MK-7145

Uncategorized Comments Off

May 172016

2D chemical structure of 1255204-84-2

MK-7145,

cas 1255204-84-2

1(3H)-Isobenzofuranone, 5,5′-(1,4-piperazinediylbis((1R)-1-hydroxy-2,1-ethanediyl))bis(4-methyl-

MF C₂₆ H₃₀ N₂ O_{6, Molecular Weight 466.53}

1(3H)-Isobenzofuranone, 5,5′-[1,4-piperazinediylbis[(1R)-1-hydroxy-2,1-ethanediyl]]bis[4-methyl-

The Renal Outer Medullary Potassium (ROMK) channel (KM .1 ) (see e.g., Ho,K., et al., Cloning and expression of an inwardly rectifying ATP -regulated potassium channel, Nature, 1993, 362(6415): p. 31-8.1, 2; and Shuck, M.E., et al., Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel, J Biol Chem, 1994, 269(39): p. 24261-70) is a member of the inward rectifier family of potassium channels expressed in two regions of the kidney: thick ascending loop of Henle (TALH) and cortical collecting duct (CCD) (see Hebert, S. C, et al., Molecular diversity and regulation of renal potassium channels, Physiol Rev, 2005, 85(1): p. 319-713). At the TALH, ROMK participates in potassium recycling across the luminal membrane which is critical for the function of the Na⁺/K⁺/2CF co-transporter, the rate-determining step for salt reuptake in this part of the nephron. At the CCD, ROMK provides a pathway for potassium secretion that is tightly coupled to sodium uptake through the amiloride-sensitive sodium channel (see Reinalter, S. C, et al., Pharmacotyping of hypokalemic salt-losing tubular disorders, Acta. Physiol Scand, 2004, 181(4): p. 513-21 ; and Wang, W., Renal potassium channels: recent developments, Curr Opin Nephrol Hypertens, 2004, 13(5): p. 549-55). Selective inhibitors of the ROMK channel (also referred to herein as inhibitors of ROMK or ROMK inhibitors) are predicted to represent novel diuretics for the treatment of hypertension and other conditions where treatment with a diuretic would be beneficial with potentially reduced liabilities (i.e., hypo- or hyperkalemia, new onset of diabetes, dyslipidemia) over the currently used clinical agents (see Lifton, R.P., A.G. Gharavi, and D.S. Geller, Molecular mechanisms of human hypertension, Cell, 2001, 104(4): p. 545-56). Human genetics (Ji, W., et al., Rare independent mutations in renal salt handling genes contribute to blood pressure variation, Nat Genet, 2008, 40(5): p. 592-9; and Tobin, M.D., et al., Common variants in genes underlying monogenic hypertension and hypotension and blood pressure in the general population, Hypertension, 2008, 51(6): p. 1658-64) and genetic ablation of ROMK in rodents (see Lorenz, J.N., et al., Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter’s syndrome, J Biol Chem, 2002, 277(40): p. 37871-80 and Lu, M., et al._s Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Banter’s) knockout mice, J Biol Chem, 2002, 277(40): p. 37881-7) support these expectations. To our knowledge, the first small molecule selective inhibitors of ROMK were reported from work done at Vanderbilt University as described in Lewis, L.M., et al., High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and KirJ.l, MoI Pharmacol, 2009, 76(5): p. 1094-1103.

PATENT

WO 2010129379

http://www.google.com/patents/WO2010129379A1?cl=ko

SCHEME 1

SCHEME 2

SCHEME 3

SCHEME 5

SCHEME 6

SCHEME 7

SCHEME 8

14 15

The preparation of compounds 16 can be achieved following the sequence detailed in Scheme 9. Treating epoxide 2-1 with commercially available 1-Boc piperazine at elevated temperatures gives rise to alcohol 2-2 (Nomura, Y. et al. Chemical & Pharmaceutical Bulletin, 1995, 43(2), 241-6). The hydroxyl group of 2-2 can be converted to the fluoride by treatment of such fluorinating reagent as DAST (Hudlicky, M. Organic Reactions, 1988, 35). Removal of the Boc group of 3-1 under acidic conditions such as TFA gives rise to piperazine 3-2. Piperazine 3-2 can be washed with an aqueous base solution followed by extraction with organic solvents to generate the free base form. The free base of 3-2 can be coupled to epoxide 5-1 at elevated temperatures to afford compound 16. The Ar-CHF- and Ar’-CHOH- groups in 16 represent examples of either Z¹ or Z².

SCHEME 9

16 General Procedures.

INTERMEDIATE (Ry-H (free base)

5-\(lR)-l -hγdroxγ-2-piperazio- 1 -ylethyl] -4-methyl-2-benzofuran- 1 f 3/f)-one To a 20 mL microwave tube charged with 4-methyl-5-[(2jS)-oxiran-2-yl]-2-benzofuran-l(3H)-one (1020 mg, 5.40 mmol) and a stir bar was added 1-Boc Piperazine (800mg, 4.3 mmol) and EtOH (15 mL). The tube was sealed and heated in a microwave apparatus to 150 ⁰C for 1 hour. The crude product was adsorbed onto silica gel, and purified by flash chromatography (Hexanes-EtOAc with 10% EtOH: 0 – 100% gradient), and solvent removed to afford terl-butyl~4-[(2R-2-hydroxy-2-(4-methyl-l -oxo-1 ,3-dihydro-2-bers2θfuran-5-yl) ethyl}piperazine-l-carboxylate. LCMS M+l (calc. 377.20, found 377.13). This product was treated with neat TFA for 15 minutes to remove the Boc group. After removal of TFA under reduced pressure, the residue was taken into aq NaHCO₃, and back-extracted with CHCl₃-IPA (3:1). The organic layers were combined, dried over sodium sulfate, and concentrated to afford 5 – [( 1 R)- 1 -hydroxy-2-piperazin- 1 -ylethyl] -4-methyl-2-benzofuran- 1 (3H)-one. ¹H NMR (OMSO-d₆, 500 MHz) δ 7.68 (d, J= 8.0 Hz, IH), 7.65 (d, J= 8.0 Hz, IH)₅ 5.38, 5.35 (AB system, J- 15.4, J= 16.7, 2H), 5.06 (dd₅ J- 3.9 Hz, J= 3.7 Hz, IH), 3.76 (m, IH)₅ 2.72 (m, 4H), 2.42 (m, 4H), 2.34 (d, J= 3.8 Hz₅ IH), 2.32 (d, J= 3.8 Hz, IH), 2.24 (s, 3H); LC/MS: (IE, m/z) [M +I]⁺ = 277.03.

EXAMPLE 2A

5, 5 ‘-{ piperazine- 1 ,4-diylbis[( 1 R)- 1 -hydroxy ethane-2 , 1 -diyl] } bis(4-methyl-2-benzofuran- 1 (3H)-one)

Method 1: To a 20 mL microwave tube charged with 4-methyl-5-[(2i?)-oxiran-2-yl]-2-benzofuran-l(3H)-one (972 mg, 5.11 mmol) and piperazine (200 mg, 2.3 mmol) was added a stir bar and EtOH (16 mL). The tube was sealed and heated in a microwave apparatus to 150 ⁰C for 90 minutes. The crude product was adsorbed onto silica gel, and purified by flash chromatography (MeOΗ-DCM 0 ~ 7% gradient). After removal of solvents, 5_»5′-{piperazine-1 ,4-diyIbi s [( 1 R)- 1 -hydroxyethane-2, 1 -diyl] } bis(4-methyl-2-benzofuran- 1 (3 H)-one) was collected. ¹H-NMR (500 MHz₉ CDCl₃) δ ppm 7.80 (s, 4H), 5.25 (s, 4H), 5.11 (d, J= 10.5 Hz₅ 2H), 4.00 (broad, 2H), 2.90 (broad, 4H)₃ 2.69-2.50 (m, 6H), 2.44 (t, J= 11 Hz, 2H), 2.29 (s, 6H); LCMS M+l (calc. 467, found 467).

Method 2: Piperazine (4.51 g, 52.4 mmol) and 4-methyl-5-[(2Λ)-oxiran-2-yl]-2-benzofuran-1 (3//)-one (20.0 g, 105 mmol) were charged to a 3-neck 500-mL roundbottom flask, equipped with a reflux condensor, under nitrogen. Toluene (80.0 mL, 751 mmol) and N,N-dimethylacetamide (80 mL, 854 mmol) were added to provide a suspension. The reaction mixture was warmed to 110 ⁰C, becoming homogeneous at 25 ⁰C. After stirring for 4.5 h at 110 ⁰C, the temperature was increased to 115 °C to drive the reaction forward. After stirring for 48 h, the reaction mixture was cooled to RT. On cooling, crystallization occurred. Water was added via addition funnel (45 mL), generating a thick slurry. The suspension was filtered and the solids were washed with 4:1 water :DMA (60 mL), followed by water (2 x 35 mL). The solid was dried on the funnel under vacuum with a nitrogen sweep to constant mass. 5,5′-{Piperazine-l,4-diylbis[(li?)-l-hydroxyethane-2,l-diyl]}bis(4-methyl-2-beiizofurari-l(3H)-one) was isolated. ¹H-NMR (500 MHz, CDCl₃) δ ppm 7.80 (s, 4H), 5.25 (s, 4H), 5.11 (d, J- 11 Hz, 2H), 4.30-3.51 (broad, 2H), 2.90 (broad, 4H), 2.69-2.50 (m, 6H), 2.44 (t, J- 11 Hz, 2H), 2.30 (s, 6H).

Compounds of the present invention are amines and can therefore be converted to a variety of salts by treatment with any of a number of acids. For example, the compound of Example 2A can be converted to several different salt forms as shown in the following representative examples. These are selected examples and are not meant to be an exhaustive list; numerous additional salts can be prepared in a similar fashion using a variety of acids. EXAMPLE 2A-1 (di-HCl salt): 5,5^t-{piperazme-l,4-diylbis[(17?)-l-hydroxyethane-2,l- diyl] } bis(4-methyl-2-benzofuran- 1 (3H)-one) dihydrochloride To a 250 mL pear shape flask charged with the free base (1.2 g, 2.6 mmol) and a stir bar was added DCM. The solution was stirred until all solids were gone. To this solution was added 4N HCl in dioxane (2.6 mL, 4.0 eq), and the mixture was allowed to stir for another 15 minutes. The solvent was removed on a rotary evaporator, and the product was left dry on a high vacuum pump until there was no weight change. The product was determined to be 5, 5 ‘-{piperazine- 1,4-diylbis [( 1 R)- 1 ~hydroxyethane-2, 1 -diyl] } bis(4-methyl-2-benzofuran- 1 (3i?)-one) dihydrochloride. EXAMPLE 2A-2 (HCl salt): 5,5’-{piperazine-l,4-diylbis[(l^)-l-hydroxyethane-2,l- diyl] } bis(4-methyl-2-benzofuran- 1 QHVone) hvdrochl oride

To a 20 dram vial charged with the free base (160 mg, 0.34 mmol) and a stir bar was added 0.1 M HCl in IPA. The solution was allowed to stir at RT for 30 minutes, and then heated to 40⁰C for 1 hour. The solvent was removed under vacuum, and the resulting product was left on a high vacuum pump for 16 hours. The product corresponded to 5,5′-{piperazine-l,4-diylbis[(li?)-l-hydroxyethane~2, 1 -diyl] } bis(4-methyl-2-benzofuran- 1 (3 H)-one) hydrochloride.

EXAMPLE 2A-3 (mono-hydrate of the di-HCl salt): 5, 5′- {piperazine- l,4-diylbis[( Ii?)- 1-hydroxyethane-2,l-diyl] Ibis^-niethyl-g-benzofuran-lfS/^-one) dihydrochloride hydrate To a flask charged with the free base (1.0 g, 2.1 rnmol) and a stir bar was added 1 N HCl (50 mL). The mixture was allowed to stir until all solids dissolved. The solvent was removed on a rotary evaporator, and the resulting product was left on a high vacuum pump for 16 hours. The product was determined to be 5,5′-{piperazine-l ,4-diylbis[(li?)-l-hydroxyethane-2,l-diyl]}bis(4-methyl-2-benzofuran-l(3H)-one) dihydrochloride hydrate.

EXAMPLE 2A-4 (H₂SO₄ salt): 5.5′-{piperaziiie-l_>4-diylbis[(lJΪ)-l-hydioxyethane-2,l- diyl] }bis(4-methyl-2-benzofuran-l(3/f)-one) sulfate (salt) To a 100 mL flask charged with a solution of the free base (154 mg, 0.330 mmol) in DMF : MeOH (3 : 1) (20 mL) and a stir bar was added 0.1 M H₂SO₄ (3.3 mL). The solution was allowed to stir at RT for 30 minutes, and then heated to 40 ⁰C for 2 hours. A lot of solids formed during that time. The solvent was removed under vacuum, and the white solids were left on high vacuum for 16 hours to afford 5₎5^l-{piperazine-l,4-diylbis[(lJ?)~l-hydroxyethane-2,l-diyl] }bis(4-methyl-2-benzofuran-l(3H)-one) sulfate (salt).

Paper

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure

Haifeng Tang^*†, et al

^†Departments of Discovery Chemistry, ^‡Ion Channels, ^§In Vivo Pharmacology, ^∥Cardiorenal, and ^⊥Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Kenilworth, New Jersey 07033, United States

ACS Med. Chem. Lett., Article ASAP

DOI: 10.1021/acsmedchemlett.6b00122

*Tel: 908-740 4932. E-mail: haifeng_tang@merck.com.

http://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.6b00122

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Cc1c(ccc2c1COC2=O)[C@H](CN3CCN(CC3)C[C@@H](c4ccc5c(c4C)COC5=O)O)O

ICH Q3D Implementation Working Group (IWG)—Training Modules

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May 172016

ICH Q3D Implementation Working Group (IWG)—Training Modules

ICH Q3D is a complex guideline. The overall requirement in terms of control is clear—there are defined limits for some 24 elements, and levels of the elements described must be controlled within these limits in the final drug product. Simple. The complexity comes when defining how this is achieved. The guideline provides a series of options to evaluate risk and effect control, ranging from control in each individual component based on a fixed dose for the product of 10 g (Option 1) to simply testing the final product (Option 3). A detailed description of the options and when/how these are applied as part of a risk assessment is beyond the scope of this review; the point is that there are significant challenges in applying the guideline practically solely using the guideline for that purpose. This was recognized by the ICH Expert Working Group responsible for the guideline, resulting in the establishment immediately after step 4 of an Implementation Working Group. A key objective of the IWG was to develop training materials to assist implementation.

In February ICH finally published the long awaited training modules.(2) These modules, produced by the ICH Q3D implementation working group, cover both safety and quality aspects, the areas covered are listed below:

Module 0

This provides an overview of the modules. Included within this is a very useful flow diagram,Figure 1, highlighting the anticipated overall process from the risk assessment through to definition of control strategy.

