂₃H₂₂FN₇O₃. ¹H NMR (400 MHz, CHLOROFORM-^ D ppm 2.15 (d, J=7.14 Hz, 3 H) 3.49 (s, 3 H) 3.80 – 3.90 (m, 2 H) 3.97 (s, 3 H) 4.27 – 4.39 (m, 2 H) 6.83 (d, J=7.73 Hz, 1 H) 7.00 – 7.13 (m, 2 H) 7.42 (d, J=7.82 Hz, 1 H) 7.61 (s, 1 H) 7.72 (s, 1 H) 8.15 (d, J=2.84 Hz, 1 H) 8.31 (s, 1 H) 8.72 (d, J=3.03 Hz, 1 H).

PATENT

WO 2015161152

https://www.google.com/patents/WO2015161152A1?cl=en

6-{ (lR)-l-[8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)[l,2,4]triazolo[4,3-a]pyridin-3-yl]ethyl}-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“Compound M”), which is a selective inhibitor of the c-Met receptor, and useful in the treatment, prevention, or amelioration of cancer:

PATENT

https://www.google.com/patents/WO2014210042A2?cl=en

The overall scheme for the preparation of Compound A is shown below. The optical purity of Compound A is controlled during the synthetic process by both the quality of the incoming starting materials and the specific reagents used for the transformations. Chiral purity is preserved during both the coupling reaction (the second step) and the dehydration reaction (the third step).

NAPH (S)-halopropionic NAPA

acid/ester

PREPARATION OF COMPOUND A

In one aspect, provided herein is a method for preparing Compound A, salts of Compound A, and the monohydrate form of Compound A. Compound A can be prepared from the NAPH, PYRH, and S-propionic acid/ester starting materials in three steps. First, NAPH and ^-propionic acid/ester undergo an S 2 alkylation reaction to result in (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid/ester. The ^-propionic acid starting material produces (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid (“NAPA”) in one step. The ^-propionic ester starting material first produces the ester analog of NAPA, and is subsequently hydrolyzed to form NAPA. During workup, the acid can optionally form a salt (e.g., HC1 or 2-naphthalenesulfonic acid).

Step 1:

NAPH (S)-2-halopropionic

acid/ester

1 2

wherein R is Br, CI, I, or OTf; and R is COOH or Ci-salkyl ester, and

when R is Ci^alkyl ester, the method of forming the NAPA or salt thereof further comprises hydrolyzing the Ci-salkyl ester to form an acid.

Second, NAPA and PYRH are coupled together to form (R)-N’-(3-fluoro-5-(lmethyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin- 6(5H)yl)propanehydrazide (“HYDZ”).

Step 2:

Third, HYDZ is dehydrated to form Compound A.

The free base form of Compound A can be crystallized as a salt or a monohydrate.

Step 1: Alkylation of NAPH to form NAPA

The first step in the preparation of Compound A is the alkylation of NAPH to form NAPA. The NAPA product of the alkylation reaction is produced as a free base and is advantageously stable.

Thus, one aspect of the disclosure provides a method for preparing NAPA comprising admixing 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (“NAPH”):

‘¹ R² , and a base, under conditions sufficient to form NAPA:

wherein R¹ is Br, CI, I, or OTf; and

R² is COOH or C^alkyl ester;

and when R² is Ci_₃alkyl ester, the method of forming the NAPA or salt thereof further comprises hydrolyzing the Ci-₃alkyl ester to form an acid.

The compound, R¹ R² , represents an (^-propionic acid and/or (S)- propionic ester

(“(S)-propionic acid/ester”). When R¹ R² is an acid (i.e., R² is COOH), NAPA is formed in one step:

-prop on c ac

When R¹ R² is an ester (i.e., R² is C_1-3 alkyl ester), then the NAPA ester analog is formed, which can be hydrolyzed to form NAPA.

The S_N2 alkylation of NAPH to form NAPA occurs with an inversion of

EXAMPLE 1

SYNTHESIS OF (R)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANOIC ACID NAPHTHALENE-2-SULFONATE (NAPA)

Scheme 1: Synthesis of naphthyridinone acid 2-napsylate (NAPA)

NAPA was synthesized according to Scheme 1 by the following procedure. A jacket reactor (60 L) was charged with 3000 g (1.0 equivalent) of 3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one and 4646 g (2.0 equivalents) of magnesium ie/t-butoxide. 12 L (4.0 Vol) tetrahydrofuran was added to the reactor and an N₂sweep and stirring were initiated. 2213 g (1.5 equivalents) of S-2-bromopropionic acid was added over at least 30 min, controlling the addition such that the batch temperature did not rise above 30 °C. The charge port was rinsed with tetrahydrofuran (0.5 Vol) after addition. The batch was then aged for at least 5 min at 25 °C. 1600 g (1.05 equivalents) of potassium iert-butoxide was added to the reactor in four portions (approximately equal) such that the batch temperature did not rise above 30 °C. The charge port was again rinsed with tetrahydrofuran (1.5 L, 0.5 Vol). The batch temperature was adjusted to 35+5 °C and the batch was aged for at least 12 h.

A separate 100 L reactor was charged with 6 L of 2-Metetrahydrofuran (2-MeTHF) (2.0 Vol), 8.4 L of water (1.5 Vol) and 9.08 L (4.0 equivalents) of 6 N HC1. The mixture from the 60 L reactor was pumped into the 100 L reactor, while maintaining the batch temperature at less than 45 °C.

The batch temperature was then adjusted to 20+5°C. The pH of the batch was adjusted with 6N HC1 (or 2N NaOH) solution until the pH was 1.4 to 1.9. The aqueous layer was separated from the product-containing organic layer. The aqueous layer was extracted with 2-MeTHF (2 Vol), and the 2-MeTHF was combined with the product stream in the reactor. The combined organic stream was washed with 20% brine (1 Vol). The organic layer was polish-filtered through a < ΙΟμιη filter into a clean vessel.

In a separate vessel, 1.1 equivalents of 2-Naphthalenesulfonic acid hydrate was dissolved in THF (2 Vol). The solution was polish-filtered prior to use. The 2-naphthalenesulfonic acid hydrate THF solution was added into the product organic solution in the vessel over at least 2 h at 25+5 °C. The batch temperature was adjusted to 60+5 °C and the batch was aged for 1+0.5 h. The batch temperature was adjusted to 20+5 °C over at least 2 h. The batch was filtered to collect the product. The collected filter cake was washed with THF (5.0 Vol) by displacement. The product cake was dried on a frit under vacuum/nitrogen stream until the water content was < lwt% by LOD.

The yield of the product (R)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin-6(5H)-yl)propanoic acid naphthalene-2-sulfonate, was 87%. The chiral purity was determined using chiral HPLC and was found to be 98-99% ee. The purity was determined using HPLC, and was found to be > 98%.

Thus, Example 1 shows the synthesis of NAPA according to the disclosure.

EXAMPLE 2

SYNTHESIS OF (R)-N’-(3-FLUORO-5-(l-METHYL-lH-PYRAZOL-4-YL)PYRIDIN-2- YL)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANEHYDRAZIDE (HYDZ)

Scheme 2: Synthesis of (R)-N’-(3-fluoro-5-(l-methyl- lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanehydrazide

HYDZ was synthesized according to Scheme 2 by the following procedure. A 60 L jacket reactor was charged with 2805.0 g (1.0 equivalent) of (R)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoic acid 2-napsylate (NAPA) and N,N-dimethylacetamide (DMAC) (4.6 mL DMAC per gram of NAPA). Stirring and an N₂ sweep were initiated. 1.05 equivalents of N,N-diisopropylethylamine (DIPEA) was added while maintaining the batch temperature at less than 35°C. Initially the NAPA dissolves. A white precipitate formed while aging, but the precipitate had no impact on the reaction performance. 2197 g (1.10 equivalents) of 3-fluoro-2-hydrazinyl-5-(l-methyl- lH-pyrazol-4-yl)pyridine (PYRH) was added to the batch. The batch temperature was adjusted to 10+5 °C. 2208 g (1.2 equivalents) of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was added in four portions (approximately equal) over at least 1 h (about 20 min interval per portion) at 10+5 °C.

The batch was aged until the amide conversion target was met. If the amide conversion target was not reached within 2 h, additional EDC was added until the conversion target was met. Once the target was met, the batch was heated to 55 °C until the solution was homogeneous. The batch was filtered through a <20 μ in-line filter into a reactor. The vessel and filter were rinsed with DMAC (0.2 mL DMAC/g of NAPA). The batch temperature was adjusted to 45+5 °C.

The reactor was charged with a seed slurry of (R)-N’-(3-fluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanehydrazide (HYDZ) (0.01 equivalents) in water (0.3 mL/g).

The batch was aged at 50+5 °C for at least 30 min. The batch temperature was adjusted to 20+5°C over at least 2 h. The batch was aged at 20+5°C for at least 30 min. 2.90 mL water per g was added at 25+5 °C over at least 2 h. The batch was aged at 20+5 °C for at least 1 h. The batch slurry was filtered to collect the product. The product was washed with 30% DMAC/H₂0 (0.5 Vol) by displacement. The product cake was washed with water (3 Vol) by displacement. The product cake was dried on the frit under vacuum/nitrogen stream until the water content was < 0.2 wt% as determined by Karl Fischer titration (KF). The product was a white, crystalline solid. The yield was about 83-84%. The ee was measured by HPLC and was found to be > 99.8%ee. The purity was determined by HPLC and was found to be >99.8 LCAP (purity by LC area percentage).

Thus, Example 2 demonstrates the synthesis of HYDZ according to the disclosure.

EXAMPLE 3

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE HYDROCHLORIDE SALT (COMPOUND A-HCL) – ROUTE 1

Scheme 3 Route 1 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl- lH-pyrazol-4-yl)- [l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride salt (Compound A-

HC1) was synthesized according to Scheme 3, Route 1 by the following procedure. A 15 L reactor, Reactor 1, was charged with 750 g HYDZ and the reactor jacket temperature was adjusted to 20+5 °C. A nitrogen sweep was initiated in Reactor 1 and the condenser coolant (at 5+5 °C) was started. Acetonitrile (3.4 L, 4.5 Vol) was added to Reactor 1 and stirring was initiated. 420 g (2.5 equivalents) of 2,6-lutidine was added to the reactor.

A solution of diphenylphosphinyl chloride Ph₂P(0)(Cl) was prepared by combining 850 g (2.3 equivalents) of Ph₂P(0)(Cl) and 300 g acetonitrile in an appropriate container. The contents of the PH₂P(0)(C1) solution were added to Reactor 1. The jacket temperature was adjusted over 60+30 min until the reflux temperature of the batch (approximately 85 °C) was reached. The reaction was stirred for 14+6 h. The batch temperature was reduced to 75+5 °C and the batch was sampled for IPT analysis. The expected result was < 2% HYDZ remaining. If the target was not met, the heating at reflux temperature was continued for 9+6 h. Sampling, analysis, and heating was repeated until a satisfactory conversion assay result was obtained (< 10% HYDZ was considered satisfactory, < 1% was actually achieved). The final sample was assayed for optical purity by HPLC, and was found to be > 99.5% ee.

A K₂CO₃/KCI quench solution (5.0 Vol) was prepared in advance by combining 555 g (3.1 equivalents) of potassium carbonate with 335 g (2.9 equivalents) of potassium chloride and 3450 g of water in an appropriate container. The quench solution was added to Reactor 1 over at least 15 min, maintaining the batch temperature at 60+5 °C. As the aqueous base reacted with excess acid some bubbling (C0₂) occurred. 3.0 L (4.0 Vol) of toluene was added to Reactor 1 at 65+5 °C. A sample of the batch was taken for IPT analysis. The lower (aqueous) phase of the sample was assayed by pH probe (glass electrode). The pH was acceptable if in the range of pH 8-11. The upper (organic) phase of the sample was assayed by HPLC.

The batch was agitated for 20+10 min at 65+5 °C. Stirring was stopped and the suspension was allowed to settle for at least 20 min. The aqueous phase was drained from Reactor 1 via a closed transfer into an appropriate inerted container. The remaining organic phase was drained from Reactor 1 via a closed transfer to an appropriate inerted container. The aqueous phase was transferred back into Reactor 1.

An aqueous cut wash was prepared in advance by combining 2.3 L (3.0 Vol) acetonitrile and 2.3 L(3.0 Vol) toluene in an appropriate container. The aqueous cut wash was added to Reactor 1. The batch was agitated for 20+10 min at 65+5 °C. The stirring was stopped and the suspension was allowed to settle for at least 20 min. The lower (aqueous) phase was drained from Reactor 1 via a closed transfer into an appropriate inerted container. The organic phase was drained from Reactor 1 via a closed transfer to the inerted container containing the first organic cut. The combined mass of the two organic cuts was measured and the organic cuts were transferred back to Reactor 1. Agitation was initiated and the batch temperature was adjusted to 60+10 °C. A sample of the batch was taken and tested for Compound A content by HPLC. The contents of Reactor 1 were distilled under vacuum (about 300-450 mmHg) to approximately 8 volumes while maintaining a batch temperature of 60+10 °C and a jacket temperature of less than 85 °C. The final volume was between 8 and 12 volumes.

The nitrogen sweep in Reactor 1 was resumed and the batch temperature adjusted to 70+5 °C. A sample of the batch was taken to determine the toluene content by GC. If the result was not within 0-10% area, the distillation was continued and concomitantly an equal volume of 2-propanol, up to 5 volumes, was added to maintain constant batch volume. Sampling, analysis, and distillation was repeated until the toluene content was within the 0-10% area window. After the distillation was complete, 540 g (450 mL, 3.5 equivalents) of hydrochloric acid was added to Reactor 1 over 45+15 min while maintaining a batch temperature at 75+5 °C.

A Compound A-HC1 seed suspension was prepared in advance by combining 7.5 g of Compound A-HC1 and 380 mL (0.5 Vol) of 3 propanol in an appropriate container. The seed suspension was added to Reactor 1 at 75+5 °C. The batch was agitated for 60+30 min at 75+5 °C. The batch was cooled to 20+5 °C over 3+1 h. The batch was agitated for 30+15 min at 20+5 °C. 2.6 L (3.5 Vol) of heptane was added to the batch over 2+1 h. The batch was then agitated for 60+30 min at 20+5 °C. A sample of the batch was taken and filtered for IPT analysis. The filtrate was assayed for Compound A-HC1. If the amount of Compound A-HC1 in the filtrate was greater than 5.0 mg/mL the batch was held at 20 °C for at least 4 h prior to filtration. If the amount of Compound A-HC1 in the filtrate was in the range of 2-5 mg.ML, the contents of Reactor 1 were filtered through a < 25 μιη PTFE or PP filter cloth, sending the filtrate to an appropriate container.

A first cake wash was prepared in advance by combining 1.5L (2.0 Vol) of 2-propanol and 1.5L (2.0 Vol) of heptane in an appropriate container. The first cake wash was added to Reactor 1 and the contents were agitated for approximately 5 min at 20+5 °C. The contents of Reactor 1 were transferred to the cake and filter. A second cake wash of 3.0L (4.0 Vol) of heptane was added to Reactor 1 and the contents were agitated for approximately 5 min at 20+5 °C. The contents of Reactor 1 were transferred to the cake and filter. The wet cake was dried under a flow of nitrogen and vacuum until the heptane content was less than 0.5 wt% as determined by GC. The dried yield was 701g, 85% as a yellow powder. The dried material was assayed for chemical purity and potency by HPLC and for residual solvent content by GC. The isolated product was 88.8% Compound A-HC1, having 99.8% ee and 0.6% water.

Thus, Example 3 shows the synthesis of Compound A-HCL according to the disclosure.

EXAMPLE 4

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE HYDROCHLORIDE SALT (COMPOUND A-HCL) – ROUTE 2

HYDZ A HCI

Scheme 4: Route 2 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl- lH-pyrazol-4-yl)- [l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one hydrochloride salt was synthesized according to Scheme 4, Route 2, by the following procedure. A clean and dry 60 L reactor was fitted with a reflux condenser, nitrogen inlet, and vented to a scrubber (Reactor 1). The jacket temperature of Reactor 1 was set to 20 °C. A scrubber was set up to the vent of Reactor 1, and aqueous bleach solution was charged to the scrubber. The circulating pump (commercial 5.25% NaOCl) was initiated. The scrubber pump was turned on and N₂ sweep on Reactor 1 was started. Reactor 1 was charged with 2597 g (0.52 equivalents) of Lawesson’s reagent. Reactor 1 was then charged with 6000 g (1.0 equivalent) of HYDZ and 30 L (5.0 vol) acetonitrile (MeCN). Agitation of Reactor 1 was initiated. The reactor was heated to 50+5 °C and aged until an LC assay showed consumption of HYDZ (> 99% conversion).

The jacket temperature of a second clean and dry reactor, Reactor 2, was set to 50 °C. The contents of Reactor 1 were transferred to Reactor 2 through a 5 micron inline filter. Reactor 1 was rinsed with MeCN, and the rinse was transferred through the inline filter to Reactor 2. Reactor 2 was charged with toluene. (31.7 Kg)

In a separate container a solution of 16.7% K₂C0₃ was prepared by adding 7200 g K₂C0₃ and 36 L water to the container and shaking the container well until all the solid was dissolved. Half of the contents of the K₂C0₃ solution was added to Reactor 2 over at least 10 min. The batch temperature of Reactor 2 was adjusted to 50+5 °C. The batch in Reactor 2 was agitated at 50+5 °C for at least 1 h. The agitation was stopped and the batch in Reactor 2 was allowed to phase separate. The aqueous phase was removed. The remaining contents of the K₂C0₃ solution was added to Reactor 2 over at least 10 min. The batch temperature in Reactor 2 was adjusted to 50+5 °C. The batch in Reactor 2 was agitated at 50+5 °C for at least 1 h. The agitation was stopped and the batch in Reactor 2 was allowed to phase separate. The aqueous phase was removed.

The jacket temperature of a clean and dry reactor, Reactor 3, was set to 50 °C. The contents of Reactor 2 were transferred to Reactor 3 through a 5 micron in-line filter. The contents of Reactor 3 were distilled at reduced pressure. Isopropyl alcohol (IP A, 23.9 kg) was charged to Reactor 3 and then the batch was distilled down. IPA (23.2 kg) was again added to Reactor 3. The charge/distillation/charge cycle was repeated. The batch temperature in Reactor 3 was adjusted to 70+15 °C. Reactor 3 was then charged with DI water (1.8 L). Concentrated HC1 (1015 mL) was added to Reactor 3 over at least 15 min at 70+15 °C.

A seed of the Compound A-HCl was prepared by combining a seed and IPA in a separate container. The Compound A-HCl seed was added to Reactor 3 as a slurry. The batch in Reactor 3 was aged at 70+15 °C for at least 15 min to ensure that the seed held. The batch in Reactor 3 was cooled to 20+5 °C over at least 1 h. Heptane (24.5 kg) was added to Reactor 3 at 20+5 °C over at least 1 h. The batch was aged at 20+5 °C for at least 15 min. The contents of Reactor 3 were filtered through an Aurora filter fitted with a <25 μιη PTFE or PP filter cloth. The mother liquor was used to rinse Reactor 3.

A 50% v/v IP A/heptane solution was prepared, in advance, in a separate container by adding the IPA and heptane to the container and shaking. The filter cake from Reactor 3 was washed with the 50% IP A/heptane solution. If needed, the IP A/heptane mixture, or heptane alone, can be added to Reactor 3 prior to filtering the contents through the Aurora filter. The cake was washed with heptane. The cake was dried under nitrogen and vacuum until there was about < 0.5 wt% heptane by GC analysis. The product was analyzed for purity and wt% assay by achiral HPLC, for wt% by QNMR, for water content by KF, for form by XRD, for chiral purity by chiral HPLC, and for K and P content by ICP elemental analysis.

Compound A-HCl had a purity of 99.56 area% and 88.3 wt% assay by achiral HPLC, and 89.9 wt% by QNMR. The water content was 0.99 wt% as determined by KF. The chiral purity was 99.9%ee as determined by chiral HPLC. The P and K content was found to be 171 ppm and 1356 ppm, respectively, as determined by ICP elemental analysis.

Thus, Example 4 shows the synthesis of Compound A-HCl according to the disclosure.

EXAMPLE 5

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A) – ROUTE 3

Scheme 5: Route 3 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one (compound A)

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)- l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 5, Route 3, by the following procedure. 0.760 g (1.6 mmol) N’-iS-fluoro-S-il-methyl-lH-pyrazol-4-yl)pyridin-2-yl)-2-(3-(2-methoxyethoxy)-5-oxo- l,6-naphthyridin-6(5H)-yl)propanehydrazide (HYDZ) and 0.62 g (2.4 mmol) triphenylphosphine were taken up in 16 mL THF. 0.31 mL (2.4 mmol) trimethylsilyl (TMS)-azide was added, followed by addition of 0.37 mL (2.4 mmol) DEAD, maintaining the reaction temperature below 33 °C. The reaction was stirred at room temperature for 50 minutes. The reaction mixture was concentrated in vacuo.

