CARBAZITAXEL

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Mar 202014

Cabazitaxel

For treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen.

4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1-hydroxy-7β,10β-dimethoxy-9-oxotax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenyl-propionate

(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-(Acetyloxy)-15-{[(2R,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxy-3-phenylpropanoyl]oxy}-1-hydroxy-9,12-dimethoxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0^3,10.0^4,7]heptadec-13-ene-2-yl benzoate

183133-96-2

Jevtana, Taxoid XRP6258, Cabazitaxelum, 183133-96-2, Xrp6258, CHEBI:63584, XRP-6258, TXD 258, XRP 6258

Molecular Formula: C₄₅H₅₇NO₁₄ Molecular Weight: 835.93238

EMA:Link, US FDA:link

Cabazitaxel is prepared by semi-synthesis from 10-deacetylbaccatin III (10-DAB) which is extracted from yew tree needles. The chemical name of cabazitaxel is (2α,5β,7β,10β,13α)-4-acetoxy-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-7,10-dimethoxy-9-oxo-5,20-epoxy-tax-11-en-2-yl benzoate and is marketed as a 1:1 acetone solvate (propan-2-one),

Cabazitaxel is an anti-neoplastic used with the steroid medicine prednisone. Cabazitaxel is used to treat people with prostate cancer that has progressed despite treatment with docetaxel. Cabazitaxel is prepared by semi-synthesis with a precursor extracted from yew needles (10-deacetylbaccatin III). It was approved by the U.S. Food and Drug Administration (FDA) on June 17, 2010.

Cabazitaxel (previously XRP-6258, trade name Jevtana) is a semi-synthetic derivative of a natural taxoid.^[1] It was developed by Sanofi-Aventis and was approved by the U.S. Food and Drug Administration (FDA) for the treatment of hormone-refractory prostate cancer on June 17, 2010. It is a microtubule inhibitor, and the fourth taxane to be approved as a cancer therapy.^[2]

Nagesh Palepu, “CABAZITAXEL FORMULATIONS AND METHODS OF PREPARING THEREOF.” U.S. Patent US20120065255, issued March 15, 2012.

US20120065255 Link out

Cabazitaxel in combination with prednisone is a treatment option for hormone-refractory prostate cancer following docetaxel-based treatment.

Clinical trials

In a phase III trial with 755 men for the treatment of castration-resistant prostate cancer, median survival was 15.1 months for patients receiving cabazitaxel versus 12.7 months for patients receiving mitoxantrone. Cabazitaxel was associated with more grade 3–4 neutropenia (81.7%) than mitoxantrone (58%).^[3]

United States	5438072	2010-06-17	exp 2013-11-22
United States	5698582	2010-06-17	2012-07-03
United States	5847170	2010-06-17	2016-03-26
United States	6331635	2010-06-17	2016-03-26
United States	6372780	2010-06-17	2016-03-26
United States	6387946	2010-06-17	2016-03-26
United States	7241907	2010-06-17	2025-12-10

JEVTANA (cabazitaxel) is an antineoplastic agent belonging to the taxane class. It is prepared by semi-synthesis with a precursor extracted from yew needles.

The chemical name of cabazitaxel is (2α,5β,7β,10β,13α)-4-acetoxy-13-({(2R,3S)-3[(tertbutoxycarbonyl) amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-7,10-dimethoxy-9oxo-5,20-epoxytax-11-en-2-yl benzoate – propan-2-one(1:1).

Cabazitaxel has the following structural formula:

JEVTANA (cabazitaxel) Structural Formula Illustration

Cabazitaxel is a white to almost-white powder with a molecular formula of C₄₅H₅₇NO₁₄^•C₃H₆O and a molecular weight of 894.01 (for the acetone solvate) / 835.93 (for the solvent free). It is lipophilic, practically insoluble in water and soluble in alcohol.

JEVTANA (cabazitaxel) Injection 60 mg/1.5 mL is a sterile, non-pyrogenic, clear yellow to brownish-yellow viscous solution and is available in single-use vials containing 60 mg cabazitaxel (anhydrous and solvent free) and 1.56 g polysorbate 80. Each mL contains 40 mg cabazitaxel (anhydrous) and 1.04 g polysorbate 80.

DILUENT for JEVTANA is a clear, colorless, sterile, and non-pyrogenic solution containing 13% (w/w) ethanol in water for injection, approximately 5.7 mL.

JEVTANA requires two dilutions prior to intravenous infusion. JEVTANA injection should be diluted only with the supplied DILUENT for JEVTANA, followed by dilution in either 0.9% sodium chloride solution or 5% dextrose solution.

The taxane family of terpenes has received much attention in the scientific and medical community, because members of this family have demonstrated broad spectrum of anti-leukemic and tumor-inhibitory activity. A well-known member of this family is paclitaxel (Taxol®).

Paclitaxel (Taxol) Paclitaxel was first isolated from the bark of the pacific yew tree (Taxus brevifolia) in 1971 , and has proved to be a potent natural anti-cancer agent. To date, paclitaxel has been found to have activity against different forms of leukemia and against solid tumors in the breast, ovary, brain, and lung in humans.

As will be appreciated, this beneficial activity has stimulated an intense research effort over recent years with a view to identifying other taxanes having similar or improved properties, and with a view to developing synthetic pathways for making these taxanes, such as paclitaxel.

This research effort led to the discovery of a synthetic analogue of paclitaxel, namely, docetaxel (also known as Taxotere®). As disclosed in U.S. Patent No. 4,814,470, docetaxel has been found to have a very good anti-tumour activity and better bioavailability than paclitaxel. Docetaxel is similar in structure to paclitaxel, having t- butoxycarbonyl instead of benzoyl on the amino group at the 3′ position, and a hydroxy group instead of the acetoxy group at the C-10 position.

As will be appreciated, taxanes are structurally complicated molecules, and the development of commercially viable synthetic methods to make taxanes has been a challenge. A number of semi-synthetic pathways have been developed over the years, which typically begin with the isolation and purification of a naturally occurring starting material, which can be converted to a specific taxane derivative of interest. Cabazitaxel (I) is an anti-tumor drug which belongs to the taxol family. It differs from docetaxel in that it has methoxy groups at positions 7 and 10 of the molecule, as opposed to the hydroxyl groups at equivalent positions in docetaxel. Cabazitaxel is obtained by semi-synthesis from an extract of Chinese yew (Taxus mairei). It is understood that cabazitaxel can be obtained via semi-synthesis from other taxus species including T.candensis, T.baccatta, T.chinensis, T. mairei etc.

Cabazitaxel is a semi-synthetic derivative of the natural taxoid 0-deacetylbaccatin III (10-DAB) with potentially unique antineoplastic activity for a variety of tumors.

Cabazitaxel binds to and stabilizes tubulin, resulting in the inhibition of microtubule depolymerization and cell division, cell cycle arrest in the G2/M phase, and the inhibition of tumor cell proliferation. This drug is a microtubule depolymerization inhibitor, which can penetrate blood brain barrier (BBB).

Cabazitaxel was recently approved by the US Federal Drug Administration (FDA) for the treatment of docetaxel resistant hormone refractory prostate cancer. It has been developed by Sanofi-Aventis under the trade name of Jevtana. The CAS number for the compound is 183133-96-2. A synonym is dimethoxydocetaxel. The compound is also known as RPR-1 16258A; XRP6258; TXD 258; and axoid XRP6258.

The free base form of cabazitaxel has the chemical name

(2aR,4S,4aS,6R,9S, 1 1 S,12S,12aR, 12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert- butoxycarbonyl)amino)-2-hydroxy-3-phenylpropanoyl)oxy)-11-hydroxy-4,6-dimethoxy- 4a,8, 13, 13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9, 10, 11 , 12, 12a, 12b-dodecahydro-1 H- 7, 1 1-methanocyclodeca[3,4]benzo[1 ,2-b]oxet-12-yl benzoate. In a first part of this description, taxel drugs including paclitaxel (taxol), docetaxel (taxotere) and cabazitaxel may be prepared starting from 10-deacetylbaccatin (known as 10-DAB) derived from Taxus plants, via semi-synthesis. Furthermore, the same inventive methodologies can be used to semi-synthesize cabazitaxel starting from 9- dihydro-13-acetylbaccatin III (9-DHB).

Patent numbers CN1213042C, CN152870, CN1179716 and CN1179775 disclose methods to prepare cabazitaxel from 10-DAB (herein compound II).

10-DAB (II)

A typical prior art synthesis route is as follows:

OCOCH₃

OCOC₆H₅

The method above which synthesizes cabazitaxel has many synthetic steps, a very low overall yield and high price.

There is therefore a need in the art to develop new methods to synthesize cabazitaxel and its intermediates to improve the yield of cabazitaxel, simplify the methodology and optimize the synthetic technology.

Cabazitaxel, chemically known as 4-acetoxy-2α-benzoyloxy-5β,20-epoxy-1-hydroxy-7β,10β-dimethoxy-9-oxotax-11-en-13α-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenyl-propionate, is represented by formula (I).

It is a microtubule inhibitor, indicated in combination with prednisone for treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing treatment regimen, under the trade name Jevtana®.

Cabazitaxel is known from U.S. Pat. No. 5,847,170. Process for preparation of Cabazitaxel as described in U.S. Pat. No. 5,847,170 involves column chromatography, which is cumbersome tedious and not commercially viable.

The acetone solvate of 4-acetoxy-2α-benzoyloxy-5β-20-epoxy-1-hydroxy-7β, 10β-dimethoxy-9-oxotan-11-en-13α-yl-(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionate (Form A) is formed by crystallization by using acetone and is characterized by XRD in U.S. Pat. No. 7,241,907.

U.S. 20110144362 describes anhydrous crystalline Forms B to Form F, ethanolates Form B, D, E and F and mono and dihydrate Forms of Cabazitaxel. All the anhydrous crystalline forms are prepared either by acetone solvate or ethanol solvate. Mono and dihydrate forms are formed at ambient temperature in an atmosphere containing 10 and 60% relative humidity, respectively.

Cabazitaxel (also called dimethoxy docetaxel) is a dimethyl derivative of docetaxel, which itself is semi-synthetic, and was originally developed by Rhone-Poulenc Rorer and was approved by the U.S. Food and Drug Administration (FDA) for the treatment of hormone-refractory prostate cancer on Jun. 17, 2010. Cabazitaxel is a microtubule inhibitor. The acetone solvate crystalline form of cabazitaxel and a process for its preparation is disclosed in the U.S. Pat. No. 7,241,907.

U.S. Pat. No. 5,847,170 describes cabazitaxel and its preparation methods. One of the methods described in U.S. Pat. No. 5,847,170 includes a step-wise methylation of 10-DAB (the step-wise methylation method is shown in FIG. 1) to provide the key intermediate (2αR,4S,4αS,6R,9S,11S,12S,12αR,12βS)-12β-acetoxy-9,11-dihydroxy-4,6-dimethoxy-4α,8,13,13-tetramethyl-5-oxo-2α,3,4,4α,5,6,9,10,11,12,12α,12β-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate, herein referred to as 7,10-di-O-methyl-10-DAB (XVa). The intermediate XVa is coupled with the 3-phenylisoserine side chain derivative VI to provide XVa′, which is followed by removal of the oxazolidine protecting group from the side chain of XVa′ to give cabazitaxel.

Another method described in U.S. Pat. No. 5,847,170 utilizes methylthiomethyl (MTM) ethers as shown in FIG. 2. MTM ethers can be prepared from alcohols using two common methods. One method comprises deprotonation of an alcohol with a strong base to form an alkoxide followed by alkylation of the alkoxide with a methylthiomethyl halide. This approach is only useful when the alcohol is stable to treatment with a strong base. 10-DAB and some of its derivatives in which C7-OH is not protected displays so instability in the presence of strong bases and epimerization of the C7-OH can occur upon contact of 10-DAB and some of its derivatives in which C7-OH is not protected with strong bases. Another method for the synthesis of MTM ethers from alcohols utilizes Ac2O and DMSO. One disadvantage of this method is that it can also lead to the oxidation of alcohols to aldehydes or ketones. For example when the synthesis of the 10-di-O-MTM derivative of 10-DAB without protecting groups at the C13 hydroxyl group is attempted undesired oxidation of the C13-OH to its corresponding ketone occurs.

U.S. Pat. No. 5,962,705 discloses a method for dialkylation of 10-DAB and its derivatives to furnish 7,10-di-O-alkyl derivatives, as shown in FIG. 3. This has been demonstrated as a one-step, one-pot reaction, however, provides the best isolated yield when potassium hydride is used at −30° C. From an industrial point of view, the use of low reaction temperature is less favorable than using ambient temperature. Furthermore the use of a strong base can cause some epimerization of the C7-OH chiral center with an associated loss of yield. Potassium hydride is a very reactive base and must be treated with great caution.

Accordingly, there is a need for an alternative processes for the preparation of cabazitaxel and its key intermediate, 7,10-di-O-methyl-10-DAB (XVa) that is short in number of synthetic steps and avoids the use of low temperatures and strong bases such as metal hydrides in the C7-O methyl ether formation step. Such a process would also be useful for the preparation of analogues of cabazitaxel wherein the C7-O and C10-O functional groups were substituted with other alkyl groups.

FIG. 1 shows the chemistry employed in the examples of U.S. Pat. No. 5,847,170.

FIG. 2 shows the chemistry employed in the examples of U.S. Pat. No. 5,847,170.

FIG. 3 shows the chemistry employed in the examples of U.S. Pat. No. 5,962,705.

FIG. 4 shows key steps of the general synthetic scheme as per Method A/A′ of the present invention for the synthesis of cabazitaxel and cabazitaxel analogues.

FIG. 5 shows key steps of the general synthetic scheme as per Method B/B′ of the present invention for the synthesis of cabazitaxel and cabazitaxel analogues.

FIG. 6 shows the general scheme for the hydrodesulfurization reaction.

FIG. 7 shows the complete synthetic route of Method A that can be used for conversion of 10-DAB to cabazitaxel.

FIG. 8 shows the complete synthetic route of Method B that can be used for conversion of 10-DAB to cabazitaxel.

FIG. 9 shows the complete synthetic route of Method A′ that can be used for conversion of 10-DAB, via XIV′, to cabazitaxel.

FIG. 10 shows the complete synthetic route of Method B′ that can be used for conversion of 10-DAB, via XVI′, to cabazitaxel.

FIG. 11 shows the synthetic relationship between two methods (A and B) used to convert 7,10-di-O-alkyl-10-DAB (XV) to cabazitaxel.

FIG. 12 shows the synthetic scheme for the preparation of XIVa.

FIG. 13 shows the synthetic scheme for the preparation of XIVb from 10-DAB.

FIG. 14 shows the synthetic scheme for the preparation of XIVb from XIVb′.

FIG. 15 shows the synthetic scheme for the preparation of XIVc.

FIG. 16 shows the synthetic scheme for the preparation of XIVa′ from XIVa.

FIG. 17 shows the synthetic scheme for the preparation of XIVa′ from XX.

FIG. 18 shows the synthetic scheme for the preparation of XIVb′.

FIG. 19 shows the synthetic scheme for the preparation of XIVc′.

FIG. 20 shows the synthetic scheme for the preparation of XVa from XIVa.

FIG. 21 shows the synthetic scheme for the preparation of XVa from XIVb.

FIG. 22 shows the synthetic scheme for the preparation of XVa from XIVc.

FIG. 23 shows the synthetic scheme for the preparation of XVa′ from XIVa.

FIG. 24 shows the synthetic scheme for the preparation of XVa′ from XIVa′.

FIG. 25 shows the synthetic scheme for the preparation of XVa′ from XIVb′.

FIG. 26 shows the synthetic scheme for the preparation of XVa′ from XIVc′.

FIG. 27 shows the synthetic scheme for the preparation of XVa′ from XVa.

FIG. 28 shows the synthesis of cabazitaxel.

FIG. 29 shows the synthesis of XVIIa.

FIG. 30 shows the synthesis of XVIIIa.

FIG. 31 shows the synthesis of XIXa.

FIG. 32 shows the synthesis of XVIa.

FIG. 33 shows the synthesis of XVa from XVIa.

see this at http://www.google.com/patents/US20130116444

………..

WO2013057260A1

Detailed description

The invention provides a new method for the preparation of cabazitaxel, one embodiment of which can be summarized as follows, showing the preparation of a protected taxane intermediate and its deprotection to taxane compounds:

OH OCOCH₃

OCOC₆H₅

This reaction is also depicted in Figure 1. The reaction of the invention reduces the number of steps and increases yield of cabazitaxel.

The deprotection methods of the invention can also be used for the preparation of paclitaxel (taxol):

The deprotection methods of the invention can also be applied to the preparation of docetaxel:

10-DAB synthetic routes

Example 12

Dissolve 100 g of 2′-THP-cabazitaxel in 1730 ml of HOAc/H₂0/THF (3:1 :1 ). under N₂ atmosphere, increase temperature to 50 degrees C and stir 4 hrs. Then cool to room temperature. Add 2L of ethyl acetate, 2 L of H₂0, stir, separate layers, wash organic layer with saturated NaHC0₃ (3 L x 2), saturated NaCI (3 L), dry with Na₂S0₄.

Concentrate to obtain white 77.8 g of cabazitaxel (yield 83%).

MS(m/z) :859(M+Na)„ _jHNMR (500MHz) δ 1.21(611, d) , 1.36(911, s) , 1.59(lH, s) , 1.64(lH,s) , 1.79(lH,m) , 1.87 (3H, s) ,2.27 (2H, m) , 2.35(3H,m) ,2.69(lH,m) ,3.30 (3H, s) ,

3.45 (3H, s) , 3.85 (2H, m) , 4.16 (1H, d) , 4.29 (1H, d) , 4.62 (1H, bs) , 4.79 (1H, s) , 5.29 (1H, m),5.42(lH, d),5.62(lH, d),6.21 (1H, t),7.2 ~ 7.4(6H, m) , 7.48 (2H, t),7.59(lH, t) , 8.11 (2H, d) ,

References

http://www.cancer.gov/drugdictionary/?CdrID=534131
“Jevtana (cabazitaxel) Injection Approved by U.S. FDA After Priority Review” (Press release). sanofi-aventis. 2010-06-17. Retrieved June 17, 2010.
“Cabazitaxel Effective for Hormone Refractory Prostate Cancer After Failure of Taxotere”.

Cabazitaxel – Official web site of manufacturer.
Cabazitaxel Prescribing Information – Official prescribing information.
U.S. National Library of Medicine: Drug Information Portal – Cabazitaxel

Patents

Patent :

Patent Number : 5438072

Country : United States

Approved : 2010-06-17

Expires : 2013-11-22

Patent :

Patent Number : 5698582

Country : United States

Approved : 2010-06-17

Expires : 2012-07-03

Patent :

Patent Number : 5847170

Country : United States

Approved : 2010-06-17

Expires : 2016-03-26

Patent :

Patent Number : 6331635

Country : United States

Approved : 2010-06-17

Expires : 2016-03-26

Patent :

Patent Number : 6372780

Country : United States

Approved : 2010-06-17

Expires : 2016-03-26

Patent :

Patent Number : 6387946

Country : United States

Approved : 2010-06-17

Expires : 2016-03-26

Patent :

Patent Number : 7241907

Country : United States

Approved : 2010-06-17

Expires : 2025-12-10

US2009069411	3-13-2009	SELF-EMULSIFYING AND SELF-MICROEMULSIFYING FORMULATIONS FOR THE ORAL ADMINISTRATION OF TAXOIDS
US2005070496	3-32-2005	Semi-solid formulations for the oral administration of taxoids
US2005025792	2-4-2005	Self-emulsifying and self-microemulsifying formulations for the oral administration of taxoids
US6403634	6-12-2002	Use of taxoid derivatives

CHEMDRAW PRO 8.0

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Mar 152014

CHEMDRAW PRO 8.0

ChemDraw is a leading chemistry molecules drawing software. ChemDraw is easy to use and best software for drawing a molecule structures and finding stereochemistry of the molecules.

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ChemDraw Pro 8.0

ChemDraw Pro 8.0 provides chemists with a rich set of easy to use tools for creating publication ready, scientifically meaningful drawings of molecules and reactions.

Free download ChemDraw Pro 8.0 full version with product key and serial number from following download link. This download link is test personally and found viruses and spam free.

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Specifications

Operating System: Windows 95/NT/98/2000/XP, Windows 7

Size: 47 MB

Nano drugs..Powder dispersion

Uncategorized Comments Off

Mar 132014

Powder dispersion

One of the main criteria for the effective drug delivery via lungs is the size of the inhaled particle. Respirable size i.e. the particle is transported to deep lungs (alveoli region) is around 1 – 5 micrometers. The particle size can be easily controlled in the powder production. The powders, however, tend to stick to each other in the collection. Therefore, the dispersion and deagglomeration behavior of the powder should be studied.