Modules 1–3 Cover Toxicology Aspects

•	Module 1—Different Routes of Administration
•	Module 2—Justification of Levels Greater than Permissible Daily Exposure Limits
•	Module 3—Non ICH Elements

Modules 4–7 Cover Chemistry (Quality) Aspects

•	Module 4—Large Volume Parenterals
•	Module 5—Risk Assessment
•	Module 6—Control
•	Module 7—Calculation Options

Highlighting some key points, module 5, relating to risk assessments, discusses the key role of GMP in assessing risk—this is an important and a helpful point relating to API manufacture. It emphasizes the importance of:

1.	Design and qualification;
2.	Maintenance procedures.

However, it also focuses on the risk arising from manufacturing equipment, making a relatively generic statement over the often more chemically aggressive nature of API manufacturing procedures compared to drug product manufacturing. It even suggests monitoring the drug substance for potential impurities arising from manufacturing equipment (e.g., stainless steel—Cr, Mn, Mo, V, Ni). It is a pity that this risk is highlighted without also making the point that it would be expected that such risk would be addressed as part of GMP and form part of the process accommodation procedure rather than rely on screening to verify.

Rightly the module makes the point that a significant potential source of elemental impurities arises from the use of metal catalysts in the synthesis of drug substances, especially if used in the latter stages of synthesis. It also states that:

“Knowledge of potential elemental impurities in synthetic steps prior to the final drug substance may provide information that can assist in the preparation of the risk assessment.”

This is an interesting point and one that cuts to the heart of the uncertainties around practical implementation. While a valid point, it raises key questions such as how many steps prior to the API should be assessed? Clearly this will be process/product specific, but it is a very real question any risk assessment will have to tackle.

Another interesting point made in the module is the potential for “platform” risk assessments. This is the concept of a risk assessment applicable across a series of products. One such platform may be for example, oligonucleotides.(3) In such instances, where the manufacturing process in terms of reagent type, equipment, and process conditions are similar irrespective of the precise end product, it should be possible to conduct an assessment based on one process and for this to relevant/transposable to comparable processes.

Figure 1

Module 6—Control of Elemental Impurities—also provides useful advice emphasizing the importance of control across the product lifecycle. In the context of the manufacture of the API, this requires oversight and governance over changes to the process that may affect the risk assessment, e.g., change in catalyst load, and so forth. Such changes require a re-evaluation and possibly confirmatory testing. Another point emphasized in the module is that routine testing of Class 1 metals, i.e., arsenic (As), mercury (Hg), cadmium (Cd), and lead (Pb), is NOT required unless there is an identified risk. This is a very important and helpful point clearly reiterating the core principle of ICH Q3D that any control strategy should be based on the risk assessment. This is especially important as several regulatory queries have been reported asking for data for Class 1 and also Class 2A metals for APIs.

One area described in Module 6 is the concept of periodic testing. This is an area of potential concern and ambiguity. It states that:

“Where the risk assessment indicates that routine testing is considered unnecessary but some additional assurance is needed post approval, periodic testing of the drug product or one or more individual components may be proposed by the applicant and implemented upon acceptance by the regional regulatory authority.”

An example is provided relating to use of a Pt catalyst in the manufacture of the API, this being the final step used in the API synthesis. In the example detectable levels of Pt at ∼ 20% of the PDE are observed (below the 30% limit stated in ICH Q3D), based on this periodic testing being proposed. In the case study described this may seem sensible but how close to reality is such an example? In such a case would an applicant simply not specify Pt on the API specification? The worry is that the option for periodic testing may be blunt instrument and be something that is regularly requested.

USP Chapter ⟨232⟩

USP very recently announced(4) a series of revisions to USP Chapter ⟨232⟩, Elemental Impurities, the revisions made being intended to align the general chapter more closely to ICH Q3D. One of the most significant is the removal of the need to routinely screen for Class 1 metals as part of any analysis, the final sentence in the text outlined below being deleted.

If, by process monitoring and supply chain control, manufacturers can demonstrate compliance, then further testing may not be needed. When testing is done to demonstrate compliance, proceed as directed in Elemental Impurities—Procedures ⟨233⟩.

This is a welcome and important amendment; the previous requirement making little scientific sense, there being no actual evidence that Class 1 metals would be more prevalent, for example, where a platinum catalyst was used than in the absence of a catalyst. Such catalysts are not a source of class 1 metals.

Overall

Overall there are likely to be challenges/uncertainties associated with ICH Q3D leading up to and for some period after the effective date as the guideline beds in, but the crucial fact is that all of the evidence to date indicates it is unlikely that there will be a widespread impact caused by issues of excessive levels of any elemental impurity whereby effective control cannot be realized.

2= http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3D/Training_package_Module_0-7.zip

/////////ICH Q3D, Elemental Impurities, Procedures ⟨233⟩, Training Modules

ICH M8 “Specification for Submission Formats for eCTD”

regulatory Comments Off

May 172016

This additional specification describes the way files should be constructed for inclusion in the eCTD.

Key Points:

It is not necessary to use a product from Adobe or from any specific company to produce PDF documents.
All ICH regional regulatory authorities are able to read and accept PDF files saved as PDF version 1.4 through 1.7, PDF/A-1, or PDF/A-2 compliant to ISO 32000-1:2008.
The size of a PDF file should not exceed 500MB.

Regulatory authorities cannot guarantee the availability of any fonts except Times New Roman, Arial, and Courier and fonts supported in the Acrobat product set itself. Therefore, all additional fonts used in the PDF files should be embedded to ensure that those fonts would always be available to the reviewer.
Times New Roman, 12-point font, is adequate in size for narrative text and should be used whenever possible. Times New Roman font sizes 9-10 or an equivalent size of other recommended fonts are considered acceptable in tables but smaller font sizes should be avoided.
The use of a black font color is recommended. Blue can be used for hypertext links. Light colors can be difficult to read on a monitor as well as when printed, and should be avoided. The use of background shadowing can be difficult to read and should be avoided.
Pages should be properly oriented so that all portrait pages are presented in portrait and all landscape pages are presented in landscape.
A sufficient margin of at least 2.0 cm on the left side of each page for portrait and top of the page for landscape should be provided to avoid obscuring information. The remaining margins should be a Page 6 of 9minimum of 0.8 cm. Header and footer information can appear within these margins but should not appear so close to the page edge to risk being lost upon printing.
All pages of a document should include a unique header or footer that briefly identifies its subject matter.
Scanning should be avoided where possible.
It is recommended that scanning be undertaken at a resolution of 300 dots per inch (dpi) to balance legibility and file size. The use of grayscale or color is discouraged because of file size. After scanning, resampling to a lower resolution should be avoided.
Paper documents containing hand-written notes should be scanned at a resolution of at least 300 dpi. Hand-written notes should be done in black ink for clarity, 600 dpi is recommended. High-pressure liquid chromatography or similar images should be scanned at a resolution of at least 300 dpi.

Applicants should validate the quality of the renditions.
Hypertext links can be designated by rectangles using thin lines or by blue text as appropriate. Bookmarks are expected even if there is no TOC In the document. The use of no more than 4 levels in the hierarchy is recommended, but additional levels could be created for study reports if such bookmarks contribute to efficient navigation.
Relative paths should be used when creating hypertext links to minimize the loss of hyperlink functionality when folders are moved between disk drives.

The bookmarks should be collapsed when document is opened so that all bookmarks are at the first level.
The first page of the document should be numbered page 1, and all subsequent pages (including appendices and attachments) should be numbered consecutively with Arabic numerals. Roman numerals should not be used to number page. The only exception should be where a document is split because of its size, the second or subsequent file should be numbered consecutively to that of the first or preceding file.
Security fields should be set to allow printing, changes to the document, selecting text and graphics, and adding or changing notes and form fields. The exception to this rule includes regulatory forms with pre-existing security and literature references that need to be copyright protected.

Reference

http://estri.ich.org/ssf/Specification_for_Submission_Formats_for_eCTD_v1_0_.pdf

////////ICH M8, Specification, Submission Formats, eCTD

Ethyl 1-(7-chloroquinolin-4-yl)-5-methyl-1H-1,2,3-triazole-4-carboxylate

Uncategorized Comments Off

May 172016

Scheme 1 General scheme of the reaction.

Scheme 2

General procedure for the synthesis of 7-chloroquinoline-1,2,3-triazoyl carboxylates

To a solution of 4-azido-7-chloroquinoline 1 (0.3 mmol, 0.061 g) in DMSO (0.3 mL), was firstly added the β-ketoesters 2a-k (0.3 mmol) and then the catalyst pyrrolidine (0.03 mmol. 0.021 g). The reaction mixture was stirred in an open vial at room temperature for 24 hours. After completion of the reaction, the crude product was purified by column chromatography on silica gel using a mixture of hexanes/ethyl acetate (5:1) as the eluent to afford the desired products 3a-k.

Ethyl 1-(7-chloroquinolin-4-yl)-5-methyl-1H-1,2,3-triazole-4-carboxylate (3a)

Yield: 0.085 g (90%); white solid; mp 128-130 °C;

¹H NMR (CDCl₃, 300 MHz) δ 9.15 (d, 1H, J4.5 Hz, HetAr-H), 8.27 (d, 1H, J1.9 Hz, HetAr-H), 7.60 (dd, 1H, J9.0 and 1.9 Hz, HetAr-H), 7.48 (d, 1H, J4.5 Hz, HetAr-H), 7.34 (d, 1H, J9.0 Hz, HetAr-H), 4.50 (qua, 2H, J7.1 Hz, OCH₂), 2.49 (s, 3H, CH₃), 1.47 (t, 3H, J7.1 Hz, CH₃);

¹³C NMR (CDCl₃, 75 MHz) δ 161.10, 151.28, 149.88, 140.20, 139.34, 137.00, 136.76, 129.67, 128.93, 123.58, 122.09, 118.75, 61.15, 14.18, 9.44;

MS m/z (relative intensity): 316 (7), 259 (15), 243 (17), 231 (19), 217 (45), 215 (100), 214 (22), 205 (16), 203 (19), 189 (28), 181 (27), 179 (27), 164 (26), 162 (80), 137 (15), 135 (44), 127 (44), 126 (27), 100 (20), 99 (65), 83 (30), 75 (15), 74 (14), 43 (25);

HRMS calcd. for C₁₅H₁₄ClN₄O₂ [M + H]⁺: 317.0805; found: 317.0788.

Journal of the Brazilian Chemical Society

On-line version ISSN 1678-4790

J. Braz. Chem. Soc. vol.27 no.1 São Paulo Jan. 2016

http://dx.doi.org/10.5935/0103-5053.20150239

ARTICLES

7-Chloroquinoline-1,2,3-triazoyl Carboxylates: Organocatalytic Synthesis and Antioxidant Properties

Maiara T. Saraiva^a, Roberta Krüger^a, Rodolfo S. M. Baldinotti^b, Eder J. Lenardão^a, Cristiane Luchese^b, Lucielli Savegnago^b, Ethel A. Wilhelm^b^*, Diego Alves^a^*

^{^a}Laboratório de Síntese Orgânica Limpa (LASOL, CCQFA), Universidade Federal de Pelotas (UFPel), CP 354, 96010-900 Pelotas-RS, Brazil

^{^b}Grupo de Pesquisa em Neurobiotecnologia (GPN), CDTec/CCQFA, Universidade Federal de Pelotas (UFPel), CP 354, 96010-900 Pelotas-RS, Brazil

ABSTRACT

We describe herein our results on the synthesis and antioxidant properties of 7-chloroquinoline-1,2,3-triazoyl-4-carboxylates. This class of compounds have been synthesized in moderated to excellent yields by the reaction of 4-azido-7-chloroquinoline with a range of β-ketoesters in the presence of a catalytic amount of pyrrolidine (10 mol%). The synthesized compounds ethyl 1-(7-chloroquinolin-4-yl)-5-methyl-1H-1,2,3-triazole-4-carboxylate and ethyl 1-(7-chloroquinolin-4-yl)-5-phenyl-1H-1,2,3-triazole-4-carboxylate were screened for their in vitro antioxidant activity and the results demonstrated that the first compound reduces the lipid peroxidation levels induced by sodium nitroprusside in liver of mice, while the second compound shown nitric oxide scavenging activity. This is an efficient method to produce new heterocyclic compounds with potential antioxidant activities.

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532016000100041&lng=en&nrm=iso&tlng=en

e-mail: diego.alves@ufpel.edu.br, ethelwilhelm@yahoo.com.br

Figure 1 Biologically important quinolines.

Key words: quinolines, 1,2,3-triazoles, organocatalysis, cycloaddition, antioxidant

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Letermovir, AIC 246

Uncategorized Comments Off

May 162016

Letermovir, MK 8828, AIC 246

2-[(4S)-8-fluoro-2-[4-(3-methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-4H-quinazolin-4-yl]acetic acid

CAS	917389-32-3

Letermovir; UNII-1H09Y5WO1F; AIC-246; 2-((4S)-8-Fluoro-2-(4-(3-methoxyphenyl)piperazin-1-yl)-3-(2-methoxy-5-(trifluoromethyl)phenyl)-4H-quinazolin-4-yl)acetic acid; 2-[(4S)-8-fluoro-2-[4-(3-methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-4H-quinazolin-4-yl]acetic acid; Letermovir [INN]

Molecular Formula:	C₂₉H₂₈F₄N₄O₄
Molecular Weight:	572.550633 g/mol

Letermovir (INN) is an antiviral drug that is being developed for the treatment of cytomegalovirus (CVM) infections. It has been tested in CMV infected patients with allogeneic stem cell transplants and may also be useful for other patients with a compromised immune system such as those with organ transplants or HIV infections.^[1]

The drug has been granted fast track status by the US Food and Drug Administration (FDA) and orphan drug status by the European Medicines Agency.^[1]

The drug candidate is under development by Merck & Co., Inc as investigative compound MK-8828.^[2]

AIC246, also known as letermovir, is a novel anti-CMV compound with IC50 value of 5.1 ± 1.2 nM. It targets the pUL56 (amino acid 230-370) subunit of the viral terminase complex [1].
The subunit pUL56 is a component of the terminase complex which is responsible for packaging unit length DNA into assembling virions.
AIC246 has a novel mode of action targets the enzyme UL56 terminase and keep active to other drug-resistant virus. The anti-HCMV activity of AIC246 was evaluated in vitro by using different HCMV laboratory strains, GCV-resistant viruses. The result showed that the inhibitory potentcy of AIC246 surpasses the current gold standard GCV by more than 400-fold with respect to EC50s (mean, ∼4.5 nM versus ∼2 μM) and by more than 2,000-fold with respect to EC90 values (mean, ∼6.1 nM versus ∼14.5 μM). In the CPE-RA strains, the EC50 values of AIC 246 ranged from 1.8 nM to 6.1 nM [2].
In mouse model with HCMV subcutaneous xenograft, oral administration of AIC246 caused significant a dose-dependent reduction of the HCMV titer. 30 mg/kg/d AIC246 for 9 days induced PFU reduction with maximum efficiency, compared with the gold standard GCV at the ED50 and ED90 level [2].
References:
[1].Verghese PS, Schleiss MR. Letermovir Treatment of Human Cytomegalovirus Infection Anti-infective Agent. Drugs Future. 2013, 38(5):291-298.
[2]. Lischka P1, Hewlett G, Wunberg T, et al.In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246.Antimicrob Agents Chemother. 2010, 54(3):1290-1297.