The crude material was taken up in dichloromethane and loaded onto silica gel. The crude material was purified via medium pressure liquid chromatography using a 90: 10: 1 DCM : MeOH : NH₄OH solvent system. 350 mg, (48% yield) of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was collected as a tan solid. The (S) isomer was also collected. The product had a purity of 97% by HPLC.

Thus, Example 5 shows the synthesis of enantiomerically pure Compound A according to the disclosure.

EXAMPLE 6

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6-NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A) AND THE HYDROCHLORIDE SALT- ROUTE 3

Scheme 6: Route 3 – Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one (compound A) and the hydrochloride salt

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 6, Route 3, by the following procedure. Benzothiazyl disulfide (3.31 g, 9.97 mmol), HYDZ (4.0 g, 8.31 mmol), and a stir bar were added to a 50 mL 3-neck flask fitted with a reflux condenser topped with a nitrogen inlet, a thermocouple and a septum. The flask headspace was purged with nitrogen, and the solids were suspended in MeCN (20.00 mL, 5 mL/g) at ambient conditions. The flask contents were heated to 50 °C on a heating mantle. Finally,

trimethylphosphine, solution in THF (9.97 ml, 9.97 mmol) was added dropwise by syringe pump with stirring over 1 h. An ice pack was affixed to the side of the flask in lieu of a reflux condenser. After about 0.5 h from addition, the resulting suspension was sampled and analyzed by, showing about 99% conversion of penultimate, and about 94% Compound A vs.

benzothiazole-2-thiol (“BtSH”) adduct selectivity.

After about 0.75 h from addition, the yellow reaction mixture was cooled to 0 °C in an ice bath, and 30% hydrogen peroxide in water (2.037 mL, 19.94 mmol) was added dropwise over 2 hours. The reaction solution was allowed to warm to room temperature overnight.

The suspension was heated to 30 °C, held at that temperature for 3 h and then cooled to room temperature. After cooling was complete, an aliquot was filtered and the filtrate was analyzed by liquid chromatography, showing 99% Compound A vs. BtSH adduct (91% purity for Compound A overall).

A Celite filtration pad about 0.5″ thick was set up on a 50 mL disposable filter frit and wetted with toluene (32.0 mL, 8 mL/g). The reaction suspension was transferred to the Celite pad and filtered to remove BtSH-related byproducts, washing with MeCN (2.000 mL, 0.5 mL/g). The filtrate was transferred to a 100 mL round bottom flask, and treated with 30 mL (7.5 Vol) of an aqueous quench solution consisting of sodium bicarbonate (7.5 ml, 8.93 mmol) and sodium thiosulfate (3.75 ml, 4.74 mmol) at overall about 5 wt% salt. The suspension was stirred for about 15 min and then the layers were allowed to separate. Once the layers were cut, the aqueous waste stream was analyzed by LC, showing 8% loss. The organic stream was similarly analyzed, showing 71% assay yield, implying about 20% loss to waste cake.

The organic cut was transferred to a 3-neck 50 mL round bottom flask with magnetic stir bar, thermocouple, and a shortpath distillation head with an ice-cooled receiving flask. The boiling flask contents were distilled at 55 °C and 300 torr pressure. The volume was reduced to 17 mL. The distillation was continued at constant volume with concomitant infusion of IPA (about 75 mL). The resulting thin suspension was filtered into a warm flask and water (0.8 mL) was added. The solution was heated to 80 °C. After this temperature had been reached, hydrochloric acid, 37% concentrated (0.512 ml, 6.23 mmol) was added, and the solution was seeded with about 30 mg (about 1 wt%) Compound A-HC1 salt. The seed held for 15 min. Next the suspension was cooled to 20 °C over 2 h. Finally heptane (17 mL, 6 Vol) was added over 2 h by syringe pump. The suspension was allowed to stir under ambient conditions overnight.

The yellow-green solid was filtered on an M-porosity glass filter frit. The wet cake was washed with 1: 1 heptane/IPA (2 Vol, 5.5 mL) and then with 2 Vol additional heptane (5.5 mL). The cake was dried by passage of air. The dried cake (3.06 g , 78.5 wt%, 94 LC area% Compound A, 62% yield) was analyzed by chiral LC showing optical purity of 99.6% ee.

Thus, Example 6 shows the synthesis of enantiomerically pure Compound A and the hydrochloric salt thereof, according to the disclosure.

EXAMPLE 7

RE-CRYSTALLIZATION OF COMPOUND A

A-HCI A monohydrate

Scheme 7: Re-crystallization of Compound A

Compound A-HCI was recrystallized to Compound A. A (60 L) jacketed reactor, Reactor 1, with a jacket temperature of 20 °C was charged with 5291 g, 1.0 equivalent of Compound A-HCI. 2 Vol (10.6 L) of IPA and 1 Vol (5.3 L) of water were added to Reactor 1 and agitation of Reactor 1 was initiated.

An aqueous NaHC0₃ solution was prepared in advance by charging NaHC0₃ (1112 g) and water (15.87 L, 3 Vol) into an appropriate container and shaking well until all solids were dissolved. The prepared NaHC0₃ solution was added to Reactor 1 over at least 30 min, maintaining the batch temperature below 30 °C. The batch temperature was then adjusted to about 60 °C. The reaction solution was filtered by transferring the contents of Reactor 1 through an in-line filter to a second reactor, Reactor 2, having a jacket temperature of 60+5 °C. Reactor 2 was charged with water (21.16 L) over at least 30 min through an in-line filter, maintaining the batch temperature at approximately 60 °C. After the addition, the batch temperature was adjusted to approximately 60 °C.

A seed was prepared by combining Compound A seed (0.01 equivalents) and IP A/water (20:80) in an appropriate container, in an amount sufficient to obtain a suspension. The seed preparation step was performed in advance. Reactor 2 was charged with the seed slurry. The batch was aged at 55-60 °C for at least 15 min. The batch was cooled to 20+5 °C over at least 1 h. The batch from Reactor 2 was recirculated through a wet mill for at least 1 h, for example, using 1 fine rotor stator at 60 Hz, having a flow rate of 4 L/min, for about 150 min.

The reaction mixture was sampled for particle size distribution during the milling operation. The solids were analyzed by Malvern particle size distribution (PSD) and

microscopic imaging. At the end of the milling operation a sample of the reaction mixture was again analyzed. The supernatant concentration was analyzed by HPLC, and the solids were analyzed by Malvern PSD and microscopic imaging to visualize the resulting crystals.

The batch temperature was adjusted to 35+5 °C and the batch was aged for at least 1 h. The batch was cooled to 20+5 °C over at least 2 h. The reaction mixture was sampled to determine the amount of product remaining in the supernatant. The supernatant concentration was analyzed by HPLC for target of <5 mg/mL Compound A in the supernatant. The contents of Reactor 2 were filtered through an Aurora filter fitted with a <25 μιη PTFE or PP filter cloth.

A 20% v/v IP A/water solution was prepared and the filter cake from Reactor 2 was washed with the 20% IP A/water solution. The cake was then washed with water. If needed, the IP A/water solution, or water alone, can be added to Reactor 2 prior to filtering to rinse the contents of the reactor. The cake was dried under moist nitrogen and vacuum until target residual water and IPA levels were reached. The product had 3.2-4.2% water by KF analysis. The product was analyzed by GC for residual IPA (an acceptable about less than or equal to about 5000 ppm). The yield and purity were determined to be 100% and 99.69% (by HPLC), respectively.

Thus, Example 6 shows the recrystallization of Compound A from the HC1 salt, Compound A-HC1, according to the disclosure.

EXAMPLE 8

SYNTHESIS OF (R)-6-(l-(8-FLUORO-6-(l-METHYL-lH-PYRAZOL-4-YL)- [l,2,4]TRIAZOLO[4,3-A]PYRIDIN-3-YL)ETHYL)-3-(2-METHOXYETHOXY)-l,6- NAPHTHYRIDIN-5(6H)-ONE (COMPOUND A)

HYDZ A

Scheme 8 Synthesis of (R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one

(R)-6-(l-(8-fluoro-6-(l-methyl-lH-pyrazol-4-yl)-[l,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-l,6-naphthyridin-5(6H)-one was synthesized according to Scheme 8 by the following procedure. A clean and dry 60 L reactor was fitted with a reflux condenser, nitrogen inlet, and vented to a scrubber (Reactor 1). The jacket temperature of Reactor 1 was set to 20 °C. A scrubber was set up to the vent of Reactor 1, and aqueous bleach solution was charged to the scrubber. The circulating pump (commercial 5.25% NaOCl) was initiated. The scrubber pump was turned on and N₂ sweep on Reactor 1 was started. Reactor 1 was charged with 1599.5 g (0.52 equivalents) of Lawesson’s reagent. Reactor 1 was then charged with 24.4 L acetonitrile (MeCN). Agitation of Reactor 1 was initiated. 3664.7 g (1.0 equivalent) of HYDZ was added to the reactor in portions over 1+0.5 h, using acetonitrile (5 L) as rinse. The reactor was heated to 50+5 °C and aged until an LC assay shows consumption of HYDZ (> 99% conversion).

The reactor was cooled to 20 °C and the reaction was assayed by HPLC for

Compound A. The assay showed a 99% crude yield of Compound A.

The contents of Reactor 1 were transferred to second reactor, Reactor 2, through a 1 micron inline filter. Reactor 2 was charged with 2 L of water. Reactor 2 was connected to a batch concentrator and vacuum distilled until a final volume of about 10 L. The jacket temperature was 50 °C during distillation and the pot temperature was maintained below 50 °C. The batch was then cooled to 20 °C.

In a separate container a solution of 10% K₂CO₃ was prepared by adding 1160 g K₂CO₃ and 10450 mL water to the container and shaking the container well until all the solid was dissolved. The K₂CO₃ solution was added to Reactor 2 through an in-line filter (5 μηι). 13 kg of purified water was added to the reactor through the in-line filter (5 μηι).

A Compound A seed was added to the reactor through an addition port. The resulting slurry was aged for one hour during which crystallization was observed. The reactor was placed under vacuum and charged with 16 L of water. The resulting slurry was aged at 20 °C overnight. The product slurry was filtered through a 25 μιη filter cloth and washed with 10 L of a 10% MeCN in water solution, followed by 12 L of water. The product was dried on a frit under a stream of ambient humidity filtered air.

Compound A was isolated as a monohydrate crystalline solid which reversibly dehydrates at < 11% RH. After drying, there was 3.9 wt.% water present in constant weight solid as determined by KF. 3.317 kg, 89% yield, of Compound A was isolated as a pale yellow solid. The product had a purity of 99.4 wt.% as determined by LCAP.

EXAMPLE 9

SYNTHESIS OF NAPH – ROUTE 1

CuBr (5-10%)

ethyl 5-bromo-2- Bromonaphthyridinone Naphthyridinone ether methylnicotinate

Scheme 9: Synthesis of NAPH – Route 1

The NAPH starting material for the synthesis of Compound A was synthesized according to Scheme 9, Route 1 by the following procedure. The jacket temperature of a 6 L jacketed reactor, Reactor 1, was set to 22 °C. 2409 g (1.0 equiv) of ethyl 5-bromo-2-methylnicotinate, 824 g (1.0 equivalent) of triazine, and 3.6 L dimethyl sulfoxide (DMSO) were added to the reactor. The jacket temperature was adjusted to 45 °C. The reactor was agitated until a homogenous solution resulted. Once complete dissolution has occurred (visually) the jacket of Reactor 1 was cooled to 22 °C.

A second, 60 mL reactor, Reactor 2, was prepared. 8.0 L of water was charged to a scrubber. 4.0 L of 10 N sodium hydroxide was added to the scrubber and the scrubber was connected to Reactor 2. The cooling condenser was started. 6411.2 g of cesium carbonate and 12.0 L of DMSO were added to Reactor 2. Agitation of Reactor 2 was initiated. The batch temperature of Reactor 2 was adjusted to 80 °C. The solution from Reactor 1 was added slowly over 1 h at 80 °C, while monitoring the internal temperature. 1.2 L of DMSO was added to Reactor 1 as a rinse. The DMSO rinse was transferred from Reactor 1 to Reactor 2 over 6 min. Reactor 2 was agitated for more than 1 h and the conversion to 3-bromo-l,6-naphthyridin-5(6H)-one was monitored by HPLC until there was < 1.0% ethyl 5-bromo-2-methylnicotinate remaining. When the reaction was complete the batch temperature was adjusted to 60 °C. 24.0 L (10V) of water was added to Reactor 2 over 2 h, maintaining a reaction temperature of 60+5 °C, using a peristaltic pump at 192 mL/min. Reactor 2 was cooled to 22 °C over 1 h 10 min. Stirring was continued at 22+5 °C until the supernatant assays for less than 3mg/mL of 3-bromo-l,6-naphthyridin-5(6H)-one (analyzed by HPLC). The crystallized product was filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The reactor and filter cake were washed with a 75 wt% H₂0-DMSO solution (3 Vol made from 1.6 L DMSO and 5.6 L water), followed by water (7.2 L, 3 Vol), and finally toluene (7.2 L, 3 Vol). The product cake was dried on the aurora filter under vacuum with a nitrogen stream at ambient temperature. The product was determined to be dry when the KF was < 2.0 wt% water. 2194 g of 3-bromo-l,6-naphthyridin-5(6H)-one was isolated as a beige solid. The chemical purity was 99.73%. The adjusted yield was 2031.6 g (91.9%).

The jacket temperature of a 100 L reactor, Reactor 3, was set to 15+5 °C. 6.45 L of 2-methoxyethanol was added to the reactor and agitation was initiated. (8107 g) lithium tert-butoxide was added portion- wise to the reactor, maintaining the reactor temperature in a range of 15 °C to 24 °C. 3795 g of 3-bromo-l,6-naphthyridin-5(6H)-one was added to the reactor. 4 mL of 2-methoxyethanol was added to rinse the solids on the wall of the reactor. The reactor contents were stirred for at least 5 min. The reaction mixture was heated to distillation to remove i-BuOH and water, under 1 atm of nitrogen (jacket temperature 145 °C). Distillation continued until the pot temperature reached 122+3 °C. The reactor contents were sampled and analyzed for water content by KF. The reaction mixture was cooled to less than 35 °C. 243 g CuBr was added to the reactor. The reaction mixture was de-gassed by applying vacuum to 50 torr and backfilling with nitrogen three times. The batch was heated to 120+5 °C while maintaining the jacket temperature below 150 °C. The batch was agitated (174 RPM) for 15.5 h. A sample of the reaction was taken and the reaction progress was monitored by HPLC. When the remaining 3-bromo-l,6-maphthyridin-5(6H)-one was less than 1%, the jacket temperature was cooled down to 25 °C.

An Aurora filter was equipped with a 25 μιη PTFE cloth and charged with Celite^®. The reactor content was transferred onto the filter cloth and the filtrate was collected in the reactor. 800 mL of 2-methoxyethanol was added to the reactor and agitated. The reactor contents were transferred onto the filter and the filtrate was collected in the reactor. 5.6 L of acetic acid was added to the reactor to adjust the pH to 6.5, while maintaining the temperature at less than 32 °C. The batch was then heated to 80 °C. The reaction mixture was concentrated to 3.0+5 Vol (about 12 L) at 80+5 °C via distillation under vacuum.

In a separate container labeled as HEDTA Solution, 589.9 g of N-(2-hydroxyethyl)ethylenediaminetriacetic acid trisodium salt hydrate and 7660 mL water were mixed to prepare a clear solution. The HEDTA solution was slowly added to the reactor while maintaining the temperature of the batch at about 80-82 °C. The batch was then cooled to 72 °C.

An aqueous seed slurry of NAPH (31.3g) in 200 mL of water was added to the reactor. The slurry was aged for 30+10 min. 20 L of water was slowly added to the reactor to maintain the temperature at 65+5 °C. The batch was aged at 65+5 °C for 30 min. The batch was cooled to 20 °C over 1 h. The reactor contents were purged with compressed air for 1 h, and then the batch was further cooled to – 15 °C and aged for 12.5 h. The batch was filtered through a centrifuge fitted with 25 μιη PTFE filter cloth. 5.31 Kg of wet cake was collected (60-62 wt ). The wet cake was reslurried in 6V HEDTA solution and filtered through the centrifuge. The collected wet cake was dried in the centrifuge, and transferred to an Aurora filter for continued drying.

2.82 kg (76% isolated yield) of NAPH was collected having a 2.7% water content by KF.

Thus, Example 8 shows the synthesis of NAPH according to the examples.

EXAMPLE 10

SYNTHESIS OF NAPH – ROUTE 2

Scheme 10: Synthesis of NAPH via Route 2

The NAPH starting material for the synthesis of Compound A was synthesized according to Scheme 10, Route 2, by the following procedure.

Preparation of protected 2-methoxy-pyridin-4ylamine. A 1600 L reactor was flushed with nitrogen and charged with 120 L of N,N-dimethylacetamide, 100.0 kg 2-methoxy-pyridin-4-ylamine, and 89.6 kg triethylamine, maintaining the temperature of the reactor at less than 20 °C. In a separate container, 103.0 kg pivaloyl chloride was dissolved in 15.0 L of N,N-dimethylacetamide and cooled to less than 10 °C. The pivaloyl chloride solution was added to the reactor using an addition funnel over 3.2 hours while maintaining the reactor temperature between 5 °C and 25 °C. The addition funnel was washed with 15.0 L of N,N-dimethylacetamide, which was added to the reactor. The reaction was stirred for 2.3 hours at 20-25 °C. A sample of the reaction was taken and analyzed for 2-methoxy-pyridin-4ylamine by TLC. No 2-methoxy-pyridin-4ylamine remained in the solution and the reaction was aged at 20-25 °C under nitrogen over night. 1200 L of deionized water was added to the reaction over 2

hours at while the reaction was maintained at 5-15 °C. The resulting mixture was stirred at 15 °C for 2 hours and then cooled to 5 °C. The reaction was centrifugated at 700-900 rpm in 3 batches. Each batch was washed 3 times with deionized water (3x 167 L) at 800 rpm. The wet solids obtained were dried under vacuum at 55 °C for 18 hours in 2 batches, sieved and dried again under vacuum at 55 °C for 21 hours until the water content was < 0.2% as determined by KF. 80.4 kg (89.7% yield) of the protected 2-methoxy-pyridin-4ylamine was collected as a white solid.

Preparation of protected 3-formyl-4-amino-2-methoxypyridine. A 1600 L reactor was flushed with nitrogen and charged with 1000 L of THF and 70.5 kg of the protected 2-methoxy-pyridin-4ylamine. The reaction was stirred for 10 min at 15-25°C. The reaction was cooled to -5 °C and 236.5 kg of w-hexyllithium (solution in hexane) was added over 11.5 hours while maintaining the temperature of the reaction at <-4°C. The reaction was maintained at <-4°C for 2 hours. A sample of the reaction was quenched with D₂0 and the extent of the ortho-lithiation was determined by 1H NMR (98.2% conversion). 61.9 kg dimethylforaiamide (DMF) was added at <-4°C over 3.2 h. After stirring 7.5 hours at <-4°C, a sample of the reaction was assayed for conversion by HPLC (98.5% conversion).

A 1600 L reactor, Reactor 2, was flushed with nitrogen and charged with 145 L THF and 203.4 kg of acetic acid. The resulting solution was cooled to -5 °C. The content of the first reactor was transferred to Reactor 2 over 2.5 hours at 0 °C. The first reactor was washed with 50 L THF and the washing was transferred into Reactor 2. 353 L deionized water was added to Reactor 2 while maintaining the temperature at less than 5 °C. After 15 min of decantation, the aqueous layer was removed and the organic layer was concentrated at atmospheric pressure over 5 hours until the volume was 337 L. Isopropanol (350 L + 355 L) was added and the reaction was again concentrated at atmospheric pressure until the volume was 337 L. Distillation was stopped and 90 L of isopropanol was added to the reactor at 75-94 °C. 350 L of deionized water was added to the reactor at 60-80 °C over 1 h (the temperature was about 60-65 °C at the end of the addition). The reaction was cooled to 0-5 °C. After 1 hour, the resulting suspension was filtered. Reactor 2 was washed twice with deionized water (2x 140L). The washings were used to rinse the solid on the filter. The wet solid was dried under vacuum at 50 °C for 15 h. 71.0 kg (80% yield) of the protected 3-formyl-4-amino-2-methoxypyridine was produced. The purity of the formyl substituted pyridine was found to be 92.7% by LCAP.