The dispersion testing of the fine powders was conducted with the novel deagglomeration apparatus [14]. Powder agglomerates, i.e. the mixture of carrier and fine powders, is fed continuously through a narrow tube with the aid of thin air flow of 1.2 l/min. At the outlet of the needle the agglomerates are subjected to the main flow rate (Q_M) from 15 to 90 l/min intending to disperse the powder i.e. deagglomeration zone, see Figure 8. These Q_M values correspond to the jet Reynolds numbers from 8000 to 48000. The flow forms a highly turbulent space, i.e. the deagglomeration zone, where possible break-up of powder agglomerates takes place. The fine powder particles are isokinetically sampled from homogeneously mixed aerosol into low-pressure impactors. Fine particle fractions (FPF), mass medium aerodynamic diameter (MMAD), and size distribution can be determined at different flow rates.

Figure 9 illustrates a common trend in the change of the particle distribution at different flow rates. As seen, all the dispersed particles, in this case salbutamol 92 w% and L-leucine 8 w%, are within the respirable size range. Also, the increase in the flow rate from 15 to 90 l/min improved the powders dispersion (the increase in particle concentration) and deagglomeration (the size of the dispersed particles decreases).

In dry powder inhaler the fine drug particles are commonly mixed with large lactose particles. These lactose carriers aid the dispersion of fine particles. Figure 10 shows the fine particle fractions of the peptide-coated drug powders that were introduced to the dispersion testing without lactose carriers. The powders with crystalline L-leucine surface exhibited good flowability that made possible to feed these powders as such i.e. without carrier particles. More importantly, they showed excellent deagglomeration performance even at very low flow rate.

Octreotide اکترئتید For treatment of acromegaly and reduction of side effects from cancer chemotherapy

Uncategorized Comments Off

Mar 122014

Octreotide

(D)-Phe-Cys-Phe-(D)-Trp-Lys-Thr-Cys-Thr-ol.

(4R,7S,10S,13R,16S,19R)-10-(4-aminobutyl)-19-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-16-benzyl-N-[(2R,3R)-1,3-dihydroxybutan-2-yl]-7-(1-hydroxyethyl)-13-(1H-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-
pentazacycloicosane-4-carboxamide

L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)].

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin.

Canada	1328402	1994-04-12	expiry 2011-04-12
United States	5922338	1997-01-13	2017-01-13
United States	5538739	1993-07-23	2013-07-23

CAS number	83150-76-9 79517-01-4 (acetate) 135467-16-2 (pamoate)

Sandostatin LAR Depot
L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-(2-hydroxy-1-(hydroxymethyl)propyl)-, cyclic(2-7)-disulfide, (R-(R*,R*))-, acetate (salt)
Octreotide Acetate Depot
AC1L1GVR
AC1Q2BPN
CCRIS 8708
Octreotide acetate [USAN:JAN]
UNII-75R0U2568I
83150-76-9 (Parent)
AC-663

Octreotide (brand name Sandostatin,^[1] Novartis Pharmaceuticals) is an octapeptide that mimics natural somatostatin pharmacologically, though it is a more potent inhibitor of growth hormone, glucagon, and insulin than the natural hormone. It was first synthesized in 1979 by the chemist Wilfried Bauer.

Since octreotide resembles somatostatin in physiological activities, it can:

inhibit secretion of many hormones, such as gastrin, cholecystokinin, glucagon, growth hormone, insulin, secretin, pancreatic polypeptide, TSH, and vasoactive intestinal peptide,
reduce secretion of fluids by the intestine and pancreas,
reduce gastrointestinal motility and inhibit contraction of the gallbladder,
inhibit the action of certain hormones from the anterior pituitary,
cause vasoconstriction in the blood vessels, and
reduce portal vessel pressures in bleeding varices.

It has also been shown to produce analgesic effects, most probably acting as a partial agonist at the mu opioid receptor.^[2]^[3]

Acromegaly is a hormonal disorder that results when the pituitary gland produces excess growth hormone (GH). It most commonly affects middle-aged adults and can result in serious illness and premature death. Once recognized, acromegaly is treatable in most patients, but because of its slow and often insidious onset, it frequently is not diagnosed correctly.

Octreotide is one drug used to treat acromegaly. Octreotide exerts pharmacologic actions similar to those of the natural hormone somatostatin. Octreotide decreases GH and IGF-1 levels, as well as glucagons and insulin. Octreotide also suppresses luteinizing hormone (LH) response to gonadotropin releasing hormone (GnRH), decreases splanchnic blood flow, and inhibits the release of serotonin, gastrin, vasoactive intestinal peptide, secretin, motilin, and pancreatic polypeptide. In many patients, GH levels fall within one hour and headaches improve within minutes after the injection of octreotide. Several studies have shown that octreotide is effective for long-term treatment. Octreotide also has been used successfully to treat patients with acromegaly caused by non-pituitary tumors. In some acromegaly patients who already have diabetes, octreotide can reduce the need for insulin and improve blood sugar control.

Octreotide is currently available as Sandostatin LAR® Depot, which is, upon reconstitution, a suspension of microspheres containing octreotide acetate. Sandostatin LAR® Depot is the only medication indicated for the long-term maintenance therapy in acromegalic patients. It is also indicated for the long-term treatment of severe diarrhea and flushing episodes associated with metastatic carcinoid tumors and profuse water diarrhea associated with VIP-secreting tumors. Sandostatin LAR® T Depot is administered via intramuscular injection every four weeks, following a titration period. Octreotide acetate has also been available in an immediate-release formulation, Sandostatin® Injection solution, which was required to be administered by injection three times daily.

Octreotide is an octapeptide with the following amino acid sequence: L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)]. The structure of octreotide is shown below.

The chemical formula is C₄₉H₆₆N₁₀O₁₀S₂and its molecular weight is 1019.3 Da. Its therapeutic category is gastric antisecretory agent.

The Food and Drug Administration (FDA) has approved the usage of a salt form of this peptide, octreotide acetate, as an injectable depot formulation for the treatment of growth hormone producing tumors (acromegaly and gigantism), pituitary tumors that secrete thyroid stimulating hormone(thyrotropinoma), diarrhea and flushing episodes associated with carcinoid syndrome, and diarrhea in patients with vasoactive intestinal peptide-secreting tumors (VIPomas).

Octreotide is used in nuclear medicine imaging by labelling with indium-111 (Octreoscan) to noninvasively image neuroendocrine and other tumours expressing somatostatin receptors.^[4] More recently, it has been radiolabelled with carbon-11^[5] as well as gallium-68, enabling imaging with positron emission tomography (PET), which provides higher resolution and sensitivity.

Octreotide can also be labelled with a variety of radionuclides, such as yttrium-90 or lutetium-177, to enable peptide receptor radionuclide therapy(PRRT) for the treatment of unresectable neuroendocrine tumours.

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Octreotide is known chemically as L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1- (hydroxy-methyl) propyl]-, cyclic (2→7)-disulfide; [R-(R*,R*)].

Sandostatin LAR Depot is available in a vial containing the sterile drug product, which when mixed with diluent, becomes a suspension that is given as a monthly intragluteal injection. The octreotide is uniformly distributed within the microspheres which are made of a biodegradable glucose star polymer, D,L-lactic and glycolic acids copolymer. Sterile mannitol is added to the microspheres to improve suspendability.

Sandostatin LAR Depot is available as: sterile 5-mL vials in 3 strengths delivering 10 mg, 20 mg, or 30 mg octreotide-free peptide. Each vial of Sandostatin LAR Depot delivers:

NAME OF INGREDIENT	10 MG	20 MG	30 MG
octreotide acetate	11.2 mg*	22.4 mg*	33.6 mg*
D, L-lactic and glycolic acids copolymer	188.8 mg	377.6 mg	566.4 mg
mannitol	41.0 mg	81.9 mg	122.9 mg
*Equivalent to 10/20/30 mg octreotide base.

Each syringe of diluent contains:

carboxymethylcellulose sodium	12.5 mg
mannitol	15.0 mg
water for injection	2.5 mL

The molecular weight of octreotide is 1019.3 (free peptide, C₄₉H₆₆N₁₀O₁₀S₂) and its amino acid sequence is

Sandostatin LAR® Depot (octreotide acetate) Amino acid sequence - Illustration

Octreotide has also been used off-label for the treatment of severe, refractory diarrhea from other causes. It is used in toxicology for the treatment of prolonged recurrent hypoglycemia after sulfonylurea and possibly meglitinides overdose. It has also been used with varying degrees of success in infants with nesidioblastosis to help decrease insulin hypersecretion.

Octreotide has been used experimentally to treat obesity, particularly obesity caused by lesions in the hunger and satiety centers of thehypothalamus, a region of the brain central to the regulation of food intake and energy expenditure.^[6] The circuit begins with an area of the hypothalamus, the arcuate nucleus, that has outputs to the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH), the brain’s feeding and satiety centers, respectively.^[7]^[8] The VMH is sometimes injured by ongoing treatment for acute lymphoblastic leukemia (ALL) or surgery or radiation to treat posterior cranial fossa tumors.^[6] With the VMH disabled and no longer responding to peripheral energy balance signals,

Octreotide has also been investigated for patients with pain from chronic pancreatitis,^[11] and it may be useful in the treatment of thymic neoplasms.

The drug has been used off-label, injected subcutaneously, in the management of hypertrophic pulmonary osteoarthropathy (HPOA) secondary to non-small cell lung carcinoma. Although its mechanism is not known, it appears to reduce the pain associated with HPOA.^{[citation needed]}

It has been used in the treatment of malignant bowel obstruction.^[12]

Octreotide may be used in conjunction with midodrine to partially reverse peripheral vasodilation in the hepatorenal syndrome. By increasing systemic vascular resistance, these drugs reduce shunting and improve renal perfusion, prolonging survival until definitive treatment with liver transplant.^[13] Similarly, octreotide can be used to treat refractory chronic hypotension.^[14]

While successful treatment has been demonstrated in case reports,^[15]^[16] larger studies have failed to demonstrate efficacy in treating chylothorax.^[17]

Octreotide is often give as an infusion for management of acute haemorrhage from esophageal varices in liver cirrhosis on the basis that it reduces portal venous pressure, though current evidence suggests that this effect is transient and does not improve survival.^[18]

A small study has shown that octreotide may be effective in the treatment of idiopathic intracranial hypertension.^[19]^[20]

Octreotide has not been adequately studied for the treatment of children, pregnant and lactating women. The drug is given to these groups of patients only if a risk-benefit analysis is positive.^[21]^[22]

Acetate

C₅₃H₇₄N₁₀O₁₄S₂ , 1139.34326

The most frequent adverse effects (more than 10% of patients) are headache, hypothyroidism, cardiac conduction changes, gastrointestinal reactions (including cramps, nausea/vomiting and diarrhoea or constipation), gallstones, reduction of insulin release, hyperglycemia^[23] or hypoglycemia, and (usually transient) injection site reactions. Slow heart rate, skin reactions such aspruritus, hyperbilirubinemia, hypothyroidism, dizziness and dyspnoea are also fairly common (more than 1%). Rare side effects include acute anaphylactic reactions, pancreatitis andhepatitis.^[21]^[22] One study reported a possible association with rheumatoid arthritis.^[24]

Some studies reported alopecia in patients who were treated by octreotide.^[25] Rats which were treated by octreotide experienced erectile dysfunction in a 1998 study.^[26]

A prolonged QT interval has been observed in patients, but it is uncertain whether this is a reaction to the drug or part of the patients’ illnesses.^[21]

Octreotide can reduce the intestinal resorption of ciclosporin, possibly making it necessary to increase the dose.^[27] Patients with diabetes mellitusmight need less insulin or oral antidiabetics when treated with octreotide. The bioavailability of bromocriptine is increased;^[22] besides being anantiparkinsonian, bromocriptine is also used for the treatment of acromegaly.

Octreotide is absorbed quickly and completely after subcutaneous application. Maximal plasma concentration is reached after 30 minutes. The elimination half-life is 100 minutes (1.7 hours) on average when applied subcutaneously; after intravenous injection, the substance is eliminated in two phases with half-lives of 10 and 90 minutes, respectively.^[21]^[22]

Conventional synthesis of octreotide may be divided into two main approaches, liquid-phase synthesis and solid-phase synthesis. · Octreotide first disclosed in US4395403, in which Octreotide is prepared by solution phase peptide synthesis. The process comprises; removing protected group from peptide;^‘ linking together by an amide bond to two peptide unit; converting a function group at the N- or C- terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate.

Since all the synthesis steps are carried out in liquid phase, US’403 process is a time- consuming, multi-step synthesis and it is difficult to separate octreotide from the reaction mixtures. Another solution phase approach described in US6987167 and WO2007110765A2, in which the cyclization of partially deprotected octreotide is carried out in the solution phase using iodine under specific conditions in presence of alcoholic solvents.

US6346601 B1 , WO2005087794A1 and WO2010089757A2 disclose a process for the preparation of octreotide by hybrid approach i. e synthesis of fragments on solid phase and condensing the obtained fragments in a liquid phase.

US6476186 describes the solid phase synthesis, in which the synthesis of octreotide using Thr(ol)(tBu)-2CI-trityl resin as starting material, followed by the cleavage of the straight chain peptide from the resin using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst.

US20040039161A1 provides a solid phase .peptide synthetic method for the preparation of C-terminal alcohols using trichloroacetimidate activated linker, making the required peptide chain on the resin support, cleaving the attached peptide; air oxidation to form said C- terminal amino alcohol containing peptide and a 36.3% yield of octreotide after HPLC purification.

Charcoal oxidation or air oxidation needs longer reaction time and results in low yield. Further, in large scale, the conversion of dithiol to disulfide bond ends in unconverted starting material.

Another solid phase approach describes in Bioconjugate chem. 2009, 20, 1323-1331. This article discloses the process of somatostatin and octreotide analogues using solid phase peptide synthesis with CTC resin.

Journal of Harbin Institute of Technology, 2008, Vol 40 (2), 292-295, discloses the process for the preparation of octreotide using CTC resin. According to this process the obtained octreotide has the purity 70.26% by HPLC. During the process of peptide bond formation which is mediated by a coupling agent, the carboxylic group of amino acid interacts with the coupling agent to form an activated intermediate, which in turn interacts with the amino group of the next amino acid.

Racemization is a side-reaction that occurs during the preparation of a peptide. In large scale production, the formations of small amounts of epimers are possible. Detection and removal of these impurities are very difficult. This constitutes one of the most serious drawbacks for the implementation of peptides in commercial scale production.

WO2005087794A1

Conventional syntheses of OCT may be divided two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Direct solid-phase synthesis comprises attachment of a C-terminal amino acid to a resin, and step-by step elongation of the peptide chain, with pre- activated amino acids.

This route is expensive because it requires large excesses of starting amino acids and additionally is quite labor consuming as the peptide size increases, necessitating complex purification procedures to separate the product from the impurities since they are very similar to the final product. These shortcomings are especially important for large scale industrial production of the product. For example, see Canadian Patent Application 2,309,312 and U.S. Patent No. 6,476,186. With each successive condensation reaction required to add an amino acid, waste of starting materials increases, and purification steps are repeated. Liquid-phase synthesis comprises condensation of amino acids in solution. Several blocks, containing from 2 to 5 amino acids may be synthesized independently, followed by condensation of these synthons to each other in the required sequence.

For example, see WO 03/097668; U.S. Patent No. 4,395,403; and RU 2196144 C1. The advantage of this kind of processes is that it is less expensive than the previous one and the product is easier to purify. This method is also more effective for scale-up. However, liquid phase synthesis of lengthy peptide blocks, for example having more than 3 amino acids, is inefficient. Liquid-phase octreotide synthesis has the drawback is that the method is extremely labor-intensive and time consuming.

U.S. Patent No. 6,346,601 describes a method for octreotide synthesis where a solid-phase method is used to obtain a 7-mer, followed by condensation in solution with the modified amino acid threoninol. However, by using solid- phase synthesis to produce a 7-mer, only one less condensation is required compared to the solid-phase process for forming octreotide itself. Thus, only a marginal efficiency is introduced.

Summary of the invention According to an embodiment of the invention, there is provided a process for obtaining octreotide or a pharmaceutically acceptable salt thereof by hybrid solid-phase – liquid-phase synthesis. The synthesis comprises the steps of condensing two or three peptide blocks using liquid phase condensation to form a condensation product followed by cyclizing the product.

Each peptide block contains two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. The condensation product comprises in sequence the amino acids residues of octreotide. In the step of cyclizing, the condensation product is cyclized to form a disulfide bridge between the two cysteine residues, thereby forming octreotide. Further, according to another embodiment of the invention, a process is provided for obtaining an intermediate in octreotide synthesis by hybrid solid- phase – liquid-phase synthesis.

The synthesis of the intermediate comprises the steps of obtaining two or three peptide blocks, each peptide block containing two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. Subsequently, the peptide blocks are condensed using liquid phase condensation to form a condensation product, wherein the condensation product comprises in sequence the amino acids residues of octreotide.

This invention provides a more cost-effective and labor-saving method for obtaining OCT and its pharmaceutically acceptable salts by means of hybrid solid-phase – liquid-phase synthesis. The invention involves liquid phase condensation of two peptide blocks, at least one of which is obtained by solid- phase synthesis, the blocks containing more two or more amino acid residue in every block, followed by formation of a disulfide bridge from the two cysteine groups. Optionally, three blocks may be condensed. This hybrid solid phase-liquid phase method involves formation of one or more blocks of the octreotide amino acid sequence by solid-phase synthesis, followed by liquid phase condensation of the block(s) with required supplementary amino acids or other block(s) of amino acids.

This method is a blend of solid-phase and liquid-phase synthesis methods, combining the efficiencies of preparing shorter (6-mer or less) peptides using a solid-phase method with relative cheapness and easiness of purification of the product, characteristic of the liquid-phase method. Generally, the methods of invention comprise synthesizing specific side- chain protected peptide fragment intermediates of OCT on a solid support or in solution, coupling of the protected fragments in solution to form a protected OCT, followed by deprotection of the side chains and oxidation to yield the final OCT. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the OCT

………………

WO2013046233A2

Stage-I: Preparation of protected octreotide anchored to 2-CTC Resin

Method -1:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of DC and DMF for the swelling. Fmoc-Thr(tBu)-OL was treated with the swelled 2- CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V/V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Method -2:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of MDC and DMF for the swelling. Fmoc-Thr-OL was treated with the swelled 2-CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V7V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Stage-ll: Cleavage of peptide from resin along with global deprotection

The peptide resin (200 g, obtained in stage I) was swelled in DCM (500 mL) for 15 to 20 minutes under nitrogen at 25-30° C. The cocktail mixture (2.0 L – TFA (1.8 L), water (80 mL) DCM (80mL) and TIPS (80 mL)) was charged to the resin at 25-30° C. and the obtained reaction mixture was stirred for 2.5 hours at 25-30°C under nitrogen atmosphere. The reaction mixture was filtered and washed the resin with TFA (250 mL). The obtained filtrate was charged into cold MTBE (4 L, pre-cooled to a temperature of 0 -5° C) under stirring and allowing the temperature to rise more than 5° C. The reaction mixture was stirred for 45-75 minutes at 0-5°C. The obtained suspension was filtered, washed the solid with MTBE (5 L) and dried the solid under nitrogen. The product was stir with 5%ethanol in ethyl acetate at 25-30°C. Filtered the product, wash ith ethyl acetate and dried under vacuum to obtain a desired product

Stage-Ill: Disulphide bridge formation

The free thiol (100 g) obtained above is dissolved in methanol (22.0 L) with small amount of acetic acid and water (4.5 L) and stirred. Iodine solution (20gm iodine in 500 mL methanol) was added to the reaction mass slowly up to yellow color persists. The reaction was maintained for another 2 hrs, and the excess iodine quenched with Indion 830-S Resin (900 g) and filtered the resin. The filtrate was evaporated and precipitated using TBE or directly taken the solution for purification using preparative HPLC.

Stage -IV: Preparative HPLC Purification

Method-1 :

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm). and eluting with a solvent system of 0.2% acetic acid in water(A) and 0.2% acetic acid in methanol(B). A linear gradient of 20- 60% B was used at a flow rate of 80mlJmin and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions were collected at regular intervals and assayed by HPLC to determine the purity of fractions. The desired purities fractions were pooled together and evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate

Method-2:

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm) and eluting with a solvent system of 0.4% acetic acid in water(A) and methanol(B). A linear gradient of 25-60% B was used at a flow rate of 80mL/min and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions are collected at regular intervals and are assayed by HPLC to determine the purity and fractions. The desired purities may be pooled together and were evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate >

……………………….

WO2010089757A2

Octreotide is a highly potent and pharmacologically selective analog of somatostatin. It inhibits growth hormone for long duration and is thereof indicated for acromegaly to control and reduce the plasma level of growth hormone. The presence of D-Phe at the N-terminal and an amino alcohol at the C-terminal, along with D-Tryptophan and a cyclic structure makes it very resistant to metabolic degradation.

Octreotide comprises 8 amino acids which has the following structural formula:

(D)Phe-Cys-Phe-{D)Trp-Lys-Thr-Cys-Thr-OL

Formula(l) wherein sulphur atoms of the Cys at the position 2 and of the Cys at the position 7 are mono-cyclic to form an -S-S- bridge.

A considerable number of known, naturally occurring small and medium-sized cyclic peptides as well as some of their artificial derivatives and analogs possessing desirable pharmacological properties have been synthesized. However, wider medical use is often hampered due to complexity of their synthesis and purification. Therefore, improved methods for making these compounds in simple, lesser steps and at lesser cost are desirable and this is the felt need of the industry and the mankind.