NMR

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

PATENT

WO 2006133822

Scheme 2:

Chromatography
on a chiral phase

Scheme 4:

Scheme 5:

Synthesis of {8-fluoro-2- [4- (3-methoxyphenyl) piperazin-l -yl] -3- [2-methoxy-5- (trifluoromethyl) phenyl] -3,4-dihydroquinazolin-4-yl }acetic acid

xample 1

N- (2-bromo-6-fluoφhenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] urea

2-methoxy-5-trifluoromethylphenyl isocyanate (274.3 g) are dissolved in acetonitrile (1 L), then 2-bromo-6-fluoroaniline (200 g) was added with acetonitrile (50 mL) flushed. The resulting clear solution is at 38 h reflux (ca. 85 ⁰ stirred C), then under vacuum at 40 ⁰ concentrated C a dogged mush. This is filtered off, with acetonitrile (260 mL, to 0-5 ⁰ C cooled) washed and incubated overnight at 45 ⁰ dried C in the VDO using entraining nitrogen. Thus, a total of 424.3 g of N- (2-bromo-6-fluorophenyl) -N ‘- get [2-methoxy-5- (trifluoromethyl) phenylJ-urea as a solid, corresponding to 99.2% of theory.

¹ H NMR (300 MHz, d ₆ -DMSO): δ = 8.93 (s, IH), 8.84 (s, IH), 8.52 (d, V = 2.3, 2H), 7, 55 (d, 2 = Vr = 7.7, IH), 7.38 to 7.26 (m, 3H), 7.22 (d, ² J = 8.5, IH), 4.00 (s, 3H) ppm;

– – MS (API-ES-pos.): M / z = 409 [(M + H) ⁺ , 100%];

HPLC (Method 1): R _τ = 22.4 and 30.6 min.

example 2

N- (2-bromo-6-fluorophenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] urea (Alterhativsynthese)

2-methoxy-5-trifluoromethylphenyl isocyanate (1.19 kg) are at about 35 ⁰ dissolved melted and C in acetonitrile (4.2 L), then 2-bromo-6-fluoroaniline (870 g) was added and with acetonitrile ( 380 mL) rinsed. The resulting clear solution is at 74-88 45 h ⁰ stirred C, then under vacuum (200 mbar) at 50 ⁰ C to a dogged mush concentrated (amount of distillate 4.4 L). This is at room temperature with diisopropylether (1.5 L), washed aspirated, with diisopropylether (1.15 L) washed and at 45 ⁰ C in the VDO using entraining nitrogen to constant weight (24 h) dried. Thus, a total of 1, 63 kg Η- (2-bromo-6-fluoro-phenyl) -W- – obtained [2-methoxy-5 (trifluoromethyl) phenyl] urea as a solid, corresponding to 87.5% of theory.

HPLC (Method 1): R _τ = 22.6 and 30.8 min.

example 3

{8-Fluor-3-[2-methoxy-5-(trifluormethyl)phenyl]-2-oxo-l,2,3,4-tetrahydrochinazolin-4-yl}essigsäuremethylester

N- (2-bromo-6-fluorophenyl) -N- [2-methoxy-5- (trifluoromethyl) phenyl] urea (300 g) under a nitrogen atmosphere in isobutyronitrile (1.2 L) was suspended, then triethylamine

(21O mL), bis (acetonitrile) dichloropalladium (7.5 g), tris- (o-tolyl) phosphine (18.0 g) and

Methyl acrylate (210 mL) were added in this order. The resulting suspension is for 16 hours at reflux (ca. 102 ⁰ stirred C) and then cooled to room temperature. Water (1.2 L) is added and the mixture 1 at room temperature stirred, then aspirated and washed with water / methanol h: washed and acetonitrile (10O mL) (1 1 30O mL). The residue is treated overnight at 45 ⁰ dried C in the VDO using entraining nitrogen. Thus, a total of 208 g as a solid, corresponding to 68.5% of theory.

¹ H NMR (300 MHz, d ₆ -DMSO): δ = 9.73 (s, IH), 7.72 (d, ² J = 7.3, IH), 7.71 (s, IH), 7 , 33 (d, ² J = 9.3, IH), 7.15 (dd, ² J = 9.6, ² J = 8.6, IH), 7.01 (d, ² J = 7.3 , IH), 6.99 to 6.94 (m, IH), 5.16 (t, ² , J = 5.9, IH), 3.84 (s, 3H), 3.41 (s, 3H) , 2.81 (dd, ² J = 15.4, ² J = 5.8, IH), 2.62 (dd, ² J = 15.4, ² J = 6.3, IH) ppm;

MS (API-ES-pos.): M / z = 413 [(M + H) ⁺ , 100%], 825 [(2M + H) ⁺ , 14%];

HPLC (Method 1): R _τ = 19.3 min; Pd (ICP): 16,000 ppm.

example 4

{8-Fluor-3-[2-methoxy-5-(trifluormethyl)phenyl]-2-oxo-l,2,3,4-tetrahydrochinazolin-4-yl}essigsäuremethylester (Alternative synthesis)

N- (2-bromo-6-fluorophenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] urea (2.5 kg) is suspended under a nitrogen atmosphere in isobutyronitrile (9 L), then triethylamine (1.31 kg), bis (acetonitrile) dichloropalladium (64.9 g), tris (o-tolyl) phosphine (149 g) and methyl acrylate (1.59 kg) were added in this order. The resulting suspension is 22 hours at 90-100 ⁰ stirred C, then cooled to room temperature. Water (9 L) is added and stirred, then aspirated and washed with water / methanol (1: 1, 2.5 L) at room temperature, the mixture for 1 hour and acetonitrile (850 mL). The residue is treated overnight at 45 ⁰ dried C in the VDO using entraining nitrogen to constant weight (21 h). Thus, a total of 1.90 kg as a solid, corresponding to 74.9% of theory.

HPLC (Method 1): R _τ = 19.4 min.

example 5

{2-Chlor-8-fluor-3-[2-methoxy-5-(trifluormethyl)phenyl]-3,4-dihydrochinazolin-4-yl}essigsäure-methylester / chlorination

A solution of 2.84 kg {8-fluoro-3- [2-methoxy-5- (trifluoromethyl) phenyl] -2-oxo-l, 2,3,4-tetrahydroquinazolin-4-yl} acetic acid methyl ester in 14.8 l of chlorobenzene is heated to reflux and the solvent is distilled off until water no longer separates. It is to 12O ⁰ cooled C. Within 10 min phosphorus oxychloride are metered in 3.17 kg, and then is added within a further 10 min 2.10 kg DBU. It is heated to reflux for 9 hours.

For working up the mixture is cooled to 40 ⁰ C., stirred overnight and dosed the reactor contents to 11.4 L of water, previously estimated at 40 ⁰ was tempered C. For dosing an internal temperature of 40-45 to ⁰ C, are satisfied. The mixture is allowed to cool to room temperature, 11.4 L of dichloromethane, filtered through a Seitz filter plate and the phases are separated. The organic phase is washed with 11.4 L of water, 11.4 L of an aqueous saturated sodium bicarbonate solution and again with 11.4 L of water. The organic phase is concentrated on a rotary evaporator in vacuo and the remaining residue (2.90 kg) is used without further treatment in the next step.

¹ H NMR (300 MHz, d ₆ -DMSO): δ = 7.93 to 7.82 (m, 2H), 7.38 (d, ² J = 8.9, IH), 7.17 (m, 2H), 6.97 to 6.91 (m, IH), 5.45 and 5.29 (m and t, ² , J = 5.4, IH), 3.91 and 3.84 (2s, 3H) , 3.48 (s, 3H), 3.0 to 2.6 (m, 2H) ppm;

MS (CI, NH ₃ ): m / z = 431 [(M + H) ⁺ , 100%];

HPLC (Method 1): R _τ = 23.5 min; typical Pd value (ICP): 170 ppm.

example 6

{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-[2-methoxy-5-(trifluormethyl)phenyl]-3,4-dihydrochinazolin-4-yl}essigsäuremethylester / Amination – –

(52.5 g) is dissolved in 1,4-dioxane (10O mL), then (25.8 g) and DBU (20.4 g) was added at room temperature 3-methoxyphenylpiperazine, whereupon the temperature rises. The mixture is stirred at reflux for 22 h, then cooled to room temperature, with ethyl acetate (500 mL) and water (200 mL) and the phases separated. The organic phase (200 mL) washed with 0.2N hydrochloric acid (three times 100 mL) and water, dried over sodium sulfate and evaporated. Thus, a total of 62.5 g obtained as a solidified foam, which is reacted as the crude product without further purification.

HPLC (Method 1): R _τ = 16.6 min.

example 7

{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-[2-methoxy-5-(trifluormethyl)phenyl]-3,4-dihydrochinazolin-4-yl}essigsäuremethylester / Pot chlorination + amination

(50.0 g) is introduced in chlorobenzene (300 mL), then chlorobenzene is partially distilled (5O mL). The mixture is heated to 120 ⁰ cooled C., DBU (36.9 g) is added, then at 120-128 is ⁰ C phosphorous oxychloride (33.4 mL) over 10 min. metered. The mixture (approximately 130 at reflux for 9 hours ⁰ C) stirred. Subsequently, at 40 ⁰cooled C, slowly at 40-45 ⁰ C with water (200 mL), cooled to room temperature and diluted with dichloromethane (200 mL), stirred and then the phases separated. The organic phase is washed with water (200 mL), saturated aqueous sodium bicarbonate solution (200 mL) and again water (200 mL), dried over sodium sulfate, concentrated by rotary evaporation and then under high vacuum at 50 ⁰ dried C. The residue (48.1 g) is dissolved in chlorobenzene (20 mL), then with 1,4-dioxane (80 mL) at room temperature and 3-methoxyphenylpiperazine (23.6 g) and DBU (18.7 g) was added, whereupon the temperature rises. The mixture is stirred at reflux for 22 h, then cooled to room temperature, with ethyl acetate (500 mL) and water (200 mL) and the phases separated. The organic phase (200 mL) washed with 0.2N hydrochloric acid (three times 100 mL) and water, dried over sodium sulfate and evaporated. Thus, a total of 55.6 g obtained as a solidified foam, which is reacted as the crude product without further purification.

HPLC (Method 1): R _τ = 16.2 min.

example 8

(^)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetate / saponification racemate

(64 g) is dissolved in 1,4-dioxane (45O mL) and IN sodium hydroxide solution (325 mL) and stirred for 2 h at room temperature, then dried in vacuo at 30 ⁰ , a part of the solvent C is distilled off (400 mL). Toluene is added (300 mL) and the phases separated. The aqueous phase is washed with toluene (15O mL twice), then the combined organic phases again with IN sodium hydroxide solution (50 mL) are extracted. The pH of the combined aqueous phases with 2N hydrochloric acid (about 150 mL) to 7.5, then MIBK (15O mL) is added. The phases are separated, the aqueous phase extracted again with MIBK (15O mL), then dried the combined MIBK phases over sodium sulfate and at 45 ⁰ concentrated C. Thus, a total of 64 g as an amorphous solid in quantitative yield.

HPLC (Method 1): R _τ = 14.9 min.

Scheme 6:

Separation of enantiomers of {8-fluoro-2- [4- (3-methoxyphenyl) piperazin-l -yl] -3- [2-methoxy-5- (tri-fluoromethyl) phenyl] -3,4-dihydroquinazolin-4-yl } acetate

x (2S, 3S) -2,3-bis [(4-methylbenzoyl) – oxyjbemsteinsäure
x EtOAc

example 9

(2S, 3 £) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (1: 1 salt) / crystallization

(62.5 g, crude product) is dissolved and filtered in ethyl acetate (495 mL). To the filtrate is (35 25 ‘,) added 2,3-bis [(4-methylbenzoyl) oxy] succinic acid (42.0 g), the mixture for 30 minutes. stirred at room temperature, then with (35 25 “) -2,3-bis [(4-methylbenzoyl) oxy] -succinic acid – (l: l salt) (165 mg) was inoculated and stirred for 3 days at room temperature, then to 0-3 ⁰ cooled C and stirred for a further 3 h, the suspension is suction filtered and washed with cold ethyl acetate (0-10. ⁰ C, 35 mL ) washed. the crystals are at 40 h 18 ⁰ C in the VDO using entraining nitrogen dried. Thus 37.1 g of the salt are obtained as a solid, corresponding to 30.4% of theory over three stages (chlorination, amination and crystallization) on the racemate, or 60.8% based on the resulting S enantiomer.

– – ¹ H NMR (300 MHz, d ₆ -DMSO): δ = 7.90 (d, ² J = 7.8, 4H), 7.56 (d, ² J = 8.3, IH), 7 , 40 (d, ² J = 7.8, 4H), 7.28 to 7.05 (m, 4H), 6.91 to 6.86 (m, 2H), 6.45 (d, ² J = 8.3, IH), 6.39 to 6.36 (m, 2H), 5.82 (s, 2H), 4.94 (m, IH), 4.03 (q, ² J = 7.1 , 2H), 3.83 (brs, 3H), 3.69 (s, 3H), 3.64 (s, 3H), 3.47 to 3.36 (m, 8H and water, 2H), 2, 98 to 2.81 (m, 5H), 2.58 to 2.52 (m, IH), 2.41 (s, 6H), 1.99 (s, 3H), 1.18 (t, ² J = 7.2, 3H) ppm;

HPLC (Method 1): R _τ = 16.6 and 18.5 min.

example 10

(25,3iS) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (1: 1 salt) / recrystallization

(2S, 3S) -2,3-bis [(4-methy lbenzoyl) oxy] succinic acid – { (l: l salt) (36.8 g) is suspended in ethyl acetate (37o mL) and (77 by heating to reflux ⁰ C) dissolved. The mixture is slowly cooled to room temperature. Here there is a spontaneous crystallization. The suspension is stirred at RT for 16 h, then 0-5 ⁰ cooled C and stirred for another 3 h. The suspension is suction filtered and washed with cold ethyl acetate (0-10 ⁰ C, twice 15 ml). The crystals are at 45 h 18 ⁰ C in the VDO using entraining nitrogen dried. Thus 33.6 g of the salt are obtained as a solid, corresponding to 91.3% of theory.

HPLC (Method 1): R _τ = 16.9 and 18.8 min .;

HPLC (Method 3): 99.9% ee

example 11

(5)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl}essigsäure

(2IS ^I , 3S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (l: l salt) (10.1 g, containing 14 ppm of Pd) are suspended in ethyl acetate (100 mL) and shaken with saturated aqueous sodium bicarbonate solution (10O mL) shaken until both phases are clear. The phases are separated, the organic phase is evaporated. The residue is dissolved in 1,4-dioxane (100 mL) and IN sodium hydroxide solution (31.2 mL) and stirred for 3 h at room temperature. Subsequently, the pH is adjusted with IN hydrochloric acid (about 17 mL) is set to 7.5, MIBK (8O mL) was added, then the pH is adjusted with IN hydrochloric acid (about 2 mL) adjusted to 7.0. The phases are separated, the organic phase dried over sodium sulfate and concentrated. The residue is dissolved in ethanol and concentrated (40 mL), then again in ethanol (40 mL) and concentrated under high vacuum at 50 ⁰ C dried. The solidified foam is at 45 h 18 ⁰ C in the VDO using entraining nitrogen dried. Thus, a total of 5.05 g as an amorphous solid, corresponding to 85.0% of theory.