A 1600 L reactor, Reactor 3, was flushed with nitrogen and successively charged with 190 L ethanol, 128.7 kg of protected 3-formyl-4-amino-2-methoxypyridine, 144 L of deionized water and 278.2 kg of sodium hydroxide. The batch was heated to 60-65°C and 329.8 kg of the bisulfite adduct was added over 1 h. After lh of stirring, a sample was taken for HPLC analysis which showed 100% conversion. The batch was aged 2 hours at 60-65 °C, then was allowed to slowly cool down to 20-25 °C. The batch was aged 12 h at 20-25 °C. The batch was filtered and the reactor was washed with water (2x 125 L). The washings were used to rinse the solid on the filter. The wet solid was transferred to the reactor with 500 L deionized water and heated to 45-50 °C for 1 h. The batch was allowed to return to 20-25 °C (24 h). The solid was filtered and the reactor was washed with deionized water (2x 250 L). The washings were used to rinse the solid on the filter. 112.5 kg of wet white solid was obtained (containing 85.1 Kg (dry) of the naphthyridine, 72.3% yield, greater than 97% purity as determined by HPLC). The wet product was used directly in the next step, without drying.

A 1600 L reactor was flushed with nitrogen and charged with 417 L of deionized water and 112.5 kg of the wet napthyridine. The scrubber was filled with 700 L of water and 92.2 kg monoethanolamine. A solution of hydrochloric acid (46.6 kg diluted in 34 L of deionized water) was added to the reactor at 15-20 °C over 10 minutes. The batch was heated to 60-65 °C for 3 h. A sample of the batch was taken and contained no remaining starting material as determined by TLC. A solution of concentrated sodium hydroxide (58.2 kg in 31 L of deionized water) was added to the reactor at 60-65 °C. 65% of the solution was added over 15 min and then the batch was seeded with crystallized NAPH. Crystallization was observed after 2.5 h and then the remaining35% of the sodium hydroxide solution was added (pH – 11.1). The batch was cooled to 25-30 °C and a solution of sodium phosphate monobasic (1.8 kg in 2.9 L of deionized water) was added over 25 min at 25-30 °C) (pH = 6.75). The batch was stirred at 15-20 °C for 12 hours and filtered. The reactor was washed twice with deionized water (2x 176 L). The washings were used to rinse the solid on the filter. The wet solid was dried under vacuum at 50 °C until the water content was < 5% (by KF), to give 78.1 kg (73.8% yield, > 95%)) of NAPH as a beige powder.

Thus, Example 9 shows the synthesis of NAPH according to the disclosure.

EXAMPLE 11

SYNTHESIS OF (R)-2-(3-(2-METHOXYETHOXY)-5-OXO-l,6-NAPHTHYRIDIN-6(5H)- YL)PROPANOIC ACID NAPHTHALENE-2-SULFONATE (NAPA)

6N HCI/ THF 80C

Scheme 11: Synthesis of NAPA, Route 3

NAPA was synthesized according to Scheme 11, Route 3 by the following procedure. 4.75 g of 3-(2-Methoxyethoxy)-l,6-naphthyridin-5(6H)-one was suspended in 45 mL of DMF. 2.58 mL (s)-methyl lactate and 9.05 g triphenylphosphine were added to the suspension. The reaction mixture was cooled to 0 °C. 5.12 mL diethyl azodicarboxylate (DEAD) was added dropwise via syringe. The mixture was stirred at 0 °C for 1 h. A sample of the reaction was taken and the reaction was determined to be complete by LCMS. The reaction mixture was concentrated under vacuum to give crude material as a yellow oil.

1 g of the crude material was loaded in dichloromethane onto a silia gel pre-column. The sample was purified using the Isco Combi-Flash System; column 40 g, solvent system hexane/ethyl acetate, gradient 0-100% ethyl acetate over 15 minutes. Product eluted at 100% ethyl acetate. The product fractions were combined and concentrated under vacuum. 256 mg of (R)-methyl 2-(3-(2-methoxyethoxy)-5-oxo-l,6-naphthyridin-6(5H)-yl)propanoate was collected as a pale yellow oil.

The remaining residue was partitioned between benzene and 6N aq hydrochloric acid (35.9 mL). The acidic layer was extracted with benzene (3x), diethyl ether (2x), ethyl acetate (2x) and dichloromethane (lx). The dichloromethane layer was back extracted with 6N aq. Hydrochloric acid (2x). The aqueous layer was diluted with THF (80 mL). The mixture was heated at 80 °C for 3 h. The reaction mixture was concentrated to remove the THF. The remaining acidic water layer was extracted with ethyl acetate and dichloromethane. The aqueous layer was concentrated under vacuum. The remaining solid was triturated with methanol. The mixture was filtered to remove the solid (naphthyridone). The methanol layer was concentrated under vacuum. The remaining solid was dried overnight on a freeze drier. 10.2 g of material was collected as a yellow solid. NAPA made up 72% of the material as determined by HPLC.

1.0 g of the crude material was dissolved in minimal hot iPrOH then filtered and cooled to RT. Crystallization didn’t occur; therefore the solution was cooled in the freezer overnight. A yellow precipitate formed. The solid was collected on a glass frit and was washed with minimal iPrOH. 171 mg of yellow solid was collected, which was NAPA with a small amount of naphthyridone by LC-MS and 1H NMR.

Acid-base extraction. About 1 g of the crude material was dissolved in saturated aqueous sodium bicarbonate. The crude material was extracted with dichloromethane. The pH of the aqueous layer was adjusted to 6-7 with acetic acid then extracted with dichloromethane. 11 mg of the product was isolated; the majority of the product remained in the aqueous layer. The pH was reduced to approximately 4-5 with additional acetic acid. The aqueous layer was extracted with dichloromethane, ethyl acetate, and 15% methanol/dichloromethane. The organic layers were concentrated under vacuum to yield 260 mg of NAPA as the free base, as determined by LC-MS.

Thus, Example 10 shows the synthesis of NAPA according to the disclosure.

EXAMPLE 12

SYNTHESIS OF BISULFITE ADDUCT

DMSO

(COCI)₂

MeCX ,ΟΗ E_t3N

aqueous solution

Scheme 12: Synthesis of bisulfite adduct

Method 1

The bisulfite adduct was synthesize according to Method 1 of Scheme 12 by the following procedure. A 2L round-bottom flask (RBF) was purged with nitrogen and charged with 73.1 mL of reagent grade oxalyl chloride and 693 mL methylene chloride. The batch was cooled to less than -40 °C. 88 mL of dimethyl sulfoxide was added to the flask via an addition funnel at less than -40 °C. After the addition, the batch was stirred for 10 in at -60 °C. 97 mL diethylene glycol monomethyl ether was added to the flask at less than -50 °C over 10 min. The resulting white slurry was stirred at -60 °C for 30 min. 229 mL triethylamine was added to the flask via an addition funnel at less than -30 °C over 1 h. The batch was warmed to RT. 300 mL MTBE was added to the flask and the batch was stirred for 15 min. The slurry was filtered through a fritted funnel and the cake was washed with 300 mL MTBE. The filtrate was concentrated to 350-400g and then filtered again to remove triethylamine-HCl salt, and the solid was rinsed with MTBE, resulting in 357.7 g of a slightly yellow filtrate solution. The solution was assayed by QNMR and comprised 19 wt (68 g) of the desired aldehyde (70% crude yield). The solution was concentrated to 150.2 g.

A 500 mL RBF was charged with 60.0 g sodium bisulfite and 150 mL of water to give a clear solution. The concentrated aldehyde solution was added to the aqueous bisulfite solution over 5 min. An exothermic temperature rising was observed up to 60 °C from 18 °C. The solution was rinsed with 15 mL water. The resulting yellow solution was cooled to RT and was stirred under a sweep of nitrogen overnight.. A QNMR of the solution was taken. The solution contained 43 wt.% of the bisulfite adduct (300 g, 70% yield).

Method 2

The bisulfite adduct was synthesized according to Method 2 of Scheme 12 by the following procedure. A 2500 L reactor was flushed with nitrogen and charged with 657.5 L of 2-methoxyethanol. 62.6 kg of lithium hydroxide monohydrate was added to the reactor while maintaining the temperature at less than 30 °C. The reactor was heated to 113+7 °C. 270 L of solvent were distilled over 1 h and then the reactor temperature was adjusted to 110 °C. 269.4 kg of bromoacetaldehyde diethyl acetal was added over 16 minutes, maintaining the temperature between 110 and 120 °C. The reaction was heated to reflux (115-127°C) for 13 hours. A sample of the reaction was assayed and conversion to 2-(2-methoxyethoxy)acetaldehyde was found to be 98.3%. The reaction was cooled to 15-20°C and 1305 L of methyl ie/t-butyl ether (MTBE) and 132 L of deionized water was added to the reactor. The reaction was stirred for 20 min and then was decanted. The aqueous layer was transferred into a 1600 L reactor and the organic layer was kept in the first reactor. The aqueous layer was extracted with 260 L of MTBE for 10 min. After 10 min decantation, the aqueous layer was removed and the organic layer was transferred to the first reactor. The mixed organic layers were washed twice, 15 min each, with a mixture of concentrated sodium hydroxide solution (2x 17.3 kg) diluted in deionized water (2x 120 L). The aqueous layers were removed, and the organic layer was concentrated at atmospheric pressure at 60-65 °C until the volume was 540 L. The organic layer was cooled down to 15-20 °C to give 2-(2-methoxyethoxy)acetaldehyde as an orange liquid solution (417.4 kg) containing 215.2 kg of pure product (87.3% yield) as determined by 1H NMR and HPLC assay.

A 1600 L reactor, Reactor 3, was flushed with nitrogen and charged with 595 L deionized water followed by 37.8 kg sulfuric acid over 25 minutes via addition funnel, while maintaining the temperature below 25 °C. The addition funnel was washed with 124 L of deionized water and the washing was added to Reactor 3.

A 2500 L reactor, Reactor 4, was flushed with nitrogen and charged with 417.4 kg of the solution of the 2-(2-methoxyethoxy)acetaldehyde. The content of Reactor 3 was transferred into Reactor 4 over 25 min while maintaining the temperature of Reactor 4 below 35 °C. The batch was aged at 30-35 °C for 3 hours. A sample of the batch was taken and assayed for 2-(2- methoxyethoxy)acetaldehyde. No 2-(2-methoxyethoxy)acetaldehyde remained. The batch was aged 5 h then cooled to 15-20 °C.

A solution of sodium carbonate (39.2 kg) in deionized water (196 L) was prepared in Reactor 3. The sodium carbonate solution was transferred to Reactor 4 over 25 min while maintaining the temperature of Reactor 4 below 30 °C. The pH of the resulting mixture was pH 5-6. 1.0 kg sodium carbonate was added by portion until the pH was about 7-8. A solution of sodium bisulfite (116.5 kg) in deionized water (218 L) was prepared in Reactor 3. The sodium bisulfite solution was transferred to Reactor 4 over 20 min while maintaining the temperature of Reactor 4 below 30 °C. Reactor 3 was washed with deionized water (15 L) and the washing was added to Reactor 4. The batch was stirred for 1.2 hours. 23.3 kg sodium bisulfite was added to Reactor 4 and the batch was aged overnight. The batch was concentrated under vacuum at 30-50 °C over 6.5 hours until precipitation was observed. The batch was cooled to 0-10°C at atmospheric pressure. After 30 min at 0-10 °C, the suspension was filtered on 2 filters. Reactor 4 was washed with deionized water (2x 23 L). The first washing was used to rinse the solid on the first filter and the second washing was used to rinse the solid on the second filter. Filtrates were joined to give 473.9 kg of an aqueous solution of the bisulfite adduct (202.5 kg of pure product, 76.3% yield) as a yellow liquid.

Thus, Example 11 shows the synthesis of the bisulfite adduct according to the invention.

EXAMPLE 13

SYNTHESIS OF 2,3-DIFLUORO-5-(l-METHYL-lH-PYRAZOL-4-YL)PYRIDINE

Scheme 13: Synthesis of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine, precursor to PYRH

2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was synthesized according to Scheme 13 by the following procedure. A boronic-ate complex slurry was prepared in a first 3-neck-2-L round-bottom flask (RBF #1). RBF #1 was charged with 141 g (66.4 wt%, 0.9 equivalents based on boronic ester) of lithium 2-hydroxy-4,4,5,5-tetramethyl-2-(l-methyl-lH-pyrazol-4-yl)-l,3,2-dioxaborolan-2-uide. 120 mL (1.6 Vol relative to 5-chloro-2,3-difluoropyridine) of nitrogen- sparged (2 h) 2-BuOH and 120 mL (1.6 Vol) nitrogen-sparged (2 h) water were added to RBF #1. Agitation and N₂ sweep were initiated. The reaction was aged at 20 °C for at least 30 min (reactions aged to 24 h were also successful).

] A second 3-neck-2-L round-bottom flask (RBF #2) was charged with 1.48 g (0.004 equivalents) of Xphos-palladacycle and 450 mL (6 Vol relative to 5-chloro-2,3-difluoropyridine) of nitrogen- sparged (2 h) 2-BuOH. Vacuum/N₂ flush was cycled through RBF #2 three times to inert the RBF with N₂. The batch in RBF #2 was heated to 80 °C. 75 g (1.0 equivalents) of 5-chloro-2,3-difluoropyridine was added to RBF #2.

The slurry of boronic-ate complex was transferred from RBF #1 to a 500 mL dropping funnel. RBF #1 was rinsed with 30 mL (0.4 Vol) 2-BuOH. Using the dropping funnel, the slurry of boronic-ate complex was added over 1 h to the hot solution mixture in RBF #2. After 1 h, 95% conversion was observed. If greater than 90% conversion was not observed, additional boronic-ate complex slurry was added (0.1 equivalents at a time with 1.6 Vol of 1: 1 2-BuOH/water relative to boronic-ate complex). After the conversion was complete, the batch was cooled to 50 °C. While cooling, 600 mL (8 Vol) of toluene was added to RBF #2. 300 mL (4 Vol) of 20% w/v NaHS0₃ in water was added to RBF #2 and the batch was stirred at 50 °C for at least 1 h. The batch was polish filtered using a 5 micron Whatman filter at 50 °C, into a 2-L Atlas reactor. RBF #2 was rinsed with 30 mL (4.0 Vol) of a 1: 1 2-BuOH:toluene solution. The temperature of the batch was adjusted to 50 °C in the Atlas reactor while stirring. The stirring was stopped and the phases were allowed to settle for at least 15 min while maintaining the batch at 50 °C. The bottom, aqueous layer was separated from the batch. The Atlas reactor was charged with 300 mL (4 Vol) of a 20% w/v NaHS0₃ solution and the batch was stirred at 50°C for 1 h. The agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous layer was removed. Agitation was initiated and the Atlas reactor was charged with 200 mL (4 Vol) of 0.5 M KF while keeping the batch at 50 °C for at least 30 min. The agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous layer was removed. Agitation was initiated and the reactor was charged with 300 mL (4 Vol) of water. The batch was aged at 50 °C for at least 30 min. Agitation was stopped and the phases were allowed to settle for at least 15 min at 50 °C. The bottom, aqueous later was removed.

The organic phase was concentrated by distillation under reduced pressure (180 torr, jacket temp 70°C, internal temp about 50 °C) to a minimal stir volume (about 225 mL). 525 mL (7 Vol) of 2-BuOH was added to the Atlas reactor. The organic batch was again concentrated using reduced pressure (85-95 torr, jacket temp 75 °C, internal temp about 55 °C) to a minimal stir volume (about 125 mL). The total volume of the batch was adjusted to 250 mL with 2-BuOH.

525 mL (7 Vol) heptane was added to the slurry mixture in the Atlas reactor. The jacket temperature was adjusted to 100 °C and the batch was aged for more than 15 min, until the batch became homogeneous. The batch was cooled to 20 °C over at least 3 h. A sample of the mixture was taken and the supernatant assayed for 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine. If the concentration was greater than 10 mg/mL, the aging was continued for at least 1 h until the supernatant concentration was less than 10 mg/mL. The batch was filtered using a medium frit. The filter cake was washed with 150 mL (2 Vol) 30% 2-BuOH/heptane solution followed by 150 mL (2 Vol) heptane. The filter cake was dried under N₂/vacuum. 76.64 g of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was isolated as a white solid (87% yield).

A 60 L jacketed reactor was fitted with a reflux condenser. The condenser cooling was initiated at 0+5 °C. The reactor was charged with 2612 g (1 equivalent) of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine and placed under an atmosphere of nitrogen. 31.7 L (12.2 Vol) water was added to the reactor and the resulting slurry was nitrogen sparged for 1 h with agitation. 7221 mL (6 equivalents) of hydrazine (35 wt% in water) was added to the reactor under a nitrogen atmosphere. The reactor was heated to 100 °C for 2+2 h until reaction was complete by HPLC analysis. The reactor was cooled to 20 °C over 2+1 h at a rate of 40°C/h. The reactor contents were stirred for 10+9 hours until the desired supernatant assay (< 2mg/mL PYRH in mother liquor). The reactor contents were filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The collected filter cake was washed with 12.0 L (4.6 V) of water in three portions. The filter cake was dried on the Aurora filter for 4-24 h at 22+5 °C, or until the product contained less than 0.5% water as determined by KF. The dry product was collected. 2.69 kg (97% yield) 2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was collected as a white crystalline solid. The solid had a water content of 12 ppm as determined by KF.

Thus, Example 12 shows the synthesis of 2,3-Difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine, a precursor to PYRH, according to the disclosure.

EXAMPLE 14

SYNTHESIS OF PYRH – ROUTE 2

Scheme 14: Synthesis of 3-fluoro-2-hydrazinyl-5-(l-methyl-lH-pyrazol-4-yl)-pyridine (PYRH)

3-fluoro-2-hydrazinyl-5-(l-methyl-lH-pyrazol-4-yl)-pyridine was synthesized according to Scheme 14 by the following procedure. A 60 L jacketed reactor was fitted with a 5 L addition funnel and the jacket temperature was set to 20+5 °C. 36.0 L (15 Vol) of 2-methyltetrahydrofuran was added to the reactor via a 20 μιη inline filter with vacuum using polypropylene transfer lines. The solution was sparged by bubbling nitrogen through a dipstick in the solution for 1+0.5 h with agitation. After 1 h the dipstick was removed but the nitrogen sweep continued. 1.55 kg of sparged 2-MeTHF was removed to be used as rinse volumes. 36.7 g of Pd₂dba₃, 75.6 g X-Phos, 259 g of tetrabutylammonium bromide, and 7397 g of potassium phosphate tribasic were added to the reactor. The manhole was rinsed with 0.125 kg of sparged 2-MeTHF. The reactor was agitated and the nitrogen sweep continued for 1+0.5 h. Then the nitrogen sweep was stopped and the reaction left under a positive pressure of nitrogen.

3.6 L (1.5 Vol) of sparged water was prepared in advance by bubbling nitrogen through a 4 L bottle of water for 1+0.5 h. The nitrogen sparged water was transferred to the 5 L addition funnel via a 20 μηι inline filter with vacuum using polypropylene transfer lines, then slowly added to the reaction while maintaining the internal temperature at 20+5 °C. The 5 L addition funnel was replaced with a 2 L addition funnel. 2412 g of 5-chloro-2,3-difluoropyridine was added to the 2 L addition funnel. The 5-chloro-2,3-difluoropyridine was then added to the reaction through the 2 L addition funnel. The 2L addition funnel was rinsed with 0.060 kg of sparged 2-MeTHF. 83.8 g (1.15 equivalents) of l-methylpyrazole-4-boronic acid, pinacol ester was added to reactor, the reactor was swept with nitrogen for 1+0.5 h, then left under a positive pressure of nitrogen. The internal temperature of the reactor was adjusted to 70+5 °C. The batch was agitated at 70+5 °C for at least 4 hours after the final reagent was added. A sample was taken from the reaction and the reaction progress assayed for conversion. The progress of the reaction was checked every 2 hours until the reaction was completed (e.g., greater than 99% conversion). The batch was cooled to 20+5 °C.

A 20% w/v sodium bisulfite solution (12.0 L, 5 Vol) was prepared by charging 12.0 L of water then 2411 g sodium bisulfite to an appropriate container and agitating until

homogeneous. The 20% sodium bisulfite solution was transferred into the reactor and agitated for 30 minutes. The agitation was stopped, the phases allowed to settle, and the aqueous phase was removed. A 0.5 M potassium fluoride solution (12.0 L, 5 Vol) was prepared by charging 12.0 L of water and 348 g of potassium fluoride to an appropriate container and agitating until homogenous. The 0.5 M potassium fluoride solution was transferred into the reactor and agitated for 30 min. The agitation was stopped, the phases were allowed to settle, and the aqueous phase was removed. A 25% w/v sodium chloride solution (12.0 L, 5 Vol) was prepared by charging an appropriate container with 12.0 L of water and 2999 g of sodium chloride and agitating until homogeneous. The 25% sodium chloride solution was transferred into the reactor and agitated for 30 min. The agitation was stopped, the phases were allowed to settle, and the aqueous phase was removed from the reactor.