Conventional synthesis of octreotide may be divided into two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Solution phase synthesis has been described by Bauer et al., (Sandoz) (Eur. Pat. Appl. 29,579 and U.S. Pat. No. 4,395,403). The process comprises: removing protected group from peptide; linking together by an amide bond two peptide unit; converting a function group at the N- or C-terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate. This process involves a time-consuming, multi-step synthesis, and it is difficult to separate octreotide from the reaction mixtures since all the synthesis steps are carried out in liquid phase.Another solution phase approach described by Chaturvedi, et al., (Wockhardt) in U.S. Pat. No. 6,987,167 and EP 1506219 A, claims the cyclization of partially deprotected octreotide in the solution phase using iodine under conditions and for a time sufficient to form the octreotide.

Synthesis in solid phase have been described subsequently (Mergler et al., Alsina et al., Neugebauer). The above prior art for solid phase peptide synthesis cites the octapeptide formation, by starting the synthesis from the threoninol residue which makes it mandatory to protect this residue. Mergler et al., (Peptides: Chemistry and Biology. Proceedings of the 12* American Peptide Symposium. Smith, J.A. And Rivier J.E. Eds ESCOM, Leiden, Poster 292 Presentation, (1991) ) describes a synthetic process, using an aminoethyl resin upon which the Threoninol residue is incorporated with the two alcohol functions protected in acetal form The synthesis is carried out following an Fmoc/tBu protection scheme, forming the disulphide bridge on resin by oxidation of the thiol groups of the previously deprotected cysteine residues and releasing and deprotecting the peptide with a 20% mixture of TFA/DCM.

In early 1997, Alsina J. et al. ( Alsina J., Chiva C, Ortiz M., Rabanal F., Giralt E., and Albericio F., Tetrahedron Letters, 38, 883-886, 1997) described the incorporation, on active carbonate resins, of a Threoninol residue with the amino group protected by the Boc group and the side chain protected by a BzI group. The synthesis was then continued by Boc/Bzl strategy. Formation of the disulfide bridge was carried out directly on resin using iodine and the peptide was cleaved from the resin and its side chain protecting groups were simultaneously removed with HF/anisole 9/1. At the final stage the formyl group was removed with a piperidine/DMF solution.

Neugebauer (Neugebauer W., Lefevre M.R., Laprise R, Escher E., Peptides: Chemistry, Structure and Biology, p 1017, Marshal G.R. And Rivier J.E. Eds. ESCOM.Leiden (1990) described a linear synthesis with a yield of only 7%.

Edwards et al., (Edwards B.W., Fields C.G., Anderson CJ., Pajeau T.S., Welch M.J., Fields G.B., J.Med.Chem. 37, 3749-3757 (1994) carried out another another solid- phase type approximation; they synthesized step-by-step on the resin, the peptide D- Phe-Cys(Acm)-Phe-D-Tφ(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-HMP-Resin. Next they proceeded to form the disulfide on resin and then release the peptide from the resin by means of aminolysis with threoninol, with obtaining a total yield of only 14%.

The solid phase synthesis described by Yao-Tsung Hsieh et. al., in U.S. Pat. No. 6,476,186 involves the synthesis of octreotide by using Thr(ol)(tBu)-2Cl-trityl resin as starting material followed by the cleavage of the straight chain peptide from the resin by using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst and a higher yield of >70%.

Another solid phase synthesis described by Berta Ponsati et.al (Lipotec) in U.S. Pat No. 6,346,601 and EP 0953577 B involve the coupling of threoninol on the protected heptapeptide in solution, after a selective acid cleavage from the chlorotrityl resin without affecting the peptide side-chain protecting groups.

A hybrid solid phase-liquid phase method for synthesis of octreotide described by Iarov et al., (Dalton Chemical Laboratories) in WO 2005087794 wherein the method comprises liquid phase condensation of two or three peptide blocks in which at least one peptide block is synthesized by solid-phase method.

EP 1511761 Bl involves cyclization on the semi-protected linear peptide wherein one of the cysteine residue is protected with an orthogonal protecting group. The radioactive isotope labeling of octreotide by the coupling of bifunctional chelating agents like DTPA or DOTA to the peptide was described by Te- Wei Lee et al., in U.S. Pat. No. 5,889,146 (Inst, of Nuclear Energy Research)

The method for cyclization of linear vapreotide by means of intramolecular cysteine formation has been described by Quattrini et. al., (Lonza AG) in WO 2006048144, wherein the process involves the synthesis of linear vapreotide peptide on Sieber-resin (from Novabiochem) by Fmoc standard groups, wherein the side chain protecting groups are D or L-Trp(Boc), Cys(Trt), Lys(Boc), Tyr(tBu). The protected peptide is cleaved off in 5% TFA in dichloromethane and then globally deprotected by acidolysis in a cleavage mix of 300 equivalents of concentrated TFA, 12 equivalents of Dithiothreitol, 12 equivalents of Dichloromethane, 50 equivalents of water forl hour at room temperature. The Boc groups are removed. The product was subjected to charcoal method using trace amounts of activated, powdered charcoal wherein a concentration of the linear cysteinyl peptide of 50 mg/ml (1 eq.) in DMF in the presence of 1 eq. Diisopropyl-ethyl-amine and that additionally air was sparged at low pressure into the liquid under stirring. After 15-20 hrs, 100% conversion was achieved with 84% (w/w) analytical yield of 79% vapreotide.

The formation of intramolecular disulphide formation in a polypeptide by reacting with hydrogen peroxide has been described by Mineo Niwa et al. (Fujisawa Pharmaceutical Co.) in U.S. Pat. No.5, 102,985 wherein the reaction is to be carried out at a pH of about 6 tol 1, wherein the molar ratio of H₂O₂ to polypeptide is within the range of 1:1 to 100:1. The above cited prior art mainly carries out the cyclization of the peptide on the resin or on partially protected or protected peptides. The use of partial or minimal protecting group strategies and improvement in the activation methods have considerable effect on limitations of poor solubility and possible danger of racemization due to the overactivation of carboxyl groups. However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules.

Example 8:

Oxidation of S-H peptide with DMSO-HCl to get S-S peptide:

(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-OL

Formula (1)

S-H peptide ( 9g) was dissolved in 6.5L DMSO and under ice-cooling 6.5L IM HCl was added slowly so that temperature is below 26°C. Stirring was continued for 6 hours. At room temperature after six hours reaction mixture was diluted with 13L of water and filtered through Whatman no. 41 through Celite bed. The filtrate was loaded on C- 18 column for concentration. The compound was eluted with 100% acetonitrile. The eluant was concentrated on rotavap and then the concentrated solution was centri-evaporated to dryness. The RP-HPLC profile of crude octreotide is depicted in Figure 1.

Weight of crude peptide =3.9g.(45%)

Purity: 44.25%

Example 9:

Purification of crude octreotide:

The crude octreotide was loaded on to cation ion exchange column and eluted using a salt gradient using a Akta Purifier (by Amersham, Sweden) low pressure chromatography system. The IEX fractions of purity >70% were further loaded for RP-HPLC purification on Kromacil C-18 column of (250x50mm,100A°.) The peptide was purified by using aqueous TF A(O-0.5%) and methanol/ethanol and/or Acetonitrile in a gradient program on a Shimadzu preparative HPLC System consisting of a controller, 2 LC8A pumps, and UV-Vis detector. The purified peptide was analysed by analytical RP-HPLC (Figure 5). Fractions of > 99% purity were subjected either by RP-HPLC or IEX to salt exchange and concentrated to remove organic solvent either by rota or reverse osmosis and subsequently lyophilized to get final API with purification step yield of 70% or above.The MS spectrum of octreotide is depicted in Figure 6.

References

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RUXOLITINIB…FOR THE TREATMENT OF INT OR HIGH-RISK MYELOFIBROSIS

Uncategorized Comments Off

Mar 102014

Ruxolitinib

(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile, cas no 941678-49-5

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

1H-Pyrazole-1-propanenitrile, beta-cyclopentyl-4-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-,(betaR)-

Formula: C₁₇H₁₈N₆
Molecular Weight: 306.37

JAKAFI® (ruxolitinib) Structural Formula Illustration

Phosphate salt

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate

INCB 018424

INCB 018424
INCB018424
Ruxolitinib
UNII-82S8X8XX8H

CAS No.: 1092939-17-7
M.Wt: 404.36
Formula: C17H21N6O4P
Ruxolitinib phosphate

LAUNCHED 2011, INCYTE FOR MYELOFIBROSIS, NDA202192, 2011-11-16

CLINICAL TRIALS.http://clinicaltrials.gov/search/intervention=INCB018424+OR+ruxolitinib

EMA:Link,

US FDA:link

HPLC, MS, NMR…http://www.medkoo.com/Product-Data/Ruxolitinib/Ruxolitinib-QC-LC20130225.pdf

http://file.selleckchem.com/downloads/nmr/S137803-INCB018424-HNMR-Selleck.pdf

http://file.selleckchem.com/downloads/hplc/S137803-INCB018424-HPLC-Selleck.pdf

Ruxolitinib phosphate is a kinase inhibitor with the chemical name (R)-3-(4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate and a molecular weight of 404.36.

Ruxolitinib is a janus-associated kinase inhibitor indicated to treat bone marrow cancer, specifically intermediate or high-risk myelofibrosis. FDA approved on November 16, 2011.

INCB018424 is the first potent, selective, JAK1/2 inhibitor to enter the clinic with IC50 of 3.3 nM/2.8 nM, >130-fold selectivity for JAK1/2 versus JAK3

Ruxolitinib phosphate has the following structural formula:

JAKAFI® (ruxolitinib) Structural Formula Illustration

Ruxolitinib phosphate is a white to off-white to light pink powder and is soluble in aqueous buffers across a pH range of 1 to 8.

Jakafi (ruxolitinib) Tablets are for oral administration. Each tablet contains ruxolitinib phosphate equivalent to 5 mg, 10 mg, 15 mg, 20 mg and 25 mg of ruxolitinib free base together with microcrystalline cellulose, lactose monohydrate, magnesium stearate, colloidal silicon dioxide, sodium starch glycolate, povidone and hydroxypropyl cellulose

.NCI: /Ruxolitinib phosphate/ The phosphate salt form of ruxolitinib, an orally bioavailable Janus-associated kinase (JAK) inhibitor with potential antineoplastic and immunomodulating activities. Ruxolitinib specifically binds to and inhibits protein tyrosine kinases JAK 1 and 2, which may lead to a reduction in inflammation and an inhibition of cellular proliferation. The JAK-STAT (signal transducer and activator of transcription) pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies. (NCI Thesaurus)

patent expiry

US pat 7598257 exp 24/12/27

US pat 8415362 exp 24/12/27

NCE.Nov 16, 2016

Discovered by Incyte, ruxolitinib phosphate is an inhibitor of Janus-associated kinase 2 (JAK2), a protein involved in signal transduction. This orally available compound was approved and launched in the U.S. in 2011 for the treatment of patients with intermediate or high-risk myelofibrosis (MF), including primary MF, post-polycythemia vera MP and post-essential thrombocythemia MF. A regulatory application in the E.U. was filed in 2011 and a positive opinion was assigned in April 2012. Final E.U. approval was obtained in August 2012. In November 2012, the product was launched in the U.K. for the treatment of disease-related splenomegaly or symptoms in primary myelofibrosis or myelofibrosis due to polycythemia vera or essential thrombocythemia. In 2012, the product has been filed for approval in Japan for the treatment of myelofibrosis.

Phase II clinical trials are also ongoing for the treatment of multiple myeloma, leukemia, pancreas cancer, thrombocytopenia and for the treatment of relapsed or refractory diffuse large B-cell or peripheral T-cell non-Hodgkin lymphoma following donor stem cell transplant. In Japan, the product is under development in phase III trials for the treatment of polycythemia vera and in phase II trials for the treatment of myelofibrosis. No recent development has been reported for clinical trials for the treatment of rheumatoid arthritis (RA), for the treatment of psoriasis or for the treatment of metastatic prostate cancer. Columbia University is evaluating the compound in preclinical studies for the treatment of alopecia areata. The University of Pennsylvania is conducting phase II clinical trials for the treatment of breast cancer.

In 2008 and 2009, the compound was assigned orphan drug designation in the U.S. and the E.U., respectively, for the treatment of myelofibrosis. This designation was assigned in Japan for this indication in 2011. Additional orphan drug designation was assigned by the FDA in 2010 for the treatment of polycythemia vera and for the treatment of essential thrombocythemia. In 2013, an orphan drug designation was assigned in U.S. for the treatment of pancreatic cancer. In 2009, fast track designation was assigned to ruxolitinib phosphate in the U.S .for the treatment of myelofibrosis.

Ruxolitinib (trade names Jakafi and Jakavi, by Incyte Pharmaceuticals and Novartis) is a drug for the treatment of intermediate or high-risk myelofibrosis, a type of bone marrow cancer.It is also being investigated for the treatment of other types of cancer (such as lymphomas and pancreatic cancer), for polycythemia vera, and for plaque psoriasis.
The phase III Controlled Myelofibrosis Study with Oral JAK Inhibitor-I (COMFORT-I) and COMFORT-II trials showed significant benefits by reducing spleen size, relieving debilitating symptoms, and improving overall survival.

INCYTE developed in cooperation with companies and NOVARTIS jak2 selective inhibitor Ruxolitinib(INCB-018424) – has been approved by the FDA successfully listed). (Safety and Efficacy of INCB018424, a JAK1 and JAK2 Inhibitor, in Myelofibrosis. Srdan Verstovsek, MD, Ph.D., Hagop Kantarjian, MD, Ruben A. Mesa. MD, et al. N Engl J Med 2010; 363: 1117-1127).

The presently disclosed a series of patent applications JAK inhibitors, including WO2001042246, WO200200066K WO2009054941 and WO2011013785, etc.

Mechanism of action

Ruxolitinib is a Janus kinase inhibitor with selectivity for subtypes 1 and 2 of this enzyme.
Side effects

Immunologic side effects have included herpes zoster (1.9%) and case reports of opportunistic infections.[10] Metabolic side effects have included weight gain (7.1%). Laboratory abnormalities have included alanine transaminase (ALT) abnormalities (25.2%), aspartate transaminase (AST) abnormalities (17.4%), and elevated cholesterol levels (16.8%).
Legal status

In November 2011, ruxolitinib was approved by the USFDA for the treatment of intermediate or high-risk myelofibrosis based on results of the COMFORT-I and COMFORT-II Trials.

Some analysts believe this to be a potential blockbuster drug.[3] As of the end of March 2012, and according to an Incyte spokesman, approximately 1000 physicians had prescribed the drug in the United States, out of a total 6500 hematologists and oncologists nationwide.

The US Food and Drug Administration had approved Incyte’s Jakafi (ruxolitinib) to treat patients with the bone marrow disease myelofibrosis (MF). Jakafi is the first and only drug granted license specifically for the treatment of the rare blood cancer.
Jakafi approved by FDA to treat rare bone marrow disease
Posted By Edward Su On November 17th, 2011

MF is a rare, potentially life-threatening blood cancer with limited treatment methods. Patients with the bone marrow disoder, characterized by bone marrow failure, enlarged spleen (splenomegaly), suffer from the symptoms of fatigue, night sweats and pruritus, poor quality of life, weight loss and shortened survival. The US drug firm Incyte estimates the disease affects about 16,000-18,500 people in the USA. Currently, the disease is treated with chemotherapy or bone marrow transplant.

Incyte’s Jakafi, the first drug to reach market from the Wilmington-based drug company, was approved by the FDA as a twice-a-day pill for the treatment of patients with intermediate or high-risk myelofibrosis (MF), including primary MF, post-polycythemia vera MF and post-essential thrombocythemia MF. The US regulators reviewed Jakafi under its priority review program for important new therapies.

The approval of Jakafi was based on the results from two clinical studies involved 528 patients with the disease. Patients in the Jakafi treatment arm experienced a significant reduction in the size of their spleen as well as a 50 percent decrease in symptoms, including pain, discomfort and night sweats.

Jakafi, generically known as ruxolitinib, works by blocking JAK1 and JAK2 enzymes associated with the disease. The company has co-developed the drug with Novartis as part of their collaboration signed in 2009. The Swiss drug firm has the rights to market Jakafi in other countries.

“The availability of Jakafi is a significant medical advancement for people living with myelofibrosis, a debilitating disease,” said Paul A. Friedman, M.D., President and Chief Executive Officer of Incyte. “This milestone marks a tremendous achievement for Incyte because a scientific discovery from our research laboratories has become the first JAK inhibitor to reach the market and provide a clinical benefit to patients.”

Richard Pazdur, director of the Office of Hematology and Oncology Drug Products in the FDA’s Center for Drug Evaluation and Research, said that Jakafi “represents another example of an increasing trend in oncology where a detailed scientific understanding of the mechanisms of a disease allows a drug to be directed toward specific molecular pathways”.

Incyte says Jakafi will be available next week, and the drug will cost $7,000 per month, or $84,000 for a year’s supply for insured patients. The company plans to provide Jakafi free to uninsured patients and will offer co-pay assistance to patients with financial need.

……………

NMR free base

http://www.google.com/patents/US8410265

For (R)-13 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

http://www.google.com/patents/US8410265

phosphate

For (R)-14 (phosphate): mp. 197.6° C.; ¹H NMR (DMSO-d₆, 500 MHz) δ ppm 12.10 (s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.36 (s 1H), 7.58 (dd, 1H, J=1.9, 3.5 Hz), 6.97 (d, 1H, J=3.6 Hz), 4.52 (td, 1H, J=3.9, 9.7 Hz), 3.25 (dd, 1H, J=9.8, 17.2 Hz), 3.16 (dd, 1H, J=4.0, 17.0 Hz), 2.41, (m, 1H), 1.79 (m, 1H), 1.59 (m, 1H), 1.51 (m, 2H), 1.42 (m, 1H), 1.29 (m, 2H), 1.18 (m, 1H); ¹³C NMR (DMSO-d₆, 125 MHz) δ ppm 152.1, 150.8, 149.8, 139.2, 131.0, 126.8, 120.4, 118.1, 112.8, 99.8, 62.5, 44.3, 29.1, 29.0, 24.9, 24.3, 22.5; C₁₇H₁₈N₆(MW, 306.37 for free base) LCMS (EI) m/e 307 (M⁺+H, base peak), 329.1 (M⁺+Na).

………………….

SYNTHESIS

http://www.google.com/patents/US8410265

Figure US08410265-20130402-C00204

Figure US08410265-20130402-C00211

Figure US08410265-20130402-C00230

………………………

SYNTHESIS

US20100190981

(R)-Methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilypethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoate ((R)-22). A solution of (E)-methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)acrylate (21, 815 mg) in tetrahydrofuran (THF, 8.0 mL) in a pressure glass tube was treated with the catalyst [Rh(COD)(−)-DuanPhos](BF₄) (4.6 mg) under nitrogen before the reaction mixture was pressurized with hydrogen gas to 50 bar pressure. The reaction mixture was stirred at 35° C. under this hydrogen pressure for 22 h. When HPLC analysis showed that the substrate was almost completely consumed, the reaction mixture was cooled down to room temperature. The enantiomeric excess of the reaction mixture was determined to be 94.7% ee (97.35% of the second peak, (R)-22; 2.65% of the first peak, (S)-22) by chiral HPLC analysis. The reaction mixture was then filtered through a thin silica gel pad and the pad was washed with tetrahydrofuran (THF, 5 mL). The filtrate was then concentrated under reduced pressure to dryness. The resultant foamy solid (778 mg) was analyzed by chiral HPLC analysis and result showed a 94.7% of enantiomeric excess favoring the second peak (97.35% of the second peak, (R)-22; 2.65% of the first peak, (S)-22).

(3R)-3-Cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23). To a stirred solution of (3R)-methyl 3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoate ((R)-22, 2.47 g, 5.26 mmol) in THF (30 mL) at room temperature was added a solution of lithium hydroxide monohydrate (LiOH—H₂O, 265 mg, 6.31 mmol, 1.2 equiv) in water (15 mL). The reaction mixture was stirred at room temperature for 3 h. When LCMS showed the reaction was complete, the reaction mixture was then acidified with 1 N aqueous HCl solution to pH 5 before it was extracted with EtOAc (2×25 mL). The combined organic layers were washed with brine, dried over magnesium sulfate (MgSO₄), filtered and concentrated under reduced pressure to afford (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23, 2.40 g, 2.40 g theoretical, 100% yield) as a colorless oil, which solidified upon standing at room temperature in vacuo. For (R)-23: ¹H NMR (CDCl₃, 300 MHz) δ ppm 8.95 (s, 1H), 8.95 (bs, 1H), 8.36 (s, 1H), 7.57 (d, 1H, J=3.7 Hz), 6.99 (d, 1H, J=3.7 Hz), 5.74 (s, 2H), 4.65 (dt, 1H, J=3.1, 10.3 Hz), 3.58 (t, 2H, J=8.2 Hz), 3.24 (dd, 1H, J=16.5, 10.3 Hz), 3.04 (dd, 1H, J=16.2, 3.1 Hz), 2.59 (m, 1H), 2.00 (m, 1H), 1.77-1.24 (m, 7H), 0.97 (t, 2H, J=8.2 Hz), 0.00 (s, 9H); C₂₃H₃₃N₅O₃Si (MW, 455.63), LCMS (EI) m/e 456.1 (M⁺+H).