¹ H NMR (300 MHz, d ₆ -DMSO): δ = 7.53 (d, ² J = 8.4, IH), 7.41 (brs, IH), 7.22 (d, ² J = 8 , 5, IH), 7.09 to 7.01 (m, 2H), 6.86 (m, 2H), 6.45 (dd, V = 8.2, ³ J = 1.8, IH) 6.39 to 6.34 (m, 2H), 4.87 (t, ² J = 7.3, IH), 3.79 (brs, 3H), 3.68 (s, 3H), 3.50 -3.38 (m, 4H), 2.96 to 2.75 (m, 5H), 2.45 to 2.40 (m, IH) ppm;

MS (API-ES-neg.): M / z = 571 [(MH), 100%];

HPLC (Method 1): R _τ = 15.1 min;

HPLC (Method 2): 99.8% ee; Pd (ICP): <1 ppm.

example 12

(2 / ?, 3Λ) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (1: 1 salt) / crystallization R-isomer from the mother liquor

The mother liquor from a crystallization of (2IS ‘, 3S) -2,3-bis [(4-methylbenzoyl) oxy] -succinic acid – {8-fluoro-2- [4- (3-methoxyphenyl) piperazin-l -yl] -3- [2-methoxy-5- (trifluoromethyl) phenyl] -3,4-dihydroquinazolin-4-yl} acetic acid methyl ester (l: l-salt) in 279 g scale is washed with saturated aqueous sodium bicarbonate solution (1.5 L ) shaken, the phases are separated and the organic phase is shaken with semi-saturated aqueous sodium bicarbonate solution (1.5 L). The phases are separated, the organic phase dried over sodium sulfate and evaporated. The residue (188.4 g) is dissolved in ethyl acetate (1.57 L), then (2R, 3R) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (121.7 g) was added and the mixture 10 min. stirred at room temperature. Is then treated with (2R, 3R) -2,3-bis [(4-methyl-benzoyl) oxy] succinic acid – (l: l salt) (0.38 g) was inoculated and stirred for 18 h at room temperature, then to 0-3 ⁰ cooled C and stirred for another 3 h. The suspension is suction filtered and washed with cold ethyl acetate (0-10 ⁰ C, 50O ml). The crystals are at 40 h 18 ⁰ C in the VDO using entraining nitrogen dried. So a total of 160 g of the salt are obtained as a solid.

HPLC (Method 1): R _τ = 16.6 and 18.5 min .;

HPLC (Method 3): -99.0% ee

example 13

(i?)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetate / production R-isomer

(2Λ, 3 /?) – 2,3-bis [(4-methylbenzoyl) oxy] succinic acid – {8-fluoro-2- [4- (3-methoxy-phenyl) pipera-tine 1-yl] -3- [ 2-methoxy-5- (trifluormethy l) pheny l] -3, 4-dihydroquinazolin-4-y 1} -acetic acid methyl ester (1: 1 salt) (170 g) are suspended in ethyl acetate (85O mL) and as long as with saturated aqueous sodium bicarbonate (850 mL) shaken until both phases are clear (about 5 min.). The phases are separated, the solvent of the organic phase under normal pressure with 1, 4-dioxane to a final temperature of 99 ⁰ exchanged C (portions distilled total 2.55 L solvent, and 2.55 L of 1,4-dioxane used). The mixture is cooled to room temperature and 18 at room temperature IN sodium hydroxide solution (525 mL) stirred. Subsequently, the pH value with concentrated hydrochloric acid (about 35 mL) is set to 7.5, MIBK (85O mL) was added, then the pH with concentrated hydrochloric acid (ca. 1O mL) adjusted to 7.0. The phases are separated, the organic phase dried over sodium sulfate and concentrated. The residue is dissolved in ethanol and concentrated (350 mL), then again in ethanol (350 mL) at 50 and ⁰ concentrated C. Thus, a total of 91.6 g as an amorphous solid, corresponding to 91.6% of theory.

HPLC (method 1): R ₇ = 14.8 min.

– – Example 14

{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetate / racemization R-enantiomer

acetic acid (50 g) is dissolved in acetonitrile (500 mL) and treated with sodium methoxide (30% in methanol, 32.4 mL) and then stirred at reflux for 60 h. After cooling to room temperature the mixture is concentrated in vacuo to half, then with hydrochloric acid (20% strength, ca. 20 ml) adjusted to pH 7.5, MIBK (200 mL) was added and hydrochloric acid (20%) on pH 7 adjusted. The phases are separated, the organic phase dried over sodium sulfate and evaporated to the hard foam. The residue is dissolved in ethanol and concentrated (15O mL), then again in ethanol (15O mL) and concentrated. Thus, 54.2 g as an amorphous solid in quantitative yield.

HPLC (Method 1): R _τ = 14.9 min .;

HPLC (method 4): 80.8 wt.%.

example 15

{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-[2-methoxy-5-(trifluormethyl)phenyl]-3,4-dihydrochinazolin-4-yl}essigsäuremethylester / Esterification racemate

acetic acid (54 g) (540 g) was dissolved in methanol, then concentrated sulfuric acid (7.85 mL) is added. The mixture is stirred at reflux for 26 h, then cooled and concentrated in vacuo to about one third of the original volume. Water (15O mL) and dichloromethane (15O mL) are added, then the phases are separated. The organic phase is washed with saturated sodium bicarbonate solution (two times 140 mL), dried over sodium sulfate and concentrated to a foamy residue. This is twice in succession in ethanol (150 mL) and concentrated, dried in vacuo using entraining nitrogen then 18 h. Thus, a total of 41.6 g as an amorphous solid, corresponding to 75.2% of theory.

HPLC (Method 1): R _τ = 16.8 min .;

HPLC (method 4): 85.3 wt.%;

HPLC (Method 3): -8.5% ee

example 16

(25 ¹ , 3S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – { (1: 1 salt) / crystallization of esterified racemate

(41.0 g) is suspended in ethyl acetate (287 mL), then (2S, 3IS) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid (27.5 g) was added. The mixture is 30 minutes. stirred at room temperature, then with (2 <S ‘, 3S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – (1: 1 salt) (0.08 g) was inoculated. The suspension is stirred at RT for 16 h, then 0-5 ⁰ cooled C and stirred for another 3 h, then filtered off with suction and washed with cold ethyl acetate (0-10 ⁰ C, four times 16 ml). The crystals are at 45 h 18 ⁰ C in the VDO using entraining nitrogen dried. So a total of 25.4 g of the salt are obtained as a solid, corresponding to 37.4% of theory.

HPLC (Method 1): R _τ = 16.9 and 18.8 min .;

HPLC (method 4): 99.5 wt.%;

HPLC (Method 3): 99.3% ee

example 17

(iS)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetate / saponification crystals

(25,3S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – (l rl salt) (25.1 g) is suspended in ethyl acetate (25O mL) and shaken with saturated aqueous sodium bicarbonate solution (250 mL) shaken until both phases are clear. The phases are separated, the organic phase is evaporated. Dissolve the residue in 1, 4-dioxane (25O mL) and IN sodium hydroxide solution (77.4 mL) and stirred for 18 h at room temperature. Subsequently, the pH is adjusted with IN hydrochloric acid (about 50 mL) is set to 7.5, was added MIBK (240 mL), then the pH is adjusted with IN hydrochloric acid (about 15 mL) adjusted to 7.0. The phases are separated, the organic phase dried over sodium sulfate and concentrated. The residue is dissolved in ethanol and concentrated (90 mL), then again in ethanol (90 mL) and concentrated. The solidified foam is at 45 h 18⁰ C in the VDO using entraining nitrogen dried. Thus, a total of 12 g as an amorphous solid, corresponding to 81.2% ^“ of the theory.

HPLC (Method 1): R _τ = 15.1 min;

HPLC (Method 2): 97.5% ee; Pd (ICP): <20 ppm.

Alternative method for the racemization:

example 18

(i)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetic acid / saponification enriched R isomer from the mother liquor after crystallization

The mother liquor from a crystallization of (2 _J S ‘, 35) -2,3-bis [(4-methylbenzoyl) oxy] -succinic acid – (l: l-salt) in 207 g scale is shaken with saturated aqueous sodium bicarbonate (500 mL), the phases are separated and the organic phase is shaken with semi-saturated aqueous sodium bicarbonate solution (500 mL). The phases are separated, the organic phase dried over sodium sulfate and evaporated. The residue is dissolved in ethanol (500 mL) and rotary evaporated to a hard foam. This is in 1,4-dioxane (1.6 L) and IN sodium hydroxide solution (1.04 L) and stirred at room temperature for 18 h, then toluene is added (1.5 L) and the phases separated. The aqueous phase is adjusted with hydrochloric acid (20% strength, ca. 155 ml) of pH 14 to pH 8, then is added MIBK (1.25 L) and hydrochloric acid (20% strength, ca. 25 mL) to pH 7 readjusted. The phases are separated, the organic phase dried over sodium sulfate and evaporated to the hard foam. This is at 45 h 18 ⁰ C in the VDO using entraining nitrogen dried. Thus, a total of 150 g obtained as (R / S) mixture as an amorphous solid.

HPLC (Method 2): 14.6% ee

– – Example 19

(i)-{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-(2-methoxy-5-trifluormethylphenyl)-3,4-dihydrochinazolin-4-yl} acetate / racemization

(150 g, R / S mixture with -14.6% ee) is dissolved in acetonitrile (1.5 L) and treated with sodium methoxide (30% in methanol, 97.2 mL) was added, then stirred at reflux for 77 h , After cooling to room temperature the mixture is concentrated in vacuo to half, then with hydrochloric acid (20% strength, ca. 80 mL) made of pH 13 to pH 7.5, was added MIBK (0.6 L) and treated with hydrochloric acid ( 20% strength, ca. 3 mL) adjusted to pH. 7 The phases are separated, the organic phase dried over sodium sulfate and evaporated to the hard foam. The residue is dissolved in ethanol and concentrated (500 mL), then again in ethanol (500 mL) and concentrated, then 18 h at 45⁰ dried C in the VDO using entraining nitrogen. Thus, a total of 148 g as an amorphous solid, corresponding to 98.7% of theory.

HPLC (Method 2): 1.5% ee

example 20

{8-Fluor-2-[4-(3-methoxyphenyl)piperazin-l-yl]-3-[2-methoxy-5-(trifluormethyl)phenyl]-3,4-dihydrochinazolin-4-yl}essigsäuremethylester (Esterification)

(±) – {8-fluoro-2- [4- (3-methoxyphenyl l) piperazin-1 -yl] -3- (2-methoxy-5-trifluormethy lphenyl) -3, 4-dihydroquinazolin-4-yl} acetic acid (148 g) (1480 g) was dissolved in methanol, then concentrated sulfuric acid (21.5 mL) is added. The mixture is stirred at reflux for 6 h, then cooled and concentrated in vacuo to about one third of the original volume. Water (400 mL) and dichloromethane (400 mL) are added, then the phases are separated. The organic phase (diluted twice 375 mL, 300 mL water) with saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated to a foamy residue. This is twice in succession in ethanol (each 400 mL) and concentrated, dried in vacuo using entraining nitrogen then 18 h. Thus, a total of 124 g as an amorphous solid, corresponding to 81.9% of theory.

HPLC (Method 1): R _τ = 16.9 min .;

example 21

(25.35) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – (1: 1 salt) / crystallization of esterified racemate

(2S, 3S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – (1: 1 salt) (123 g, 14.4% ee) is suspended in ethyl acetate (861 mL) and filtered, then (2IS ‘, 3IS) -2,3-bis [(4-methylbenzoyl) oxy ] succinic acid (82.5 g). The mixture 30 min. stirred at room temperature, then with (2 £, 3 <S) -2,3-bis [(4-methylbenzoyl) oxy] succinic acid – (1: 1 salt) (0.24 g) was inoculated. The suspension is stirred for 4 days at RT, then concentrated to approximately 600 mL and again with (25 ‘, 3 ₁ -2,3-bis [(4-methylbenzoyl) oxy] succinic acid S) – (l: l salt) (0.24 g) was inoculated. The suspension is stirred for 1 week at RT, to 0-5 ⁰ cooled C and further stirred for 3 hours, then filtered off with suction and washed with cold ethyl acetate (0-10 ⁰ C, 4 x 40 ml). The crystals are at 45 h 18 ⁰ C in the VDO using entraining nitrogen dried. So a total of 1 1.8 g of salt are obtained as a solid, corresponding to 5.8% of theory.

Scheme 7:

example 22

N- (2-Fluoφhenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] urea

2-methoxy-5-trifluoromethylphenyl isocyanate (1057.8 g) is dissolved in acetonitrile (4240 mL), then 2-fluoro aniline (540.8 g) was added with acetonitrile (50 mL) flushed.The resulting clear solution is stirred for 4 h at reflux (about 82 ° C), then seeded at about 78 ° C and about 15 min. touched. The suspension is on 0 ⁰ cooled C, aspirated and the product with acetonitrile (950 mL, to 0-5 ⁰ cooled C) washed. The product is dried overnight at 45 ° C in a vacuum drying oven using entraining nitrogen. Thus, a total of 1380.8 g of N- (2-fluorophenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] -harnstqff ^‘ obtained as a solid, corresponding to 86.4% of theory.

¹ H NMR (500 MHz, d ₆ -DMSO): δ = 9.36 (s, IH), 9.04 (s, IH), 8.55 (d, 1.7 Hz, IH), 8.17 ( t, 8.2 Hz, IH), 7.33 (d, 8.5 Hz, IH), 7.20 to 7.26 (m, 2H), 7.14 (t, 7.6 Hz, IH), 7, 02 (m, IH), 3.97 (s, 3H) ppm;

MS (API-ES-pos.): M / z = 329 [(M + H) ⁺ , 100%];

HPLC: R _τ = 48.7 min.

Instrument: HP 1100 Multiple Wavelength detection; Column: Phenomenex-Prodigy ODS (3) 100A, 150 mm x 3 mm, 3 microns; Eluent A: (1.36 g KH ₂ PO ₄ +0.7 mL H ₃PO ₄ ) / L water, eluent B:

acetonitrile; Gradient: 0 min 20% B, 40 min 45% B, 50 min 80% B, 65 min 80% B; Flow: 0.5 mL / min; Temp .: 55 ⁰ C; UV detection: 210 nm.

example 23

Methyl (2E) -3- {3-fluoro-2 – [({[2-methoxy-5 – (trifluormethy l) pheny 1] amino} carbonylation l) amino] pheny 1} acrylate

N- (2-fluorophenyl) -N ‘- [2-methoxy-5- (trifluoromethyl) phenyl] urea (0.225 kg) is dissolved in acetic acid (6.75 L) and (30.3 g) was added with palladium acetate. Then 65% oleum is (247.5 g) is added and then methyl acrylate (90 g). The solution is stirred overnight at room temperature. Then, at about 30 ⁰ C and about 30 mbar acetic acid (3740 g) were distilled off. The suspension is treated with water (2.25 L) and stirred for about 1 hour. The product is drained, washed twice with water (0.5 L) and incubated overnight at 50 ⁰ dried C in a vacuum drying oven using entraining nitrogen. Thus, a total of 210.3 g of methyl (2E) -3- {3-fluoro-be 2 – [({[2-methoxy-5- (trifluoromethyl) phenyl] amino} carbonyl) amino] phenyl} acrylate obtained as a solid, corresponding to 72.2% of theory.