The organic phase was distilled at constant volume (36 L, 15 Vol) while maintaining the internal temperature of the reactor at 50+5 °C by adjusting the vacuum pressure until no more than 0.3% of water remained. 2-Methyltetrahydrofuran was added to the reactor as needed to

maintain constant volume. The batch was cooled to 20 °C and transferred into drums. The batch was transferred using a polish filter (using a 5 μιη inline filter) into a 60 L jacketed reactor with a batched concentrator attached. 1.2 L of 2-MeTHF was used to rinse the drums. The batch was concentrated to about 9 Vol while maintaining the internal temperature of the vessel at 50+5 °C by adjusting the vacuum pressure. The batch was then distilled at constant volume (22.0 L, 9Vol) while maintaining the internal temperature of the vessel at 50+5 °C by adjusting the vacuum pressure. Heptane was added with residual vacuum until a 15% 2-MeTHF:heptane supernatant mixture was obtained. The pressure was brought to atmospheric pressure under nitrogen. The reactor was cooled to 20+5 °C over 2+2 h. The batch was agitated at 20+5 °C until an assay of the supernatant indicated that the amount of product was 7 mg/mL 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine.

A 10% 2-MeTHF:heptane (7.2 L, 3 Vol) wash solution was prepared by mixing 720 mL of 2-MeTHF and 6.5 L of heptane. The batch slurry was filtered through an Aurora filter fitted with a 25 μιη polypropylene filter cloth, resulting in heavy crystals that required pumping with a diaphragm pump using polypropylene transfer lines through the top of the reactor while stirring. The mother liquor was recycled to complete the transfer. The reactor and filter cake were washed with two portions of the 10% 2-MeTHF:heptane wash solution (3.6 L each). The product cake was dried on a frit under a nitrogen stream at ambient temperature. The 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine was determined to be dry when the 1H NMR assay was < 0.05+0.05. 2.635 kg was isolated as an off white crystalline solid (85% yield).

A 60 L jacketed reactor was fitted with a reflux condenser. The condenser cooling was initiated at 0+5 °C. The reactor was charged with 2612 g (1 equivalent) of 2,3-difluoro-5-(l-methyl-lH-pyrazol-4-yl)pyridine and placed under an atmosphere of nitrogen. 31.7 L (12.2 Vol) water was added to the reactor and the resulting slurry was nitrogen sparged for 1 h with agitation. 7221 mL (6 equivalents) of hydrazine (35 wt% in water) was added to the reactor under a nitrogen atmosphere. The reactor was heated to 100 °C for 2+2 h until reaction was complete by HPLC analysis. The reactor was cooled to 20 °C over 2+1 h at a rate of 40°C/h. The reactor contents were stirred for 10+9 hours until the desired supernatant assay was reached (< 2mg/mL PYRH in mother liquor). The reactor contents were filtered through an Aurora filter fitted with 25 μιη polypropylene filter cloth. The collected filter cake was washed with 12.0 L

(4.6 V) of water in three portions. The filter cake was dried on the Aurora filter for 4-24 h at 22+5 °C, or until the product contained less than 0.5% water as determined by KF. The dry product was collected. 2.69 kg was isolated as a white crystalline solid (97% yield). The water content was determined to be 12 ppm by KF.

WO2007075567A1 *	Dec 18, 2006	Jul 5, 2007	Janssen Pharmaceutica, N.V.	Triazolopyridazines as tyrosine kinase modulators
WO2007138472A2 *	May 18, 2007	Dec 6, 2007	Pfizer Products Inc.	Triazolopyridazine derivatives
WO2008008539A2 *	Jul 13, 2007	Jan 17, 2008	Amgen Inc.	Fused heterocyclic derivatives useful as inhibitors of the hepatocyte growth factor receptor
WO2008051805A2 *	Oct 18, 2007	May 2, 2008	Sgx Pharmaceuticals, Inc.	Triazolo-pyridazine protein kinase modulators
WO2008155378A1 *	Jun 19, 2008	Dec 24, 2008	Janssen Pharmaceutica Nv	Polymorphic and hydrate forms, salts and process for preparing 6-{difluoro[6-(1-methyl-1h-pyrazol-4-yl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl]methyl}quinoline

References:
1. Hughes, P. E.; et. al. Abstract 728: AMG 337, a novel, potent and selective MET kinase inhibitor, has robust growth inhibitory activity in MET-dependent cancer models. Cancer Res 2014, 74, 728.
2. Boezio, A. A.; et. al. Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors. Bioorg Med Chem Lett 2009, 19(22), 6307-6312.
3. ClinicalTrials.gov Phase 2 Study of AMG 337 in MET Amplified Gastric/Esophageal Adenocarcinoma or Other Solid Tumors. NCT02016534 (retrieved 10-06-2015)
4. ClinicalTrials.gov A Study of AMG 337 in Subjects With Advanced Solid Tumors. NCT01253707 (retrieved 10-06-2015)

/////////// AMG-337, AMG337, AMG 337, 1173699-31-4, AMGEN, ESOPHAGUS

O=C1C2=C(N=CC(OCCOC)=C2)C=CN1[C@@H](C3=NN=C4C(F)=CC(C5=CN(C)N=C5)=CN43)C

CRD 1152, CURADEV PHARMA PRIVATE LTD

cancer, Uncategorized Comments Off

Apr 052016

Several candidates……One is …..CRD1152

ONE OF THEM IS CRD 1152

Kynurenine pathway regulators (solid tumors)

Compound 2

CAS1638121-21-7

US159738837

N³-(3-Chloro-4- fluorophenyl) furo[2,3- c]pyridine-2,3- diamine

COMPD 190

CAS 1638118-99-6

US159738837

COMPD248

US159738837

7-Chloro-N3- (3-chloro-4- fluorophenyl) furo[2,3- c]pyridine-2,3- diamine, 166

DMSO-d₆: δ 7.87 (d, J = 5.1 Hz, 1H), 7.25 (s, 2H), 7.16-7.10 (m, 2H), 6.88 (d, J = 5.1 Hz, 1H), 6.59 (dd, J′ = 6.2 Hz, J″ = 2.6 Hz, 1H), 6.48 (dt, J′ = 8.8 Hz, J″ = 6.7 Hz, J′′′ = 3.4 Hz, 1H) M + H] 312

US159738837

N³-(3,4- difluorophenyl)- 7-(pyridin-4- yl)furo[2,3- c]pyridine-2,3- diamine, 184

CD₃CN: δ 8.72 (s, 2H), 8.26 (s, 3H), 7.07-7.03 (m, 2H), 6.47-6.40 (m, 2H), 5.74 (s, 1H), 5.55 (s, 2H) M + H] 339

US159738837

COMPD73

CAS 1638117-85-7

US159738837

Several candidates………..CRD1152

Company	Curadev Pharma Pvt. Ltd.
Description	Small molecule dual indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO1; IDO) inhibitor
Molecular Target	Indoleamine 2,3-dioxygenase (INDO) (IDO) ; Tryptophan 2,3-dioxygenase (TDO2) (TDO)
Mechanism of Action	Indoleamine 2,3-dioxygenase (INDO) inhibitor
Therapeutic Modality	Small molecule

Latest Stage of Development	Preclinical
Standard Indication	Cancer (unspecified)
Indication Details	Treat cancer
Regulatory Designation
Partner	Roche

Hoffmann-La Roche partners with Curadev Pharma Ltd. for IDO1 and TDO inhibitors (April 20, 2015)

Curadev Pharma Pvt Ltd., founded in 2010 and headquartered in New Delhi, announced that it has entered into a research collaboration and exclusive license agreement with Roche for the development and commercialization of IDO1 and TDO inhibitors to treat cancer. The agreement covers the development of CRD1152, the lead preclinical immune tolerance inhibitor and a research collaboration with Roche’s research and early development organization to further explore the IDO and TDO pathways.

IDO1 (indoleamine-2,3-dioxygenase-1) and TDO (tryptophan-2,3-dioxygenase) are enzymes that mediate cancer-induced immune suppression. This mechanism is exploited by tumor cells as well as certain type of immune cells, limiting the anti-tumor immune response. Dual inhibition of the IDO1 and TDO pathways promises to maintain the immune response, prevent local tumor immune escape and potentially avoid resistance to other immunotherapies when used in combination, and could lead to new treatment options for cancer patients. Curadev’s preclinical lead-compound, a small-molecule that shows potent inhibition of the two rate-limiting enzymes in the tryptophan to kynurenine metabolic pathways, has the potential for mono therapy as well as combination with Roche’s broad oncology pipeline and portfolio.

Under the terms of agreement, which includes a research collaboration with Roche’s research and early development organization, Curadev will receive an upfront payment of $25 million and will be eligible to receive up to $530 million in milestone payments, as well as escalating royalties potentially reaching double digits for the first product from the collaboration developed and commercialized by Roche. Curadev is also eligible for milestones and royalties on any additional products resulting from the research collaboration.

Curadev Announces Research Collaboration and Licensing Agreement to Develop Cancer Immunotherapeutic

Curadev’s dual IDO and TDO immune tolerance inhibitor – a novel approach in cancer immunotherapy

Apr 20, 2015, 06:30 ET from Curadev

NEW DELHI, India, April 20, 2015 /PRNewswire/ —

Curadev Pharma Private Ltd. today announced that it has entered into a research collaboration and exclusive license agreement with Roche for the development and commercialization of IDO1 and TDO inhibitors. The agreement covers the development of the lead preclinical immune tolerance inhibitor and a research collaboration with Roche’s research and early development organization to further explore the IDO and TDO pathways.

Dual inhibition of the IDO1 and TDO pathways promises to maintain the immune response, prevent local tumor immune escape and potentially avoid resistance to other immunotherapies when used in combination, and could lead to new treatment options for cancer patients. Curadev’s preclinical lead-compound, a small-molecule that shows potent inhibition of the two rate-limiting enzymes in the tryptophan – to kynurenine metabolic pathways, has the potential for mono therapy as well as combination with Roche’s broad oncology pipeline and portfolio.

“We are very excited to be working with the global leader in oncology with their unrivalled expertise in clinical development,” said Arjun Surya, PhD, Chief Scientific Officer, Curadev. “The collaboration acknowledges our focused research efforts on patient-critical drug targets that have yielded a drug candidate that could make a significant difference in the development of novel treatments for patients suffering from cancer.”

Under the terms of agreement, which includes a research collaboration with Roche’s research and early development organization to further extend Curadev’s findings, Curadev will receive an upfront payment of $25 million and will be eligible to receive up to $530 million in milestone payments based on achievement of certain predetermined events and sales levels as well as escalating royalties potentially reaching double digits for the first product from the collaboration developed and commercialized by Roche. Curadev would also be eligible for milestones and royalties on any additional products resulting from the research collaboration. Roche will fund future research, development, manufacturing and commercialization costs and will also provide additional research funding to Curadev for support of the research collaboration.

About Curadev

Headquartered in New Delhi, India, Curadev Pharma Private Limited was founded in 2010 by a team of professionals from the pharmaceutical and biotech sectors with the mission to improve human health and enhance the quality of human life by accelerating the discovery and delivery of new drugs. Curadev focuses on the creation and out-licensing of pre-IND assets and IND packages for drug development.

For further information:

Curadev Partnering

Manish Tandon – VP and Chief Financial Officer, manish@curadev.in

PATENT

US20160046596) INHIBITORS OF THE KYNURENINE PATHWAY

https://patentscope.wipo.int/search/en/detail.jsf?docId=US159738837&recNum=2&maxRec=17&office=&prevFilter=&sortOption=Pub+Date+Desc&queryString=FP%3A%28curadev%29&tab=PCTDescription

Monali Banerjee
Sandip Middya
Ritesh Shrivastava
Sushil Raina
Arjun Surya
Dharmendra B. Yadav
Veejendra K. Yadav
Kamal Kishore Kapoor
Aranapakam Venkatesan
Roger A. Smith
Scott K. Thompson

ONE ………….Example 2

Synthesis of N³-(3-Chloro-4-fluoro-phenyl)-furo[2,3-c]pyridine-2,3-diamine (Compound 2)

Step 1: 3-Methoxymethoxy-pyridine

To a stirred solution of 3-hydroxypyridine (60 g, 662.9 mmol) in THF:DMF (120:280 mL) at 0° C. was added t-BuOK (81.8 gm, 729.28 mmol) portion-wise. After stirring the reaction mixture for 15 min, methoxymethyl chloride (52 mL, 696.13 mmol) was added to it at 0° C. and the resulting mixture was stirred for 1 hr at 25° C. Reaction mixture was diluted with water and extracted with ethyl acetate (4×500 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure to afford 100 g crude which was purified by column chromatography using silica (100-200 mesh) and 10% EtOAc-hexane as eluent to afford 3-methoxymethoxy-pyridine (54 g) as pale brown liquid. LCMS: 140 (M+H).

Step 2: 3-Methoxymethoxy-pyridine-4-carbaldehyde

To a stirred solution of 3-methoxymethoxypyridine (2 g, 14.3885 mmol) in anhydrous THF (40 mL) was added TMEDA (1.83 g, 15.82 mmol) at 25° C. The reaction mixture was cooled to −78° C., n-BuLi (7.3 mL, 15.82 mmol, 2.17 M in hexane) was added dropwise manner maintaining the temperature −78° C. After stirring for 2 hr at −78° C., DMF (1.52 g, 20.86 mmol) was added to it and stirred for 2 hr at 25° C. Reaction mixture was cooled to −40° C. and saturated ammonium chloride solution was added drop wise. The reaction mass was extracted with ethyl acetate (250 mL×2), EtOAc part was washed with water followed by brine, dried over sodium sulfate and concentrated under reduced pressure to afford 3 g of crude product which was passed through a pad of silica (100-200 mesh) using 10% EtOAc-hexane as eluent to afford 1.6 g of 3-methoxymethoxy-pyridine-4-carbaldehyde as pale yellow liquid. GC-MS: 167 (m/z).

Step 3: 3-Hydroxy-pyridine-4-carbaldehyde

To a stirred solution of 3-methoxymethoxypyridine-4-carbaldehyde (11 g, 65.83 mmol) in THF (50 mL) was added 3N HCl (100 mL) and stirred at 60° C. for 1 hr. The reaction mixture was cooled under ice bath and pH was adjusted to 7 with solid K₂CO₃. Resulting mixture was extracted with EtOAc (250 mL×5). The organic layer was dried over sodium sulfate, concentrated under reduced pressure to afford 15 g of crude which was purified by column chromatography using silica gel (100-200 mesh) and 23% EtOAc/hexane as eluent to afford 4 g of 3-hydroxy-pyridine-4-carbaldehyde as pale yellow solid. GC-MS: 123 (m/z), ¹H-NMR (DMSO-d₆, 400 MHz): δ 11.04 (bs, 1H), 10.37 (s, 1H), 8.46 (s, 1H), 8.20 (d, 1H, J=4.88 Hz), 7.46 (d, 1H, J=4.88 Hz). GC-FID: 99.51%.

Step 4: 4-{[3-Chloro-4-fluoro-phenylimino]-methyl}-pyridin-3-ol

3-Hydroxypyridine-4-carbaldehyde (3 g, 24.39 mmol) was taken in mixed solvent (TFE (20 mL):MeCN (20 mL)) and 4-fluoro-3-chloroaniline (3.55 g, 24.39 mmol) was added to it at 25° C. The resulting mixture was stirred at this temperature for 1 hr. The reaction mass was concentrated and purified by triturating with n-pentane to afford 6 g of 4-{[3-chloro-4-fluoro-phenylimino]-methyl}-pyridin-3-ol). LCMS: 251.2 (M+H).

Step 5: N³-(3-Chloro-4-fluoro-phenyl)-furo[2,3-c]pyridine-2,3-diamine

To a stirred solution of 4-{[3-chloro-4-fluoro-phenylimino]-methyl}-pyridin-3-ol (6 g, 24 mmol) in mixed solvent [DCM (10 mL):TFE (10 mL)] was added TMSCN (10.5 mL, 84 mmol) at 25° C. The reaction mixture was stirred 3 hr at 25° C., concentrated, and the crude material was triturated with n-pentane to provide 4.9 g (73% yield) of N³-(3-chloro-4-fluoro-phenyl)-furo[2,3-c]pyridine-2,3-diamine as pale pink solid. LCMS: 278 (M+H), HPLC: 98.65%, ¹H-NMR (DMSO-d₆, 400 MHz): δ 8.41 (s, 1H), 8.06 (d, 1H, J=5.08 Hz), 7.14-7.10 (m, 2H), 6.91 (s, 2H), 6.86 (d, 1H, J=5.08 Hz), 6.56-6.54 (m, 1H), 6.48-6.45 (m, 1H).

Monali Banerjee – Director, R&D

Ms. Banerjee has more than 10 years of research experience, during which she has held positions of increasing responsibility. Her past organizations include TCG Lifesciences (Chembiotek) and Sphaera Pharma. Ms. Banerjee is a versatile scientist with a deep understanding of the fundamental issues that underlie various aspects of drug discovery. At Curadev, she has been responsible for target selection, patent analysis, pharmacophore design, assay development, ADME/PK and in vivo and in vitro pharmacology. Ms. Banerjee holds a Masters in Biochemistry and a Bachelors in Chemistry both from Kolkata University.

writeup

The essential amino acid Tryptophan (Trp) is catabolized through the kynurenine (KYN) pathway. The initial rate-limiting step in the kynurenine pathway is performed by heme-containing oxidoreductase enzymes, including tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase-1 (IDO1), and indoleamine 2,3-dioxygenase-2 (IDO2). IDO1 and IDO2 share very limited homology with TDO at the amino acid level and, despite having different molecular structures, each enzyme has the same biochemical activity in that they each catalyze tryptophan to form N-formylkynurenine. IDO1, IDO2, and/or TDO activity alter local tryptophan concentrations, and the build-up of kynurenine pathway metabolites due to the activity of these enzymes can lead to numerous conditions associated with immune suppression.

IDO1 and TDO are implicated in the maintenance of immunosuppressive conditions associated with the persistence of tumor resistance, chronic infection, HIV infection, malaria, schizophrenia, depression as well as in the normal phenomenon of increased immunological tolerance to prevent fetal rejection in utero. Therapeutic agents that inhibit IDO1, IDO2, and TDO activity can be used to modulate regulatory T cells and activate cytotoxic T cells in immunosuppressive conditions associated with cancer and viral infection (e.g. HIV-AIDS, HCV). The local immunosuppressive properties of the kynurenine pathway and specifically IDO1 and TDO have been implicated in cancer. A large proportion of primary cancer cells have been shown to overexpress IDO1. In addition, TDO has recently been implicated in human brain tumors.

The earliest experiments had proposed an anti-microbial role for IDO1, and suggested that localized depletion of tryptophan by IDO1 led to microbial death (Yoshida et al., Proc. Natl. Acad. Sci. USA, 1978, 75(8):3998-4000). Subsequent research led to the discovery of a more complex role for IDO1 in immune suppression, best exemplified in the case of maternal tolerance towards the allogeneic fetus where IDO1 plays an immunosuppressive role in preventing fetal rejection from the uterus. Pregnant mice dosed with a specific IDO1 inhibitor rapidly reject allogeneic fetuses through induction of T cells (Munn et al., Science, 1998, 281(5380): 1191-3). Studies since then have established IDO1 as a regulator of certain disorders of the immune system and have discovered that it plays a role in the ability of transplanted tissues to survive in new hosts (Radu et al., Plast. Reconstr. Surg., 2007 June, 119(7):2023-8). It is believed that increased IDO1 activity resulting in elevated kynurenine pathway metabolites causes peripheral and ultimately, systemic immune tolerance. In-vitro studies suggest that the proliferation and function of lymphocytes are exquisitely sensitive to kynurenines (Fallarino et al., Cell Death and Differentiation, 2002, 9(10):1069-1077). The expression of IDO1 by activated dendritic cells suppresses immune response by mechanisms that include inducing cell cycle arrest in T lymphocytes, down regulation of the T lymphocyte cell receptor (TCR) and activation of regulatory T cells (T-regs) (Terness et al., J. Exp. Med., 2002, 196(4):447-457; Fallarino et al., J. Immunol., 2006, 176(11):6752-6761).

IDO1 is induced chronically by HIV infection and in turn increases regulatory T cells leading to immunosuppression in patients (Sci. Transl. Med., 2010; 2). It has been recently shown that IDO1 inhibition can enhance the level of virus specific T cells and concomitantly reduce the number of virus infected macrophages in a mouse model of HIV (Potula et al., 2005, Blood, 106(7):2382-2390). IDO1 activity has also been implicated in other parasitic infections. Elevated activity of IDO1 in mouse malaria models has also been shown to be abolished by in vivo IDO1 inhibition (Tetsutani K., et al., Parasitology. 2007 7:923-30.