(3R)-3-Cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24). To a stirred solution of (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanoic acid ((R)-23, 20 mg, 0.044 mmol) in DMF (1 mL) at room temperature was added N,N-carbonyldiimidazole (CDI, 21 mg, 0.13 mmol, 3.0 equiv). The reaction mixture was then stirred at room temperature and TLC was used to follow the reaction for formation of acyl imidazole (consumption of acid to a higher Rf spot with 30% EtOAc/hexane). When TLC showed that the acyl imidazole transformation was complete, ammonia gas was then bubbled through the stirred solution for 30 min to afford the amide (followed by LCMS). The excess amount of ammonia gas was evaporated by bubbling nitrogen vigorously through the solution. The crude product, (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24), in DMF was used directly to the following reaction to convert amide ((R)-24) into the corresponding nitrile ((R)-20).

(3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20). Method A. To a stirred solution of (3R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanamide ((R)-24, 20 mg, 0.044 mmol) in DMF (1 mL) at 0° C. was added methylene chloride (1 mL) and triethylamine (0.12 mL, 0.88 mmol, 20.0 equiv), followed by trichloroacetyl chloride (0.052 ml, 0.462 mmol, 10.5 equiv). The resulting reaction mixture was stirred at 0° C. for 1 h. When LCMS showed the reaction was complete, the reaction mixture was quenched with saturated sodium bicarbonate solution (NaHCO₃, 5 mL) before being extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine, dried over magnesium sulfate (MgSO₄), filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography with 0-75% EtOAc/hexane gradient elution to give (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20, 10 mg, 19 mg theoretical, 53% yield). For (R)-20: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 8.83 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.77 (d, 1H, J=3.7 Hz), 7.09 (d, 1H, J=3.7 Hz), 5.63 (s, 2H), 4.53 (td, 1H, J=19.4, 4.0 Hz), 3.51 (t, 2H, J=8.1 Hz), 3.23 (dq, 2H, J=9.3, 4.3 Hz), 2.41 (m, 1H), 1.79 (m, 1H), 1.66-1.13 (m, 7H), 0.81 (t, 2H, J=8.2 Hz), 0.124 (s, 9H); C₂₃H₃₂N₆OSi (MW, 436.63), LCMS (EI) m/e 437 (M⁻+H) and 459 (M⁺+Na).

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-13, free base). Method B. To a solution of (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-20, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L) was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF₄, 987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The reaction temperature was observed to decrease from ambient to 12° C. upon addition of the water and then increase to 33° C. during the addition of lithium tetrafluoroborate (LiBF₄). The resulting reaction mixture was heated to reflux (about 80° C.) for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC analyses showed both the hydroxyl methyl intermediate ((R)-25) and fully de-protected material ((R)-13, free base) produced but no starting material ((R)-20) left, the reaction mixture was cooled gradually to <5° C. before a 20% aqueous solution of ammonium hydroxide (NH₄OH, 450 mL) was added gradually to adjust the pH of the reaction mixture to 9 (checked with pH strips). The cold bath was removed and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v) to confirm complete de-protection. When LCMS and TLC showed the reaction was deemed complete, the reaction mixture was filtered and the solids were washed with acetonitrile (1 L). The combined filtrates were then concentrated under reduce pressure, and the residue was partitioned between ethyl acetate (EtOAc, 6 L) and half-saturated brine (3 L). The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layers were washed with half-saturated sodium bicarbonate (NaHCO₃, 3 L) and brine (3 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure to give the crude product as an orange oil. The crude material was then purified by flash column chromatography (SiO₂, 40 to 100% ethyl acetate/heptane gradient elution) to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-13, free base, 273 g, 324.9 g theoretical, 84% yield) as a white foam. This material was checked by ¹⁹F NMR to ensure no lithium tetrafluoroborate (LiBF₄) remained and by chiral HPLC (Chiralcel OD, 90:10 hexane/ethanol) to confirm enantiomeric purity and was used without further purification to prepare the corresponding phosphate salt. For (R)-13 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

3-Cyclopentylacrylonitrile (8). A solution of diethyl cyanomethylphosphonate (7, 742.5 g, 4.2 mol, 1.1 equiv) in dry THF (5.75 L) was stirred under nitrogen on an ice-water-methanol bath and a solution of 1 M potassium tert-butoxide in THF (4 L, 4.0 mol, 1.05 equiv) was added at such a rate as to keep the temperature below 0° C. After addition of 1 M potassium tert-butoxide in THF was complete, the stirring was continued on the cold bath for 1 h and a solution of cyclopentanecarbaldehyde (6, 374 g, 3.81 mol) in dry THF (290 mL) was added at such a rate as to maintain the temperature below 0° C. The cold bath was removed, and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. When the reaction was deemed complete, the reaction mixture was partitioned between methyl tent-butyl ether (MTBE, 14 L), water (10 L) and brine (6 L). The two layers were separated, and the combined organic phase was washed with brine (6 L). The aqueous phase was extracted with MTBE (10 L) and washed with brine (6 L). The combined organic extracts were concentrated under reduced pressure and the residue was distilled (65-78° C./6 torr) to afford 3-cyclopentylacrylonitrile (8, 437.8 g, 461.7 g theoretical, 94.8% yield) as a colorless oil, which was found to be a mixture of E- and Z-isomer. For 8: ¹H NMR (DMSO-d₆, 400 MHz, for Z-isomer) δ ppm 6.58 (t, 1H, J=10.6 Hz), 5.55 (dd, 1H, J=10.8, 0.59 Hz), 2.85 (m, 1H), 1.9-1.46 (m, 6H), 1.34 (m, 2H) and (for E-isomer) δ ppm 6.83 (q, 1H, J=8.3 Hz), 5.66 (dd, 1H, J=16.5, 1.4 Hz), 2.60 (m, 1H), 1.9-1.46 (m, 6H), 1.34 (m, 2H); ¹³C NMR (DMSO-d₆, 100 MHz, for Z-isomer) δ ppm 159.8, 116.6, 97.7, 42.3, 32.3, 25.1 and (for E-isomer) δ ppm 160.4, 118.1, 97.9, 43.2, 31.5, 24.8; C₈H₁₁N (MW, 121.18), GCMS (EI) m/e 120 (M⁺−H).

4-(1H-Pyrazol-4-yl)-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5). Method A. To a flask equipped with a reflux condenser, a nitrogen inlet, mechanical stirrer, and a thermowell was added 4-chloro-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (3a, 817 g, 2.88 mol) and dioxane (8 L). To this solution was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (4, 728 g, 3.75 mol, 1.30 equiv) followed by a solution of potassium carbonate (K₂CO₃, 1196 g, 8.67 mol, 3.0 equiv) in water (4 L). The solution was degassed by passing a stream of nitrogen through the solution for 15 minutes before being treated with tetrakis(triphenylphosphine)palladium(0) (167 g, 0.145 mol, 0.05 equiv) and the resulting reaction mixture was heated at reflux (about 90° C.) for 2 hours. When the reaction was deemed complete by TLC (1:1 heptane/ethyl acetate) and LCMS, the reaction mixture was cooled to room temperature, diluted with ethyl acetate (24 L) and water (4 L). The two layers were separated, and the aqueous layer was extracted with ethyl acetate (4 L). The combined organic layers were washed with water (2×2 L), brine (2 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The residue was suspended in toluene (4 L) and the solvent was removed under reduced pressure. The residue was finally triturated with methyl tert-butyl ether (MTBE, 3 L) and the solids were collected by filtration and washed with MTBE (1 L) to afford 4-(1H-pyrazol-4-yl)-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5, 581.4 g, 908.5 g theoretical, 64% yield) as white crystalline solids. For 5: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 13.41 (bs, 1H), 8.74 (s, 1H), 8.67 (bs, 1H), 8.35 (bs, 1H), 7.72 (d, 1H, J=3.7 Hz), 7.10 (d, 1H, J=3.7 Hz), 5.61 (s, 2H), 3.51 (t, 2H, J=8.2 Hz), 0.81 (t, 2H, J=8.2 Hz), 0.13 (s, 9H); C₁₅H₂₁N₅OSi (MW, 315.45), LCMS (EI) m/e 316 (M⁺+H).

Racemic 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound). Method A. 3-Cyclopentylacrylonitrile (8, 273.5 g, 2.257 mol, 1.20 equiv) and DBU (28 mL, 0.187 mol, 0.10 equiv) was added to a suspension of 4-(1H-pyrazol-4-yl)-7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (5, 591.8 g, 1.876 mol) in acetonitrile (4.7 L) at room temperature. The resulting reaction mixture was heated to 50-60° C. for 17 hours (a clear solution developed midway through heating) then to 70-80° C. for 8 hours. When LCMS analysis showed the reaction was deemed complete, the reaction mixture was cooled to room temperature. The cooled solution was then concentrated under reduced pressure to give the crude product (9) as a thick amber oil. The crude product was dissolved in dichloromethane (DCM) and absorbed onto silica gel then dry-loaded onto a silica column (3 Kg) packed in 33% EtOAc/heptanes. The column was eluted with 33% EtOAc/heptanes (21 L), 50% EtOAc/heptanes (28 L), 60% EtOAc/heptanes (12 L) and 75% EtOAc/heptanes (8 L). The fractions containing the desired product (9) were combined and concentrated under reduced pressure to generate a yellow oil, which was transferred to a 3 L flask with EtOAc. The solvent was removed under reduced pressure and the residual EtOAc by co-evaporating with heptanes. The residue was further dried under high vacuum for overnight to afford racemic 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound, 800 g, 819.1 g theoretical, 97.7% yield) as an extremely viscous yellow oil. For 9: ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 8.83 (s, 1H), 8.75 (s, 1H), 8.39 (s, 1H), 7.77 (d, 1H, J=3.7 Hz), 7.09 (d, 1H, J=3.7 Hz), 5.63 (s, 2H), 4.53 (td, 1H, J=19.4, 4.0 Hz), 3.51 (t, 2H, J=8.1 Hz), 3.23 (dq, 2H, J=9.3, 4.3 Hz), 2.41 (m, 1H), 1.79 (m, 1H), 1.66-1.13 (m, 7H), 0.81 (t, 2H, J=8.2 Hz), 0.124 (s, 9H); C₂₃H₃₂N₆OSi (MW, 436.63), LCMS (EI) m/e 437 (M⁺+H) and 459 (M⁺+Na).

(3R)-Cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo [2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10) and (3S)-Cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((S)-10) A slurry of 1.5 Kg of 20-micron Chiralcel® OD chiral stationary phase (CSP) made by Daicel in 3.0 L of isopropanol (IPA) was packed into a PROCHROM Dynamic Axial Compression Column LC110-1 (11 cm ID×25 cm L; Column Void Vol.: approximate 1.5 L) under 150 bar of packing pressure. The packed column was then installed on a Novasep Hipersep HPLC unit. The column and the Hipersep unit were flushed with methanol (17 L) followed by the mobile phase made of a mixture of isopropanol and hexane (2:8 by volume, 17 L). The feed solution was then prepared by dissolving 3-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile (9, racemic SEM-protected compound, 2795 g, 6.4 mol) in the mobile phase to a concentration of 80 g/L. The feed solution was then sequentially injected into the preparative chiral column for separation. Each injection was 120 ml in volume. The chiral column was eluted with the mobile phase at a flow rate of 570 mL/min at room temperature. The column elution was monitored by UV at a wavelength of 330 nm. Under these conditions a baseline separation of the two enantiomers was achieved. The retention times were 16.4 minutes (Peak 1, the undesired (S)-enantiomer (S)-10) and 21.0 minutes (Peak 2, the desired (R)-enantiomer (R)-10), respectively. The cycle time for each injection was 11 minutes and a total of 317 injections were performed for this separation process. Fractions for Peak 1 (the undesired (S)-enantiomer, (S)-10) and Peak 2 (the desired (R)-enantiomer, (R)-10) were collected separately from each injection. The collected fractions collected were continuously concentrated in the 1-square feet and 2-square feet ROTOTHERM evaporator, respectively, at 40° C. under reduced pressure (40-120 bar). The residue from each evaporator was further dried under high vacuum to constant weight to afford (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10, 1307 g, 1397.5 g theoretical, 93.5%) from Peak 2 as a light yellow oil and (3S)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((S)-10, 1418 g, 1397.5 g theoretical, 101.5%) from Peak 1 as an yellow oil.

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base). Method A. To a solution of (3R)-cyclopentyl-3-{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-yl}propionitrile ((R)-10, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L) was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF₄, 987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The reaction temperature was observed to decrease from ambient to 12° C. upon addition of the water and then increase to 33° C. during the addition of lithium tetrafluoroborate (LiBF₄). The resulting reaction mixture was heated to reflux (about 80° C.) for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC analyses showed both the hydroxyl methyl intermediate ((R)-11) and fully de-protected material ((R)-12, free base) produced but no starting material ((R)-10) left, the reaction mixture was cooled gradually to <5° C. before a 20% aqueous solution of ammonium hydroxide (NH₄OH, 450 mL) was added gradually to adjust the pH of the reaction mixture to 9 (checked with pH strips). The cold bath was removed and the reaction mixture was gradually warmed to room temperature and stirred at room temperature for overnight. An aliquot was quenched into ethyl acetate/water and checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v) to confirm complete de-protection. When LCMS and TLC showed the reaction was deemed complete, the reaction mixture was filtered and the solids were washed with acetonitrile (1 L). The combined filtrates were then concentrated under reduce pressure, and the residue was partitioned between ethyl acetate (6 L) and half-saturated brine (3 L). The two layers were separated and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layers were washed with half-saturated sodium bicarbonate (NaHCO₃, 3 L) and brine (3 L), dried over sodium sulfate (Na₂SO₄), and concentrated under reduced pressure to give the crude product as an orange oil. The crude material was then purified by flash column chromatography (SiO₂, 40 to 100% ethyl acetate/heptane gradient elution) to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base, 273 g, 324.9 g theoretical, 84% yield) as a white foam. This material was checked by ¹⁹F NMR to ensure no lithium tetrafluoroborate (LiBF₄) remained, and by chiral HPLC (Chiralcel® OD-H, 90:10 hexane/ethanol) to confirm enantiomeric purity (98.7% ee), and was used without further purification to prepare the corresponding phosphate salt. For (R)-12 (free base): ¹H NMR (DMSO-d₆, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J=0.42 Hz), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, 1H, J=2.34, 3.51 Hz), 6.98 (dd, 1H, J=1.40, 3.44 Hz), 4.53 (td, 1H, J=19.5, 4.63 Hz), 3.26 (dd, 1H, J=9.77, 17.2 Hz), 3.18 (dd, 1H, J=4.32, 17.3 Hz), 2.40 (m, 1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H); C₁₇H₁₈N₆(MW, 306.37) LCMS (EI) m/e 307 (M⁺+H).

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (R)-10. A solution of (R)-3-cyclopentyl-3-(4-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile ((R)-10, 75.0 g, 0.172 mol, 98.8% ee) in acetonitrile (600 mL) was cooled to 0-5° C. To the cooled solution was added boron trifluoride diethyl etherate (54.4 mL, 0.429 mol) over 10 minutes while maintaining the internal reaction temperature below 5° C. Following the addition, the cold bath was removed and the reaction mixture was allowed to warm to room temperature. When HPLC analysis indicated that the level of (R)-10 was below 1%, the initial phase of the deprotection reaction was considered complete. The reaction was then cooled to 0-5° C., followed by the slow addition of water (155 mL). Following the water addition, the cold bath was removed and the resulting reaction mixture was allowed to warm to 13-17° C., and stirred for an additional 2-3 hours. The resulting reaction mixture was cooled again to 0-5° C. To the cooled reaction mixture was added slowly a solution of ammonia in water [prepared by mixing aqueous 28% ammonia solution (104.5 mL) and water (210.5 mL)] while maintaining the internal reaction temperature at below 5° C. After the aqueous ammonia solution was added, the cold bath was removed and the reaction was allowed to warm to room temperature. The hydrolysis was deemed complete when the level of the hydroxylmethyl intermediate was below 1% by HPLC analysis.

The resulting reaction mixture was diluted with ethyl acetate (315 mL) and washed with 20% brine (315 mL). The aqueous fraction was back extracted with ethyl acetate (315 mL). The organic fractions were combined and concentrated under vacuum with a bath temperature of 40° C. to a volume of 380 mL. The concentrated residue was diluted with ethyl acetate (600 mL) and washed with 1M NaHCO₃(2×345 mL) and 20% brine (345 mL). The aqueous washes were combined and back extracted with ethyl acetate (345 mL). The organic fractions were combined and polish filtered into a clean 2L round bottom flask. The organic fraction was washed with warm water (50° C., 2×450 mL) and then treated with activated charcoal at 65° C. with stirring for 1.5 hours. The slurry was filtered through a celite bed. The filtrate was concentrated under vacuum with a bath temperature of 40° C. The resulting syrup was placed under high vacuum to provide (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile [(R)-12, 54.2g, 103% yield] as a light yellow foam. This material was checked by ¹⁹F NMR to ensure that the product was not contaminated by any fluorinated impurities. The chemical purity of the isolated free base was 96.3%. The chiral purity of the free base was 98.8% by chiral HPLC (chiralcel OD, 90:10 hexane/ethanol). The free base was used without further purification to prepare the phosphate salt. ¹H NMR (DMSO-d₆, 400 MHz) δ 12.11(bs, 1H), 8.79(d, 1H, J=0.43 Hz), 8.67(s, 1H), 8.37(s, 1H), 7.59(q, 1H, J=2.3 Hz), 6.98(q, 1H, J=1.6 Hz), 4.53(td, 1H, J=19.2, 4.1 Hz), 3.22(dq, 2H, J=9.8, 4.3 Hz), 2.40(m, 1H), 1.79(m, 1H), 1.65-1.13(m, 7H). C₁₇H₁₆N₆(MW, 306.37), LCMS (EI) m/e 307 (M⁺+H).

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile phosphate salt ((R)-13, phosphate).

Method A. To a solution of (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile ((R)-12, free base, 572 g, 1.87 mol) in isopropanol (IPA, 8 L) at 60-65° C. was added a solution of phosphoric acid (186.2 g, 1.9 mol, 1.10 equiv) in isopropanol (1.6 L). No exotherm was observed while adding a solution of phosphoric acid, and a precipitate was formed almost immediately. The resulting mixture was then heated at 76° C. for 1.5 hours, then cooled gradually to ambient temperature and stirred at room temperature for overnight. The mixture was filtered and the solids were washed with a mixture of heptanes and isopropanol (1/1, v/v, 3 L) before being transferred back to the original flask and stirred in heptanes (8 L) for one hour. The solids were collected by filtration, washed with heptanes (1 L), and dried in a convection oven in vacuum at 40° C. to a constant weight to afford (3R)-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile phosphate salt ((R)-13, phosphate, 634.2 g , 755 g theoretical, 84% yield) as white to off-white crystalline solids. For (R)-13, phosphate: mp. 197.6° C.; ¹H NMR (DMSO-d₆, 500 MHz) δ ppm 12.10 (s, 1H), 8.78 (s, 1H), 8.68 (s, 1H), 8.36 (s 1H), 7.58 (dd, 1H, J=1.9, 3.5 Hz), 6.97 (d, 1H, J=3.6 Hz), 4.52 (td, 1H, J=3.9, 9.7 Hz), 3.25 (dd, 1H, J=9.8, 17.2 Hz), 3.16 (dd, 1H, J=4.0, 17.0 Hz), 2.41, (m, 1H), 1.79 (m, 1H), 1.59 (m, 1H), 1.51 (m, 2H), 1.42 (m, 1H), 1.29 (m, 2H), 1.18 (m, 1H); ¹³C NMR (DMSO-d₆, 125 MHz) δ ppm 152.1, 150.8, 149.8, 139.2, 131.0, 126.8, 120.4, 118.1, 112.8, 99.8, 62.5, 44.3, 29.1, 29.0, 24.9, 24.3, 22.5; C₁₇H₁₈N₆(MW, 306.37 for free base) LCMS (EI) m/e 307 (M⁺+H, base peak), 329.1 (M⁺+Na).

Method B. To a solution of (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H -pyrazol-1-yl)-3-cyclopentylpropanenitrile ((R)-12, 54.2 g, 177 mol) in dichloromethane (782 mL) and 2-propanol (104 mL) at reflux was added a solution of phosphoric acid (19.9 g, 0.173 mol, 1.15 equiv) in 2-propanol (34.0 mL) over a period of 47 minutes. Following the acid addition, the resulting mixture was heated to reflux for an additional 1 hour. The mixture was gradually cooled to ambient temperature and stirred for 3 hours. The solids were collected by filtration and washed with dichloromethane (390 mL), followed by n-heptane (390 mL). The solids were partially dried under vacuum at room temperature and then under vacuum at 62° C. to afford (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate (60.1 g, 84% yield) as white to off-white crystalline solids. Analysis by chiral HPLC (chiralcel OD, 90:10 hexane/ethanol) gave the enantiopurity as 99.2% ee.