¹ H NMR (300 MHz, d ₆ -DMSO): δ = 9.16 (s, IH), 8.84 (s, IH), 8.45 (d, 1.7 Hz, IH), 7.73 ( m, 2H), 7.33 (m, 3H), 7.22 (d, 8.6 Hz, IH), 6.70 (d, 16Hz, IH), 3.99 (s, 3H), 3.71 (s, 3H) ppm;

MS (API-ES-pos.): M / z = 429.9 [(M + NH,) ⁺ ]; 412.9 [(M + H) ⁺ ]

HPLC: R _τ = 46.4 min.

Instrument: HP 1100 Multiple Wavelength detection; Column: Phenomenex-Prodigy ODS (3) 100A, 150 mm x 3 mm, 3 microns; Eluent A: (1.36 g KH ₂ PO ₄ +0.7 mL H ₃PO ₄ ) / L water, eluent B: acetonitrile; Gradient: 0 min 20% B, 40 min 45% B, 50 min 80% B, 65 min 80% B; Flow: 0.5 mL / min; Temp .: 55 ⁰ C; UV detection: 210 nm.

example 24

{8-FluorO-[2-methoxy-5-(trifluormethyl)phenyl]-2-oxo-l,2,3,4-tetrahydrochinazolin-4-yl}essigsäuremethylester

Methyl (2E) -3- {3-fluoro-2 – [({[2-methoxy-5- (trifluoromethyl) phenyl] amino} carbonyl) amino] phenyl} acrylate (50 g) is dissolved in acetone (1.2 L) was suspended and 3.7 g) was added l, 8-diazabicyclo [5.4.0] undec-7-ene (. The suspension is heated to reflux (ca..56 ° C) and stirred for 4 h. The resulting clear solution is hot through diatomaceous earth (5 g) was filtered. The diatomaceous earth is rinsed with warm acetone (100 ml). Subsequently, acetone (550 g) was distilled off. The resulting suspension is in 3 h at O ⁰ cooled and stirred C. The product is drained, washed twice with cold acetone (50 ml) and incubated overnight at 45 ⁰ dried C in a vacuum drying oven using entraining nitrogen. Thus, a total of 44.5 g of {8-fluoro-3- [2-methoxy-5- (trifluoromethyl) phenyl] -2-oxo-1, 2, 3, 4-tetrahydrochinazo-lin-4-yl} acetic acid methyl ester as a solid, corresponding to 89% of theory.

5.9, IH), 3.84 (s, 3H), 3.41 (s, 3H), 2.81 (dd, ¹ J = 15.4, V = 5.8, IH), 2.62 (dd, 2 Vr = = 15.4, V = 6.3, IH) ppm;

MS (API-ES-pos.): M / z = 413 [(M + H) ⁺ , 100%], 825 [(2M + H) ⁺ , 14%];

HPLC: R _τ = 37.1 min.

PATENT

WO 2015088931

Human cytomegalovirus (HCMV) is ubiquitously distributed in the human population. In immunocompetent adults infections are mainly asymptomatic, but in

immunocompromised patients, such as transplant recipients or AIDS patients, life threatening infections occur at a high rate. HCMV is also the leading cause of birth defects among congenitally transmitted viral infections.

Various substituted heterocyclic compounds are inhibitors of the HCMV terminase enzyme. Included in these heterocycles are quinazolines related to Compound A, as defined and described below. These compounds and pharmaceutically acceptable salts thereof are useful in the treatment or prophylaxis of infection by HCMV and in the treatment, prophylaxis, or delay in the onset or progression of HCMV infection. Representative quinazoline compounds that are useful for treating HCMV infection are described, for example, in US Patent Patent No. 7, 196,086. Among the compounds disclosed in US7, 196,086, is (S)-2-(8-fluoro-3-(2-methoxy-5-(trifluoromethyl)phenyl)-2-(4-(3-methoxyphenyl)piperazin-l-yl)-3,4-dihydroquinazolin-4-yl)acetic acid, hereinafter referred to as Compound A. Compound A is a known inhibitor of HCMV terminase. The structure of Compound A is as follows:

Compound A

US Patent Nos. 7,196,086 and 8,084,604 disclose methodology that can be employed to prepare Compound A and related quinazoline-based HCMV terminase inhibitors. These methods are practical routes for the preparation of Compound A and related heterocyclic compounds.

EXAMPLE 6

Preparation of Compound A

To a slurry of compound 7 (20g, 18.9 mmol) in MTBE (40.0 mL) at room temperature was added a solution of sodium phosphate dibasic dihydrate (8.42 g, 47.3 mmol) in water (80 mL) and the resulting slurry was allowed to stir at room temperature for 40 minutes. The reaction mixture was transferred to a separatory funnel and the organic phase was collected and washed with a solution of sodium phosphate dibasic dihydrate (3.37 g, 18.91 mmol) in water (40.0 mL). A solution of KOH (4.99 g, 76 mmol) in water (80 mL) and methanol (10.00 mL) was then added to the organic phase and the resulting mixture was heated to 50 °C and allowed to stir at this temperature for 6 hours. MTBE (20 mL) and water (40 mL) were then added to the

reaction mixture and the resulting solution was transferred to a separatory funnel and the aqueous layer was collected and washed with MTBE (20 mL). Additional MTBE (40 mL) was added to the aqueous layer and the resulting solution was adjusted to pH 4-5 via slow addition of concentrated HCl. The resulting acidified solution was transferred to a separatory funnel and the organic phase was collected, concentrated in vacuo and solvent switched with acetone, maintaining a 30 mL volume. The resulting acetone solution was added dropwise to water and the precipitate formed was filtered to provide compound A as a white solid (10 g, 92%). ^XH NMR (500 MHz, d₆-DMSO): δ_Η 12.6 (1H, s), 7.52 (1H, dd, J= 8.6, 1.3 Hz), 7.41 (1H, brs), 7.22 (1H, d, J= 7.2 Hz), 7.08-7.02 (2H, m), 6.87-6.84 (2H, m), 6.44 (1H, dd, J= 8.3, 1.8 Hz), 6.39 (1H, t, J= 2.1 Hz), 6.35 (1H, dd, J= 8.1, 2.0 Hz), 4.89 (1H, t, J= 7.3 Hz), 3.79 (3H, br s), 3.68 (3H, s), 3.47 (2H, br s), 3.39 (2H, br s), 2.96-2.93 (2H, m), 2.82-2.77 (3H, m), 2.44 (1H, dd, J = 14.8, 7.4 Hz).

XAMPLE 1

Preparation of Intermediate Compound 2

N,N-dicyclohexylmethylamine

IPAC, 80°C

To a degassed solution of 2-bromo-6-fluoroaniline (1, 99.5 g, 0.524 mol), methyl acrylate (95.0 mL, 1.05 mol), Chloro[(tri-tert-butylphosphine)-2-(2-aminobiphenyl)] palladium(II) (0.537 g, 1.05 mmol) in isopropyl acetate (796 mL), was added degassed N,N-dicyclohexylmethylamine (135 mL, 0.628 mol). The resulting reaction was heated to 80 °C and allowed to stir at this temperature for 5 hours. The resulting slurry was cooled to 20 °C and filtered. The filtrate was washed with 1 M citric acid to provide a solution that contained compound 2 (99.3 g, 97% assay yield) in isopropyl acrylate, which was used without further purification. ‘H NMR (500 MHz, d-CHCl₃): δ_Η 7.79 ppm (1H, d, J= 15.9 Hz), 7.17 ppm (1H, d, J= 8.2 Hz), 7.00 ppm (1H, ddd, J= 10.7, 8.2, 1.2 Hz), 6.69 ppm (1H, td, J = 8.2, 5.1 Hz), 6.38 ppm (1H, d, J= 15.9 Hz), 4.06 ppm (2H, br s), 3.81 ppm (3H, s).

EXAMPLE 2

Preparation of Intermediate Compound 3

To a solution of compound 2 (48.8 g, 0.250 mol) in 683 mL of isopropyl acetate was added 244 mL of water, followed by di-sodium hydrogen phosphate (53.2 g, 0.375 mol). To the resulting solution was added phenyl chloroformate (39.2 mL, 0.313 mol) dropwise over 30 minutes. The resulting reaction was heated to 30 °C and allowed to stir at this temperature for 5 hours for 4 hours and then was heated to 60 °C and allowed to stir at this temperature for 5 hours for an additional 2 hours to remove excess phenyl chloroformate. An additional 293 mL of isopropyl acetate was then added and the reaction mixture was allowed to stir at room temperature until the solids completely dissolved into solution. The resulting reaction mixture was transferred to a separatory funnel and the organic phase was washed with 98 mL of water and collected to provide a solution of compound 3 in isopropyl acetate, which was used without further purification. ^XH NMR (500 MHz, d-acetonitrile): δ_Η 7.91 ppm (1H, d, J= 15.9 Hz), 7.85 ppm (1H, br s), 7.63 ppm (1H, d, J= 7.9 Hz), 7.45-7.39 ppm (3H, m), 7.33-7.27 ppm (2H, m), 7.21 ppm (2H, br), 6.60 ppm (1H, d, J= 16.0 Hz).

EXAMPLE 3

Preparation of Intermediate Compound 4

A solution of compound 3 (79.0 g, 0.250 mol), 2-methoxy-5-(trifluoromethyl)aniline (52.7 g, 0.276 mol), and 4-dimethylaminopyridine (0.92 g, 0.0075 mol) in isopropyl acetate (780 mL) was heated to reflux and allowed to stir at this temperature for 5 hours. The resulting slurry was cooled to 20 °C, then allowed to stir at this temperature for for two hours at this temperature, then filtered. The collected filter cake was dried in vacuo to provide compound 5 (95.0 g, 0.230 mol) as a white solid, which was used without further purification. ¾ NMR (500 MHz, d-TFA): δ_Η 7.98 ppm (1H, d, J= 16.1 Hz), 7.87 ppm (1H, s), 7.47 ppm (1H, d, J = 7.9 Hz), 7.41 ppm (1H, d, J= 8.5 Hz), 7.35 ppm (1H, q, J= 8.5 Hz), 7.19 ppm (1H, t, J= 8.6 Hz), 6.98 ppm (1H, d, J= 8.6 Hz), 6.56 ppm (1H, d, J= 16.0 Hz), 3.85 ppm (6H, br s).

EXAMPLE 4

Preparation of Intermediate Compound 6

To a stirred suspension of compound 4 (14.0 g, 34.0 mmol) in toluene (140 mL) at room temperature was added 2-picoline (10.1 mL, 102 mmol) followed by PCI5 (8.19 g, 37.3 mmol). The resulting reaction was heated to 40 °C and allowed to stir at this temperature for 4 hours, then was cooled to 0 °C and cautiously (internal temperature kept <15 °C) quenched with KOH (2 M, 102 mL). The resulting solution was allowed to warm to room temperature, allowed to stir for 30 minutes, then was filtered and the filtrate transferred to a separatory funnel. The organic phase was washed sequentially with H₃PO₄ (1M, 50 mL) and H₂0 (50 mL) to provide a solution of compound 5 in toluene, which was used without further purification. ^XH NMR (500 MHz, d₆-DMSO): δ_Η 7.96 (1H, d, J= 16.2 Hz), 7.74 (1H, d, J= 7.9 Hz), 7.61 (1H, dd, J= 6.7, 1.6 Hz), 7.50 (1H, d, J= 1.9 Hz), 7.43 (1H, t, J= 9.2 Hz), 7.30 (1H, d, J= 8.4 Hz), 7.28 (1H, m), 6.79 (1H, d, J= 16.2 Hz), 3.91 (3H, s), 3.74 (3H, s).

To the solution of compound 5 at room temperature was added an aqueous solution of piperazine hydrochloride (0.40 M, 93.3 mL, 37.3 mmol) followed by Na₂HP0₄ (14.5 g, 102 mmol). The resulting reaction was allowed to stir for 1 hour at room temperature, then transferred to a separatory funnel. The organic phase was washed sequentially with aH₂P0₄ (50 mL) and H₂0 (50 mL). Salicylic acid (5.16 g, 37.3 mmol) was then added to the organic phase, and the resulting solution was cooled to 0 °C and allowed to stir at this temperature for 1 hour to provide a slurry which was filtered and washed with cold toluene (50 mL). The filter cake was dried under air to provide compound 6 (23.0 g, 31.7 mmol, 93 %) as a white crystalline solid: ^XH NMR (500 MHz, d₆-DMSO): δ_Η 12.9 (1H, br s), 7.75 (1H, dd, J= 7.8, 1.8 Hz), 7.72 (1H, d, J= 16.1 Hz), 7.40 (1H, td, J= 7.2, 1.7 Hz), 7.27 (1H, d, J= 7.8 Hz), 7.17 (1H, m), 7.16 (1H, t, J= 8.2 Hz), 7.02 (1H, br s), 6.95 (1H, t, J= 8.6 Hz), 6.88-6.81 (3H, m), 6.78 (1H, br s), 6.60 (1H, dd, J= 8.2, 2.0 Hz), 6.54 (1H, m), 6.48 (1H, d, J= 16.1 Hz), 6.43 (1H, dd, J= 8.0, 2.1 Hz), 3.73 (3H, s), 3.71 (3H, s), 3.69 (4H, br s), 3.68 (3H, s).

Free Base: ^XH NMR (500 MHz, CD₃CN): δ_Η 7.91 (1H, d, J= 16.1 Hz), 7.29 (1H, d, J= 8.0 Hz), 7.24 (1H, d, J= 1.4 Hz), 7.20 (1H, t, J= 8.1 Hz), 7.15 (1H, dd, J= 8.6, 1.4 Hz), 6.94 (1H, m), 6.92 (1H, t, J= 8.1 Hz), 6.80 (1H, td, J= 8.1, 5.4 Hz), 6.60 (1H, dd, J= 8.3, 2.2 Hz), 6.54 (1H, t, J= 2.2 Hz), 6.50 (1H, d, J= 16.1 Hz), 6.47 (2H, m), 3.80 (3H, s), 3.79 (3H, s), 3.72 (3H, s), 3.63 (4H, t, J= 5.1 Hz), 3.25 (4H, t, J= 5.0 Hz).