More recently, numerous reports published by a number of different groups have focused on the ability of tumors to create a tolerogenic environment suitable for survival, growth and metastasis by activating IDO1 (Prendergast, Nature, 2011, 478(7368):192-4). Studies of tumor resistance have shown that cells expressing IDO1 can increase the number of regulatory T cells and suppress cytotoxic T cell responses thus allowing immune escape and promoting tumor tolerance.

Kynurenine pathway and IDO1 are also believed to play a role in maternal tolerance and immunosuppressive process to prevent fetal rejection in utero (Munn et al., Science, 1998, 281(5380):1191-1193). Pregnant mice dosed with a specific IDO1 inhibitor rapidly reject allogeneic fetuses through suppression of T cells activity (Munn et al., Science, 1998, 281(5380):1191-1193). Studies since then have established IDO1 as a regulator of immune-mediated disorders and suggest that it plays a role in the ability of transplanted tissues to survive in new hosts (Radu et al., Plast. Reconstr. Surg., 2007 June, 119(7):2023-8).

The local immunosuppressive properties of the kynurenine pathway and specifically IDO1 and TDO have been implicated in cancer. A large proportion of primary cancer cells overexpress IDO1 and/or TDO (Pilotte et al., Proc. Natl. Acad. Sci. USA, 2012, Vol. 109(7):2497-2502). Several studies have focused on the ability of tumors to create a tolerogenic environment suitable for survival, growth and metastasis by activating IDO1 (Prendergast, Nature, 2011, 478:192-4). Increase in the number of T-regs and suppression of cytotoxic T cell responses associated with dysregulation of the Kynurenine pathway by overexpression of IDO1 and/or TDO appears to result in tumor resistance and promote tumor tolerance.

Data from both clinical and animal studies suggest that inhibiting IDO1 and/or TDO activity could be beneficial for cancer patients and may slow or prevent tumor metastases (Muller et al., Nature Medicine, 2005, 11(3):312-319; Brody et al., Cell Cycle, 2009, 8(12):1930-1934; Witkiewicz et al., Journal of the American College of Surgeons, 2008, 206:849-854; Pilotte et al., Proc. Natl. Acad. Sci. USA, 2012, Vol. 109(7):2497-2502). Genetic ablation of the IDO1 gene in mice (IDO1−/−) resulted in decreased incidence of DMBA-induced premalignant skin papillomas (Muller et al., PNAS, 2008, 105(44):17073-17078). Silencing of IDO1 expression by siRNA or a pharmacological IDO1 inhibitor 1-methyl tryptophan enhanced tumor-specific killing (Clin. Cancer Res., 2009, 15(2). In addition, inhibiting IDO1 in tumor-bearing hosts improved the outcome of conventional chemotherapy at reduced doses (Clin. Cancer Res., 2009, 15(2)). Clinically, the pronounced expression of IDO1 found in several human tumor types has been correlated with negative prognosis and poor survival rate (Zou, Nature Rev. Cancer, 2005, 5:263-274; Zamanakou et al., Immunol. Lett. 2007, 111(2):69-75). Serum from cancer patients has higher kynurenine/tryptophan ratio, a higher number of circulating T-regs, and increased effector T cell apoptosis when compared to serum from healthy volunteers (Suzuki et al., Lung Cancer, 2010, 67:361-365). Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase has been studied by Pilotte et al. (Pilotte et al., Proc. Natl. Acad. Sci. USA, 2012, Vol. 109(7):2497-2502). Thus, decreasing the rate of kynurenine production by inhibiting IDO1 and/or TDO may be beneficial to cancer patients.

IDO1 and IDO2 are implicated in inflammatory diseases. IDO1 knock-out mice don’t manifest spontaneous disorders of classical inflammation and existing known small molecule inhibitors of IDO do not elicit generalized inflammatory reactions (Prendergast et al. Curr Med Chem. 2011; 18(15):2257-62). Rather, IDO impairment alleviates disease severity in models of skin cancers promoted by chronic inflammation, inflammation-associated arthritis and allergic airway disease. Moreover, IDO2 is a critical mediator of autoantibody production and inflammatory pathogenesis in autoimmune arthritis. IDO2 knock-out mice have reduced joint inflammation compared to wild-type mice due to decreased pathogenic autoantibodies and Ab-secreting cells (Merlo et al. J. Immunol. (2014) vol. 192(5) 2082-2090). Thus, inhibitors of IDO1 and IDO2 are useful in the treatment of arthritis and other inflammatory diseases.

Kynurenine pathway dysregulation and IDO1 and TDO play an important role in the brain tumors and are implicated in inflammatory response in several neurodegenerative disorders including multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, stroke, amyotrophic lateral schlerosis, dementia (Kim et al., J. Clin. Invest, 2012, 122(8):2940-2954; Gold et al., J. Neuroinflammation, 2011, 8:17; Parkinson’s Disease, 2011, Volume 2011). Immunosuppression induced by IDO1 activity and the Kynurenine metabolites in the brain may be treated with inhibitors of IDO1 and/or TDO. For example, circulating T-reg levels were found to be decreased in patient with glioblastoma treated with anti-viral agent inhibitors of IDO1 (Soderlund, et al., J. Neuroinflammation, 2010, 7:44).

Several studies have found Kynurenine pathway metabolites to be neuroactive and neurotoxic. Neurotoxic kynurenine metabolites are known to increase in the spinal cord of rats with experimental allergic encephalomyelitis (Chiarugi et al., Neuroscience, 2001, 102(3):687-95). The neurotoxic effects of Kynurenine metabolities is exacerbated by increased plasma glucose levels. Additionally, changes in the relative or absolute concentrations of the kynurenines have been found in several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease and Parkinson’s disease, stroke and epilepsy (Németh et al., Central Nervous System Agents in Medicinal Chemistry, 2007, 7:45-56; Wu et al. 2013; PLoS One; 8(4)).

Neuropsychiatric diseases and mood disorders such as depression and schizophrenia are also said to have IDO1 and Kynurenine dysregulation. Tryptophan depletion and deficiency of neurotransmitter 5-hydroxytryptamine (5-HT) leads to depression and anxiety. Increased IDO1 activity decreases the synthesis of 5-HT by reducing the amount of Tryptophan availability for 5-HT synthesis by increasing Tryp catabolism via the kynurenine pathway (Plangar et al. (2012) Neuropsychopharmacol Hung 2012; 14(4): 239-244). Increased IDO1 activity and levels of both kynurenine and kynurenic acid have been found in the brains of deceased schizophrenics (Linderholm et al., Schizophrenia Bulletin (2012) 38: 426-432)). Thus, inhibition of IDO1, IDO1, and TDO may also be an important treatment strategy for patients with neurological or neuropsychiatric disease or disorders such as depression and schizophrenia as well as insomnia.

Kynurenine pathway dysregulation and IDO1 and/or TDO activity also correlate with cardiovascular risk factors, and kynurenines and IDO1 are markers for Atherosclerosis and other cardiovascular heart diseases such as coronary artery disease (Platten et al., Science, 2005, 310(5749):850-5, Wirlietner et al. Eur J Clin Invest. 2003 July; 33(7):550-4) in addition to kidney disease. The kynurenines are associated with oxidative stress, inflammation and the prevalence of cardiovascular disease in patients with end-stage renal disease (Pawlak et al., Atherosclerosis, 2009, (204)1:309-314). Studies show that kynurenine pathway metabolites are associated with endothelial dysfunction markers in the patients with chronic kidney disease (Pawlak et al., Advances in Medical Sciences, 2010, 55(2):196-203).

///////CRD1152, CRD-1152, CRD 1152, CURADEV PHARMA PRIVATE LTD, ROCHE, IDO1 and TDO inhibitors, COLLABORATION, CANCER, indoleamine-2,3-dioxygenase-1, Hoffmann-La Roche, kynurenine pathway regulators, solid tumors

PF 06650808

cancer, MONOCLONAL ANTIBODIES, Uncategorized Comments Off

Mar 252016

Picture credit….Bethany Halford

PF 06650808

	Phase 1

Phase 1

$PFE compound inspired by auristatins

https://clinicaltrials.gov/ct2/show/NCT02129205

http://www.pfizer.com/sites/default/files/product-pipeline/8_7_2014_Pipeline_Update.pdf

ALL DATA COMING………

Notch-3 receptor antagonists

Neoplasms
Breast

Pfizer

Cancer

PF-06650808, is currently being examined in a Ph1 clinical trial (Protocol B7501001).

Notch3
Researchers are also exploring the use of Notch3 targeting. “The Notch pathway plays an important role in the growth of several solid tumours, including breast and ovarian cancer and melanoma,” explained Joerger. “In particular, Notch3 alterations such as gene amplification and upregulation are associated with poor patient survival. Research using Notch3 targeting as an innovative approach to treat solid malignancies included 27 patients unselected for Notch3 who received increasing doses of the anti-Notch3 antibody-drug conjugate PF-06650808. Responses were seen in two breast cancer patients (LBA 30). While preliminary, targeting Notch3 may become a new treatment approach in patients with selected solid tumours.”

The anti-Notch3 antibody-drug conjugate PF-06650808 is being developed by Pfizer.

31 Jul 2014 Phase-I clinical trials in Solid tumours (Late-stage disease) in USA (Parenteral)
30 Apr 2014 Preclinical trials in Solid tumours in USA (Parenteral)
30 Apr 2014 Pfizer plans a phase I trial for Solid tumours (late-stage disease, second-line therapy or greater) in USA (NCT02129205)

251st Am Chem Soc (ACS) Natl Meet (March 13-17, San Diego) 2016, Abst MEDI 262

/////////PF 06650808, PF-06650808, PF-6650808, monoclonal antibody, pfizer, phase 1, Solid tumours , Notch-3 receptor antagonists

C1(C(N(C(C1)=O)CCCCCC(=O)NC([C@H](C)C)C(=O)NC(C(=O)Nc2ccc(cc2)COC(=O)NC(C)(C)C(=O)N[C@@H](C(C)C)C(=O)[N@](C)C(C(CC)C)[C@@H](OC)CC(=O)N3CCC[C@H]3C(OO)C(C)C(=O)N[C@H](c4nccs4)CC)CCCNC(=O)N)=O)SC

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Trioxacarcin A

cancer Comments Off

Mar 182016

Trioxacarcin A, DC-45A

CAS No. 81552-36-5

Molecular FormulaC₄₂H₅₂O₂₀
Average mass876.850 Da
17′-[(4-C-Acetyl-2,6-dideoxyhexopyranosyl)oxy]-19′-(dimethoxymethyl)-10′,13′-dihydroxy-6′-methoxy-3′-methyl-11′-oxospiro[oxirane-2,18‘-[16,20,22]trioxahexacyclo[17.2.1.0^2,15.0^5,14.0^7,12.0^17,21 ]docosa[2(15),3,5(14),6,12]pentaen]-8′-yl 4-O-acetyl-2,6-dideoxy-3-C-methylhexopyranoside

(1S,2R,3aS,4S,8S,10S,13aS)-13a-(4-C-Acetyl-2,6-dideoxy-alpha-L-xylo-hexopyranosyloxy)-2-(dimethoxymethyl)-10,12-dihydroxy-7-methoxy-5-methyl-11-oxo-4,8,9,10,11,13a-hexahydro-3aH-spiro[2,4-epoxyfuro[3,2-b]naphtho[2,3-h]-1-benzopyran-1,2′-oxiran]-8-yl 4-O-acetyl-2,6-dideoxy-3-C-methyl-alpha-L-xylo-hexopyranoside
Kyowa Hakko Kirin INNOVATOR

Trioxacarcin B

Trioxacarcin B; Antibiotic DC 45B1; DC-45-B1; Trioxacarcin A, 14,17-deepoxy-14,17-dihydroxy-; AC1MJ5N1; 81534-36-3;

Molecular Formula:	C₄₂H₅₄O₂₁
Molecular Weight:	894.86556 g/mol

Trioxacarcin C

(CAS NO.81781-28-4):C₄₂H₅₄O₂₀
Molecular Weight: 878.8662 g/mol
Structure of Trioxacarcin C :

The trioxacarcins are polyoxygenated, structurally complex natural products that potently inhibit the growth of cultured human cancer cells

Natural products that bind and often covalently modify duplex DNA figure prominently in chemotherapy for human cancers. The trioxacarcins are a new class of DNA- modifying natural products with antiproliferative effects. The trioxacarcins were first described in 1981 by Tomita and coworkers (Tomita et al. , J. Antibiotics, 34( 12): 1520- 1524, 1981 ; Tamaoki et al., J. Antibiotics 34( 12): 1525- 1530, 1981 ; Fujimoto et al. , J. Antibiotics 36(9): 1216- 1221 , 1983). Trioxacarcin A, B, and C were isolated by Tomita and coworkers from the culture broth of Streptomyces bottropensis DO-45 and shown to possess anti-tumor activity in murine models as well as gram-positive antibiotic activity. Subsequent work led to the discovery of other members of this family. Trioxacarcin A is a powerful anticancer agent with subnanmolar IC70 values against lung (LXFL 529L, H-460), mammary (MCF-7), and CNS (SF-268) cancer cell lines. The trioxacarcins have also been shown to have antimicrobial activity {e.g., anti-bacterial and anti-malarial activity) (see, e.g. , Maskey et al., J. Antibiotics (2004) 57:771 -779).

trioxacarcin A

An X-ray crystal structure of trioxacarcin A bound to N-7 of a guanidylate residue in a duplex DNA oligonucleotide substrate has provided compelling evidence for a proposed pathyway of DNA modification that proceeds by duplex intercalation and alkylation (Pfoh et al, Nucleic Acids Research 36( 10):3508-3514, 2008).

All trioxacarcins appear to be derivatives of the aglycone, which is itself a bacterial isolate referred to in the patent literature as DC-45-A2. U.S. Patent 4,459,291 , issued July 10, 1984, describes the preparation of DC-45-A₂ by fermentation. DC-45-A2 is the algycone of trioxacarcins A, B, and C and is prepared by the acid hydrolysis of the fermentation products trioxacarcins A and C or the direct isolation from the fermentation broth of Streptomyces bottropensis.

Based on the biological activity of the trioxacarcins, a fully synthetic route to these compounds would be useful in exploring the biological and chemical activity of known trioxacarcin compounds and intermediates thereto, as well as aid in the development of new trioxacarcin compounds with improved biological and/or chemical properties.

PAPER

Component-Based Syntheses of Trioxacarcin A, DC-45-A1, and Structural Analogs
T. Magauer, D. Smaltz, A. G. Myers, Nat. Chem. 2013, 5, 886–893. (Link)

Component-based syntheses of trioxacarcin A, DC-45-A1 and structural analogues

Nature Chemistry5,886–893(2013)

doi:10.1038/nchem.1746

PAPER

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

PAPER

PATENT

http://www.google.com/patents/EP2550285A1?cl=en

(S)-9-Hvdrox v- 10-methoxy-5-(4-methoxybenzylox v)- 1 -(methoxymethox y)-3- methyl-8-oxo-5,6.7.8-tetrahvdroanthracene-2-carbaldehvde. Potassium osmate dihydrate (29 mg, 0.079 mmol, 0.05 equiv) was added to an ice -cooled mixture of (S,£)-9-hydroxy- 10- methoxy-4-(4-methoxybenzyloxy)-8-(methoxymethoxy)-6-methyl-7-(prop- l -enyl)-3,4- dihydroanthracen-l -one (780 mg, 1.58 mmol, 1 equiv), 2,6-lutidine (369 μί, 3.17 mmol, 2.0 equiv), and sodium periodate ( 1.36 g, 6.33 mmol, 4.0 equiv) in a mixture of tetrahydrofuran (20 mL) and water ( 10 mL). After 10 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 1.5 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed with aqueous sodium chloride solution (50 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (20% ethyl acetate- hexanes) to provide 498 mg of the product, (5)-9-hydroxy- 10-methoxy-5-(4- methoxybenzyloxy)- l -(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8-tetrahydroanthracene-2- carbaldehyde, as an orange foam (65%). Ή NMR (500 MHz, CDC1₃): 15.17 (s, 1 H), 10.74 (s, 1 H), 7.66 (s, 1 H), 7.27 (d, 2H, 7 = 8.5 Hz), 6.86 (d, 2H, 7 = 8.6 Hz), 5.30-5.18 (m, 3H), 4.63 (d, 1H,7= 11.1 Hz), 4.52 (d, 1H,7 = 12.0 Hz), 3.86 (s, 3H), 3.79 (s, 3H), 3.62 (s, 3H), 3.22 (m, 1H), 2.75 (s, 3H), 2.63 (m, 1H), 2.54 (m, 1H), 2.08 (m, 1H). ^I3C NMR (125 MHz, CDC1₃): 204.9, 193.2, 163.2, 161.7, 159.2, 144.4, 141.7, 137.0, 130.1, 129.4, 120.7, 117.9, 113.8, 110.0, 102.8, 70.4, 67.2, 62.9, 58.3, 55.2, 32.3, 26.3, 22.2. FTIR, cm^-1 (thin film): 2936 (m), 2907 (m), 1684 (s), 1611 (s), 1377 (s), 1246 (s). HRMS (ESI): Calcd for

(C₂₇H2808+K)⁺: 519.1416; Found 519.1368. TLC (20% ethyl acetate-hexanes): R,= 0.17 (CAM).

86% yield

[00457] (S)-l,9-Dihvdroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde. A solution of B-bromocatecholborane (418 mg, 2.10 mmol, 2.0 equiv) in dichloromethane (15 mL) was added to a solution of (S)-9-hydroxy-10- methoxy-5-(4-methoxybenzyloxy)-l-(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (490 mg, 1.05 mmol, 1 equiv) in dichloromethane (15 mL) at -78 °C. After 50 min, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (25 mL) and dichloromethane (100 mL). The cooling bath was removed, and the partially frozen mixture was allowed to warm to 23 °C. The biphasic mixture was diluted with 0.2 M aqueous sodium hydroxide solution (100 mL). The layers were separated. The aqueous layer was extracted with dichloromethane (100 mL). The organic layers were combined. The combined solution was washed sequentially with 0.1 M aqueous hydrochloric acid solution (100 mL), water (2 x 100 mL), then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide 380 mg of the product, (S)-\ ,9- dihydroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde, as a yellow foam (86%). Ή NMR (500 MHz, CDCI3):

15.89 (brs, 1H), 12.81 (br s, 1H), 10.51 (s, 1H), 7.27-7.26 (m, 3H), 6.86 (d, 2H, J = 9.2 Hz), 5.14 (app s, 1H),4.62 (d, \H,J= 11.0 Hz), 4.51 (d, 1H,7= 11.0 Hz), 3.85 (s, 3H), 3.80 (s, 3H), 3.21 (m, 1H), 2.73 (s, 3H), 2.62 (m, 1H), 2.54 (m, 1H), 2.07 (m, 1H). ^I3C NMR (125 MHz, CDCI3): 204.4, 192.7, 166.6, 164.3, 159.3, 144.4, 142.7, 137.9, 130.4, 130.2, 129.4, 114.9, 114.2, 113.9, 113.8, 109.4, 70.4, 67.1,62.8, 55.3, 31.8, 26.5. FTIR, cm^-1 (thin film): 3316 (brw), 2938 (m), 1678 (m), 1610 (s), 1514 (m), 1393 (m), 1246 (s). HRMS (ESI): Calcd for (C₂₅H₂₄0₇+Na)⁺ 459.1414; Found 459.1354. TLC (50% ethyl acetate-hexanes): R = 0.30 (CAM).

[00458] (5)-2,2-Di-/erf-butyl-7-methoxy-8-(4-methoxybenzyloxy)-5-methyl- 1 1 -oxo- 8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de \ 1 ,3,21dioxasiline-4-carbaldehyde. Όι^‘-tert- butyldichlorosilane (342 μL·, 1.62 mmol, 1.8 equiv) was added to a solution of (5)-l ,9- dihydroxy- 10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (380 mg, 0.90 mmol, 1 equiv), hydroxybenzotriazole (60.8 mg, 0.45 mmol, 0.50 equiv) and diisopropylethylamine (786 μί, 4.50 mmol, 5.0 equiv) in dimethylformamide (30 mL). The reaction flask was heated in an oil bath at 55 °C. After 2 h, the reaction flask was allowed to cool to 23 °C. The reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution (100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed sequentially with water (2 x 100 mL) then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate- hexanes) to provide 285 mg of the product, (S)-2,2-di-/<?ri-butyl-7-methoxy-8-(4- methoxybenzyloxy)-5-methyl- 1 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de] [ 1 ,3,2]dioxasiline-4- carbaldehyde, as a yellow foam (56%). The enantiomeric compound (/?)-2,2-di-½ri-butyl-7- methoxy-8-(4-methoxybenzyloxy)-5-methyl- l 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 – i/e][ l ,3,2]dioxasiline-4-carbaldehyde has been prepared using the same route by utilizing R- (4-methoxybenzyloxy)cyclohex-2-enone as starting material. Ή NMR (500 MHz, CDCI3): 10.84 (s, 1 H), 7.37 (s, 1 H), 7.25 (d, 2H, J = 8.8 Hz), 6.85 (d, 2H, = 8.7 Hz), 5.20 (app s, 1 H), 4.62 (d, 1 H, 7 = 10.0 Hz), 4.51 (d, 1H, J = 1 1.4 Hz), 3.88 (s, 3H), 3.78 (s, 3H), 3.03 (m, 1H), 2.73 (s, 3H), 2.57-2.53 (m, 2H), 2.07 (m, 1H), 1.16 (s, 9H), 1.14 (s, 9H). ¹³C NMR (125 MHz, CDCl₃): 195.6, 190.9, 160.5, 159.2, 150.4, 145.7, 140.4, 134.0, 133.9, 130.3, 129.4, 1 19.5, 1 16.6, 1 15.8, 1 15.3, 1 13.8, 70.4, 67.8, 62.9, 55.2, 34.0, 26.0, 26.0, 22.5, 21.3, 21.1. FTIR, cm^“1 (thin film): 2936 (m), 2862 (m), 1682 (s), 1607 (s), 1371 (s), 1244 (s) 1057 (s). HRMS (ESI): Calcd for (C₃3H₄o0₇Si+H)⁺ 577.2616; Found 577.2584. TLC (10% ethyl acetate-hexanes): R/ = 0.19 (CAM). Alternative Routes to (4S,6S)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy) cyclohex-2-enone.