¹H NMR (DMSO-d₆, 400 MHz) δ 12.11(bs, 1H), 8.79(d, 1H, J=0.59 Hz), 8.67(s, 1H), 8.36(s, 1H), 7.59(q, 1H, J=2.3 Hz), 6.98(q, 1H, J=1.6 Hz), 4.53(td, 1H, J=19.6, 4.4 Hz), 3.22(dq, 2H, J=9.6, 4.3 Hz), 2.40(m, 1H), 1.79(m, 1H), 1.65-1.13(m, 7H). C₁₇H₂₁N₆O₄P (MW, 404.36), LCMS (EI) m/e 307 (M⁺+H) and m/e 329 (M⁺+Na).

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate.

Into a 1L round bottom flask, equipped with stir bar, distillation head, addition funnel and heating mantle, were charged methanol (520 mL) and (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate ((R)-13, phosphate, 40.0 grams, 98.92 mmol). The slurry was heated to 55° C. to generate a slightly pink solution. The solution was cooled to 50° C. and filtered into a 2 L flask equipped with an overhead stirrer, distillation head, addition funnel and heating mantle. The 1 L round bottom flask and the filter funnel were rinsed with additional methanol (104.0 mL). The filtrate solution was heated to reflux to distill methanol (281 mL) over 1 hour under atmospheric pressure. Isopropyl alcohol (IPA) (320 mL) was charged slowly via the addition funnel over 80 minutes while maintaining the internal temperature approximately at 65° C. Precipitation of the phosphate salt was observed during IPA addition. After the addition of IPA was complete, n-heptane (175 mL) was added slowly at the same temperature. Distillation was continued under atmospheric pressure. Additional n-heptane (825 mL) was added at approximately the same rate as the distillation rate while maintaining the internal temperature at approximately 65° C. The distillation was complete when the volume of the distillate reached 742 mL (excluding the volume of 281 mL of methanol from the previous distillation). The distillation took approximately 1 hour. The vapor temperature during the distillation was in the range of 54-64° C. and the internal temperature was 67° C. at the end of the distillation. The mixture was slowly cooled to room temperature and stirred for an additional 3 hours. The solids were collected by filtration. The wet cake was washed with 16.7% (v/v) of isopropyl alcohol in n-heptane (384.0 mL), followed by n-heptane (280.0 mL), and dried under vacuum at 55° C. to provide 36.1 grams of the desired product as white solids in 90% yield. The chemical purity is 99.79% by HPLC analysis. The chiral purity is 99.8% by chiral HPLC analysis.

¹H NMR (499.7 MHz, DMSO-d6) δ (ppm): 12.21 (s, 1H), 10.71 (s, 3H), 8.80 (s, 1H), 8.72 (s, 1H), 8.40 (s, 1H), 7.60 (d, J=3.5 Hz, 1H), 7.00 (d, J=3.5 Hz, 1H), 4.51 (td, J=9.75, 4.0 Hz, 1H), 3.25 (dd, J=17.3, 9.75 Hz, 1H), 3.14 (dd, J=17.0, 4.0 Hz, 1H), 2.43-2.35 (m, 1H), 1.79-1.73 (m, 1H), 1.58-1.42 (m, 3H), 1.41-1.33 (m, 1H), 1.30-1.23 (m, 2H), 1.19-1.12 (m, 1H);

¹³C NMR (125.7 MHz, DMSO-d6) δ (ppm): 152.8, 151.2, 150.3, 140.0, 131.8, 127.7, 120.8, 118.8, 113.5, 100.7, 63.3, 45.0, 29.8, 25.6, 25.0, 23.2;

LCMS m/z: calculated for C₁₇H₁₈N₆(M+H)⁺:=307.2. Found (M+H)⁺: 307.0.

……………………….

US8410265

(JAK1, JAK2) inhibitor, developed by the Incyte Corporation, trade name Jakafi.
Ruxolitinib synthetic route as shown below. 4 – bromo-pyrazole ( 1 ) with ethyl vinyl ether ( 2 ) to protect, and then with a Grignard reagent to a halogen – exchanged with isopropyl magnesiumpinacol ester ( 3 ) quenching to obtain 4 . Compound 5 is obtained consisting of hydrogen is protected 6 , and then with a boronic acid ester 4 Suzuki coupling occurs under acidic conditions after removal of the protecting group pyrazolyl 7 , 7 and α, β-unsaturated aldehyde 8 chiral catalyst 9 of under the catalysis of asymmetric Michael addition to give ( R ) -10 (90% EE). ( R) -10 , after reaction with ammonia to obtain an imine oxidation with iodine nitrile 11 , respectively, with different conditions for the final removal of the protecting group to afford Ruxolitinib.

Ruxolitinib <wbr> 2011 年 11 月 FDA approved drugs treat myelofibrosis

…………………………………

Bioorganic and Medicinal Chemistry Letters, 2013 , vol. 23, # 9 p. 2793 – 2800

http://www.sciencedirect.com/science/article/pii/S0960894X13001868

Figure 1.

Structures of tofacitinib (1a) and ruxolitinib (1b).

………………………….

Organic Letters, 2009 , vol. 11, 9 p. 1999 – 2002

http://pubs.acs.org/doi/abs/10.1021/ol900350k

An enantioselective synthesis of INCB018424 via organocatalytic asymmetric aza-Michael addition of pyrazoles (16 or 20) to (E)-3-cyclopentylacrylaldehyde (23) using diarylprolinol silyl ether as the catalyst was developed. Michael adducts (R)-24 and (R)-27 were isolated in good yield and high ee and were readily converted to INCB018424

http://pubs.acs.org/doi/suppl/10.1021/ol900350k/suppl_file/ol900350k_si_001.pdf

COMPD 1 IS RUXOLITINIB

(3R)-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol
-1-yl]propionitrile (1, INCB018424).

. Method A. To a solution of (3R)-cyclopentyl-3-
{4-[7-(2-trimethylsilanylethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]pyrazol-1-
yl}proprionitrile ((R)-25, INCB032306, 463 g, 1.06 mol, 98.6% ee) in acetonitrile (4.5 L)
was added water (400 mL) followed immediately by lithium tetrafluoroborate (LiBF4,
987.9 g, 10.5 mol, 10.0 equiv) at room temperature. The resulting reaction mixture was
heated to reflux for overnight. An aliquot was quenched into ethyl acetate/water and
checked by LCMS and TLC (95:5 ethyl acetate/methanol, v/v). When LCMS and TLC
analyses indicated that both the hydroxyl methyl intermediate (R)-26, INCB021499) and
the fully deprotected product (1, INCB018424) SEE LINKhttp://pubs.acs.org/doi/suppl/10.1021/ol900350k/suppl_file/ol900350k_si_001.pdf

:1H NMR
(DMSO-d6, 400 MHz) δ ppm 12.1 (bs, 1H), 8.80 (d, 1H, J = 0.4 Hz), 8.67 (s, 1H), 8.37
(s, 1H), 7.59 (dd, 1H, J = 2.3, 3.5 Hz), 6.98 (dd, 1H, J = 1.4, 3.4 Hz), 4.53 (td, 1H, J =
19.5, 4.6 Hz), 3.26 (dd, 1H, J = 9.8, 17.2 Hz), 3.18 (dd, 1H, J = 4.3, 17.3 Hz), 2.40 (m, S-12
1H), 1.79 (m, 1H), 1.65 to 1.13 (m, 7H);

13C NMR (DMSO-d6, 100MHz) δ ppm 152.1,
151.0, 149.9, 139.3, 131.0, 126.8, 120.6, 118.2, 112.8, 99.8, 62.5, 44.3, 29.1, 25.0, 24.3,
22.5; IR (KBr) 3197, 3118, 2956, 2865, 1731, 1581, 1448, 1344, 1251 cm-1;

HRMS (CI)
m/z calculated for C17H19N6 (M + H)+
307.1671, found 307.1665

REFERENCES

Jakafi (ruxolitinib) dosing, indications, interactions, adverse effects, and more”. Medscape Reference. WebMD. Retrieved 16 February 2014.
Mesa, Ruben A.; Yasothan, Uma; Kirkpatrick, Peter (2012). “Ruxolitinib”. Nature Reviews Drug Discovery 11 (2): 103–4.doi:10.1038/nrd3652. PMID 22293561.
Harrison, C; Mesa, R; Ross, D; Mead, A; Keohane, C; Gotlib, J; Verstovsek, S (2013). “Practical management of patients with myelofibrosis receiving ruxolitinib”. Expert Review of Hematology 6 (5): 511–23. doi:10.1586/17474086.2013.827413. PMID 24083419.
Natoli, Cori Anne (May 5, 2012), “Incyte looks to ride on drug’s success”, The News Journal, retrieved May 6, 2012
Harrison, C.; Kiladjian, J. J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.; Stalbovskaya, V.; McQuitty, M.; Hunter, D. S.; Levy, R.; Knoops, L.; Cervantes, F.; Vannucchi, A. M.; Barbui, T.; Barosi, G. (2012). “JAK Inhibition with Ruxolitinib versus Best Available Therapy for Myelofibrosis”. New England Journal of Medicine 366 (9): 787–798.doi:10.1056/NEJMoa1110556. PMID 22375970. edit
Verstovsek, S.; Mesa, R. A.; Gotlib, J.; Levy, R. S.; Gupta, V.; Dipersio, J. F.; Catalano, J. V.; Deininger, M.; Miller, C.; Silver, R. T.; Talpaz, M.; Winton, E. F.; Harvey Jr, J. H.; Arcasoy, M. O.; Hexner, E.; Lyons, R. M.; Paquette, R.; Raza, A.; Vaddi, K.; Erickson-Viitanen, S.; Koumenis, I. L.; Sun, W.; Sandor, V.; Kantarjian, H. M. (2012). “A Double-Blind, Placebo-Controlled Trial of Ruxolitinib for Myelofibrosis”. New England Journal of Medicine 366 (9): 799–807. doi:10.1056/NEJMoa1110557.PMID 22375971. edit
Tefferi, A. (2012). “Challenges Facing JAK Inhibitor Therapy for Myeloproliferative Neoplasms”. New England Journal of Medicine 366(9): 844–846. doi:10.1056/NEJMe1115119. PMID 22375977. edit
ASCO Annual Meeting 2011: JAK Inhibitor Ruxolitinib Demonstrates Significant Clinical Benefit in Myelofibrosis
Mesa, RA (2010). “Ruxolitinib, a selective JAK1 and JAK2 inhibitor for the treatment of myeloproliferative neoplasms and psoriasis”. IDrugs : the investigational drugs journal 13 (6): 394–403.PMID 20506062. edit
Pardanani, A.; Tefferi, A. (2011). “Targeting myeloproliferative neoplasms with JAK inhibitors”. Current Opinion in Hematology 18 (2): 1. doi:10.1097/MOH.0b013e3283439964. PMID 21245760. edit
Wysham, NG; Allada G, Sullivan DR (2013). Chest 143 (5): 1478–9.PMID 23648912.
“FDA Approves Incyte’s Jakafi(TM) (ruxolitinib) for Patients with Myelofibrosis” (Press release). Incyte. Retrieved 2012-01-02.
Harrison, C.; Kiladjian, J.-J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.;Stalbovskaya, V.; McQuitty, M.; Hunter, D. S.; Levy, R.; Knoops, L.;Cervantes, F.; Vannucchi, A. M.; Barbui, T.; Barosi, G. N. Eng. J. Med. 2012,366, 787.Zhou, J.; Liu, P.; Lin, Q.; Metcalf, B. W.; Meloni, D.; Pan, Y.; Xia, M.; Li, M.; Yue,T.-Y.; Rodgers, J. D.; Wang, H. WO 2010083283 A2, 2010.Rodgers, J. D.; Shepard, S.; Maduskuie, T. P.; Wang, H.; Falahatpisheh, N.;Rafalski, M.; Arvanitis, A. G.; Storace, L.; Jalluri, R. K.; Fridman, J. S.; Vaddi, K.U.S. 20070135461 A1, 2007.Lin, Q.; Meloni, D.; Pan, Y.; Xia, M.; Rodgers, J.; Shepard, S.; Li, M.; Galya, L.;Metcalf, B.; Yue, T.-Y.; Liu, P.; Zhou, J. Org. Lett. 1999, 2009, 11.http://www.google.com/patents/US8410265

US7598257 *	Dec 12, 2006	Oct 6, 2009	Incyte Corporation	Heteroaryl substituted pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrimidines as janus kinase inhibitors
US20090233903 *	Mar 10, 2009	Sep 17, 2009	Incyte Corporation	Azetidine and cyclobutane derivatives as jak inhibitors
WO2007070514A1 *	Dec 12, 2006	Jun 21, 2007	Incyte Corp	Heteroaryl substituted pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrimidines as janus kinase inhibitors
WO2007117494A1 *	Apr 5, 2007	Oct 18, 2007	Vertex Pharma	Deazapurines useful as inhibitors of janus kinases

US8309718	Nov 13, 2008	Nov 13, 2012	Incyte Corporation	4-pyrazolyl-N-arylpyrimidin-2-amines and 4-pyrazolyl-N-heteroarylpyrimidin-2-amines as janus kinase inhibitors
US8410265	Jan 14, 2010	Apr 2, 2013	Incyte Corporation	Processes for preparing JAK inhibitors and related intermediate compounds
US8415362	Jun 12, 2008	Apr 9, 2013	Incyte Corporation	Pyrazolyl substituted pyrrolo[2,3-b]pyrimidines as Janus kinase inhibitors
US8486902	Oct 8, 2010	Jul 16, 2013	Incyte Corporation	Hydroxyl, keto, and glucuronide derivatives of 3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile
US8530485	Mar 30, 2011	Sep 10, 2013	Incyte Corporation	Heteroaryl substituted pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrimidines as Janus kinase inhibitors
US8541425	Aug 27, 2009	Sep 24, 2013	Incyte Corporation	Heteroaryl substituted pyrrolo[2,3-b]pyridines and pyrrolo[2,3-b]pyrimidines as Janus kinase inhibitors
US8604043	May 21, 2010	Dec 10, 2013	Incyte Corporation	3-[4-(7H-pyrrolo[2,3-D]pyrimidin-4-yl)-1H-pyrazol-1-yl]octane- or heptane-nitrile as jak inhibitors

RIDAFOROLIMUS

Uncategorized Comments Off

Mar 072014

Ridaforolimus

572924-54-0

(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^4,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

42-(dimethylphosphinate) Rapamycin

Deforolimus, MK-8669, AP-23573, S1022_Selleck, AP23573, AP23573, MK-8669, Ridaforolimus, Deforolimus, 572924-54-0, MK 8669

AP 23573
AP23573
Deforolimus
MK 8669
MK-8669
MK8669
Ridaforolimus
Taltorvic
UNII-48Z35KB15K

Molecular Formula: C₅₃H₈₄NO₁₄P Molecular Weight: 990.206122

An mTOR inhibitor for the treatment of cancer.

Ridaforolimus (MK-8669; AP23573; formerly Deforolimus)

Merck, under exclusive worldwide license agreement with Ariad Pharmaceuticals

Method of Action: Oral inhibitor of mammalian target of rapamycin inhibitor (mTOR)

Indications/Phase of Trial: Maintenance therapy for metastatic soft-tissue sarcoma and bone sarcomas after at least four chemotherapy cycles (under review after receiving Complete Response letter from FDA in June; NME)

Ridaforolimus is an investigational small-molecule inhibitor of the protein mTOR, a protein that acts as a central regulator of protein synthesis, cell proliferation, cell cycle progression and cell survival, integrating signals from proteins, such as PI3K, AKT and PTEN, known to be important to malignancy.

TARGET- mTOR

Ridaforolimus (also known as AP23573 and MK-8669; formerly known as Deforolimus^[1]) is an investigational targeted and small-molecule inhibitor of the protein mTOR, a protein that acts as a central regulator of protein synthesis, cell proliferation, cell cycle progression and cell survival, integrating signals from proteins, such as PI3K, AKT and PTEN known to be important to malignancy. Blocking mTOR creates a starvation-like effect in cancer cells by interfering with cell growth, division, metabolism, and angiogenesis.

It has had promising results in a clinical trial for advanced soft tissue and bone sarcoma.

RIDAFOROLIMUS

NMR….http://file.selleckchem.com/downloads/nmr/S102201-Deforolimus-HNMR-Selleck.pdf

HPLC . http://file.selleckchem.com/downloads/hplc/S102201-Deforolimus-HPLC-Selleck.pdf

MSDS..http://www.selleckchem.com/msds/Deforolimus-MSDS.html

Commercial arrangements

Ridaforolimus is being co-developed by Merck and ARIAD Pharmaceuticals. On May 5, 2010, Ariad Pharmaceuticals and Merck & Company announced a clinical development and marketing agreement. With this agreement, Ariad received $125 million in upfront payments from Merck and $53 million in milestone payments. Future payments are triggered upon acceptance of the NDA by the FDA with another payment when the drug receives marketing approval. There are similar milestones for acceptance and approval in both Europe and Japan. Other milestone payments are tied to revenue goals for the drug.^[2] ARIAD has opted to co-promote ridaforolimus in the U.S. Merck plans to submit a New Drug Application (NDA) for ridaforolimus to the U.S. Food and Drug Administration (FDA) and a marketing application in the European Union in 2011.^[3]

Clinical trials

Phase III SUCCEED

On June 6, 2011, Ariad and Merck announced detailed results from the largest randomized study ever in the soft tissue and bone sarcoma population, the Phase III SUCCEED clinical trial. SUCCEED evaluated oral ridaforolimus, in patients with metastatic soft-tissue or bone sarcomas who previously had a favorable response to chemotherapy. In this patient population, ridaforolimus improved progression-free survival (PFS) compared to placebo, the primary endpoint of the study. The complete study results were presented by Sant P. Chawla, M.D., director, Sarcoma Oncology Center, Santa Monica, CA, during the 2011 American Society of Clinical Oncology (ASCO) annual meeting.
The SUCCEED (Sarcoma Multi-Center Clinical Evaluation of the Efficacy of Ridaforolimus) trial was a randomized (1:1), placebo-controlled, double-blind study of oral ridaforolimus administered at 40 mg/day (five of seven days per week) in patients with metastatic soft-tissue or bone sarcomas who previously had a favorable response to chemotherapy. Oral ridaforolimus was granted a Special Protocol Assessment (SPA) by the FDA for the SUCCEED trial.
Based on 552 progression-free survival (PFS) events in 711 patients, (ridaforolimus (N=347), placebo (N=364) determined by an independent radiological review committee, the study achieved its primary endpoint of improvement in PFS, with a statistically significant (p=0.0001) 28 percent reduction in the risk of progression or death observed in those treated with ridaforolimus compared to placebo (hazard ratio=0.72).

Median PFS was 17.7 weeks for those treated with ridaforolimus compared to 14.6 weeks in the placebo group. Furthermore, based on the full analysis of PFS determined by investigator assessment, there was a statistically significant (p<0.0001) 31 percent reduction by ridaforolimus in the risk of progression or death compared to placebo (hazard ratio=0.69). In the investigator assessment analysis, median PFS was 22.4 weeks for those treated with ridaforolimus compared to 14.7 weeks in the placebo group ^[4

EU WITHDRAWAL IN NOV 2012

Merck, known as MSD outside the U.S. and Canada, announced today that it has formally notified the European Medicines Agency (EMA) of Merck’s decision to withdraw the Marketing Authorisation Application (MAA) for ridaforolimus.

The application for Marketing Authorisation for ridaforolimus was accepted by the EMA in August 2011. At the time of the withdrawal it was under review by the Agency’s Committee for Medicinal Products for Human Use (CHMP). In its letter to the EMA, Merck said that the withdrawal of ridaforolimus was based on the provisional view of the CHMP that the data available to date and provided in the Marketing Authorisation Application were not sufficient to permit licensure of ridaforolimus in the European Union for the maintenance treatment of patients with soft tissue sarcoma or primary malignant bone tumor.

Although the application for these uses was withdrawn, Merck is studying ridaforolimus in combination with other drugs in other tumor types. The withdrawal of the European application of ridaforolimus for the maintenance treatment of patients with soft tissue sarcoma or primary malignant bone tumor does not change Merck’s commitment to the ongoing clinical trials with ridaforolimus.

Ridaforolimus

Description

42-(dimethylphosphinate) Rapamycin (Ridaforolimus) represented by the following formula I:

2. Description of RelatedArt

The mammalian target of Rapamycin (mTOR) is known as a mechanistic target of Rapamycin (H), which is found in the studies of Rapamycin. On the other hand, 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) is a derivative of Rapamycin (II), which is also a kind of mTOR inhibitor. Ridaforolimus (I) can inhibit cell division and possibly lead to tumor cell death. Hence, there are many studies related to solid tumor treatments and blood cancer treatments using Ridaforolimus (I). In addition, in 2011, Merck also applied a certification of this compound against soft tissue and bone cancer.

U.S. Pat. No. 7,091,213 discloses a process for preparing 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I), and the process thereof is shown in the following Scheme I.