2: 1 NDSA Salt: ‘H NMR (500 MHz, d₆-DMSO): δ_Η 10.2 (2H, br s), 8.86 (1H, d, J= 8.6 Hz), 7.92 (1H, d, J= 7.0 Hz), 7.47-7.37 (4H, m), 7.27-7.14 (4H, m), 6.96 (1H, d, J= 8.6 Hz), 6.65 (1H, d, J= 8.3 Hz), 6.59 (1H, s), 6.54 (1H, d, J= 15.9 Hz), 6.47 (1H, d, J= 8.3 Hz), 3.91 (4H, m), 3.77 (3H, s), 3.76 (3H, s), 3.74 (3H, s), 3.43 (4H, m). 1,5 -naphthalene disulfonic acid

EXAMPLE 5

Preparation of Intermediate Compound 7

To a suspension of compound 6 (12.5 g, 16.6 mmol) in 125 mL of toluene was added 50 mL of 0.43M aqueous K₃P0₄. The resulting reaction was allowed to stir for 1 hour at room temperature and the reaction mixture was transferred to a separatory funnel. The organic phase was collected, washed once with 30 mL 0.43M aqueous K₃P0₄then cooled to 0 °C and aqueous K₃P0₄ (60 mL, 0.43 M, 25.7 mmol) was added. To the resulting solution was added a room temperature solution of ((lS,2S,4S,5R)-l-(3,5-bis(trifluoromethyl)benzyl)-2-((R)-

hydroxy( 1 -(3 -(trifluoromethyl)benzyl)quinolin- 1 -ium-4-yl)methyl)-5-vinylquinuclidin- 1 -ium bromide) (0.704 g, 0.838 mmol) in 1.45 mL of DMF. The resulting reaction was allowed to stir at 0 °C until the reaction was complete (monitored by HPLC), then the reaction mixture was transferred to a separatory funnel and the organic phase was collected and washed sequentially with 1M glycolic acid (25 mL) and water (25 mL). The organic phase was filtered through solka flok and concentrated in vacuo to a total volume of 60 mL. Ethyl acetate (20 mL) was added to the resulting solution, followed by (S,S)-Di-P-Toluoyl-D-tartaric acid (5.61 g, 14.1 mmol). Penultimate seed (0.2 g) was added the resulting solution was allowed to stir at room

temperature for 12 hours. The solution was then filtered and the collected solid was washed twice with ethyl acetate, then dried in vacuo to provide compound 7 as its DTTA salt ethyl acetate solvate (13.8 g, 78%) . ‘H NMR (500 MHz, d₆-DMSO): δ_Η 13.95 (2H, br s), 7.90 (4H, d, J= 8.1 Hz), 7.55 (1H, dd, J= 8.6, 1.3 Hz), 7.38 (4H, d, J= 8.1 Hz), 7.26 (1H, d, J= 7.8 Hz), 7.09-7.05 (3H, m), 6.91-6.86 (2H, m), 6.44 (1H, dd, J= 8.2, 1.7 Hz), 6.39 (1H, t, J= 2.0 Hz), 6.36 (1H, dd, J= 8.2, 2.0 Hz), 5.82 (2H, s), 4.94 (1H, t, J= 7.1 Hz), 4.02 (2H, q, J= 7.1 Hz), 3.83 (3H, br s), 3.68 (3H, s), 3.64 (3H, s), 3.47 (2H, br s), 3.37 (2H, br s), 2.95 (2H, br s), 2.87- 2.80 (3H, m), 2.56 (1H, dd, J= 14.3, 7.0 Hz), 2.39 (6H, s), 1.98 (3H, s), 1.17 (3H, t, J= 7.1 Hz).

PAPER

Asymmetric Synthesis of Letermovir Using a Novel Phase-Transfer-Catalyzed Aza-Michael Reaction

Guy R. Humphrey^*, Stephen M. Dalby^*, Teresa Andreani, Bangping Xiang, Michael R. Luzung, Zhiguo Jake Song, Michael Shevlin, Melodie Christensen, Kevin M. Belyk, and David M. Tschaen

Department of Process Chemistry, Merck and Co., Inc., P.O. Box 2000, Rahway, New Jersey 07065, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00076

Publication Date (Web): May 13, 2016

*E-mail: stephen.dalby@merck.com., *E-mail: guy_humphrey@merck.com.

ACS Editors’ Choice – This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Abstract

The development of a concise asymmetric synthesis of the antiviral development candidate letermovir is reported, proceeding in >60% yield over a total of seven steps from commercially available materials. Key to the effectiveness of this process is a novel cinchonidine-based PTC-catalyzed aza-Michael reaction to configure the single stereocenter.

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00076

Supporting Info

(S)-2-(8-Fluoro-3-(2-methoxy-5-(trifluoromethyl)phenyl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)-3,4-dihydroquinazolin-4-yl)acetic Acid (Letermovir, 1)

letermovir (1, 20.2 g, 35.3 mmol, 100 wt %, 94%) as an amorphous white powder. ¹H NMR (DMSO-d₆, 600 MHz) δ_H 7.52 (dd, J = 8.7, 1.7 Hz, 1H), 7.40 (brs, 1H), 7.21 (m, 1H), 7.07 (t, J = 8.2 Hz, 1H), 7.04 (m, 1H), 6.87 (m, 2H), 6.44 (dd, J = 8.2, 1.9 Hz, 1H), 6.40 (t, J = 2.3 Hz, 1H), 6.36 (dd, J = 8.0, 2.0 Hz, 1H), 4.89 (t, J = 7.2 Hz, 1H), 3.80 (brs, 3H), 3.68 (s, 3H), 3.39–3.48 (m, 4H), 2.82–2.95 (m, 4H), 2.80 (dd, J = 14.8, 7.4 Hz, 1H), 2.46 (dd, J = 14.9, 7.4 Hz, 1H); ¹³C NMR (DMSO-d₆, 150 MHz) δ_C 171.8, 160.2, 156.5, 154.6 (d, J_CF = 246.3 Hz), 153.2, 152.2, 134.2, 132.3 (d, J_CF = 11.2 Hz), 129.6, 124.1 (q, J_CF = 271.3 Hz), 123.8 (q, J_CF = 3.7 Hz), 122.4, 122.1 (q, J_CF = 7.1 Hz), 121.4 (q, J_CF = 29.2 Hz), 120.8, 114.5 (d, J_CF = 19.5 Hz), 113.3, 108.3, 104.6, 101.9, 59.0, 56.3, 54.8, 47.9, 45.6, 40.0; HR-MS calcd for C₂₉H₂₉F₄N₄O₄⁺ [M + H]⁺ 573.2119, found 573.2117 (Δ = 0.2 mmu).

References

“Neues Virostatikum Letermovir” (in German). Deutsche Apothekerzeitung. 2011-08-29.

Masangkay, Estel Grace (July 29, 2014). “Merck Kicks Off Phase 3 Study Of CMV Drug Letermovir”. Retrieved 8 Oct 2014.

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Letermovir
Systematic (IUPAC) name

{(4S)-8-Fluoro-2-[4-(3-methoxyphenyl)-1-piperazinyl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydro-4-quinazolinyl}acetic acid
Clinical data
Routes of administration	Oral
Legal status
Legal status	Investigational
Identifiers
ATC code	None
PubChem	CID 45138674
ChemSpider	26352849
UNII	1H09Y5WO1F
ChEMBL	CHEMBL1241951
Synonyms	AIC246
Chemical data
Formula	C₂₉H₂₈F₄N₄O₄
Molar mass	572.55 g/mol

/////Letermovir, MK 8828, AIC 246, fast track status, US Food and Drug Administration, orphan drug status , European Medicines Agency

COC1=C(C=C(C=C1)C(F)(F)F)N2[C@H](C3=C(C(=CC=C3)F)N=C2N4CCN(CC4)C5=CC(=CC=C5)OC)CC(=O)O

BMS-520, a Potent and Selective Isoxazole-Containing S1P1 Receptor Agonist

PRECLINICAL, Uncategorized Comments Off

May 132016

BMS-520
CAS: 1236188-38-7
MF: C23H17F3N4O4
MW: 470.1202

Synonym: BMS-520; BMS 520; BMS520.

INNOVATOR Bristol-Myers Squibb Company

INVENTORS

Scott Hunter Watterson, Alaric J. Dyckman,William J. Pitts, Steven H. Spergel

1-[4-[5-[3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl]-1,2,4-oxadiazol-3-yl]benzyl]azetidine-3-carboxylic acid

1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid

US2011300165

¹H NMR (500 MHz, DMSO-d₆) δ: 3.20–3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J = 8.25 Hz, 2H), 7.60–7. 70 (m, 5H), and 8.06 (d, J = 7. 70 Hz, 2H);

MS m/e 471(M+H⁺);

HPLC (XBridge 5 μ C18 4.6 × 50 mm, 4 mL/min, solvent A: 10% MeOH/water with 0.2% H₃PO₄, solvent B: 90% MeOH/water with 0.2% H₃PO₄, gradient with 0–100% B over 4 min): 3.14 min;

Anal. Calcd for C₂₃H₁₇N₄O₄F₃•0.01 EtOH: C, 58.72; H, 3.65; N, 11.90. Found: C, 58.63; H, 3.41; N, 11.84.

BMS-520 is a potent and selective S1P1 agonist. BMS-520 demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Agonism of S1P1, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression.

Sphingosine-1 -phosphate (SlP) has been demonstrated to induce many cellular effects, including those that result in platelet aggregation, cell proliferation, cell morphology, tumor cell invasion, endothelial cell and leukocyte chemotaxis, endothelial cell in vitro angiogenesis, and lymphocyte trafficking. SlP receptors are therefore good targets for a wide variety of therapeutic applications such as tumor

15 growth inhibition, vascular disease, and autoimmune diseases. SlP signals cells in part via a set of G protein-coupled receptors named SlPi or SlPl, SIP₂ or S1P2, SIP3 or S1P3, SlP₄ Or S1P4, and SlP₅ or S1P5 (formerly called EDG-I, EDG-5, EDG-3, EDG-6, and EDG-8, respectively). [0003] SlP is important in the entire human body as it is also a major regulator of

20 the vascular and immune systems. In the vascular system, SlP regulates angiogenesis, vascular stability, and permeability. In the immune system, SlP is recognized as a major regulator of trafficking of T- and B-cells. SlP interaction with its receptor SlPi is needed for the egress of immune cells from the lymphoid organs (such as thymus and lymph nodes) into the lymphatic vessels. Therefore, modulation

25 of SlP receptors was shown to be critical for immunomodulation, and SlP receptor modulators are novel immunosuppressive agents.

The SlPi receptor is expressed in a number of tissues. It is the predominant family member expressed on lymphocytes and plays an important role in lymphocyte trafficking. Downregulation of the SlPi receptor disrupts lymphocyte

30 migration and homing to various tissues. This results in sequestration of the lymphocytes in lymph organs thereby decreasing the number of circulating lymphocytes that are capable of migration to the affected tissues. Thus, development of an SlPi receptor agent that suppresses lymphocyte migration to the target sites associated with autoimmune and aberrant inflammatory processes could be efficacious in a number of autoimmune and inflammatory disease states. [0005] Among the five SlP receptors, SlPi has a widespread distribution and is highly abundant on endothelial cells where it works in concert with S IP3 to regulate cell migration, differentiation, and barrier function. Inhibition of lymphocyte recirculation by non-selective SlP receptor modulation produces clinical immunosuppression preventing transplant rejection, but such modulation also results in transient bradycardia. Studies have shown that SlPi activity is significantly correlated with depletion of circulating lymphocytes. In contrast, SIP₃ receptor agonism is not required for efficacy. Instead, SIP3 activity plays a significant role in the observed acute toxicity of nonselective SlP receptor agonists, resulting in the undesirable cardiovascular effects, such as bradycardia and hypertension. (See, e.g., Hale et al, Bioorg. Med. Chem. Lett., 14:3501 (2004); Sanna et al, J. Biol. Chem., 279: 13839 (2004); Anliker et al., J. Biol. Chem., 279:20555 (2004); Mandala et al., J. Pharmacol. Exp. Ther., 309:758 (2004).)

An example of an SlPi agonist is FTY720. This immunosuppressive compound FTY720 (JPI 1080026-A) has been shown to reduce circulating lymphocytes in animals and humans, and to have disease modulating activity in animal models of organ rejection and immune disorders. The use of FTY720 in humans has been effective in reducing the rate of organ rejection in human renal transplantation and increasing the remission rates in relapsing remitting multiple sclerosis (see Brinkman et al., J. Biol. Chem., 277:21453 (2002); Mandala et al., Science, 296:346 (2002); Fujino et al., J. Pharmacol, and Exp. Ther., 305:45658 (2003); Brinkman et al., Am. J. Transplant, 4: 1019 (2004); Webb et al., J.

Neuroimmunol, 153: 108 (2004); Morris et al., Eur. J. Immunol, 35:3570 (2005); Chiba, Pharmacology & Therapeutics, 108:308 (2005); Kahan et al., Transplantation, 76: 1079 (2003); and Kappos et al., N. Engl. J. Med, 335: 1124 (2006)). Subsequent to its discovery, it has been established that FTY720 is a prodrug, which is phosphorylated in vivo by sphingosine kinases to a more biologically active agent that has agonist activity at the SlPi, SIP3, SlP₄, and SIP5 receptors. It is this activity on the SlP family of receptors that is largely responsible for the pharmacological effects of FTY720 in animals and humans.

Clinical studies have demonstrated that treatment with FTY720 results in bradycardia in the first 24 hours of treatment (Kappos et al., N. Engl. J. Med., 335: 1124 (2006)). The observed bradycardia is commonly thought to be due to agonism at the SIP₃ receptor. This conclusion is based on a number of cell based and animal experiments. These include the use of SIP3 knockout animals which, unlike wild type mice, do not demonstrate bradycardia following FTY720 administration and the use of SlPi selective compounds. (Hale et al., Bioorg. Med. Chem. Lett., 14:3501 (2004); Sanna et al., J. Biol. Chem., 279: 13839 (2004); and Koyrakh et al., Am. J. Transplant., 5:529 (2005)).

The following applications have described compounds as SlPi agonists: WO 03/061567 (U.S. Publication No. 2005/0070506), WO 03/062248 (U.S. Patent No. 7,351,725), WO 03/062252 (U.S. Publication No. 2005/0033055), WO 03/073986 (U.S. Patent No. 7,309,721), WO 03/105771, WO 05/058848, WO

06/047195, WO 06/100633, WO 06/115188, WO 06/131336, WO 2007/024922, WO 07/116866, WO 08/023783 (U.S. Publication No. 2008/0200535), and WO 08/074820. Also see Hale et al., J. Med. Chem., 47:6662 (2004). [0009] There still remains a need for compounds useful as SlPi agonists and yet having selectivity over Sl P3.

BMS 520

Paper

Journal of Medicinal Chemistry (2016), 59(6), 2820-2840

Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P₁): Discovery and SAR of a Novel Isoxazole Based Series

Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States

J. Med. Chem., 2016, 59 (6), pp 2820–2840

DOI: 10.1021/acs.jmedchem.6b00089

Publication Date (Web): February 28, 2016

*Phone: 609-252-6778. E-mail: scott.watterson@bms.com.

Abstract

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P_1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P₁, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P₁. Isoxazole 6d demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.