Alternative Route 1.

[00459] (25,45,55)-2,4-Bis(ferf-butyldimethylsilyloxy)-5-hvdroxycvclohexanone. Dess- Martin periodinane (6.1 1 g, 14.4 mmol, 1.1 equiv) was added to a solution of diol (5.00 g, 13.3 mmol, 1 equiv) in tetrahydrofuran (120 mL) at 23 °C (Lim, S. M.; Hill, N.; Myers, A. G. J. Am. Chem. Soc. 2009, 131, 5763-5765). After 40 min, the reaction mixture was diluted with ether (300 mL). The diluted solution was filtered through a short plug of silica gel (-5 cm) and eluted with ether (300 mL). The filtrate was concentrated. The bulk of the product was transformed as outlined in the following paragraph, without purification. Independently,

an analytically pure sample of the product was obtained by flash-column chromatography (20% ethyl acetate-hexanes) and was characterized by Ή NMR, ^{l 3}C NMR, IR, and HRMS. TLC: (17% ethyl acetate-hexanes) R = 0.14 (CAM); Ή NMR (500 MHz, CDCI3) δ: 4.41 (dd, 1 H, 7 = 9.8, 5.5 Hz), 4.05 (m, l H), 4.00 (m, 1H), 2.81 (ddd, 1 H, 7 = 14.0, 3.7, 0.9 Hz), 2.52 (ddd, 1 H, 7 = 14.0, 5.3, 0.9 Hz), 2.29 (br s, 1 H), 2.18 (m, 1H), 1.98 (m, 1 H), 0.91 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.1 1 (s, 3H), 0.09 (s, 3H), 0.04 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 207.9, 73.9, 73.3, 70.5, 43.3, 39.0, 25.7, 25.6, 18.3, 17.9, -4.7, -4.8, -4.9, -5.4; FTIR (neat), cm^“‘ : 3356 (br), 2954 (m), 2930 (m), 2857 (m), 1723 (m), 1472 (m). 1253 (s), 1 162 (m), 1 105 (s), 1090 (s), 1059 (s), 908 (s), 834 (s), 776 (s), 731 (s); HRMS (ESI): Calcd for (C|₈H380₄Si2+H)⁺ 375. 2381 , found 375.2381.

[00460] (4 ,6 )-4.6-Bis(fcr/-butyldimethylsilyloxy)cvclohex-2-enone. Trifluoroacetic anhydride (6.06 mL, 43.6 mmol, 3.3 equiv) was added to an ice-cooled solution of the alcohol ( 1 equiv, see paragraph above) and triethylamine ( 18.2 mL, 131 mmol, 9.9 equiv) in dichloromethane (250 mL) at 0 °C. After 20 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 18 h, the reaction flask was cooled in an ice bath at 0 °C, and the product solution was diluted with water ( 100 mL). The cooling bath was removed and the reaction flask was allowed to warm to 23 °C. The layers were separated. The aqueous layer was extracted with dichloromethane (2 x 200 mL). The organic layers were combined. The combined solution was washed with saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (6% ethyl acetate-hexanes) to provide 3.02 g of the product, (4S,65)-4,6-bis(/eri-butyldimethylsilyloxy)cyclohex-2-enone, as a colorless oil (64% over two steps). TLC: (20% ethyl acetate-hexanes) R = 0.56 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.76 (dd, 1 Η, / = 10.1 , 3.6 Hz), 5.88 (d, 1 H, 7 = 10.1 Hz), 4.66 (ddd, 1 H, 7 = 5.6, 4.1 , 3.6 Hz), 4.40 (dd, 1 H, 7 = 8.1 , 3.7 Hz), 2.26 (ddd, 1 H, / = 13.3, 8.0, 4.1 Hz), 2.1 1 (ddd, 1 H, J = 13.2, 5.6, 3.8 Hz), 0.91 (s, 9H), 0.89 (s, 9H), 0.12 (s, 3H), 0. 1 1 (s, 3H), 0. 10 (s, 3H), 0.10 (s, 3H); ¹³C NMR ( 125 MHz, CDC1₃) δ: 197.5, 150.3, 127.0, 71 .0, 64.8, 41.6, 25.7, 25.7, 18.3, 18.1 , -4.7, -4.8, -4.8, -5.4; FTIR (neat), cm^-1 : 3038 (w), 2955 (m), 2930 (m), 1705 (m), 1472 (m), 1254 (m), 1084 (m), 835 (s), 777 (s), 675 (s); HRMS (ESI): Calcd for (C,8H360₂Si2+Na)⁺ 379. 2095, found 379. 2080.

[00461] (4S,6S)-6-(/er/-Butyldimethylsilyloxy)-4-hydroxycvclohex-2-enone. Tetra- j- butylammonium fluoride ( 1 .0 M solution in tetrahydrofuran, 8.00 mL, 8.00 mmol, 1 .0 equiv) was added to an ice-cooled solution of the enone (2.85 g, 8.00 mmol, 1 equiv) and acetic acid (485 ί, 8.00 mmol, 1 .0 equiv) in tetrahydrofuran (80 mL) at 0 °C. After 2 h, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 22 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (300 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 300 mL). The organic layers were combined. The combined solution was washed sequentially with saturated aqueous sodium bicarbonate solution ( 100 mL) then saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (25% ethyl acetate-hexanes) to provide 760 mg of the product, (4S,6S)-6-(ferNbutyldimethylsilyloxy)-4-hydroxycyclohex-2-enone, as a white solid (39%). TLC: (20% ethyl acetate-hexanes) R/ = 0.20 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.87 (dd, 1 Η, 7 = 10.2, 3.2 Hz), 5.95 (dd, 1H, J = 10.3, 0.9 Hz), 4.73 (m, 1 H), 4.35 (dd, 1 H, 7 = 7.6, 3.7 Hz), 2.39 (m, 1 H), 2. 13 (ddd, 1 H, J = 13.3, 6.2, 3.4 Hz), 1.83 (d, 1 H, J = 6.2), 0.89 (s, 9H), 0.10 (s, 3H), 0. 10 (s, 3H); ¹³C NMR ( 125 MHz, CDCb) δ: 197.3, 150.0, 127.5, 70.9, 64.2, 41 .0, 25.7, 18.2, -4.8, -5.4; FTIR (neat), cm^“1 : 2956 (w), 293 1 (w), 2858 (w), 1694 (m); HRMS (ESI): Calcd for (C |₂H220₃Si+H)⁺ 243.141 1 , found 243. 1412.

82″:.

[00462] (45.6S)-6-(fgrf-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. Triphenylmethyl tetrafluoroborate ( 16 mg, 50 μπιοΐ, 0.050 equiv) was added to a solution of 4-methoxybenzyl-2,2,2-trichloroacetimidate (445 μΙ_, 2.5 mmol, 2.5 equiv) and alcohol (242 mg, 1 .0 mmol, 1 equiv) in ether ( 10 mL) at 23 °C. After 4 h, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 15 mL) and ethyl acetate (50 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL). The organic layers were combined. The combined solution was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 297 mg of the product, (4S,6S)-6-(im-butyldimethylsilyloxy)-4-(4- methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%).

Alternative Route 2.

[00463] (5)-?erf-Butyl(4-(4-methoxybenzyloxy)cvclohexa- 1.5-dienyloxy)dimethylsilane. rerr-Butyldimethylsilyl trifluoromethanesulfonate (202 iL, 0.94 mmol, 2.0 equiv) was added to an ice-cooled solution of triethylamine (262 μί, 1.88 mmol, 4.0 equiv) and enone ( 109 mg, 0.47 mmol, 1 equiv) in dichloromethane (5.0 mL). After 30 min, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 10 mL), water (30 mL), and dichloromethane (40 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous ammonium chloride solution (20 mL) then saturated aqueous sodium chloride solution (20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography with triethylamine-treated silica gel (5% ethyl acetate-hexanes), to provide 130 mg of the product, (5)-ierr-butyl(4-(4- methoxybenzyloxy)cyclohexa- l ,5-dienyloxy)dimethylsilane, as a colorless oil (80%). Ή

NMR (500 MHz, CDC1₃): 7.27 (d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.6 Hz), 5.96 (dd, 1 H, J = 9.9, 3.5 Hz), 5.87 (d, 1 H, 7 = 9.6 Hz), 4.94 (m, l H), 4.46 (s, 2H), 4.14 (m, 1 H), 3.81 (s, 3H), 2.49 (m, 2H), 0.93 (s, 9H), 0. 16 (s, 3H), 0.15 (s, 3H). ^{, 3}C NMR ( 125 MHz, CDC1₃): 159.1 , 147.5, 130.9, 129.2, 128.6, 128.1 , 1 13.8, 101.4, 70.2, 69.0, 55.3, 28.5, 25.7, 18.0, ^1.5, -4.5. FTIR, cm^-1 (thin film): 2957 (m), 2931 (m), 2859 (m), 1655 (w), 1613 (w), 1515 (s), 1248 (s), 1229 (s), 1037 (m), 910 (s). HRMS (ESI): Calcd for (C₂oH3o0₃Si+H)⁺ 347.2037; Found 347.1912. TLC (20% ethyl acetate-hexanes): R = 0.74 (CAM).

OP B OPMB DM 00 ,,Α,,

c Ύ’^“ -ietone ii ·η- ) ‘”OH

OTBS 82 ^Q

[00464] (4S,6S)-6-Hvdroxy-4-(4-methoxybenzyloxy)cvclohex-2-enone. A solution of dimethyldioxirane (0.06 M solution in acetone, 2.89 mL, 0.17 mmol, 1.2 equiv) was added to an ice-cooled solution of (S)-ieri-butyl(4-(4-methoxybenzyloxy)cyclohexa- l ,5- dienyloxy)dimethylsilane (50 mg, 0.14 mmol, 1 equiv). After 10 min, the reaction mixture was partitioned between dichloromethane ( 15 mL) and 0.5 M aqueous hydrochloric acid ( 10 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous sodium bicarbonate solution ( 10 mL) then water ( 10 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 30 mg of the product, (4S,6S)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%). Ή NMR (500 MHz, CDC1₃): 7.28 (d, 2H, J = 8.2 Hz), 6.89 (m, 3H), 6.09 (d, 1 H, J = 10.1 Hz), 4.64 (m, 2H), 4.53 (d, 1 H, 7 = 1 1 .4 Hz), 4.24 (m, 1 H), 3.81 (s, 3H), 3.39 (d, 1 H, 7 = 1.4 Hz), 2.67 (m, 1 H), 1 .95 (ddd, 1 H, 7 = 12.8, 12.8, 3.6 Hz). ^{I 3}C NMR ( 125 MHz, CDC1₃): 200.4, 159.5, 146.6, 129.7, 129.4, 127.8, 1 14.0, 71.6, 69.8, 68.9, 55.3, 35.1 . FTIR, cm^-1 (thin film): 3474 (br), 2934 (m), 2864 (m), 1692 (s), 1613 (m), 1512 (s), 1246 (s), 1059 (s), 1032 (s). HRMS (ESI): Calcd for (C,₄H_l6O₄+Na)⁺ 271.0941 ; Found 271.0834. TLC (50% ethyl acetate-hexanes): R/ = 0.57 (CAM).

[00465] (45,65)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. rerr-Butyldimethychlorosilane (26 mg, 0.18 mmol, 1.5 equiv) was added to an ice- cooled solution of (45,65)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone (29 mg, 0.12 mmol, 1 equiv) and imidazole (24 mg, 0.35 mmol, 3 equiv) in dimethylformamide (0.5 mL). After 45 min, the reaction mixture was partitioned between water (15 mL), saturated aqueous sodium chloride solution (15 mL), and ethyl acetate (20 mL). The layers were separated. The organic layer was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 29 mg of the product, (4S,6S)-6-(rm-butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cyclohex-2- enone, as a colorless oil (87%).

Glycosylation experiments

[00466] Glycosylation experiments demonstrate that the chemical process developed allows for the preparation of synthetic, glycosylated trioxacarcins. Specifically, the C4 or CI 3 hydroxyl group may be selectively glycosylated with a glycosyl donor (for example, a glycosyl acetate) and an activating agent (for example, TMSOTf), which enables preparation of a wide array of trioxacarcin analogues.

Selective Glycosylation of the C4 Hydroxyl Group

[00467] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 19.9 mg, 88 μιτιοΐ, 1.1 equiv) was added to a vigorously stirring, biphasic solution of differentially protected trioxacarcin precursor (60 mg, 80 μιτιοΐ, 1 equiv) in dichloromethane ( 1.1 mL) and pH 7 phosphate buffer (220 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 3 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (50 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μιτι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 33 mg of the product as a yellow-green powder (65%).

[00468] Trimethylsilyl triflate ( 10% in dichloromethane, 28.3 μί, 16 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected trioxacarcin precursor (33 mg, 52 μπιοΐ, 1 equiv), 1 -0- acetyltrioxacarcinose A ( 14.1 mg, 57 μιτιοΐ, 1.1 equiv), and powdered 4- A molecular sieves (-50 mg) in dichloromethane (1 .0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between

dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 20 mg of the product as a yellow-green powder (47%). TLC: (5% methanol-dichloromethane) R = 0.40 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 7.47 (s, 1H), 5.38 (d, 1H, J = 3.6 Hz), 5.35 (app s, 1 H), 5.26 ppm (d, 1 H, 7 = 4.0 Hz), 4.84 (d, 1 H, J = 4.0 Hz), 4.78 (dd, 1 H, 7 = 12.3, 5.2 Hz), 4.75 (s, 1H), 4.71 (s, 1 H), 4.52 (q, 1H, J = 6.6 Hz), 3.86 (s, 1 H), 3.83 (s, 3H), 3.62 (s, 3H), 3.47 (s, 3H), 3.15 (d, l H, y = 5.3 Hz), 3.05 (d, 1 H, 7 = 5.3 Hz), 2.60 (s, 3H), 2.58 (m, 1H), 2.35 (m, 1 H), 2.14 (s, 3H), 1.96 (dd, 1 H, 7 = 14.6, 4.1 Hz), 1.62 (d, 1 H, 7 = 14.6 Hz), 1.26 (s, 1 H), 1.23 (d, 3H, J = 6.6 Hz), 1.08 (s, 3H), 0.95 (s, 9H), 0.24 (s, 3H), 0.16 (s, 3H); ‘³C NMR ( 125 MHz, CDC1₃) 6: 202.8, 170.5, 163.2, 151.8, 144.4, 142.4, 135.2, 126.6, 1 16.8, 1 15.2, 1 15.1 , 108.3, 104.0, 100.3, 98.6, 98.3, 74.6, 73.4, 69.8, 69.5, 69.5, 68.9, 69.5, 69.5, 68.9, 68.4, 62.9, 62.7, 57.2, 56.8, 50.7, 38.8, 36.8, 26.0, 25.9, 21.1 , 20.6, 18.6, 17.0, -4.2, -5.3; FTIR (neat), cm^“‘ : 2953 (w), 2934 (w), 2857 (w), 1749 (w), 1622 (m), 1570 (w), 1447 (w), 1391 (m), 1321 (w), 1294 (w), 1229 (m), 1 159 (m), 1 121 (s), 1084 (s), 1071 (m), 1020 (m), 995 (s), 943 (s), 868 (m), 837 (m), 779 (m); HRMS (ESI): Calcd for (C₄oH₅₄0i₆Si+Na)⁺ 841.3073, found

841.3064.

Glycosylation of a Cycloaddition Coupling Partner

[00469] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 14.3 mg, 63 μπιοΐ, 1.2 equiv) was added to a vigorously stirring, biphasic solution of differentially protected aldehyde (37 mg, 52 μιτιοΐ, 1 equiv) in dichloromethane (870 μί) and water (175 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 2 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (40 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 28 mg of the product as a yellow powder (91 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.37 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1H), 7.30 (s, 1 H), 5.45 (m, 1H), 4.68 (dd, 1H, / = 10.3, 4.2 Hz), 3.97 (s, 3H), 3.31 (brs, 1H), 2.72 (s, 3H), 2.51-2.45 (m, 1H), 2.41-2.37 (m, 1H), 1.15 (s, 9H), 1 , 13 (s, 9H), 0.88 (s, 9H), 0.15 (s, 3H), 0.1 1 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 194.6, 191 , 160.5, 150.2, 146, 140.8, 135.8, 134, 1 19.6, 1 16.2, 1 15.4, 1 14.7, 72.7, 63.7, 62.4, 38.8, 29.9, 62.4, 38.8, 63.7, 62.4, 38.8, 63.7, 62.4, 38.8, 29.9, 26.2, 26.1 , 26, 22.7, 21.4; FTIR (neat), cm^“1 : 3470 (br, w), 2934 (w), 2888 (w), 1684 (s), 1607 (s), 1560 (w), 1472 (m), 1445 (w), 1392 (m), 1373 (s), 1242 (s), 1 153 (s), 1 1 19 (w), 1074 (m), 1044 (s), 1013 (s), 982 (w), 934 (m), 907 (w), 870 (m), 827 (s), 795 (s), 779 (s), 733 (s), 664 (s); HRMS (ESI): Calcd for (C₃iH₄₆0₇Si₂+H)⁺ 587.2855, found 587.2867.

[00470] Trimethylsilyl triflate (10% in dichloromethane, 25.9 μί, 14 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected aldehyde (28 mg, 48 μηιοΐ, 1 equiv), 1-0- acetyltrioxacarcinose A (12.9 mg, 52 μπιοΐ, 1.1 equiv), and powdered 4-A molecular sieves (-50 mg) in dichloromethane ( 1.0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 80→98% acetonitrile in water, flow rate: 15 mL/min) to provide 15 mg of the product as a yellow powder (41 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.29 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1 H), 7.32 (s, 1 H), 5.43 (d, 1 H, J = 3.9 Hz), 5.32 (m, 1H), 4.74 (s, 1 H), 4.67 (dd, 1 H, J = 12.3, 5.0 Hz), 4.54 (q, 1H, J = 6.6 Hz), 3.91 (s, 1H), 3.88 (s, 3H), 2.72 (s, 3H), 2.59 (ddd, 1 H, J = 13.8, 5.0, 3.2 Hz), 2.34 (m, 1H), 2.14 (s, 3H), 1.97 (dd, 1H, J = 14.2, 4.2 Hz), 1.71 (d, 1 Η, / = 14.6 Hz), 1.22 (d, 3H, J = 6.3 Hz), 1.15 (s, 9H), 1.15 (s, 9H), 1.08 (s, 3H), 0.93 (s, 9H), 0.23 (s, 3H), 0.13 (s, 3H); ¹³C NMR (125 MHz, CDC1₃) δ: 193.9, 191.0, 170.5, 146.4, 140.9, 134.0, 132.4, 1 19.8, 1 16.8, 1 15.8, 1 15.0, 1 10.8, 99.6, 74.6, 71.5, 70.4, 68.9, 62.9, 62.7, 39.1 , 36.9, 26.2, 26.1 , 26.1 , 25.9, 24.1 , 22.7, 21.5, 21.3, 21.1 , 18.7, 16.9, -4.1 , -5.3; FTIR (neat), cm^-1 : 3524 (br, w), 2934 (m), 2861 (m), 1749 (m), 1686 (s), 1607 (s), 1560 (m), 1474 (m), 1447 (m), 1424 (w), 1375 (s), 1233 (s), 1 159 (s), 1 1 17 (m), 1080 (m), 1049 (s), 1015 (s), 997 (s), 937 (m), 883 (m), 872 (m), 827 (s), 797 (m), 781 (m), 737 (w), 677 (w), 667 (m); HRMS (ESI): Calcd for (C₄₀H₆₀O, ,Si₂+H)⁺773.3747, found 773.3741.