In this process, a solution of Rapamycin (II) in dichloromethane (DCM) was respectively added with 2,6-di-tert-butyl-4-methylpyridine or 3,5-lutidine as a base, and followed by the addition of a solution of dimethylphosphinic chloride (DMP-Cl) to perform a phosphorylation reaction at 0° C., under a stream of N_2(g). The crude product was purified by flash chromatography (eluted with MeOH/DCM/EtOAc/hexane=1:10:3:3) to provide 42-(dimethyl- phosphinate) Rapamycin (Ridaforolimus) (I), which is a phosphorylated compound at 42-hydroxyl position of Rapamycin (II). In addition, this patent also disclosed a side product of 31,42-bis(dimethyl phosphinate) Rapamycin (III), which is a phosphorylated compound at both 31- hydroxyl position and 42- hydroxyl position of Rapamycin (II).

…………………..

SYNTHESIS

US7091213

Some additional transformations of potential interest to the practitioner are shown below, including the preparation of reagents for generating the described C-43 phosphorus-containing rapalogs:

Preparation of Diakyl/diaryl Chlorophoshates

Preparation of Alkyl Halide Phosphonates

Illustrative routes for using the foregoing sorts of reagents to prepare certain rapalogs of this invention are shown below.

The synthesis of compounds of this invention often involves preparation of an activated form of the desired moiety “J”, such as a phosphoryl chloride as shown above (e.g. (R)(RO)P—Cl or RR′P(═O)—Cl, etc), and reaction of that reagent with rapamycin (or the appropriate rapalog) under conditions yielding the desired product, which may then be recovered from residual reactants and any undesired side products. Protecting groups may be chosen, added and removed as appropriate using conventional methods and materials.

Purification of Compounds of the Invention

A variety of materials and methods for purifying rapamycin and various rapalogs have been reported in the scientific and patent literatures and may be adapted to purification of the rapalogs disclosed herein. Flash chromatography using a BIOTAGE prepacked cartridge system has been particularly effective. A typical protocol is disclosed in the Examples which follow.

Physicochemical Characterization of Compounds of the Invention

The identity, purity and chemical/physical properties of the rapalogs may be determined or confirmed using known methods and materials, including HPLC, mass spectral analysis, X ray crystallography and NMR spectroscopy. High resolution 1D ¹H and ³¹P NMR spectra acquired using a typical relaxation delay of 3 seconds have proved useful, as has reverse phase HPLC analysis (analytical column, 3 micron particle size, 120 ansgstrom pore size, thermostatted to 50° C. with a mobile phase of 50% acetonitrile, 5% methanol and 45% water (all % s by volume), for example, in an isocratic elution system, with elution of product and impurity peaks followed by UV detection at 280 nanometers). Normal phase HPLC may also be used, especially to evaluate the level of residual rapamycin or rapalog by-products. The presence of residual solvent, heavy metals, moisture and bioburden may be assessed using conventional methods.

Example 9

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

Dimethyl-phosphinic Acid C-43 Rapamycin Ester

To a cooled (0° C.) solution of rapamycin (0.1 g, 0.109 mmol) in 1.8 mL of dichloromethane was added 0.168 g (0.82 mmol) of 2,6-di-t-butyl-4-methyl pyridine, under a stream of N₂, followed immediately by a solution of dimethylphosphinic chloride (0.062 g, 0.547 mmol) in 0.2 mL of dichloromethane. The slightly yellow reaction solution was stirred at 0° C., under an atmosphere of N₂, for 3.5 h (reaction monitored by TLC). The cold (0° C.) reaction solution was diluted with ˜20 mL EtOAc then transferred to a separatory funnel containing EtOAc (150 mL) and saturated NaHCO₃(100 mL). Upon removing the aqueous layer, the organic layer was washed successively with ice cold 1N HCl (1×100 mL), saturated NaHCO₃(1×100 mL), and brine (1×100 mL), then dried over MgSO₄and concentrated. The crude product was purified by silica gel flash chromatography (eluted with 1:10:3:3 MeOH/DCM/EtOAc/hexane) to provide 0.092 g of a white solid:

¹H NMR (300 MHz, CDCl₃) d 4.18 (m, 1H), 4.10 (m, 1H), 3.05 (m, 1H), 1.51 (m, 6H);
³¹P NMR (121 MHz, CDCl₃) d 53.6; 1013 m/z (M+Na).

Example 9

Alternative Synthesis

Rapamycin and dichloromethane are charged into a nitrogen-purged reaction flask. The stirred solution is cooled to approximately 0° C. (an external temperature of −5±5° C. is maintained throughout the reaction). A solution of dimethylphosphinic chloride (2.0 molar equivalents) in dichloromethane is then added over a period of approximately 8–13 minutes.

This is followed immediately by the addition of a solution of 3,5-lutidine (2.2 molar equivalents) in dichloromethane over a period of approximately 15–20 minutes. Throughout both additions, the internal temperature of the reaction sssstays below 0° C. The cooled reaction solution is stirred for 1 hour and then transferred, while still cold, to an extractor containing saturated aqueous NaHCO₃and methyl-t-butyl ether (MTBE), ethyl acetate or diethyl ether. In-process samples are removed at 30 and 60 minute time points.

Samples are prepared in a similar fashion to that described for the reaction workup. Reaction progress is monitored by TLC (1:10:3:3 MeOH/DCM/EtOAc/hexanes) and reverse-phase HPLC analyses. The isolated organic layer is successively washed with ice cold 1N HCl, saturated aqueous NaHCO₃(2×), saturated aqueous NaCl, and dried over sodium sulfate. Upon filtration and solvent removal, the residue undergoes solvent exchange with acetone followed by concentration in vacuo to provide crude product, which may be analyzed for purity by normal- and reversed-phase HPLC.

…………………….

SYNTHESIS

US20140058081

The process of the present invention is shown in the following Scheme II.

EXAMPLE 7

Preparation of 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I)

42-(dimethylphosphinate)-31-triethylsilylether Rapamycin (VI-b) (2.312 g, available from 1.945 mmole of Rapamycin -28-triethylsilylether) and tetrahydrofuran (60 mL) was placed into a flask, and the resulting solution was cooled to 0˜−5° C. Next, a sulthric acid solution (2 N, 6 mL) was slowly added into the resulting solution dropwise. When the 42-(dimethylphosphinate)-31-triethylsilylether Rapamycin (VI-b) was less than 2%, ethyl acetate (1000 mL) was added into the resulting solution. Then, the organic layer was successively washed with a NaCl saturated solution (300 mL), a NaHCO₃saturated solution (200 mL) and a NaCl saturated solution (200 mL), dried over anhydrous sodium sulfate and concentrated to obtain a crude product of 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) (2.341 g). The crude product was then purified by Licrhoprep RP-18 silica gel chromatography (eluted with acetonitrile: 0.02 M ammonium formate solution=6:4, wherein the pH of the ammonium formate solution was adjusted to 4.0 with formic acid), extracted with ethyl acetate, concentrated and dried to obtain a white foam solid 42-(dimethylphosphinate) Rapamycin (Ridaforolimus) (I) (1.840 g, purity=99.48%). The yield thereof was 95.55% based on 2.0 g of 31-triethylsilyl ether Rapamycin.

¹H-NMR(400 MHz, CDCl₃)d 4.18(m, 1H), 4.10(m, 1H), 3.05(m, 1H),1.51(m, 6H); ³¹P-NMR(161 MHz, CDCl₃)d 53.33; 1012.6 m/z [M+Na]⁺.

“ARIAD Reports First Quarter 2009 Development Progress and Financial Results- Ridaforolimus New USAN Name to Replace Deforolimus”. ARIAD Pharmaceuticals. 2009. Retrieved 2009-05-07.
“ARIAD – News release”. Phx.corporate-ir.net. Retrieved 2012-10-07.
“ARIAD – News release”. Phx.corporate-ir.net. 2011-03-17. Retrieved 2012-10-07.
“ARIAD – News release”. Phx.corporate-ir.net. 2011-06-06. Retrieved 2012-10-07.

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Cidofovirסידופוביר سيدوفوفير

Uncategorized Comments Off

Mar 072014

CIDOFOVIR

(S)-1-(3-Hydroxy-2-phosphonylmethoxypropyl)cytosine
[(S)-2-(4-Amino-2-oxo-1,2-dihydropyrimidin-2-yl)-1-(hydroxymethyl)ethoxymethyl]phosphonic acid

113852-37-2 CAS

120362-37-0 (Na salt)
149394-66-1 (dihydrate)

launched 1996 Gilead

EMA:Link,

US FDA:link

SYNTHESIS.. CHEMDRUG

Rega Instituut (Originator)

For the treatment of CMV retinitis in patients with acquired immunodeficiency syndrome (AIDS)

US5142051 PATENT

Canada	1340856	1999-12-21	EXPIRY 2016-12-21
United States	5142051	1993-06-26	2010-06-26

Cidofovir is a DNA polymerase inhibitor that was launched in 1996 by Gilead for the intravenous treatment of cytomegaloviral (CMV) retinitis in AIDS patients. Early clinical trials are underway at the National Institute for Allergy & Infectious Disease (NIAID) for the treatment of BK virus nephropathy (BKVN) in patients who have undergone kidney transplants.

Cidofovir suppresses CMV replication by selective inhibition of viral DNA synthesis. Biochemical data support selective inhibition of CMV DNA polymerase by cidofovir diphosphate, the active intracellular metabolite of cidofovir. Cidofovir diphosphate inhibits herpesvirus polymerases at concentrations that are 8- to 600-fold lower than those needed to inhibit human cellular DNA polymerases alpha, beta, and gamma1, 2, 3. Incorporation of cidofovir into the growing viral DNA chain results in reductions in the rate of viral DNA synthesis.

Cidofovir was originally developed under a collaboration between the Academy of Sciences of the Czech Republic and the Rega Institute for Medical Research. In 1991 and 1992, Gilead entered into license agreements with the Rega Institute that covered a large number of nucleotide analogue compounds and structures, including cidofovir. The drug became the subject of a marketing collaboration between Gilead and Pfizer (formerly Pharmacia & Upjohn) in August 1996 that covers all countries outside the U.S.

Cidofovir (brand name Vistide) is an injectable antiviral medication primarily used as a the treatment for cytomegalovirus (CMV) retinitis (an infection of the retina of the eye) in patients with AIDS.^[1]^[2]

Its only indication that has received regulatory approval worldwide is cytomegalovirus retinitis.^[1]^[2] Cidofovir has also shown efficacy in the treatment ofaciclovir-resistant HSV infections.^[3] Cidofovir has also been investigated as a treatment for progressive multifocal leukoencephalopathy with successful case reports of its use.^[4] Despite this meta-analyses have failed to demonstrate any efficacy in AIDS patients,^[5] and the limited data in non-AIDS patients fail to demonstrate any efficacy either.^[6] Cidofovir might have anti-smallpox efficacy and might be used on a limited basis in the event of a bioterror incident involving smallpox cases.^[7] A cidofovir derivative with much higher activity against smallpox that can be taken orally has been developed.^[8] It has inhibitory effects on varicella-zoster virus replication in vitro although no clinical trials have been done to date, likely due to the abundance of safer alternatives such as aciclovir.^[9] Cidofovir shows anti-BK virus activity in a subgroup of transplant patients.^[10] Cidofovir is being investigated as a complementary intralesional therapy against papillomatosis caused by HPV.^[11]^[12]
It first received FDA approval on the 26th of June 1996,^[13] TGA approval on the 30th of April 1998^[2] and EMA approval on the 23rd of April 1997.^[14]

Other

It has been suggested as an antitumour agent, due to its suppression of FGF2.^[15]^[16]

Cidofovir was discovered at the Institute of Organic Chemistry and Biochemistry, Prague, by Antonín Holý, and developed by Gilead Sciences^[20] and is marketed with the brand name Vistide by Gilead in the USA, and by Pfizerelsewhere.

The chemical name of cidofovir is 1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate (HPMPC), with the molecular formula of C8H14N3O6P•2H2O and a molecular weight of 315.22 (279.19 for anhydrous). The chemical structure is:

Cidofovir is a white crystalline powder with an aqueous solubility of ≥ 170 mg/mL at pH 6 to 8 and a log P (octanol/aqueous buffer, pH 7.1) value of -3.3.
Cidofovir Injection is a sterile, hypertonic aqueous solution for intravenous infusion only. The solution is clear and colorless. It is supplied in clear glass vials, each containing 375 mg of anhydrous cidofovir in 5 mL aqueous solution at a concentration of 75 mg/mL.
The formulation is pH-adjusted to 7.4 (range 7.1 to 7.7) with sodium hydroxide and/or hydrochloric acid and contains no preservatives. The appropriate volume of Cidofovir Injection must be removed from the single-use vial and diluted prior to administration

INTRODUCTION

Cidofovir’s chemical formula is C₈H₁₄N₃O₆P and its IUPAC name is ({[(S)-1-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3-hydroxypropan-2-yl]oxy}methyl)phosphonic acid. Cidofovir has also been described as (S)-(1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yloxy)methylphosphonic acid as well as possibly by other chemical names. Its chemical structure is:

Cidofovir was discovered at the Institute of Organic Chemistry and Biochemistry, Prague, and developed by Gilead Sciences. Today, cidofovir is an injectable antiviral medication for the treatment of cytomegalovirus (CMV) retinitis in patients with AIDS. It suppresses CMV replication by selective inhibition of viral DNA polymerase and therefore prevention of viral replication and transcription. It is an acyclic nucleoside phosphonate, and is therefore independent of phosphorylation by viral enzyme, in contrast to, for instance, acyclovir.

Cidofovir is marketed with the brand name Vistide® by Gilead in the United States and by Pfizer in other parts of the world. Vistide® is a sterile, hypertonic aqueous solution for intravenous infusion only. The solution is clear and colorless. It is supplied in clear glass vials, each containing 375 mg of anhydrous cidofovir in 5 mL aqueous solution at a concentration of 75 mg/mL. The formulation is pH-adjusted to 7.4 with sodium hydroxide and/or hydrochloric acid and contains no preservatives. Renal impairment is the major toxicity of Vistide®.

Presently, there are no Orange Book patents listed as having claims which cover Vistide®, although previously U.S. Pat. No. 5,142,051 was listed in the Orange Book for Vistide®. The ‘051 patent is not directed specifically to cidofovir or its crystalline forms. Instead, it broadly discloses N-phosphonylmethoxyalkyl derivatives of pyrimidine and purine bases.

Cytomegalovirus (Cytomegaoviyns, CMV) is one of the biggest dangers of the herpes virus, the body’s infection rates as high as 50% to 80% of the current adult prevalence rate of more than 95%, generally showed a latent infection, most infections had no clinical symptoms, but under certain conditions, the invasion of organs and systems to produce more severe disease. The virus can invade the lung, liver, kidney, salivary gland, mammary gland and other polymorphonuclear leukocytes and lymphocytes, and, since the long-term or intermittent saliva, milk sweat, blood, urine, semen, exclude uterine secretions of the virus. Spread through a variety of ways in the mouth, genital tract, placenta, blood transfusion or organ transplantation.

When the body’s immune dysfunction, such as infected with HIV, cancer patients undergoing radiotherapy, chemotherapy, organ or bone marrow transplantation immunosuppressive anti-rejection etc will stimulate active infection, can cause acute retinitis, interstitial pneumonia, gastroenteritis and encephalitis, blindness or death without treatment rate of over 70%. With the rise in HIV infection rates and organ transplants extensively for anti-CMV drugs is also increasing demand.

cidofovir (cidofovir, HPMPC) are novel ether derivatives of cytidine phosphono chemical name

[5]-NL [(3 – hydroxy-2 – methoxy-phosphonic acid) glycerol]-N4-cytosine, Molecular structure of the formula (I):

Gilead developed by the United States, in May 1996 the FDA approved injectable celecoxib Duofu Wei listed, France and Canada also continued with the approval of the use of the trade name Vistide. Its CAS number is 113852-37-2, formula C8H14N3O6P, the structure of formula (I). Cidofovir for CMV is highly inhibitory activity of certain ganciclovir or foscarnet resistant strains of the virus are also active. And herpes simplex virus (HSV), herpes zoster virus (VZV), human papillomavirus (HPV), also has a strong activity.

Its mechanism of action: cidofovir having a phosphoric acid group, a ring-opening mechanism of the antiviral nucleoside phosphonate compound (ANP) and the consistent cyclic nucleoside analogues are nucleosides or virus in vivo kinase activation into triphosphate metabolite, thereby inhibiting viral replication by DNA polymerase and reverse transcriptase. Unlike the three-step cyclic nucleoside analogues must phosphorylation reaction, ring opening nucleoside phosphonate group containing phosphorus compound itself, eliminating the first step of the phosphorylation reaction speed, and thus a higher activity. Cidofovir is absorbed when the cells in the cell pyrimidine nucleoside phosphorylase kinase (P bandit kinase and NDP kinase) to effect conversion of the active metabolite monophosphate (HPMPCp), diphosphate (HPMPCpp) and a bile acid base adducts. Cidofovir diphosphate inhibits viral DNA polymerase or reverse transcriptase activity, and its corresponding natural dNTP incorporated into the viral DNA chain competition, since no 3 – hydroxy end, continue to extend the DNA chain termination. Can slow the synthesis of DNA, viral DNA and to the loss of stability, thereby inhibiting viral replication, transcription of the ability to reduce viral DNA to exert antiviral activity. Compared with other anti-CMV drugs, cidofovir characteristics: significant and lasting effect, started the first two weeks administered once a week, then only administered once every two weeks, easy to use, and to reduce its toxicity side effects.

Several major techniques are based on the synthesis of cidofovir cytosine as starting material, mainly carried out to improve the synthesis of the side chain.

(I) J. Med Chem, 1989,32,1457 ~ 1463 discloses a synthetic process:

The route to cytosine as the raw material, with a chiral side chain by condensation, deprotection and reduction can be obtained in three steps cidofovir.However, chiral side chain subject to a six-step reaction system. The total yield is low, adverse side. And using Me3SiBr, so that the costs and the risk of surge, is not conducive to industrial production.

(2) US 5591852,1995-1-7; US 2005/023833 & WO 2006/014429 and US 2009/0270618, Tetrahedron Lett 1994,35,3243-3246 and “Chinese Journal of New Drugs”, 2007,16. , 1272-1274 for the synthesis of a lot of improvements:

Benzoyl cytosine with a chiral starting material and trityloxymethyl ethylene oxide condensation, deprotection and hydrolysis was then prepared by deprotection cidofovir group. The synthetic steps to make some shorter, but still use expensive Me3SiBr, adverse ones, the low yield of the security at the cost of industrial production is still unfavorable. (Several different patent protection only in the order of the amino cytosine different!)

(3) Patent Publication No. CN1690065A, CN1690066A, CN1690067A (2005 年 11 月 2 Publication Date) and the “Chinese Journal of Medicinal Chemistry” 2007,17,41-46, reported a new synthetic route:

The route of process steps is too long, the total yield is low, side effects side. But not conducive to industrial production.

(4) Patent No. CN 101205215A (25 June 2008 publicly) announced a halogen epoxy propane as a starting material for the synthesis route:

Use of the route (R) – epihalohydrin reaction with cytosine, cytosine ring because alkaline easily cause epoxy ring-opening reaction of the ring, but side reactions, the purified product is not, nor is suitable for industrial production.

Subsequently, the patent number CN 101525352A (2009 年 9 月 9 Publication Date) discloses (4) based on the modified route through epoxypropionate alkane ether in the form of a direct reaction with cytosine, after a series of similar steps obtain the final product cidofovir.

In view of the clinical application of cidofovir more favorable therapeutic effect in, looking for a high yield and because of economic and practical, easy to control, the risk of small synthetic methods and technology is now more urgent needs.

Synthesis

Brodfuehrer, P; Howell, Henry G.; Sapino, Chester; Vemishetti, Purushotham (1994). “A practical synthesis of (S)-HPMPC”. Tetrahedron Letters 35 (20): 3243. doi:10.1016/S0040-4039(00)76875-4.

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CN 102268040

Figure CN102268040AD00112

, Example 1:

1 Synthesis of 4,4 ‘- dimethoxytrityl methyl – (R) – glycidol (Compound III): The 5 04 g (15 mmoDDMT-Cl grain port 0 20 g (1 52 mmol… ) 4_ dimethylaminopyridine (DMAP) was dissolved in 100 mL CH2C12 cooled to 0 ° C, was added dropwise 10 mL TEA was slowly added 2. 00 g (27mmol) hydroxymethyl chiral oxirane (Compound II ) addition was completed, the reaction warmed to room temperature naturally. fly 4 h, until TLC until the disappearance of the detection DMT-Cl, the reaction was stopped by filtration, the filtrate was washed with saturated NaHC03 solution (50mLX2), saturated NaCl solution (50 mLX2), anhydrous Na2S04 dried, filtered, and concentrated to a viscous colorless directly, i.e., 5 08 g of 4,4 ‘-dimethoxy-triphenylmethyl _ -.. (R) – glycidol (Compound III), yield 90 %, HPLC purity 99%.