SEE…..http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00089

PAPER

This article reports an efficient scale-up synthesis of 1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (BMS-520), a potent and selective isoxazole-containing S1P₁ receptor agonist. This process features a highly regioselective cycloaddition leading to a key intermediate, ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate, a chemo-selective hydrolysis of its regioisomers, as well as an improved method for 1,2,4-oxadiazole formation, relative to the original synthesis. The improved process was applied to the preparation of multiple batches of BMS-520 for preclinical toxicological studies.

An Efficient Scale-Up Synthesis of BMS-520, a Potent and Selective Isoxazole-Containing S1P₁ Receptor Agonist

Xiaoping Hou^*, Juliang Zhu, Bang-Chi Chen, Scott H. Watterson, William J. Pitts, Alaric J. Dyckman, Percy H. Carter, Arvind Mathur, and Huiping Zhang

Discovery Chemistry, Bristol-Myers Squibb Research and Development, Route 206 and Provinceline Road, Princeton, New Jersey 08543, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00112

Publication Date (Web): May 05, 2016

*xiaoping.hou@bms.com.

SEE……http://pubs.acs.org/doi/abs/10.1021/acs.oprd.6b00112

.HPLC purity 99.8%; t_R= 7.62 min (method A); 99.9%; t_R = 8.45 min (method B);

LCMS (ESI) m/z calcd for C₂₃H₁₇F₃N₄O₄ [M + H]⁺ 445.2. Found: 471.3.

¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.20–3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J = 8.25 Hz, 2H), 7.60–7.70 (m, 5H), and 8.06 (d, J = 7.70 Hz, 2H).

Anal. Calcd for C₂₃H₁₇N₄O₄F₃, 0.44% water: C, 58.42; H, 3.70; N, 11.83. Found: C, 58.52; H, 3.43; N, 11.86.

PATENT

WO 2010085581

https://www.google.com/patents/WO2010085581A1?cl=en

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Example 1

l-(4-(5-(3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol-3- yl)benzyl)azetidine-3-carboxylic acid

1-A. 4,4,4-Trifluorobut-2-yn-l-ol

To a solution of diisopropylamine (24.7 mL, 176 mmol) in ether (100 mL) at -78 ⁰C was added a 1OM solution of butyllithium in ether (17.6 mL, 176 mmol) over 5 min. After 10 min. at -78°C, 2-bromo-3,3,3-trifluoroprop-l-ene (14.0 g, 80 mmol) was added to the pale yellow solution. After an additional 10 min., paraformaldehyde (2.40 g, 80 mmol) was added, the dry-ice bath was removed, and the reaction mixture was stirred at room temperature overnight. As the reaction mixture approached room temperature, it became dark in color. The reaction was quenched with a IN aqueous solution of hydrochloric acid (100 mL), diluted with ether (500 mL), washed with a IN aqueous solution of hydrochloric acid (2 x 100 mL), washed with brine 100 mL, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a dark liquid which was distilled under low-vacuum (-50 Torr, ~50 ⁰C) to give 4,4,4-trifluorobut-2-yn-l-ol (7.1 g, 57.2 mmol, 72 % yield) as a pale yellow liquid. ¹H NMR (500 MHz, CDCl₃) δ ppm 2.31 (br. s., IH) and 4.38 – 4.42 (m, 2H).An Alternative Preparation of 1 -A: 4,4,4-Trifluorobut-2-yn- 1 -ol

-CF, (1-A) [00117] To an ether (pre-dried over magnesium sulfate) solution of phenanthroline (2.16 mg, 0.012 mmol) (indicator) at -78°C under nitrogen was added a 2M solution of n-butyl lithium in pentane. An orange color immediately appeared. Trifluoromethylacetylene gas was bubbled through the solution at -78°C. After ~4 min. of gas introduction, the orange color almost completely disappeared, the reaction solution became cloudy (due to some precipitation), and a pale light orange color persisted. Paraformaldehyde was added, and the dry ice/isopropanol bath was removed after 5 min. and replaced with a 0⁰C ice-bath. Stirring was continued for 45 min., the ice bath was removed, and stirring was continued for an additional 1.25 h. The reaction flask was immersed in a 0⁰C ice bath, and a saturated aqueous solution of ammonium chloride (20.0 mL) was added. The layers were separated, and the organic layer was washed with water (2x), washed with brine, and dried over anhydrous sodium sulfate. Concentration under low-vacuum (~50 Torr) without heat afforded a dark brown liquid which was purified by vacuum distillation (~50 Torr, -50 ⁰C) to give 4,4,4-trifluorobut-2-yn-l-ol (7.1 g, 57.2 mmol, 72 % yield) as a colorless liquid.

1-B. N-Hydroxybenzimidoyl chloride

This compound was prepared according to the method of Liu, K.C. et al, J. Org. Chem., 45:3916-1918 (1980).To a colorless, homogeneous solution of (E)-benzaldehyde oxime (24.4 g, 201 mmol) in N,N-dimethylformamide (60 mL) at room temperature was added N- chlorosuccinimide (26.9 g, 201 mmol) portion-wise over 30 min. During each addition, the reaction mixture would turn yellow and then gradually return to near colorlessness. Additionally, an exotherm was noted with each portion added to ensure that the reaction initiated after the addition of N-chlorosuccinimide. An ice bath was available, if required, to cool the exotherm. After the addition was complete, the homogeneous reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 250 mL of water and extracted with ether (3 x 100 mL). The organic layers were combined, washed with water (2 x 100 mL), washed with a 10% aqueous solution of lithium chloride (2 x 100 mL), and washed with brine (100 mL). The aqueous layers were back extracted with ether (100 mL), and the combined organic layers (400 mL) were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded (Z)-N-hydroxybenzimidoyl chloride (30.84 g, 198 mmol, 98 % yield) as a fluffy, pale yellow solid. The product had an HPLC ret. time = 1.57 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 155.8. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 7.30 – 7.64 (m, 3H), 7.73 – 7.87 (m, 2H), and 12.42 (s, IH).

l-C. 3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

To a pale yellow, homogeneous mixture of N-hydroxybenzimidoyl chloride (5.50 g, 35.4 mmol) and 4,4,4-trifluorobut-2-yn-l-ol (5.46 g, 39.6 mmol) in dichloroethane (85 mL) in a 250 mL round bottom flask at 70⁰C was added triethylamine (9.85 mL, 70.7 mmol) in 22 mL of dichloroethane over 2.5 h via an addition funnel (the first -50% over 2 h and the remaining 50% over 0.5 h). After the addition was complete, the reaction mixture was complete by HPLC (total time at 70⁰C was 3 h). The reaction mixture was stirred at room temperature overnight. [00121] The reaction mixture was diluted with dichloromethane (100 mL), washed with water (100 mL), and the organic layer was collected. The aqueous layer was extracted with dichloromethane (2 x 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. Analysis indicated that the product mixture was composed of a 86: 14 mixture of the desired regioisomer (1-C), (3-phenyl-4-(trifluoromethyl)isoxazol-5- yl)methanol, and the undesired regioisomer, (3-phenyl-5-(trifluoromethyl)isoxazol-4- yl)methanol. The mixture was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1% to pack and load – 5% – 9% – 12%) to afford (3- phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (5.34 g, 21.96 mmol, 62.1 % yield) as a pale yellow oil. The compound had an HPLC ret. time = 1.91 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ =244.2. ¹H NMR (500 MHz, CDCl₃) δ ppm 2.21 (br. s., IH), 4.97 (s, 2H), 7.47 – 7.56 (m, 3H), and 7.65 (d, J=6.60 Hz, 2H).

1-D. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid

Preparation of Jones’ Reagent

To an orange, homogeneous solution of chromium trioxide (12.4 g, 0.123 mol) in water (88.4 mL) at 0⁰C was added sulfuric acid (10.8 mL) dropwise via addition funnel over 30 min. with stirring. The addition funnel was rinsed with water

(1 mL) to give 1.23 M solution of Jones’ Reagent (0.123 mol of reagent in 100 mL of solvent).

To a solution of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

(5.24 g, 21.6 mmol) in acetone (75 mL) at room temperature (immersed in a water bath) was added Jones’ Reagent (43.8 mL, 53.9 mmol) via addition funnel slowly over 1.5 h. The dark reaction mixture was stirred at room temperature overnight. By HPLC, the reaction was 93% complete. An additional 0.5 equivalents (9 mL) of the Jones’ Reagent was added. After 1 h, the reaction was 95% complete. After an additional 3h, the reaction was 96% complete. An additional 0.5 equivalents (9 mL) of the Jones’ Reagent was added. The reaction mixture was stirred for an additional 2.5 h. By HPLC, the reaction was 97% complete. Isopropyl alcohol (6 mL) was added, and the mixture was stirred for 90 min, resulting in a dark green precipitate. The mixture was diluted with ether (600 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite (5 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with ether (2 x 100 mL). By HPLC, there was no additional product in the aqueous layer. The combined organic layers were washed with water (100 mL), washed with a saturated aqueous solution of brine (100 mL), and dried over anhydrous sodium sulfate. The aqueous layer was back-extracted with ether (100 mL), and the organic layer was added to the previous organic layers. The solution was concentration under reduced pressure to give 3-phenyl-4-

(trifluoromethyl)isoxazole-5-carboxylic acid as an off-white solid. The solid was diluted with dichloromethane (200 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid (3.84 g, 14.93 mmol, 69.3 % yield) as a pale yellow solid. The product was 96% pure by HPLC with a ret. time = 1.60 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.2. [00124] The sodium hydrogen sulfite aqueous layer still contained a significant amount of product. The brine layer contained no additional product and was discarded. The aqueous layer was saturated with sodium chloride, the pH was adjusted to -3.5, and the solution was extracted with ether (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to afford additional 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (1.12 g, 4.36 mmol, 20.21 % yield) as a white solid. The product was >99% pure by HPLC with a ret. time = 1.60 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.1. ¹H NMR (500 MHz, DMSO-(I₆) δ ppm 7.55 – 7.63 (m, 5H). The products were combined to give 4.96 g (90% yield) of 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid.

An Alternative Preparation of 1-D: 3 -Phenyl -4-(trifluoromethyl)isoxazole-5- carboxylic acid starting with (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

A mixture of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (2.1 g, 8.64 mmol), TEMPO (0.094 g, 0.604 mmol), and a sodium phosphate buffer (0.67M) (32.2 mL, 21.59 mmol) was heated to 35°C. A solution of sodium phosphate buffer (40 mL, pH -6.5) consisting of a 1: 1 solution OfNaH₂PO₄ (20 mL, 0.67M) and Na₂HPO₄ (20 mL, 0.67M) was prepared in acetonitrile (30 mL) was prepared prior to use. Solutions of sodium chlorite (3.91 g, 34.5 mmol) in water (4.5 mL) and bleach (4.3 mL, 6% wt.) were added simultaneously over 40min. The reaction was monitored by HPLC, and after 2 h, -30% of the starting material remained. After 6 h, 10% remained. Additional bleach (100 μL) was added, and the reaction mixture was left at room temperature overnight. [00127] Additional bleach (100 μL) was added. The resulting mixture was allowed to stir at 35°C for additional 2 h. HPLC indicated complete conversion. The reaction was quenched by the slow addition of a solution of sodium sulfite (2.07 mL, 43.2 mmol) in water (90 mL) at 0⁰C, resulting in the disappearance of the brown reaction color. The solvent was removed under reduced pressure, and the remaining aqueous residue was extracted with ethyl acetate (3 x 40 mL). The organic layers were combined, washed with water (8 mL), washed with brine (8 mL), and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 3 -phenyl – 4-(trifluoromethyl)isoxazole-5-carboxylic acid (2.2 g, 8.55 mmol, 99 % yield) as a pale yellow solid. An alternative procedure for the for the preparation of 3-phenyl-4-(trifluoromethyl) isoxazole-5-carboxylic acid starting with 4,4,4-trifluorobut-2ynoate (1-D)

Alt.1 -D- 1. Ethyl 3 -phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate

To a pale yellow mixture of (Z)-N-hydroxybenzimidoyl chloride (1.04 g, 6.68 mmol) and ethyl 4,4,4-trifluorobut-2-ynoate (1.238 g, 7.45 mmol) in diethyl ether (20 mL) at room temperature was added triethylamine (1.86 mL, 13.4 mmol) over 15 min., resulting in a precipitant. After the addition was complete, the pale yellow slurry was stirred at room temperature over a weekend. The heterogeneous reaction mixture was filtered under reduced pressure to remove the triethylamine hydrochloride salt, and the filtrate was concentrated to give the product mixture as a dark yellow, viscous oil (2.03 g). By HPLC, the reaction mixture was composed of a mixture of the desired regioisomer, ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5- carboxylate, and the undesired regioisomer, ethyl 3-phenyl-5- (trifluoromethyl)isoxazole-4-carboxylate, in an approximately 15:85 ratio. The compound mixture was dissolved in hexane and sonicated for 5 min. The hexane was decanted off, and the dark red, oily residue was found to have only trace product by HPLC. The hexane was removed under reduced pressure, and the residue (1.89 g) was purified by preparative HPLC. The desired fractions containing ethyl 3-phenyl- 4-(trifluoromethyl)isoxazole-5-carboxylate were concentrated, and the residue was diluted with dichloromethane, washed with a saturated aqueous solution of sodium bicarbonate, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded ethyl 3-phenyl-4-(trifluoromethyl) isoxazole-5-carboxylate (0.087 g, 0.305 mmol, 4.6 % yield) as a pale yellow solid. The compound had an HPLC ret. time = 2.88 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.46 (t, J=7.15 Hz, 3H), 4.53 (q, J=7.03 Hz, 2H), 7.48 – 7.55 (m, 3H), and 7.58 (d, J=7.53 Hz, 2H).

An Alternative Preparation of 1-D-l : Ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5- carboxylic acid starting with ethyl 4,4,4-trifluorobut-2-enoate

1-D-l. Ethyl 2,3-dibromo-4,4,4-trifluorobutanoate

Br L _/COOEt

Br (1-D-l) [00129] Bromine (18.4 mL, 357 mmol) was added dropwise over 30 minutes to a solution of (E)-ethyl 4,4,4-trifluorobut-2-enoate (50 g, 297 mmol) in carbon tetrachloride (50 mL) at room temperature under nitrogen. The resulting dark red solution was refluxed for 4 hours. Additional bromine (2ml) was added and heating was continued until the HPLC analysis showed that the starting material had been consumed. The reaction mixture was concentrated under reduced pressure to give light brown oil which used in the next step without purification. HPLC (XBridge 5μ Cl 8 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0-100 % B over 4 minutes): 2.96 and 3.19 minutes.

l-D-2. (Z/E)-Ethyl 2-bromo-4,4,4-trifluorobut-2-enoate

,COOEt

F₃C

Br (l-D-2)

To a solution of ethyl 2,3-dibromo-4,4,4-trifluorobutanoate (1-B-l) in hexane (200 mL) cooled to 0⁰C was added triethylamine (49.7 ml, 357mmol) drop- wise over 35 minutes, during which time a white precipitate formed. The reaction mixture was stirred for an additional 2 hours until LC indicated complete conversion. The solid was filtered and rinsed with hexane (3 x 5OmL), and the filtrate was concentrated and passed through a short silica gel pad eluting with 10% ethyl acetate/hexane to give (Z/E)-ethyl 2-bromo-4,4,4-trifluorobut-2-enoate (65.5 g, 265mmol, 89 % yield for two steps) as a colorless oil. Alternatively, the crude product can be purified by distillation (85 ⁰C / -60 mmHg). ¹H NMR (CDCl₃, 400 MHz) 5 7.41 (q, IH, J= 7.28 Hz), 4.35 (q, 2H, J= 7.11 Hz), 1.38 (t, 3H, J= 7.15 Hz); HPLC (XBridge 5μ Cl 8 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0- 100 % B over 4 minutes): 3.09 minutes.