General Glycosylation Procedure of the C13 Hydroxyl Group

[00471] Crushed 4-A molecular sieves (-570 mg / 1 mmol sugar donor) was added to a stirring solution of the sugar acceptor (1 equiv.) and the sugar donor (30.0 equiv.) in dichloromethane ( 1.6 mL / 1 mmol sugar donor) and diethylether (0.228 mL / 1 mmol sugar donor) at 23 °C. The bright yellow mixture was stirred for 90 min at 23 °C and finally cooled to -78 °C. TMSOTf (10.0 equiv.) was added over the course of 10 min at -78 °C. After 4 h, a second portion of TMSOTf (5.0 equiv.) was added at -78 °C and stirring was continued for 1 h. The last portion of TMSOTf (5 equiv.) was added. After 1 h, triethylamine (20 equiv.) was added and the reaction the product mixture was filtered through a short column of silica gel deactivated with triethylamine (30% ethyl acetate-hexanes initially, grading to 50% ethyl acetate-hexanes). H NMR analysis of the residue showed minor sugar donor remainings and that the sugar acceptor had been glycosylated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→100% acetonitrile in water, flow rate: 15 mL/min) to provide the glycosylation product as a bright yellow oil

Three Specific Compounds Prepared by the General Glycosylation Procedure for the CI 3 Hydroxyl Group:

[00472] 10% yield; TLC: (50% ethyl acetate-hexane) R = 0.58 (UV, CAM); Ή NMR (600 MHz, CDC1₃) δ: 7.43 (s, 1 H), 5.84 (t, J = 3.6 Hz, 1 H), 5.29 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.01 (q, J = 6.6 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.73 (s, 1 H), 3.88 (s, OH), 3.77 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 3.03 (app q, J = 5.4 Hz, 2H), 2.84 (d, J = 6.0 Hz, 1 H), 2.77 (d, J = 6.0 Hz, 1 H), 2.72 (t, J = 6.6 Hz, 2H), 2.58 (s, 3H), 2.36 (s, 3H), 2.33 (t, J = 3.0 Hz, 2H), 2.23 (s, 3H), 2.1 1 -2.06 (m, 2H), 1.08 (d, J = 6.0 Hz, 3H). ^‘

[00473] 81 % yield, TLC: (50% ethyl acetate-hexane) R = 0.30 (UV, CAM); Ή NMR (600 MHz, CDCI3) δ: 7.46 (s, 1 H), 7.28 (d, J = 9 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2 H), 5.83 (dd, J = 3.6, 1.8 Hz, 1 H), 5.30 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.19 (m, 1 H), 5.00 (q, J = 6.0 Hz, 1 H), 4.96 (dd, J = 12.0, 4.8 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.74 (s, l H), 4.70 (d, y = 10.8 Hz, 1 H), 4.59 (d, J = 10.8 Hz, 1 H), 3.86 (s, OH), 3.83 (s, 3H), 3.80 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 2.81 (d, J = 6.0 Hz, 1 H), 2.73-2.68 (m, 1 H), 2.70 (d, J = 6.0 Hz, 1 H), 2.59 (s, 3H), 2.35 (s, 3H), 2.33-2.28 (m, 2H), 2.22 (s, 3H), 2.19- 2.1 3 (m, 1 H), 1 .08 (d, J = 6.0 Hz, 3H), 0.97 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H); HRMS (ESI): Calcd for (C₄9H₆₂0₁₈Si+H)⁺ 967.3778, found 967.3795; HRMS (ESI): Calcd for (C ¾₂0,₈Si+Na)⁺ 989.3598, found 989.3585.

[00474] Compound Detected by ESI Mass Spectrometry: Calculated Mass for

[C52H₇| N₃02i Si-Hr^l = 1 100.4277, Measured Mass = 1 100.4253.

PATENT

US 4511560

https://www.google.com/patents/US4511560

The physico-chemical characteristics of DC-45-A and DC-4-5-B₂ according to this invention are as follows:

(1) DC-45-A

(1) Elemental analysis: H:5.74%, C:55.11%

(2) Molecular weight: 877

(3) Molecular formula: C₄₂ H₅₂ O₂₀

(4) Melting point: 180° C.±3° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 1 (in 50% methanol)

(6) Infrared absorption spectrum: As shown in FIG. 2 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-15.3° (c=1.0, ethanol)

(8) PMR spectrum (in CDC]₃ ; ppm): 1.07 (3H,s); 1.10 (3H, d, J=6.8); 1.24 (3H,d, J=6.5); many peaks between 1.40-2.30; 2.14 (3H,s); 2.49 (3H,s); 2.63 (3H,s); many peaks between 2.30-2.80; 2.91 (1H,d, J=5.6); 3.00 (1H,d, J=5.6); 3.49 (3H,s); 3.63 (3H,s); 3.85 (3H, s); many peaks between 3.60-4.00; 4.18 (1H,s); 4.55 (1H,q, J=6.8); many peaks between 4.70-4.90; 5.03 (1H, q, J=6.5); 5.25 (1H,d, J=4.0); 5.39 (1H, d, J=4.0); 5.87 (1H, m); 7.52 (1H,s); 14.1 (1H,s)

(9) CMR spectrum (in CDCl₃ ; ppm): 210.9; 203.8; 170.3; 162.1; 152.5; 145.2; 142.3; 135.3; 126.7; 117.0; 114.2; 108.3; 105.3; 99.7; 97.2; 93.7; 85.1; 79.0; 74.6; 71.1; 69.6; 69.3; 68.8; 67.9; 66.3; 64.0; 62.8; 57.3; 55.9; 36.5; 32.2; 28.0; 25.7; 20.9; 20.2; 17.0; 14.7

(10) Solubility: Soluble in methanol, ethanol, water and chloroform; slightly soluble in acetone and ethyl acetate, and insoluble in ether and n-hexane

(2) DC-45-B₂

(1) Elemental analysis: H: 6.03%, C: 54.34%

(2) Molecular weight: 879

(3) Molecular formula: C₄₂ H₅₄ O₂₀

(4) Melting point: 181°-182° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 5 (in 95% ethanol)

(6) Infrared absorption spectrum: As shown in FIG. 6 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-10° (c=0.2, ethanol)

(8) PMR spectrum (in CDCl₃ ; ppm): 1.07 (3H,s); many peaks between 1.07-1.5; many peaks between 1.50-2.80; 2.14 (3H,s); 2.61 (3H, broad s); 2.86 (1H, d, J=5.7); 2.96 (1H, d, J=5.7); 3.46 (3H,s); 3.63 (3H, s); 3.84 (3H, s); many peaks between 3.65-4.20; many peaks between 4.40-5.00; many peaks between 5.10-5.50; 5.80 (1H, broad s); 7.49 (1H, d, J=1.0); 14.1 (1H, s)

(9) CMR spectrum (in CDCl₃ ; ppm): 202.8; 170.2; 163.1; 151.8; 144.8; 142.9; 135.4; 126.5; 116.8; 114.9; 107.3; 104.6; 101.5; 99.6; 98.0; 94.4; 74.4; 72.5; 71.4; 70.4; 69.1; 68.8; 68.3; 67.9; 67.5; 66.4; 62.9; 62.7; 56.8; 56.5; 48.0; 36.7; 32.3; 25.7; 20.8; 20.3; 18.2; 16.9; 15.5

(10) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform; slightly soluble in benzene, ether and water; and insoluble in n-hexane.

//////

CC1C(C(CC(O1)OC2CC(C(=O)C3=C(C4=C5C(=C(C=C4C(=C23)OC)C)C6C7C(O5)(C8(CO8)C(O6)(O7)C(OC)OC)OC9CC(C(C(O9)C)(C(=O)C)O)O)O)O)(C)O)OC(=O)C

Some new cancer drugs are available in other countries, but not in India. BY E. Kumar Sharma, Dec 22, 2013

cancer Comments Off

Mar 102015

E. Kumar Sharma Follow @EKumarSharma Edition:Dec 22, 2013

http://businesstoday.intoday.in/story/some-new-cancer-drugs-still-not-available-in-india/1/201095.html

Cancer, Chemistry, and the Cell: Molecules that Interact with the Neurotensin Receptors

cancer Comments Off

Sep 262014

Cancer, Chemistry, and the Cell: Molecules that Interact with the Neurotensin Receptors

R.M. Myers,* J.W. Shearman, M.O. Kitching, A. Ramos-Montoya, D.E. Neal, S.V. Ley, ACS Chem. Biol.2009, 4, 503-525.

see

http://pubs.acs.org/doi/abs/10.1021/cb900038e

The literature covering neurotensin (NT) and its signalling pathways, receptors, and biological profile is complicated by the fact that the discovery of three NT receptor subtypes has come to light only in recent years. Moreover, a lot of this literature explores NT in the context of the central nervous system and behavioral studies. However, there is now good evidence that the up-regulation of NT is intimately involved in cancer development and progression. This Review aims to summarize the isolation, cloning, localization, and binding properties of the accepted receptor subtypes (NTR1, NTR2, and NTR3) and the molecules known to bind at these receptors. The growing role these targets are playing in cancer research is also discussed. We hope this Review will provide a useful overview and a one-stop resource for new researchers engaged in this field at the chemistry−biology interface.

Keywords:

Agonist: A molecule that binds to a specific receptor and triggers a response in the cell mimicking the action of an endogenous ligand.; Antagonist: A molecule that binds to a specific receptor yet does not provoke a biological response itself upon binding but blocks or dampens the agonist-mediated response.; Homology and identity: Measures of similarity between protein sequences. Homology implies an evolutionary link and is qualitative, whereas identity is quantifiable.; Site-directed mutagenesis: A process where specific single nucleotide changes within a gene can be made resulting in the change of a specific amino acid within a protein sequence.; Prognostic factor: A biomarker that can be used to estimate the chance of recovery from a disease or the chance of the disease recurring.; Predictive factor: A biomarker used to predict the response of a cancer to a given therapy.

Aldoxorubicin…CytRx is pouring money into R&D of cancer-fighting drugs

Uncategorized Comments Off

Sep 012014

Aldoxorubicin, DOXO-EMCH

N’-[1-[4(S)-(3-Amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyloxy)-2(S),5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydronaphthacen-2-yl]-2-hydroxyethylidene]-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanohydrazide

1H-Pyrrole-1-hexanoic acid, 2,5-dihydro-2,5-dioxo-, (2E)-2-[1-[(2S,4S)-4-[(3-amino-2,3,6-trideoxy-α-L-lyxo– hexopyranosyl)oxy]-1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7-methoxy-6,11-dioxo-2-naphthacenyl]-2- hydroxyethylidene]hydrazide

CytRx is pouring money into R&D of cancer-fighting drugs see article

Los Angeles Times

s most promising cancer-fighting drug, aldoxorubicin, is “sort of like a guided … Phase 3 clinical trial of a second-line treatment for soft-tissue sarcoma.

Aldoxorubicin-INNO206 structure

Aldoxorubicin

http://www.ama-assn.org/resources/doc/usan/aldoxorubicin.pdf

in phase 3 Cytrx Corporation

(E)-N’-(1-((2S,4S)-4-(((2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride

1H-Pyrrole-1-hexanoic acid, 2,5-dihydro-2,5-dioxo-, (2E)-2-[1-[(2S,4S)-4-[(3-amino-
2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-
7-methoxy-6,11-dioxo-2-naphthacenyl]-2-hydroxyethylidene]hydrazide

N’-[(1E)-1-{(2S,4S)-4-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-2,5,12-
trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl}-2-
hydroxyethylidene]-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanohydrazide
MOLECULAR FORMULA C37H42N4O13

MOLECULAR WEIGHT 750.7

SPONSOR CytRx Corp.

CODE DESIGNATION

Aldoxorubicin
INNO 206
INNO-206
UNII-C28MV4IM0B

CAS REGISTRY NUMBER 1361644-26-9

CAS: 151038-96-9 (INNO-206); 480998-12-7 (INNO-206 HCl salt), 1361644-26-9

QC data:	View NMR, View HPLC, View MS
Safety Data Sheet (MSDS):	View Material Safety Data Sheet (MSDS)

hydrochloride

CAS: 151038-96-9

Chemical Formula: C37H42N4O13

Exact Mass: 750.27484

Molecular Weight: 750.75

Certificate of Analysis:	View current batch of CoA
QC data:	View NMR, View HPLC, View MS
Safety Data Sheet (MSDS):	View Material Safety Data Sheet (MSDS)

Chemical structure for Aldoxorubicin (USAN)

In vitro protocol:	Clin Cancer Res. 2012 Jul 15;18(14):3856-67
In vivo protocol:	Clin Cancer Res. 2012 Jul 15;18(14):3856-67.Invest New Drugs. 2010 Feb;28(1):14-9.Invest New Drugs. 2012 Aug;30(4):1743-9.Int J Cancer. 2007 Feb 15;120(4):927-34.
Clinical study:	Expert Opin Investig Drugs. 2007 Jun;16(6):855-66.

Aldoxorubicin (INNO-206): Aldoxorubicin, also known as INNO-206, is the 6-maleimidocaproyl hydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) with antineoplastic activity. Following intravenous administration, doxorubicin prodrug INNO-206 binds selectively to the cysteine-34 position of albumin via its maleimide moiety. Doxorubicin is released from the albumin carrier after cleavage of the acid-sensitive hydrazone linker within the acidic environment of tumors and, once located intracellularly, intercalates DNA, inhibits DNA synthesis, and induces apoptosis. Albumin tends to accumulate in solid tumors as a result of high metabolic turnover, rapid angiogenesis, hyervasculature, and impaired lymphatic drainage. Because of passive accumulation within tumors, this agent may improve the therapeutic effects of doxorubicin while minimizing systemic toxicity.

“Aldoxorubicin has demonstrated effectiveness against a range of tumors in both human and animal studies, thus we are optimistic in regard to a potential treatment for Kaposi’s sarcoma. The current standard-of-care for severe dermatological and systemic KS is liposomal doxorubicin (Doxil®). However, many patients exhibit minimal to no clinical response to this agent, and that drug has significant toxicity and manufacturing issues,” said CytRx President and CEO Steven A. Kriegsman. “In addition to obtaining valuable information related to Kaposi’s sarcoma, this trial represents another opportunity to validate the value and viability of our linker technology platform.” The company expects to announce Phase-2 study results in the second quarter of 2015.

Kaposi’s sarcoma is an orphan indication, meaning that only a small portion of the population has been diagnosed with the disease (fewer than 200,000 individuals in the country), and in turn, little research and drug development is being conducted to treat and cure it. The FDA’s Orphan Drug Act may grant orphan drug designation to a drug such as aldoxorubicin that treats a rare disease like Kaposi’s sarcoma, offering market exclusivity for seven years, fast-track status in some cases, tax credits, and grant monies to accelerate research

The widely used chemotherapeutic agent doxorubicin is delivered systemically and is highly toxic, which limits its dose to a level below its maximum therapeutic benefit. Doxorubicin also is associated with many side effects, especially the potential for damage to heart muscle at cumulative doses greater than 450 mg/m2. Aldoxorubicin combines doxorubicin with a novel single-molecule linker that binds directly and specifically to circulating albumin, the most plentiful protein in the bloodstream. Protein-hungry tumors concentrate albumin, thus increasing the delivery of the linker molecule with the attached doxorubicin to tumor sites. In the acidic environment of the tumor, but not the neutral environment of healthy tissues, doxorubicin is released. This allows for greater doses (3 1/2 to 4 times) of doxorubicin to be administered while reducing its toxic side effects. In studies thus far there has been no evidence of clinically significant effects of aldoxorubicin on heart muscle, even at cumulative doses of drug well in excess of 2,000 mg/m2.

INNO-206 is an anthracycline in early clinical trials at CytRx Oncology for the treatment of breast cancer, HIV-related Kaposi’s sarcoma, glioblastoma multiforme, stomach cancer and pancreatic cancer. In 2014, a pivotal global phase 3 clinical trial was initiated as second-line treatment in patients with metastatic, locally advanced or unresectable soft tissue sarcomas. The drug candidate was originally developed at Bristol-Myers Squibb, and was subsequently licensed to KTB Tumorforschungs. In August 2006, Innovive Pharmaceuticals (acquired by CytRx in 2008) licensed the patent rights from KTB for the worldwide development and commercialization of the drug candidate. No recent development has been reported for research that had been ongoing for the treatment of small cell lung cancer (SCLC).

INNO-206 is a doxorubicin prodrug. Specifically, it is the 6-maleimidocaproyl hydrazone of doxorubicin. After administration, the drug candidate rapidly binds endogenous circulating albumin through the acid sensitive EMCH linker. Circulating albumin preferentially accumulates in tumors, bypassing uptake by other non-specific sites including the heart, bone marrow and the gastrointestinal tract. Once inside the acidic environment of the tumor cell, the EMCH linker is cleaved and free doxorubicin is released at the tumor site. Like other anthracyclines, doxorubicin inhibits DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, thus preventing the replication of rapidly-growing cancer cells. It also creates iron-mediated free oxygen radicals that damage the DNA and cell membranes. In 2011, orphan drug designation was assigned in the U.S. for the treatment of pancreatic cancer and for the treatment of soft tissue sarcoma.

CytRx Corporation (NASDAQ:CYTR) has announced it has initiated a pivotal global Phase 3 clinical trial to evaluate the efficacy and safety of aldoxorubicin as a second-line treatment for patients with soft tissue sarcoma (STS) under a Special Protocol Assessment with the FDA. Aldoxorubicin combines the chemotherapeutic agent doxorubicin with a novel linker-molecule that binds specifically to albumin in the blood to allow for delivery of higher amounts of doxorubicin (3.5 to 4 times) without several of the major treatment-limiting toxicities seen with administration of doxorubicin alone.

According to a news from Medicalnewstoday.com; CytRx holds the exclusive worldwide rights to INNO-206. The Company has previously announced plans to initiate Phase 2 proof-of-concept clinical trials in patients with pancreatic cancer, gastric cancer and soft tissue sarcomas, upon the completion of optimizing the formulation of INNO-206. Based on the multiple myeloma interim results, the Company is exploring the possibility of rapidly including multiple myeloma in its INNO-206 clinical development plans.

According to CytRx’s website, In preclinical models, INNO-206 was superior to doxorubicin with regard to ability to increase dosing, antitumor efficacy and safety. A Phase I study of INNO-206 that demonstrated safety and objective clinical responses in a variety of tumor types was completed in the beginning of 2006 and presented at the March 2006 Krebskongress meeting in Berlin. In this study, doses were administered at up to 4 times the standard dosing of doxorubicin without an increase in observed side effects over historically seen levels. Objective clinical responses were seen in patients with sarcoma, breast, and lung cancers.

INNO-206 – Mechanism of action:

According to CytRx’s website, the proposed mechanism of action is as the follow steps: (1) after administration, INNO-206 rapidly binds endogenous circulating albumin through the EMCH linker. (2) circulating albumin preferentially accumulates in tumors, bypassing uptake by other non-specific sites including heart, bone marrow and gastrointestinal tract; (3) once albumin-bound INNO-206 reaches the tumor, the acidic environment of the tumor causes cleavage of the acid sensitive linker; (4) free doxorubicin is released at the site of the tumor.

INNO-206 – status of clinical trials:

CytRx has announced that, in December 2011, CytRx initiated its international Phase 2b clinical trial to evaluate the preliminary efficacy and safety of INNO-206 as a first-line therapy in patients with soft tissue sarcoma who are ineligible for surgery. The Phase 2b clinical trial will provide the first direct clinical trial comparison of INNO-206 with native doxorubicin, which is dose-limited due to toxicity, as a first-line therapy. (source:http://cytrx.com/inno_206, accessed date: 02/01/2012).

Results of Phase I study:

In a phase I study a starting dose of 20 mg/m2 doxorubicin equivalents was chosen and 41 patients with advanced cancer disease were treated at dose levels of 20–340 mg/m2 doxorubicin equivalents . Treatment with INNO-206 was well tolerated up to 200 mg/m2 without manifestation of drug-related side effects which is a ~3-fold increase over the standard dose for doxorubicin (60 mg/kg). Myelosuppression and mucositis were the predominant adverse effects at dose levels of 260 mg/m2 and became dose-limiting at 340 mg/m2. 30 of 41 patients were assessable for analysis of response. Partial responses were observed in 3 patients (10%, small cell lung cancer, liposacoma and breast carcinoma). 15 patients (50%) showed a stable disease at different dose levels and 12 patients (40%) had evidence of tumor progression. (source: Invest New Drugs (2010) 28:14–19)

phase 2

CytRx Corporation (CYTR), a biopharmaceutical research and development company specializing in oncology, today announced that its oral presentation given by Sant P. Chawla, M.D., F.R.A.C.P., Director of the Sarcoma Oncology Center, titled “Randomized phase 2b trial comparing first-line treatment with aldoxorubicin versus doxorubicin in patients with advanced soft tissue sarcomas,” was featured in The Lancet Oncology in its July 2014 issue (Volume 15, Issue 8) in a review of the major presentations from the 2014 American Society of Clinical Oncology (ASCO) Annual Meeting.