2, Synthesis (S)-N1_ [(2 – hydroxy-3 – (dimethoxytrityl) propyl] cytosine (Compound IV):. Under nitrogen to 3 56 g (32 mmol) of cytosine was added 150 mL of anhydrous N, N-dimethylformamide (DMF), and at room temperature, was added portionwise 1. 24 g (31 mmol, molar concentration of 60%) NaH, 0. 5 h after adding 11 92 g (31 mmol) 4,4 ‘-. dimethoxytrityl methyl – (R) – glycidol (Compound III), plus finished warming up to 10 (Tll (TC reaction . 6-8 h and then filtered, and the filtrate evaporated under reduced pressure DMF, the remaining solid phase was added 500 mL of ethyl acetate and 50 mL of water, separated and the organic layer was washed with saturated NaHC03 solution (50 mL X 2), saturated NaCl solution (50 mL X 2), dried over anhydrous Na2S04 filtered and dried, and concentrated to give 13 90 g of a white solid, S Jie (S)-Nl-[(2 -.. hydroxy-3 – (methoxy-dimethoxytrityl ) propyl] cytosine (Compound IV), yield 92%, HPLC purity 98%.

3 Synthesis ⑶-Nl-{[2_ (phosphonic acid methoxy diethoxy) -3 – (methoxy-dimethoxytrityl)] propyl} cytosine (Compound V):

75 ~ 80 ° C under the conditions, 48 76 g (0 100 mol.) (S) _N1_ [(2 – hydroxy-3 – (dimethoxytrityl) propyl]. Cytosine (Compound IV) was added to 150 mL anhydrous DMF, and then inputs 8. 5g (0. 050 mol) tert-butoxide, magnesium reaction 0.5-1 h, tosyloxy added diethyl 32 methylsulfinyl . 2 g (0. 100 mol), the reaction epileptic 8 h, p-toluenesulfonic acid was added to neutralize the excess alkali to neutral distilled DMF, ethyl acetate (300 mLX 3) washing the combined ethyl acetate phase was concentrated to give a solid, i.e., synthetic 58 18 g (S)-Nl-. {[2 – (diethoxy-phosphono-methoxy) -3 – (methoxy-dimethoxytrityl)] propyl} cytosine (Compound V), yield 89%, HPLC purity greater than 95%.

4 Synthesis of (S)-Nl-{[2_ (phosphonic acid methoxy diethoxy) -3 – hydroxy] propyl} cytosine (Compound VI): The 10 g (S)-Nl- {[2 – (phosphono-methoxy ethoxy) -3 – (methoxy-dimethoxytrityl)] propyl}-cell

Pyrimidine (compound V) was dissolved in a concentration of 70 mL of 80% acetic acid solution, 90 ° C reaction. After 5 h, cooled to room temperature, 50 mL of water and 30 mL of dichloromethane, and the organic phase washed with water (30 mL X2) and the combined aqueous phase was concentrated to give crude 9. 5 g, can be performed directly in the next reaction.

can also be separated by flash column chromatography (CH2C12 = MeOH = 10: 1), 4.6 g obtained as a pale yellow oil, i.e. (S)-Nl-{[2 – (methoxy diethoxy phosphono ) -3 – hydroxy] propyl} cytosine (Compound VI), yield 90%.

5 was synthesized ⑶-Nl-{[2_ (diphosphonic acid methoxy) -3 – hydroxy] propyl} cytosine (Compound I):

The 9.5g (S)-Nl-{[2 – (methoxy diethoxy phosphonomethyl) -3 – hydroxy] propyl} cytosine (Compound VI) into a crude product containing 5 76 g (0.. 045 mol) solution of hydrogen iodide, hydroiodic acid, and after reflux for 4-5 h. (50 mLX 2) wash solution was separated with ethyl acetate. The aqueous phase was added sodium hydroxide to adjust pH between 3 Γ3 6, filtered, recrystallized from methanol to give 3.81 g of white crystalline solid, S Jie (S)-Ni-{[2 -.. (Diphosphonic acid methoxy yl) -3 – hydroxy] propyl} cytosine (Compound I), yield 88% (containing two crystal water), HPLC purity greater than 99%.

…………………………………………

POLYMORPHS

US20120277191

Example 7 Amorphous Cidofovir

Intermediate 5 (FIG. 7; 0.5 g, 0.054 mol) was heated with a solution of sodium methoxide in methanol (0.5 M, 15 mL, 7.5 mmol) at 72° C. for 14.5 h then at 90° C. for 5.5 h. The reaction mixture was quenched with water (10 mL) and filtered through a bed of ion exchange resin Dowex® 50WX8 100-200 (H). The filtrate was cycled through the ion exchange bed (2 times) then washed successively with 1:1 methanol:water (40 mL), methanol (40 mL) and 4% triethylamine:methanol (50 mL). This ion-exchange bed was further washed with 48:48:4 methanol:water:triethylamine (100 mL) until no UV absorbance was detected in the filtrate. This reaction produced intermediate 7 (FIG. 7) together with cyclic cidofovir impurity. This mixture was then dissolved in 6 N HCl and heated to 65° C. After cooling the reaction mixture to room temperature, ethyl acetate was charged and stirred and the aqueous layer separated. The aqueous was stirred with ethanol (50 mL). The precipitated material was filtered and the solid was washed with ethanol. The ethanol filtrate was concentrated. The concentrated material was taken up in acetonitrile and stirred with trimethylsilyl bromide (19 mL) at room temperature for 18 h. The reaction mixture was filtered and the filtrate concentrated. The residue was taken up in toluene (30 mL) and ammonium hydroxide (28%, 50 mL) was charged and stirred at room temperature. The organic phase was separated and the aqueous phase was concentrated to dryness. Water (20 mL) and ethanol (15 mL) were added to the residue. The mixture pH was 6 and was adjusted to pH 3 with concentrated HCl (2 mL) then adjusted to pH 4 to 4.5 with 28% NH₄OH. After stirring for 0.5 h, the mixture was cooled, filtered and the solids washed with 2:1 EtOH:H₂O and dried under vacuum for 18 h. The isolated solid was taken up in water (10 mL) and 28% NH₄OH added to give a solution. Concentrated HCl was added to the solution until pH 4 was reached. Ethanol (13 mL) was charged and the mixture stirred at −17° C. for 18 h, filtered and the solids washed with 2:1 EtOH:water (2×8 mL), dried under vacuum at 35° C. The cidofovir isolated in this manner was determined to be in the amorphous form by XRPD.

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Journal of the American Chemical Society, 2011 , vol. 133, 7 p. 2264 – 2274

http://pubs.acs.org/doi/abs/10.1021/ja109823e

Abstract Image

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Uncialamycin

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Mar 052014

Uncialamycin

(1aS,11S,11aR,18R)-3,18-Dihydroxy-11a-[1(R*)-hydroxyethyl]-9,10,11,11a-tetrahydro-4H-11,1a-[3]heptene[1,5]diynonaphtho[2,3-h]oxireno[c]quinoline-4,9-dione

439.4163

C26 H17 N O6

870471-83-3 cas

WO2007038868A2, WO2013122823A1,

University of British Columbia (Originator)

uncialamycin, an enediyne natural product isolated from the Streptomyces uncialis, bacteria present on the surface of the lichen Cladonia uncialis.

Laboratory cultures of an undescribed streptomycete obtained from the surface of a British Columbia lichen produce uncialamycin (1), a new enediyne antibiotic.Uncialamycin exhibits potent in vitro antibacterial activity against Gram-positive and Gram-negative human pathogens, including Burkholderia cepacia, a major cause of morbidity and mortality in patients with cystic fibrosis.

Uncialamycin is an enediyne antibiotic with some unprecedented activity. The isolationists have filed a patent application almost right away. The total synthesis by Nicolaou [ACIE2007, 46, 4704] goes along nearly the same lines that have been predicted, and similar to Myers’ synthesis of dynemicin A [JACS 1997, 119, 6072], only it is not paper chemistry but the real one.

They have easily constructed the quinoline system with required functionality and subjected it to AllocCl-assisted acetylide addition (if I interpreted correctly “92% yield based on 80% conversion”). 5-alkoxyquinoline system was later advanced to iminoquinone and the two remaining rings were again attached by Hauser annulation with 3-cyanophthalide. The final product turned out be different from the one reported, more precisely, it was a C26-epimer. It is funny that I have accidentally drawn the correct structure with R-configuration at C-26 in the previous post.

The synthetic scheme allowed to easily invert this stereocenter via oxidation/reduction sequence on the last compound shown on the scheme below. The spectral properties of the final product thus obtained matched the reported data, and the structure of uncialamycin was confirmed by X-ray, despite it was isolated as an oil. The structure on the right is the revised one. The remaining details, including the chemistry behind DNA-cleaving Bergmann cycloaromatization,

Total Synthesis and Stereochemistry of Uncialamycin

K. C. Nicolaou, Hongjun Zhang, Jason S. Chen, James Crawford, Laxman Dasunoori

¹Department of Chemistry and, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
²Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

A new tot synth of Uncialamycin by Nicolaou. This is a natural occurring enediyne. Because the stereochemistry of C26 was unknown, both diastereomers as shown were synthesized. The retrosyn led back to simpler fragments 2, 3, and 4.

The following scheme illustrates the route to fragment 2. The key transformation was the two-step Friedlander quinoline synthesis (7 to 9).

Then fragment 2 was used in the following sequence. The key steps in the sequence involved installation of enediyne fragment 3 to give 11, the closure of the macrocycle to give 15, and the Hauser annulation in the last step to give 1a from 16.

In this case, it was found that the final product’s spectrum (1a) did not match the reported value. And therefore, the other isomer was synthesized. This was easily done using fragment 12 through oxidation-reduction sequence to give 18 with the opposite stereochemistry at C26. Sequence in Scheme 3 was then repeated on this fragment.

And 1b was found to match spectrum of the natural isomer. This natural compound was found to be stable as a solid and as solutions in a variety of solvents. But in presence of dray HCl in CH2Cl2 solution at rt, it rapidly converts to hexacyclic 19 through a cascade of Bergman cycloaromatization reaction. This cascade of reactions is believed to be responsible mode of action in damaging DNA and killing cells.

The enediynes are a family of antibiotics that possess a distinctive strained nine- or ten-member ring system comprising a Z-carbon-carbon double bond and two carbon- carbon triple bonds, usually arranged with the latter two flanking the former. The enediynes are potent damagers of DNA, causing single and double strand cuts. Their potency is attributed to their ability to bind to DNA and undergo a Bergmann rearrangement in which the strained ring system is converted into a highly reactive 1 ,4-benzenoid diradical, which damages the DNA by abstracting hydrogens from it.

Uncialamycin is an enediyne isolated from a Streptomyces strain found on the lichen Cladonia uncialis (Davies et al. 2005; 2007). (Full citations for references cited in this specification by first named author or inventor and year are provided in the section entitled “REFERENCES” later herein.)

Uncialamycin

The structure of uncialamycin has been confirmed by total synthesis (Nicolaou et al. 2007a; 2007b). In the course of the synthesis, it was noted that the unnatural 26(S) epimer was almost as active as the natural 26(R) epimer – that is, the stereochemistry of the C27 methyl had a minor effect on biological activity. Both epimers were active against several ovarian tumor cell lines. The IC₅₀ values rang ed from 9 x 10^“12 to 1 x 10^“10, depending on the epimer and cell line or sub-line (Nicolaou et al, 2008).

Conjugates are an important method for the delivery of anti-cancer drugs, which are often highly cytotoxic and might otherwise be problematic to administer due to the risk of systemic toxicity. In a conjugate, the drug is conjugated (covalently linked) to a targeting moiety that specifically or preferentially binds to a chemical entity characteristic of the cancer cell, thus delivering the drug there with high specificity. Further, the drug is held in an inactive form until released from the conjugate, usually by cleavage of the covalent linker.

Typically, the targeting moiety is an antibody or an antigen-binding portion thereof, whose antigen is overexpressed or uniquely expressed by a cancer cell (“tumor associated antigen”). In such instances, the resulting conjugate is sometimes refered to as an “immunoconjugate” or an “antibody-drug conjugate” (ADC). Preferably the tumor associated antigen is located on the surface of the cancer cell, but also can be one that is secreted into the vicinal extracellular space. Upon binding, the antigen-conjugate complex is internalized and eventually finds its way inside a vesicular body such as a lysosome, where the covalent linker is cleaved, liberating active drug to exert its chemotherapeutic effect.

Advantageously, the covalent linker is designed such that cleavage is caused by a factor prevalent inside a cancer cell but not in plasma. One such factor is the low lysosomal pH, so that the covalent linker can be an acid-sensitive group such as a hydrazone. Another such factor is the generally higher intracellular concentration of glutathione, allowing for the cleavage of a disulfide covalent linker by a disulfide exchange mechanism. Yet another such factor is the presence of lysosomal enzymes such as cathepsin B, which can cleave peptide linkers designed to be preferred substrates (Dubowchik et al. 2002).

Conjugates have been used to deliver enediyne drugs in oncology. Gemtuzumab ozogamicin (Mylotarg®) is a conjugate of an anti-CD33 monoclonal antibody and a derivative of the enediyne calicheamicin. It was approved for treatment of acute

myelogenous leukemia but was later withdrawn from the market. Several other enediyne drugs, especially in the conjugated form, have been the subject of development efforts

If handled carefully, enediynes make powerful cancer drugs.

Inventors N. S. Chowdari, S. Gangwar, and B. Sufi synthesized enediyne compounds with general formula 1 that are based on the natural enediyne uncialamycin (2) scaffold (Figure 1). These compounds, used alone or in conjugates, are potent cytotoxins that may be useful in cancer chemotherapy.

Enediynes are a class of natural antibiotics that are characterized by 9- or 10-membered rings that contain two C≡C bonds separated by a cis (Z)-substituted C=C bond. Enediynes can undergo Bergman cyclization to form 1,4-benzenoid diradicals, which abstract hydrogen atoms from other molecules. When the diradical is generated near DNA, it abstracts hydrogen atoms from the sugar backbone of the DNA molecule and results in single- and double-strand lesions.

The high reactivity of enediynes toward DNA makes them very toxic. Their potent activity may be beneficial, however, if they are used to target the DNA of cancerous tumors. Most enediynes inhibit the proliferation of various cancer cells, including those that resist other chemotherapeutic drugs. Several naturally occurring enediynes are in clinical trials against cancer.

Both epimers at C26 of the natural enediyne uncialamycin are active against several ovarian tumor cell lines, with IC50 values ranging from 9 × 10–12 to 1 × 10–10 M, depending on the epimer and the cell line or subline. The synthetic enediynes described by the inventors are derivatives of uncialamycin.

Using these toxic molecules demands specific delivery systems. Conjugates are innovative drug-delivery systems designed to target tumor cells precisely and minimize the risk of systemic toxicity. Typically, drugs are linked covalently to conjugates that act as targeting moieties, which specifically or preferentially bind to a chemical entity characteristic of the cancer cell.

The covalent linker is designed to be cleaved only by a factor that exists inside a cancer cell and not in plasma, so that the drug remains in an inactive form until it is released from the conjugate. A typical targeting moiety may be a polymer or an antibody. Polymer-conjugated and antibody-linked enediyne drugs such as gemtuzumab ozogamicin (Mylotarg) were used to deliver enediyne drugs to cancer cells. Mylotarg, however, has been withdrawn from the market because of high patient mortality.

General formula of compounds of the invention and natural enediyne uncialamycin

Compounds of structure 1 may be conjugated to a targeted moiety through a chemical bond to substituent R1. Compounds 3 and 4, shown in Figure 2, are examples of the synthetic enediynes with structure 1.

The investors tested the antiproliferative activities of several compounds against cancer cell lines. EC50 data for compounds 3 and 4 against 786-0 renal cancer cells and H226 lung cancer cells are shown in the table:

Example	786-0 cells, EC50 (nM)	H226 cells, EC50 (nM)
3	1.275	0.986
4	0.058	0.873

Representative examples of synthetic enediynes

Several assays were also conducted on conjugates derived from other compounds of formula 1. (Bristol-Myers Squibb [Princeton, NJ]. WIPO Publication 2013122823, Aug 22, 2013;

DAVIES ET AL.: ‘UNCIALAMYCIN, A NEW ENEDIYNE ANTIBIOTIC‘ ORGANIC LETTERS vol. 7, no. 23, 13 October 2005, pages 5233 – 5236

http://pubs.acs.org/doi/abs/10.1021/ol052081f

Production cultures of the producing strain were grown as lawns on solid agar medium (ISP4, 16 L) for 14−21 days at 30 °C. The solid agar cultures were extracted repeatedly with EtOAc. Concentration of the combined EtOAc extracts in vacuo gave a gummy residue that was partitioned between EtOAc and H2O. The EtOAc soluble material was fractionated by sequential application of flash C-18 reversed-phase chromatography (eluent: step gradient from H2O to MeOH) and reversed-phase HPLC (column-Inertsil ODS-2; eluent: CH3CN/H2O 40:60) to give pure uncialamycin (1) (

300 μg) as a bright purple [UV(MeOH): λmaxnm (ε) 206 (25000), 254 (33000), 280 (shoulder), 320 (shoulder), 539 (9400)] optically active ([α]D +3300 (c 0.005, MeOH)) oil.

Uncialamycin (1) gave a [M + Na]+ ion at m/z 462.0956 in the HRESIMS appropriate for a molecular formula of C26H17NO6 (calcd for C26H17NO6Na 462.0954) requiring 19 sites of unsaturation. NMR data for uncialamycin were recorded in DMSO-d6 at 600 MHz using a cryoprobe. The 13C NMR spectrum (Table 1) showed well-resolved resonances for 26 carbon atoms, and the 1H NMR spectrum contained resonances integrating for 17 protons, in agreement with the HRMS data. Inspection of the HSQC data revealed that four of the protons (δ 5.39, 6.66, 10.0, and 13.2) were not attached to carbon atoms. Two major fragments A and B (Figure 1) of uncialamycin could be identified from analysis of the COSY, HSQC, and HMBC data obtained for the molecule.

Table 1. 13C and 1H NMR Assignments for Uncialamycin (1). Data were Recorded in DMSO-d6 at 600 MHz for 1H

position	δ 13C	δ 1H (mult., J (Hz))
1		10.0 (d, 4.6)
2	143.6
3	110.4
4	187.0a
5	134.4b
6	126.1c	8.23 (dd, 1.4, 7.6)c
7	133.6d	7.88 (ddd, 1.4, 7.6, 7.6)d
8	134.9d	7.94 (ddd, 1.4, 7.6, 7.6)d
9	126.6c	8.24 (dd, 1.4, 7.6)c
10	132.2b
11	182.2a
12	112.7
13	154.9
14	129.9	8.51 (s)
15	135.6
16	63.5
17	63.0	5.14 (d, 3.3)
18	100.4
19	89.7
20	123.4	6.05 (dd, 0.8, 10)
21	124.0	5.97 (ddd, 1.4, 1.5, 10)
22	87.4
23	98.9
24	43.2	5.04 (dd, 1.5, 4.6)
25	76.0
26	63.6	4.31 (qd, 6.0, 6.0)
27	22.1	1.30 (d, 6.0)
13-OH		13.2 (brd.s)
17-OH		6.66 (brd.s)
26-OH		5.39 (d,6.0)

a−d May be interchanged.http://pubs.acs.org/doi/suppl/10.1021/ol052081f/suppl_file/ol052081fsi20051004_065853.pdf

……………..

WO2007038868A2

Isolation of Uncialamvcin

[0034] As part of a screening program aimed at discovering new antibiotics active against Bcc, it was found that crude organic extracts of cultures of a previously undescribed Streptomycete showed potent in vitro inhibition of Bcc. Bioassay guided fractionation of the crude extracts led to the identification of uncialamycin (1), a new enediyne antibiotic, as the active component. Bioactivity-guided fractionation involves thin layer chromatography of the extracts and fractions thereof and detection of the activity by overlaying a sensitive tester strain. A zone of inhibition identifies the active fraction containing the active compound.

The producing strain was extracted from the surface of the lichen Cladonia uncialis collected near Pitt River, British Columbia. Characterisation by 16S RNA sequencing showed the strain to be related, but not identical, to Streptomyces cyanogenus. Antibiotic activity of the strain was assayed by cutting plugs from solid agar cultures of the strain and placing them on lawns of tester strains of bacteria. Good inhibitory activity was detected against Gram-positive and Gram-negative bacteria (including Bcc), but not against yeasts.

Production cultures of the producing strain were grown as lawns on solid agar medium ISP4 for 14 to 21 days at room temperature. The solid agar cultures were lyophilized and extracted repeatedly with EtOAc. Concentration of the combined EtOAc extracts in vacuo gave a gummy residue that was partitioned between EtOAc and H₂O. The EtOAc soluble material was fractionated by sequential application of flash C- 18 reversed-phase chromatography (eluent: step gradient from H₂O to MeOH) and reversed-phase HPLC (column-Inertsil ODS-2; eluent: CH₃CN/H₂O 40:60) to give pure uncialamycin (1) (~ 300 μg) as a bright purple [UV(MeOH): λ_maxnm (ε) 206 (25,000), 254 (33,000), 280 (shoulder), 320 (shoulder), 539 (9,400)], optically active ([α]_D +3,300 (c 0.005, MeOH)) oil.