1-D-l. Ethyl 3 -phenyl -4-(trifluoromethyl)isoxazole-5-carboxylate

(Z/E)-Ethyl 2-bromo-4,4,4-trifluorobut-2-enoate, l-D-3, (39.7 g, 161 mmol) and N-hydroxybenzimidoyl chloride (30 g, 193mmol) were dissolved in ethyl acetate (15OmL). Indium (III) chloride (8.89 g, 40.2mmol) was added and the resulting mixture stirred for 60 minutes at RT under N₂. Potassium hydrogen carbonate (32.2 g, 321mmol) was added to the reaction mixture which was allowed to stir overnight for 14 hours at RT. The solvent was removed in vacuo. The residue was re-suspended in 30OmL hexane and stirred for lOmiutes then filtered. The filter cake was washed with hexane (3X3 OmL) and the combined filtrate was concentrated in vacuo to give crude product, which was further purified with flash chromatography to generate 33g product (72%) as light yellowish oil as a mixture of the desired isomer 1-D-l and undesired isomer 1-D-la in a ratio of -30/1. MS m/e 286.06(M+H⁺); ¹H NMR (CDCl₃, 400 MHz) δ 7.56 (m, 5H), 4.53 (q, 2H, J= 7.3 Hz), 1.46 (t, 3H, J= 7.2 Hz); HPLC (XBridge 5μ C18 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0-100 % B over 4 minutes): 3.57 minutes.

Alt.1-D. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid, lithium salt

A mixture of ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate, 1-D-l, (0.085 g, 0.298 mmol) and lithium hydroxide hydrate (0.013 g, 0.298 mmol) in methanol (2.0 mL) and water (1.0 mL) was stirred at room temperature overnight. The reaction mixture was concentrated to dryness to give 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid, lithium salt (0.079 g, 0.299 mmol, 100 % yield) as a pale yellow solid. The compound had an HPLC ret. time = 1.72 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.0. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.49 – 7.57 (m, 3H) and 7.58 – 7.62 (m, 2H).1-E. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride

To a mixture of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (3.00 g, 11.7 mmol) and pyridine (1.132 mL, 14.0 mmol) in dichloromethane (100 mL) at room temperature was added 2,4,6-trifluoro-l,3,5-triazine (cyanuric fluoride) (1.18 mL, 14.0 mmol). The reaction mixture was stirred at room temperature overnight, diluted with dichloromethane (300 mL), washed with an ice-cold solution of 0.5N aqueous hydrochloric acid (2 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane (200 mL), and the combined organic layers were dried anhydrous sodium sulfate and concentrated to afford 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (2.91 g, 11.2 mmol, 96 % yield) as a yellow, viscous oil. The product was found to react readily with methanol and on analysis was characterized as the methyl ester, which had an HPLC ret. time = 2.56 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 272.3 (methyl ester).1-F. tert-Butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol- 3-yl)-benzyl)azetidine-3-carboxylate

A suspension of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (2.91 g, 11.2 mmol), (Z)-tert-butyl 1-(4-(N’- hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (Int. l, 3.43 g, 11.2 mmol), and Hunig’s Base (2.55 mL, 14.6 mmol) in acetonitrile (20 mL) was stirred at room temperature over the weekend. The reaction mixture had completely solidified (pinkish-tan in color), but was judged complete by HPLC and LCMS. The reaction mixture was partitioned between a saturated aqueous of sodium bicarbonate (150 mL) and dichloromethane (150 mL). The aqueous layer was extracted with dichloromethane (2 x 100 mL), and the combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a tan solid which was purified by flash silica gel chromatography using a mixture of ethyl acetate in hexane (0-50%) to afford tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl) isoxazol-5-yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (4.60 g; 78%) as a white, crystalline solid. The material was suspended in methanol (-75 mL) and was sonicated for 5 minutes. The MeOH was removed under reduce pressure, and the residue was re-suspended in methanol (-50 mL) with sonication. Vacuum filtration and drying afforded tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)- l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (4.04 g, 7.67 mmol, 68 % yield) as a white, crystalline solid. The methanol filtrate was concentrated to afford additional tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)- 1,2,4- oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (570 mg; 10%) as a slightly off- white solid. The compound had an HPLC retention time = 3.12 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ =527.1. ¹H NMR (500 MHz, CDCl₃) δ ppm 1.47 (s, 9H) 3.28 – 3.37 (m, 3H), 3.60 (br. s., 2H), 3.74 (br. s., 2H), 7.49 (d, J=7.70 Hz, 2H), 7.53 – 7.62 (m, 3H), 7.69 (d, J=7.15 Hz, 2H), and 8.16 (d, J=7.70 Hz, 2H). 1. Preparation of l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4- oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid

A mixture of tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5- yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (6.12 g, 11.6 mmol) and trifluoroacetic acid (50.1 mL, 651 mmol) was stirred at room temperature for 1.5 h. By HPLC, the deprotection appeared to be complete after 1 h. The TFA was removed under reduced pressure, and the oily residue was diluted with water (100 mL) and sonicated for 5 min. The resulting suspension was stirred for an additional 10 min until a consistent white suspension was observed. A IN aqueous solution of sodium hydroxide was added portion-wise until the pH was ~4.5 (42 mL of IN NaOH). Over time, the pH drifted back down to 3-4, and additional IN aqueous sodium hydroxide had to be added. The suspension was stirred overnight at room temperature. Several drops of IN aqueous sodium hydroxide were added to re-adjust the pH to 4.5, and after 60 min., the pH appeared to be stable. The solid was collected by vacuum filtration, washed with water several times, and dried under reduced pressure for 5 h. The solid was then suspended in methanol (110 mL) in a 150 mL round bottom flask and sonicated for 15 min. During the sonication, the solution became very thick. An additional 25 mL of methanol was added, and the suspension was stirred overnight. The product was collected by vacuum filtration, washed with methanol (-50 mL), and dried under reduced pressure. The solid was transferred to a 250 mL round bottom flask, re-suspended in methanol (115 mL), sonicated for 5 min., and stirred for 60 min. The solid was collected by vacuum filtration, washed with methanol (~50 mL), and dried over well under reduced pressure to give l-(4-(5-(3- phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3- carboxylic acid (5.06 g, 10.7 mmol, 92 % yield) as a crystalline, white solid. The product had an HPLC ret. time = 2.79 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 471.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.20 – 3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J=8.25 Hz, 2H), 7.60 – 7.70 (m, 5H), and 8.06 (d, J=7.70 Hz, 2H).

HPLC purity 100/99.8%, ret. time = 7.62 min. (A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95% acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = l2 min., 100% B (15 min.) was employed on a SunFire C18 3.5u 4.6 x 150 mm column. Flow rate was 2 ml/min and UV detection was set to 220/254 nm.).

HPLC purity 100/99.9%, ret. time = 8.45 min. (A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95% acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = l2 min., 100% B (15 min.) was employed on a XBridge Ph 3.5u 4.6 x 150 mm column. Flow rate was 2 ml/min and UV detection was set to 220/254 nm.).

CONSTRUCTION

Alt.1 -D- 1. Ethyl 3 -phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate

ADDITIONAL INFORMATION

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation

REFERENCES

Watterson, S. H.; Guo, J.; Spergel, S. H.; Langevine, C. L.; Moquin, R. V.; Shen, D.
R.; Yarde, M.; Cvijic, M. E.; Banas, D.; Liu, R.; Suchard, S. J.; Gillooly, K.; Taylor,
T.; Rex-Rabe, S.; Shuster, D. J.; McIntyre, K. W.; Cornelius, G.; Darienzo, C.;
Marino, A.; Balimane, P.; Warrack, B.; Saltercid, L.; McKinnon, M.; Barrish, J. C.;
Carter, P. C.; Pitts, W. J.; Xie, J.; Dyckman, D. J. J. Med. Chem. 2016, 59, 2820.

Watterson, S.H.; Guo, J.; Spergel, S.H.; et al.
Potent and selective agonists of Sphingosine-1-Phosphate 1 (S1P1): The discovery and SAR of a novel isoxazole based series
241st Am Chem Soc (ACS) Natl Meet (March 27-30, Anaheim) 2011, Abst MEDI 96

/////Potent and Selective Isoxazole-Containing S1P₁ Receptor Agonist, BMS 520, Sphingosine-1-Phosphate 1 (S1P1)

O=C(C1CN(CC2=CC=C(C3=NOC(C4=C(C(F)(F)F)C(C5=CC=CC=C5)=NO4)=N3)C=C2)C1)O

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

Uncategorized Comments Off

May 132016

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

READ……http://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.6b00107

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

Bob Milley^*^†, Radwan Kiwan^†, Gary S. Ott^†, Carlo Calacsan^†, Melissa Kachura^†, John D. Campbell^†, Holger Kanzler^‡, and Robert L. Coffman^†

^† Dynavax Technologies Corporation, 2929 Seventh Street, Suite 100, Berkeley, California 94710, United States

^‡ MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland 20878, United States

Bioconjugate Chem., Article ASAP

DOI: 10.1021/acs.bioconjchem.6b00107

Publication Date (Web): April 13, 2016

*E-mail: bmilley@dynavax.com. Phone: (510) 665-7227. Fax: (510) 848-1327.

//////Optimization, Production, Characterization, CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant, Enhanced Immunogenicity, Anthrax Protective Antigen

Solvates, Salts, and Cocrystals: A Proposal for a Feasible Classification System

Uncategorized Comments Off

May 132016

The design of pharmaceutical cocrystals has initiated widespread debate on the classification of cocrystals. Current attempts to classify multicomponent crystals suffer from ambiguity, which has led to inconsistent definitions for cocrystals and for multicomponent crystals in general. Inspired by the work of Aitipamula et al. (Cryst. Growth Des. 2012, 12, 2147–2152), we present a feasible classification system for all multicomponent crystals. The present classification enables us to analyze and classify multicomponent crystal structures present in the Cambridge Structural Database (CSD). This reveals that all seven classes proposed are relevant in terms of frequency of occurrence. Lists of CSD refcodes for all classes are provided. We identified over 5000 cocrystals in the CSD, as well as over 12 000 crystals with more than two components. This illustrates that the possibilities for alternative drug formulations can be increased significantly by considering more than two components in drug design.

READ ………….http://pubs.acs.org/doi/full/10.1021/acs.cgd.6b00200

Solvates, Salts, and Cocrystals: A Proposal for a Feasible Classification System

E. Grothe^†, H. Meekes^†, E. Vlieg^†, J. H. ter Horst^‡, and R. de Gelder^*^†

^† Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands

^‡ EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallisation, Strathclyde Institute of Pharmacy and Biomedical Sciences, Technology and Innovation Centre, University of Strathclyde, 99 George Street, Glasgow G1 1RD, United Kingdom

Cryst. Growth Des., Article ASAP

DOI: 10.1021/acs.cgd.6b00200

Publication Date (Web): April 21, 2016

*E-mail: r.degelder@science.ru.nl.

Synopsis

A classification system for multicomponent crystals is applied to the organic crystals in the Cambridge Structural Database (CSD) in an attempt to reveal the population of structures within each subclass. Seven subclasses of multicomponent crystals are presented, each illustrated by an example of an isonicotinamide crystal structure that can be found in the CSD.

//////////Solvates, Salts, Cocrystals, Proposal, Feasible Classification System

Teslaphoresis of Carbon Nanotubes

Uncategorized Comments Off

May 132016

This paper introduces Teslaphoresis, the directed motion and self-assembly of matter by a Tesla coil, and studies this electrokinetic phenomenon using single-walled carbon nanotubes (CNTs). Conventional directed self-assembly of matter using electric fields has been restricted to small scale structures, but with Teslaphoresis, we exceed this limitation by using the Tesla coil’s antenna to create a gradient high-voltage force field that projects into free space. CNTs placed within the Teslaphoretic (TEP) field polarize and self-assemble into wires that span from the nanoscale to the macroscale, the longest thus far being 15 cm. We show that the TEP field not only directs the self-assembly of long nanotube wires at remote distances (>30 cm) but can also wirelessly power nanotube-based LED circuits. Furthermore, individualized CNTs self-organize to form long parallel arrays with high fidelity alignment to the TEP field. Thus, Teslaphoresis is effective for directed self-assembly from the bottom-up to the macroscale.

SEE………..http://pubs.acs.org/doi/full/10.1021/acsnano.6b02313

Teslaphoresis of Carbon Nanotubes

^†Department of Chemistry, ^‡Department of Materials Science and NanoEngineering, ^§Smalley-Curl Institute, Rice University, 6100 Main Street, Houston, Texas 77005, United States

^∥ Department of Chemistry and Physics, University of Tennessee—Chattanooga, 615 McCallie Avenue, Chattanooga, Tennessee 37403, United States

^⊥ Department of Biomedical Engineering, Texas A&M University, 101 Bizzell Street, College Station, Texas 77843,United States

^# Second Baptist School, 6410 Woodway Drive, Houston, Texas 77057, United States

ACS Nano, 2016, 10 (4), pp 4873–4881

DOI: 10.1021/acsnano.6b02313

Publication Date (Web): April 13, 2016

*E-mail: paul.cherukuri@rice.edu.

Keywords:

dielectrophoresis, directed self-assembly, carbon nanotubes, Tesla, wireless energy

/////////

Palladium- and Nickel-Catalyzed Amination of Aryl Fluorosulfonates

spectroscopy, SYNTHESIS Comments Off

May 132016

Examples of the palladium- and nickel-catalyzed amination of aryl fluorosulfonates with aromatic and alkyl amines are described. Aniline is coupled to a diverse series of aryl fluorosulfonates catalyzed by the combination of CpPd(cinammyl) and Xantphos, and the relative reactivity of aryl fluorosulfonates to undergo Pd-catalyzed amination was compared with other common aryl electrophiles. In addition, we report the direct amination of a phenol by in situ formation of an aryl fluorosulfonate by reaction with sulfuryl fluoride and base followed by subsequent amination to form a new C–N bond. Finally, we report examples of the nickel-catalyzed amination of aryl fluorosulfonates catalyzed by the combination of Ni(COD)₂ and DPPF in the presence of MeCN. The high reactivity of the aryl fluorosulfonate electrophile with generic palladium and nickel catalyst systems, combined with its simple preparation from sulfuryl fluoride will enable commercial amination reactions of abundant phenolic raw materials.

SEE…….http://pubs.acs.org/doi/full/10.1021/acscatal.6b00865