“We are honored to have been included in The Lancet Oncology’s review of major presentations from ASCO and pleased that these important clinical findings are being recognized by one of the world’s premier oncology journals,” said Steven A. Kriegsman, CytRx President and CEO. “In clinical trials, aldoxorubicin has been shown to be a well-tolerated and efficacious single agent for the treatment of soft tissue sarcoma (STS) that lacks the cardiotoxicity associated with doxorubicin therapy, the current standard of care. We remain on track to report the full overall survival results from this trial prior to year-end 2014.”

The data presented at ASCO 2014 were updated results from CytRx’s ongoing multicenter, randomized, open-label global Phase 2b clinical trial investigating the efficacy and safety of aldoxorubicin compared with doxorubicin as first-line therapy in subjects with metastatic, locally advanced or unresectable STS. The updated trial results demonstrated that aldoxorubicin significantly increases progression-free survival (PFS), PFS at 6 months, overall response rate (ORR) and tumor shrinkage, compared to doxorubicin, the current standard-of-care, as a first-line treatment in patients with STS. The data trended in favor of aldoxorubicin for all of the major subtypes of STS

phase 3

Aldoxorubicin is currently being studied in a pivotal global Phase 3 clinical trial evaluating the efficacy and safety of aldoxorubicin as a second-line treatment for patients with STS under a Special Protocol Assessment with the FDA. CytRx is also conducting two Phase 2 clinical trials evaluating aldoxorubicin in patients with late-stage glioblastoma (GBM) and HIV-related Kaposi’s sarcoma and expects to start a phase 2b study in patients with relapsed small cell lung cancer

PATENTS WO 2000076551, WO 2008138646, WO 2011131314,

…………………….

WO 2014093815

http://www.google.com/patents/WO2014093815A1?cl=en

Anthracyclines are a class of antibiotics derived from certain types of Streptomyces bacteria. Anthracyclines are often used as cancer therapeutics and function in part as nucleic acid intercalating agents and inhibitors of the DNA repair enzyme topoisomerase II, thereby damaging nucleic acids in cancer cells, preventing the cells from replicating. One example of an anthracycline cancer therapeutic is doxorubicin, which is used to treat a variety of cancers including breast cancer, lung cancer, ovarian cancer, lymphoma, and leukemia. The 6-maleimidocaproyl hydrazone of doxorubicin (DOXO-EMCH) was originally synthesized to provide an acid-sensitive linker that could be used to prepare immunoconjugates of doxorubicin and monoclonal antibodies directed against tumor antigens (Willner et al., Bioconjugate Chem 4:521-527 (1993)). In this context, antibody disulfide bonds are reduced with dithiothreitol to form free thiol groups, which in turn react with the maleimide group of DOXO-EMCH to form a stable thioether bond. When administered, the doxorubicin-antibody conjugate is targeted to tumors containing the antigen recognized by the antibody. Following antigen-antibody binding, the conjugate is internalized within the tumor cell and transported to lysosomes. In the acidic lysosomal environment, doxorubicin is released from the conjugate intracellularly by hydrolysis of the acid-sensitive hydrazone linker. Upon release, the doxorubicin reaches the cell nucleus and is able to kill the tumor cell. For additional description of doxorubicin and

DOXO-EMCH see, for example, U.S. Patents 7,387,771 and 7,902,144 and U.S. Patent Application No. 12/619,161, each of which are incorporated in their entirety herein by reference.

[0003] A subsequent use of DOXO-EMCH was developed by reacting the molecule in vitro with the free thiol group (Cys-34) on human serum albumin (HSA) to form a stable thioether conjugate with this circulating protein (Kratz et al, J Med Chem 45:5523-5533 (2002)). Based on these results, it was

hypothesized that intravenously-administered DOXO-EMCH would rapidly conjugate to HSA in vivo and that this macromolecular conjugate would preferentially accumulate in tumors due to an “enhanced permeability and retention” (EPR) intratumor effect (Maeda et al., J Control Release 65:271-284 (2000)).

[0004] Acute and repeat-dose toxicology studies with DOXO-EMCH in mice, rats, and dogs identified no toxicity beyond that associated with doxorubicin, and showed that all three species had significantly higher tolerance for DOXO-EMCH compared to doxorubicin (Kratz et al, Hum Exp Toxicol 26: 19-35 (2007)). Based on the favorable toxicology profile and positive results from animal tumor models, a Phase 1 clinical trial of DOXO-EMCH was conducted in 41 advanced cancer patients (Unger et al, Clin Cancer Res 13:4858-4866 (2007)). This trial found DOXO-EMCH to be safe for clinical use. In some cases, DOXO-EMCH induced tumor regression.

[0005] Due to the sensitivity of the acid-cleavable linker in DOXO-EMCH, it is desirable to have formulations that are stable in long-term storage and during reconstitution (of, e.g., previously lyophilized compositions) and administration. DOXO-EMCH, when present in compositions, diluents and administration fluids used in current formulations, is stable only when kept at low temperatures. The need to maintain DOXO-EMCH at such temperatures presents a major problem in that it forces physicians to administer cold (4°C) DOXO-EMCH compositions to patients. Maintaining DOXO-EMCH at low temperatures complicates its administration in that it requires DOXO-EMCH to be kept at 4°C and diluted at 4°C to prevent degradation that would render it unsuitable for patient use. Further, administration at 4°C can be harmful to patients whose body temperature is significantly higher (37°C).

[0006] Lyophilization has been used to provide a stable formulation for many drugs. However, reconstitution of lyophilized DOXO-EMCH in a liquid that does not maintain stability at room temperature can result in rapid decomposition of DOXO-EMCH. Use of an inappropriate diluent to produce an injectable composition of DOXO-EMCH can lead to decreased stability and/or solubility. This decreased stability manifests itself in the cleavage of the linker between the doxorubicin and EMCH moieties, resulting in degradation of the DOXO-EMCH into two components: doxorubicin and linker-maleimide. Thus, stable,

reconstituted lyophilized solutions of anthracycline-EMCH (e.g., DOXO-EMCH), and injectable compositions containing the same, are required to solve these problems and to provide a suitable administration vehicle that can be used reasonably in treating patients both for clinical trials and commercially.

DOXO-EMCH. The term “DOXO-EMCH,” alone or in combination with any other term, refers to a compound as depicted by the following structure:

DOXO-EMCH is also referred to as (E)-N’-(l-((2S,4S)-4-(4-amino-5-hydroxy-6- methyl-tetrahydro-2H-pyran-2-yloxy-2,5 , 12-trihydroxy-7-methoxy-6, 11- dioxol,2,3,4,6,l l-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2H- pyrrol- 1 (5H)yl)hexanehydrazide^»HCl.

………………………………

CN 102675385

http://www.google.com/patents/CN102675385A?cl=en

According to literature reports, (eg see David Willner et al, “(6_Maleimidocaproyl) hydrazoneof Doxorubicm-A New Derivative for the Preparation ofImmunoconjugates oiDoxorubicin,” Bioconjugate Chem. 1993,4, 521-527; JK Tota Hill, etc. man, “The method of preparation of thioether compounds noir,” CN1109886A, etc.), adriamycin 13 – bit hydrazone derivative synthesis and the main process are as follows:

[0004]

[0005] First, maleic anhydride and 6 – aminocaproic acid was refluxed in a large number of acid reaction ko ni acid I; agent under the action of the ring after the cyclization maleimidocaproic acid 2 (yield 30-40% ), cyclic acid anhydride mixture is generally ko, trimethyl silyl chloride and tri-amines such ko; maleimido aminocaproic acid tert-butyl ester with hydrazine to condensation to give 2 – (6 – aminocaproic maleimido ) hydrazine carboxylic acid tert-butyl ester 3 (yield 70-85%), the condensing agent is N-methylmorpholine and isobutyl chloroformate; 3 in a large number of trifluoroacetic acid deprotection ko maleimido ko has trifluoroacetic acid hydrazide 4 (yield 70%); the doxorubicin hydrochloride salt with a ko in trifluoroacetic acid catalyzed condensation in methanol solvent to doxorubicin hydrazone product was obtained (yield 80%) .

[0006] The synthetic method the yield is low (in particular, by maleic acid imido step 2), the total yield of not more than 20%, and the solvent consumption is large, adriamycin hydrazone product per Malek consumes about ko acid reaction solvent, 70mL, tetrahydrofuran 300mL, ko trifluoroacetic acid 40mL, and because the 2 – (6 – maleimido hexanoyl)-hydrazine carboxylic acid tert-butyl ester was purified by column chromatography required, but also to consume a large amount of Solvent. This has resulted in synthesis post-processing complex process, complicated operation. And because the end product of the synthesis of doxorubicin hydrazone ko using trifluoroacetic acid, inevitably there will be in the product ko trifluoroacetic acid impurities, not divisible. Based on the high cost of such a route exists, yield and production efficiency is low, Eri Arts route operational complexity and other shortcomings, is obviously not suitable for mass production, it is necessary to carry out improvements or exploring other Eri Arts synthesis methods.

doxorubicin hydrazone derivative,

Wherein n is an integer of 1-15, characterized in that said method comprises the steps of: (1) the maleic acid chloride of the formula H2N-(CH2) n-COOH amino acid I b in the presence of a base prepared by condensation of maleimido group steps I c acid,

(2) maleic acid imido group I c and then with an acylating reagent of tert-butyl carbazate in the presence of a base in the reaction of step I d,

(3) I d deprotection with trifluoroacetic acid, the alkali and removing trifluoroacetic acid to obtain the maleimido group I e hydrazide steps

(4) an imido group of maleic hydrazide I e and doxorubicin hydrochloride catalyzed condensation of hydrogen chloride to obtain a final product hydrazone derivative of doxorubicin,

[0028]

[0049] The butene-ni chloride 15. 2g (0. Imol) was dissolved in 25mL of chloroform was dried by adding anhydrous potassium carbonate 27. 6g (0. 2mol), the gas and gas protection and conditions of 0 ° C was added dropwise 6 – aminocaproic acid 13. 2g (0. ImoI) in chloroform (50mL) solution, add after reaction at room temperature for 3 hours. Washed with saturated brine, dried over anhydrous magnesium sulfate, suction filtered, concentrated under reduced pressure. The residue was recrystallized from alcohol ko maleimido acid (Compound c) 18. 8g, 90% yield, m.p. :85-87 ° C.

[0050] Compound c 10. 5g (50mmol) and thionyl chloride crab 5. 3mL (75mmol) was heated under reflux the mixture I. 5 hours and concentrated under reduced pressure in an argon atmosphere under the conditions of 0 ° C and added dropwise to the hydrazine carboxylic acid tert-butyl ester 6.6g (50mmol) amine with a three ko

10. 8mL (75mmol) in anhydrous ko ether (50mL) solution added after the reaction was continued at room temperature for I. 5 hours. Washed with 5% hydrochloric acid, 5% sodium bicarbonate, and saturated brine, dried over anhydrous magnesium sulfate overnight, filtered with suction to give the compound of d ko ether solution. The solution was cooled to 0 ° C, was added dropwise trifluoroacetic acid ko 7. 4mL (100mmOl), After the addition the reaction was continued for 10 minutes, suction filtered, the filter cake was washed twice with ether, ko and dried in vacuo to give 6 – maleic acid sub-aminocaproic acid hydrazide trifluoro-ko 12. 2g, 72% yield, m.p. 99-102 ° C. IOmL this salt is added to sodium hydroxide (10%) solution, stirred for a while, with ko extracted with ether, the organic layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated to give 6 – aminocaproic maleimido hydrazide (compound e) 7. Sg, 70% yield.

[0051] The doxorubicin hydrochloride 0. 58g (Immol) with compound e 0. 45g (2mmol) was dissolved in 150mL of anhydrous methanol, passing about 2mmol of dry hydrogen chloride, under argon, at room temperature protected from light and reaction conditions 24 inches. Concentrated under reduced pressure at room temperature, the solid was washed with about IOOmL ko nitrile, and dried in vacuo doxorubicin 6 – aminocaproic maleimido hydrazone O. 63g, 80% yield. 1H NMR (CD3OD) δ: 7. 94 (bd, 1H), 7. 82 (t, 1H), 7. 55 (d, 1H), 6. 78 (s, 2H), 5. 48 (s, 1H ), 5. 07 (t, 1H), 4 · 59 (d, 1H), 4 · 21 (m, 1Η), 4 · 02 (s, 3H), 3 · 63-3. 30 (m, 5H) , 2 · 55-2. 26 (m, 4H), 2. 19-1. 88 (m, 3Η), I. 69-1. 18 (m, 12Η, I. 26). [0052] Although specific reference to the above embodiments of the present invention will be described, it will be understood that in the appended claims without departing from the invention as defined by the spirit and scope of the skilled person can be variously truncated, substitutions and changes. Accordingly, the present invention encompasses these deletions, substitutions and changes.

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US 5622929

http://www.google.co.in/patents/US5622929

http://www.google.co.in/patents/EP0554708A1

Method A:

As noted below, Method A is the preferred method when the Michael Addition Receptor is a maleimido moiety.

[0077]

Alternatively, the Formula (IIa) compound may be prepared by reaction of the drug with a hydrazide to form an intermediate hydrazone drug derivative followed by reaction of this compound with a Michael Addition Receptor containing moiety according to the general process described in Method B:

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http://www.google.co.in/patents/WO2012167255A1?cl=en

Synthesis of DOXO-EMCH

The synthesis of DOXO-EMCH was done initially in accordance with that previously published by Willner and co-workers (Bioconjugate Chem., 4:521-527, 1993). Problems arose in the initial addition of the 6-maleimidocaproylhydrazine to the C-13 ketone of doxorubicin. HPLC results did not give a good yield of product, only 50-60%. Upon further analysis, we determined TFA was not needed to catalyze the reaction, and instead used pyridine. With pyridine, chromatograms from the HPLC showed 90% DOXO-EMCH relative to 10% DOX. The pyridine may have improved the yield by serving as a base to facilitate formation of the hydrazone. Another problem we encountered in the synthesis was purification of the final product. According to Willner’ s method, 5 volumes of acetonitrile (ACN) were to be added to a concentrated methanolic solution of crude DOXO-EMCH to achieve crystallization after 48 h at 4 °C. By this method, only 10-20%) of the desired product precipitated. To obtain a better yield, the crystallization step was done 4 times with 6 volumes of ACN used in each step. A lesser amount of methanol was needed each time, as less product remained in solution. Even with the multiple crystallizations, a final yield of only 59% was obtained. Various other methods for crystallization were explored, including using different solvents and increasing the initial solubility in methanol by heat, but none gave better results. 1.2 Rate of Hydrolysis of DOXO-EMCH at Varying pH

Subsequent pH studies to determine the rate of hydrolysis of the hydrazone were carried out as a benchmark for later hydrolysis experiments with PPD-EMCH. The results of the hydrolysis experiments showed that at lower pH, the hydrolysis reaction proceeded very quickly in the formation of DOX. Moreover, at higher pH the hydrazone proved to be very robust in that its degradation is very slow.

General HPLC instruments and methods

Analytical HPLC methods were performed using a Hewlett-Packard/ Aligent 1050/1100 chromatograph with an auto injector, diode array UV-vis absorption detector. Method 1.1 : Analytical HPLC injections were onto an Aligent Zorbax Eclipse XDB-C18 reversed phase column, 4.6 mm x 150 mm, eluting at 1.0 mL/min. A gradient of acetonitrile/20 mM sodium phosphate buffer (pH 6.9), 80% buffer to 55% at 10 min, 55% to 40% at 12 min, 40% to 80% at 13 min. Retention times: at 480 nm, DOX (9.4 min), DOXO-EMCH (1 1.2 min).

Synthesis of DOXO-EMCH

The synthesis of DOXO-EMCH was accomplished using the procedure reported by Willner et al, with several changes to improve the yield (Willner, D., et al.,

Bioconjugate Chem., 4:521-27, 1993). DOX’HCl (20 mg, 34 μιηοΐ) was dissolved in 6 mL of methanol. Pyridine (12.53 μί) was added to the solution, followed by 35.4 mg

EMCH’TFA. The reaction was stirred at room temperature overnight. By HPLC, the reaction was 90% complete. The solvent was evaporated to dryness by rotary evaporation. A minimal amount of methanol was used to dissolve the solid, and six volumes of acetonitrile at 4 °C were added to the solution. The resulting solution was allowed to sit undisturbed at 4 °C for 48 h for crystallization. The precipitate was collected, and the crystallization method was repeated 4 times. The resulting solids were combined and washed three times with 1 : 10 methanokacetonitrile. The final yield of DOXO-EMCH was 11.59 mg, 58%. HPLC Method 1.1 was used. NMR spectra corresponded to those previously given by Willner (Bioconjugate Chem. 4:521-27. 1993).

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http://www.google.co.in/patents/US20070219351

DOXO-EMCH, the structural formula of which is shown below,

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SEE

(6-Maleimidocaproyl)hydrazone of doxorubicin – A new derivative for the preparation of immunoconjugates of doxorubicin
Bioconjugate Chem 1993, 4(6): 521

References

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5: Kratz F, Elsadek B. Clinical impact of serum proteins on drug delivery. J Control Release. 2012 Jul 20;161(2):429-45. doi: 10.1016/j.jconrel.2011.11.028. Epub 2011 Dec 1. Review. PubMed PMID: 22155554.

6: Elsadek B, Kratz F. Impact of albumin on drug delivery–new applications on the horizon. J Control Release. 2012 Jan 10;157(1):4-28. doi: 10.1016/j.jconrel.2011.09.069. Epub 2011 Sep 16. Review. PubMed PMID: 21959118.

7: Kratz F, Fichtner I, Graeser R. Combination therapy with the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin achieves complete remissions and improves tolerability in an ovarian A2780 xenograft model. Invest New Drugs. 2012 Aug;30(4):1743-9. doi: 10.1007/s10637-011-9686-5. Epub 2011 May 18. PubMed PMID: 21590366.

8: Boga C, Fiume L, Baglioni M, Bertucci C, Farina C, Kratz F, Manerba M, Naldi M, Di Stefano G. Characterisation of the conjugate of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin with lactosaminated human albumin by 13C NMR spectroscopy. Eur J Pharm Sci. 2009 Oct 8;38(3):262-9. doi: 10.1016/j.ejps.2009.08.001. Epub 2009 Aug 18. PubMed PMID: 19695327.

9: Graeser R, Esser N, Unger H, Fichtner I, Zhu A, Unger C, Kratz F. INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model. Invest New Drugs. 2010 Feb;28(1):14-9. doi: 10.1007/s10637-008-9208-2. Epub 2009 Jan 8. PubMed PMID: 19148580.

10: Kratz F. Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release. 2008 Dec 18;132(3):171-83. doi: 10.1016/j.jconrel.2008.05.010. Epub 2008 May 17. Review. PubMed PMID: 18582981.

11: Unger C, Häring B, Medinger M, Drevs J, Steinbild S, Kratz F, Mross K. Phase I and pharmacokinetic study of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin. Clin Cancer Res. 2007 Aug 15;13(16):4858-66. PubMed PMID: 17699865.

12: Lebrecht D, Walker UA. Role of mtDNA lesions in anthracycline cardiotoxicity. Cardiovasc Toxicol. 2007;7(2):108-13. Review. PubMed PMID: 17652814.

13: Kratz F. DOXO-EMCH (INNO-206): the first albumin-binding prodrug of doxorubicin to enter clinical trials. Expert Opin Investig Drugs. 2007 Jun;16(6):855-66. Review. PubMed PMID: 17501697.

14: Kratz F, Ehling G, Kauffmann HM, Unger C. Acute and repeat-dose toxicity studies of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the anticancer agent doxorubicin. Hum Exp Toxicol. 2007 Jan;26(1):19-35. PubMed PMID: 17334177.

15: Lebrecht D, Geist A, Ketelsen UP, Haberstroh J, Setzer B, Kratz F, Walker UA. The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage. Int J Cancer. 2007 Feb 15;120(4):927-34. PubMed PMID: 17131338.

16: Di Stefano G, Lanza M, Kratz F, Merina L, Fiume L. A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate. Eur J Pharm Sci. 2004 Dec;23(4-5):393-7. PubMed PMID: 15567293.

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EP0269188A2 *	Jun 18, 1985	Jun 1, 1988	Elf Sanofi	Cytotoxic conjugates useful in therapy, and process for obtaining them
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EP0457250A2 *	May 13, 1991	Nov 21, 1991	Bristol-Myers Squibb Company	Novel bifunctional linking compounds, conjugates and methods for their production