Chemical Characterization of Uncialamycin

Uncialamycin (1) gave a [M + Na]⁺ ion at m/z 462.0956 in the

HRESIMS appropriate for a molecular formula Of C₂₆H₁₇NO₆ (calc’d for C₂₆H₁₇NO₆Na 462.0954) requiring 19 sites of unsaturation. NMR data for uncialamycin was recorded in DMSO-^₆ at 600 MHz using a cryoprobe. The ¹³C NMR spectrum (Table 1) showed well-resolved resonances for 26 carbon atoms and the ¹H NMR spectrum contained resonances integrating for 17 protons in agreement with the HRMS data. Inspection of the HMQC data revealed that four of the protons (δ 5.39, 6.66, 10.0, and 13.2) were not attached to carbon atoms. Two major fragments A and B (Figure 1) of uncialamycin could be identified from analysis of the COSY, HMQC, and HMBC data obtained for the molecule.

Position δ ¹W WH^mult, J(Hz)) ,

1 10.0 (d, 4.6)

2 143.6

3 110.4

4 187.0

5 134.4

6 126.1 8.23 (dd, 1.4, 7.6)

7 133.6 7.88 (ddd, 1.4, 7.6, 7.6)

8 134.9 7.94 (ddd, 1.4, 7.6, 7.6)

9 126.6 8.24 (dd, 1.4, 7.6)

10 132.2

11 182.2

12 112.7

13 154.9

14 129.9 8.51 (s)

15 135.6

16 63.5

17 63.0 5.14 (d, 3.3)

18 100.4

19 89.7

20 123.4 6.05 (dd, 0.8, 10)

21 124.0 5.97 (ddd, 1.4, 1.5, 10)

22 87.4

23 98.9

24 43.2 5.04 (dd, 1.5, 4.6)

25 76

26 63.6 4.31 (qd, 6.0, 6.0)

27 22.1 1.30 (d, 6.0)

13-OH 13.2 (brd.s)

17-OH 6.66 (brd.s)

26-OH 5.39 (d,6.0)

Table 1. C and H NMR assignments for uncialamycin (1). Data were recorded in OMSO-d₆ at 600 MHz for ¹H. [0038] A pair of olefinic resonances at δ 5.97 (H-21 ) and 6.05 (H-20), that were strongly correlated to each other in the COSY spectrum and had a coupling constant of 10 Hz, were assigned to a cis disubsituted olefin. The upfield olefinic resonance at δ 5.97 (H-21) showed strong HMBC correlations to non-protonated carbon resonances at δ 89.7 (C- 19) and 98.9 (C-23), and the downfield olefinic resonance at δ 6.05 (H-20) showed strong correlations to non-protonated carbon resonances at δ 87.4 (C-22) and 100.4 (C- 18). This suite of HMBC correlations identified an enediyne substructure in 1 (see Fragment A in Figure 1). The olefinic resonance at δ 5.97 (H-21) showed a long range COSY correlation to a methine resonance at δ 5.04 (H- 24), indicating that the carbon bearing the methine proton (C-24: δ 43.2) was attached to the C-23 alkyne carbon. A COSY correlation observed between the methine (δ 5.04, H-24) and a broad singlet at 10.0, that was not correlated to a carbon in the HMQC spectrum, and the chemical shift of the methine carbon (C-24, δ 43.2) suggested that C-24 had an NH substituent. HMBC correlations observed between the H-24 methine (δ 5.04) and the two alkyne carbon resonances at δ 87.4 (C-22) and 98.9 (C-23) confirmed the attachment of C-24 to the C-23 alkyne carbon.

A singlet methine resonance at δ 5.14 (H- 17) showed HMBC correlations to the alkyne carbon resonances at δ 89.7 (C- 19) and 100.4 (C- 18), which demonstrated that the methine carbon (C- 17: δ 63.0) was linked to the second alkyne at C-18. Both of the methine resonances at δ 5.04 (H- 24) and 5.14 (H- 17) showed HMBC correlations to a pair of deshielded resonances at δ 63.5 (C- 16) and 76.0 (C-25), assigned to non-protonated oxygen bearing carbons. This set of four HMBC correlations indicated that the two oxygenated carbons bridged the C- 17 and C-24 carbons to form a ten membered ring (C- 16 to C-25) containing the enediyne substructure. A COSY correlation between the methine resonance at δ 5.14 and a broad singlet at 6.66 (17-OH) revealed an alcohol funtionality attached to the methine carbon.

A methyl doublet at δ 1.30 (Me-27, J = 6 Hz) was correlated in the COSY spectrum to a methine at 4.31 (H-26, q, J = 6.0 Hz)) that was further correlated to a broad singlet at 5.39 (OH-26), assigned to an alcohol. The methyl resonance (δ 1.30, Me-27) showed an HMBC correlation to the carbon resonance at 76.0 (C-25), indicating that the hydroxyethyl fragment (C-26 and C-27) was the fourth subsituent on the non-protonated carbon C- 25. Both the NH-I proton (δ 10.0) and the H-17 methine (5.14) were correlated to a carbon at δ 135.6 (C- 15), and the H-24 methine (δ 5.04) was correlated to a carbon at 143.6 (C-2) in the HMBC spectrum indicating that the NH and C- 16 were vicinal substituents on an olefin or aromatic ring. A deshielded singlet at δ 8.51 showed strong HMBC correlations into carbon resonances at δ 63.5 (C-16), 143.6 (C-2), and 112.7 (C- 12) and a weak correlation into the carbon resonance at 154.9 (C- 13). This set of HMBC correlations confirmed that the NH and C-16 were attached to a benzene ring. Based on the assumption that the intense HMBC correlations were through three bonds, these correlations also indicated that the aromatic methine (δ 8.51, H-14) was ortho to C-16 (δ 63.5) and meta to the NH (C-2, δ 143.6). The weak HMBC correlation between δ 8.51 and 154.9 was attributed to a two bond coupling, placing the carbon at 154.9 (C-13) ortho to the methine carbon (C- 14) and its chemical shift required an oxygen substituent. [0041] The second fragment B of uncialamycin contained an isolated

¹H spin system comprised of four contiguous aromatic protons (δ 8.23, dd, J = 1.4, 7.6 Hz H-6; 7.88, ddd, 1.4, 7.6, 7.6 Hz H-7; 7.94, ddd, J = 1.4, 7.6, 7.6 Hz H-8; 8.24, dd, J = 1.4, 7.6 Hz H-9). HMBC correlations observed between the proton resonance at δ 8.23 (H-6) and a carbon resonance at 187.0 (C-4) and between the proton resonance at 8.24 (H-8) and a carbon resonance at 182.2 (C-11) suggested that the other two subsituents on the benzene ring were quinone carbonyls. Fragments A and B shown in Figure 1 accounted for all of the carbon, hydrogen, and nitrogen atoms in the molecular formula of uncialamycin (1), but contained one extra oxygen atom. In order to complete the quinone and satisfy the remaining aromatic valences in Fragment A, the two carbonyl carbons of fragment B (C-4 and C-I l) had to be attached to the two substituted aromatic carbons (C-3 and C- 12) of fragment A. Finally, it was apparent that the two oxygentated carbons C- 16 and C-25 had to be bridged by an epoxide to account for the number of oxygen atoms and sites of unsaturation required by the molecular formula of 1. This implied that the C- 13 oxygen substituent had to be part of a phenol functionality that would engage in intramolecular hydrogen bonding with the C-I l carbonyl consistent with the observed OH chemical shift of δ 13.2.

A ROESY correlation between δ 5.14 (H- 17) and 4.31 (H-26) showed that C-26 and C- 17 were cis oriented about the C-16/C-25 epoxide and also defined the relative stereochemistry of H- 17 as shown. Molecular models revealed that due to steric and bond angle strain the C- 17 to C-23 enediyne containing bridge could only reasonably be cis fused to the piperidine ring. Uncialamycin (1) shares structural features with dynemicin A (2) and deoxydynemicin A (3) isolated from Micromonospora chersina. The H-24 resonance in uncialamycin (1) has a chemical shift of δ 5.04 and a 4.6 Hz coupling to the NH-I proton, which is nearly identical to the chemical shift (δ 5.05) and coupling (J = 4.3 Hz) of the corresponding methine proton (H-2) in dynemicin A (2), in agreement with the relative stereochemical assigment at C-24 in 1. Comparison of the additional NMR assigments reported for dynemicin A (2) and its triacetate derivative provided further strong support for the assigned structure of uncialamycin

…………….

Angewandte Chemie – International Edition, 2008 , vol. 47, 1 p. 185 – 189

http://onlinelibrary.wiley.com/doi/10.1002/anie.200704577/abstract

The highly potent DNA-cleaving molecule uncialamycin (1) was prepared in an asymmetric total synthesis featuring an enantioselective Noyori reduction. Compound 1 and its C26 epimer exhibit impressive broad-spectrum antibacterial properties and highly potent antitumor activities against a variety of cell lines.

LUPIN.. giant leap forward

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Mar 022014

Lupin Limited

news

Expanding domestic portfolio, ability to maintain market share in current products and new launches are key growth triggers
The lipid control or cholesterol lowering segment is emerging as a key growth driver for Lupin’s sales in the US. The first among three products is Tricor. The Lupin stock had corrected about five per cent in May on the announcement that Mylan would launch the $1.2 billion (Rs 7,200 crore) drug in tablet form.

Lupin has been able to maintain its generic market share so far with a share of 34 per cent vis-a-vis Mylan (market share of one-two per cent). While Balaji Prasad and Rohit Goel of Barclays estimate the drug will contribute about $29 million to Lupin’s revenues in the June 2014 quarter, Ebitda margins are expected to expand by 210 basis points, feel analysts at Kotak Securities.

Though the launch of generics in capsule form by Mylan has to be watched carefully, Lupin’s Antara, along with authorised generics, continues to dominate with 70 per cent share, observes Hitesh Mahida at Fortune Research.

In addition to Tricor, two other products in the cholesterol lowering segment the company is eyeing are Trilipix and Niaspan. Instead of a launch in January 2014 as was anticipated earlier, the company is now likely to launch the generics version of this $550 million drug Trilipix this month.

While the company says it is mulling options about the launch, Edelweiss analysts believe an early launch is likely to add $11 million in FY14 and about $15 million in FY15 to the company’s revenues. The third drug in this segment is Niaspan, to be launched in March 2014 and expected to add about $35 million to the company’s revenues in FY15. Together, the three cholesterol controlling drugs are expected to contribute $85-90 million to FY15 sales for Lupin.

Lupin Limited is a transnational pharmaceutical company based in Mumbai. It is the 2nd largest Indian pharma company by market capitalization;^[14] the 14th largest generic pharmaceutical company globally^[15] and; the 5th largest generic pharmaceutical company in the US by prescription-led market share.^[16] It has the distinction of being the fastest growing generic pharmaceutical player in the two largest pharmaceutical markets of the world – the US^[17] and Japan;^[18] and is the 5th largest ^[19] and the fastest growing generic pharmaceutical player in South Africa.^[20]

Market Position in the US gradually improving

Lupin 5thlargest generic company in the US in terms of prescriptions

14 products are market leader and 27 (among top 3) out of 30


Type	Public
Traded as	BSE: 500257 NSE: LUPIN
Industry	Pharmaceuticals
Founded	1968 ^[1]
Founder(s)	Dr. Desh Bandhu Gupta^[2]
Headquarters	Mumbai, Maharashtra^[3], India
Key people	Dr. Kamal K Sharma, Vice Chairman;^[4]……………………………………..Vinita Gupta, Chief Executive Officer, Lupin Limited;^[5] …………………. Nilesh Gupta, Managing Director;^[6] …………… Shakti Chakraborty, Group President – India Region Formulations & CIS;^[7] ……………………. Vinod Dhawan, Group President AAMLA & Business Development;^[8] ……………………. Ramesh Swaminathan, President Finance & Planning.^[9] New leadership team at Lupin from September 2013^[10]
Products	Pharmaceuticals, branded andgeneric drugs, biotechnology, Advanced Drug Delivery Systems, New Chemical Entity Research, vaccines, Over-the-Counter drugs
Revenue	9461 crore (US$1.5 billion) (2012-2013)^[11]^[12]
Profit	1314 crore(US$210 million) (2013)
Employees	11355^[13]
Subsidiaries	Lupin Pharmaceuticals, Inc. Kyowa Pharmaceutical Industry Co. Ltd. I’rom Pharmaceutical Co. Ltd. Pharma Dynamics Multicare Pharmaceuticals Generic Health Pte. Ltd. Hormosan Pharma GmbH
Website	www.lupinworld.com

2012 .http://icra.in/Files/ticker/Indian%20Pharmaceutical%20Sector.pdf

History and Evolution

Lupin was founded in 1968 by Dr. Desh Bandhu Gupta,^[21] then an Associate Professor at BITS-Pilani, Rajasthan. Named after the Lupin flowerbecause of its inherent qualities and what it personifies and stands for, the company was created with a vision to fight life threatening infectious diseases and to manufacture drugs of the highest social priority.

Lupin first gained recognition when it became one of the world’s largest manufacturers of tuberculosis drugs.^[22] The company today has a significant market share in key markets in the Cardiovascular (prils and statins), Diabetology, Asthma, Pediatrics, CNS, GI, Anti-Infectives and NSAIDs therapy segments. It also has a global leadership position in the Anti-TB and Cephalosporin segments. The company’s R&D endeavours have resulted in significant progress in its NCE program. Lupin’s foray into Advanced Drug Delivery Systems has resulted in the development of platform technologies that are being used to develop value-added generic pharmaceuticals. Its manufacturing facilities, spread across India and Japan, have played a critical role in enabling the company realize its global aspirations. Benchmarked to International standards, these facilities are approved by international regulatory agencies including the US FDA, UK MHRA, Japan’s MHLW, TGA Australia, WHO, and the MCC South Africa.

Research and Development

Lupin’s Research Program covers the entire pharma value chain. The company’s global R&D program is headquartered out of the Lupin Research Park located near Pune that houses over 1200 scientists. Lupin’s R&D covers:

Generics Research

Process Research
Pharmaceutical Research

Advanced Drug Delivery Systems (ADDS) Research
Intellectual Property Management
Novel Drug Discovery and Development (NDDD)
Biotechnology Research

Differentiation is the heart of our research efforts at Lupin. We have created a truly unique world-class research program, designed to ensure a sustainable pipeline of high-value opportunities to maximise growth.

Research and Development is at the core and is the most critical part of any pharmaceutical company. At Lupin we see R&D differently. It is fundamentally about creativity, originality and being aware of what is really required. At Lupin, our Research & Development program has been the key to our sustained growth over the past ten years; growth that has made us one of the most exciting research driven pharmaceutical companies globally; a hotbed of differentiation and innovation. Today we are building the future by strengthening our research foundation through prudent investments that position us at the cutting-edge of technology, helping us deliver complex products that very few in the world can.

Headquartered at the state-of-the-art Lupin Research Park in Pune, India, the Company’s research program is home to over 1,400 scientists. The Company’s global research operations are spread over multiple research facilities in India and Japan. During FY 2013, the Company invested 7.5% of its net sales in Research & Development and related spends, amounting to 7,098 million. FY 2013 was a record year in terms of progress made all around, be it our pace of filing DMFs (Drug Master Files) and ANDAs (Abbreviated New Drug Applications), progress in our drug discovery and development program, milestones in our drug delivery program and approvals in our biotechnology program.

NOVEL DRUG DISCOVERY & DEVELOPMENT

Long-term, one of the Company’s biggest differentiators will be its Novel Drug Discovery and Development (NDDD) program. The Program focuses on the discovery, development and commercialisation of new drugs that address disease areas with significantly unmet medical need. Lupin’s NDDD efforts are directed towards identifying and developing new therapies for disease areas that include metabolic/endocrine disorders, pain and inflammation, autoimmune diseases, CNS disorders, cancer and infectious diseases.

Scientists at NDDD have been able to create a portfolio of novel compounds that are moving through a robust pipeline from discovery to development. This steady movement will ensure that at least one compound enters the clinical phase in terms of first-in-human studies each year. Lupin has adopted a ‘Quick-win, fail-fast’ cost-efficient development approach, in which novel compounds are filtered at every stage before entering development and differentiated by efficacy with a focus on enhanced safety.
Highlights, FY 2013

Successfully completed Phase I studies in Europe for a program in the CNS area, which is being advanced to Phase II clinical trials in Europe now

Candidates from two programs in the area of endocrine disorders and cancer will enter clinical development in FY 2014
Six other programs in various stages of discovery across different therapy areas
Strong intellectual property creation and management strategy in place, with a total of over 80 patent applications filed to date

Therapeutic Targets

Therapy Area	Differentiated Pipeline
METABOLIC / ENDOCRINE DISEASES	NOVEL MECHANISMS FOR NEW ANTI-DIABETICS
PAIN / INFLAMMATION	HOLY GRAIL OF PAIN REMEDY
AUTO-IMMUNE DISEASES	TARGETED FOR RHEUMATOID ARTHRITIS AND OTHER IMMUNE DISORDERS
CNS DISORDERS	ROBUST TREATMENTS FOR COGNITIVE DEFICITS IN DIFFERENT CNS CONDITIONS
ONCOLOGY	HIGHLY DIFFERENTIATED ANTI-CANCER TREATMENT (HITTING ONLY CANCER CELLS)
INFECTIOUS DISEASES	HIGHLY POTENT ANTI-VIRAL THERAPY

Businesses

Lupin’s businesses encompass the entire pharmaceutical value chain, ranging from branded and generic formulations, APIs, advanced drug deliverysystems to biotechnology. The company’s drugs reach 70 countries^[23] with a footprint that covers Advanced Markets such as USA, Europe, Japan,^[24] Australia as well as Emerging Markets including India,^[25] the Philippines and South Africa to name a few.

KEY MARKETS AND BUSINESSES

USA
Headquartered in Baltimore, Maryland, Lupin Pharmaceuticals, Inc. (LPI), the company’s US subsidiary is a $ 706 million enterprise.^[26] LPI has a presence in the branded and generics markets of the US. In the branded business, Lupin operates in the CVS and Pediatric segments. The company is the market leader in 24 products out of the 46 products marketed in the US generics market, of which it is amongst the Top 3 by market share in 37 of these products (IMS Health, March 2013): Suprax (Cefixime), a paediatric antibiotic, is Lupin’s top-selling product here. The company is also the 5th largest and fastest growing generics player in the US (5.3% market share by prescriptions, IMS Health). Lupin’s US brands business contributed 21% of total US sales whereas the generics business contributed 79% during FY 2012-13.^[26]

In the US market, since December 2012, Lupin has posted a 40-80 per cent growth rate on the back of new launches, as well as growing sales of its existing drugs. For May, the company has posted growth of 50 per cent year-on-year in US sales. The outlook is also good. On the whole, Lupin has one of the strongest pipelines of 18-20 products for the US market over the next 18 months.

Robust US sales continue
Despite the 51 per cent rise in share price over six months, most analysts continue to be bullish on the company due to its strong showing in the US market. Among leading Indian pharma majors, this geography contributes nearly 40 per cent of its revenues, second only to Sun (43 per cent) and a good performance rubs off well on the company’s overall show.

About half its US sales are contributed by Antara, the generic form of the cholesterol lowering drugTricor, antibiotics Suprax and Cefdinir and the generic form of antipsychotic drug Geodon. Among other segments expected to drive growth are oral contraceptives ($100 million estimated sales in FY14), dermatology, ophthalmology and asthama.

India Region Formulations (IRF)
Lupin’s IRF business focuses on lifestyle and chronic therapy segments. The company has emerged as one of the fastest growing players in therapies like Cardiology, Central Nervous System(CNS), Diabetology, Anti-Asthma, Anti-Infective, Gastro Intestinal and Oncology. The IRF business contributed 25% of the company’s overall revenues for FY 2012-13, growing by 24% and recording revenues of 2364 crore (US$380 million) for FY 2012-13 as compared to 1905 crore (US$300 million) for FY 2011-12.^[26]

There are 9 manufacturing plant and 2 Research pant in India, such as Jammu(J&K),Mandideep & Indore(Madhya pradesh), Ankaleswar & Dabasa (Gujarat), Tarapur, Aurangabad and Nagpur (Maharastra) and Goa; where research centre at Pune and Aurangabad.^[27] Among these the baby plant is Nagpur plant which will the the biggest formulation unit for Lupin in coming year.

Lupin is also gradually expanding its domestic portfolio through expansion into more segments and tie-ups. On Thursday, it announced a non-exclusive tie-up with US-basedMerck Sharp and Dohme (MSD) for marketing the latter’s pneumococcal vaccine (preventive care for diabetes and, chronic heart, lung and liver diseases) for adults.

Given its growth, Bank of America Merrill Lynch analysts believe the valuation multiple at 20 times FY15 earnings estimates is likely to expand (closer to larger peers) due to stronger and sustainable growth rates, both on the net profit front (22 per cent annually over the next two years) and return ratios, expected at 30 per cent versus 26 per cent for the peers. Most analysts have a target price of Rs 875-900 for the stock. Though a re-rating could be on the cards, given the surge in share price, investors should look at corrections to add the scrip to their portfolio.

Europe
Lupin’s focus in the European Union encompasses Anti-Infectives, Cardiovascular, and CNS therapy areas, along with niche opportunities in segments like Oral Contraceptives, Dermatology and Ophthalmics. The company’s presence in France is by way of a trade partnership; in Germany, it operates through its acquired entity Hormosan Pharma GmbH (Hormosan);^[28] while the UK business is a direct-to-market initiative.

Japan
Lupin is the fastest-growing Top 10 generic pharmaceuticals player in Japan (IMS). Lupin operates in Japan through its subsidiary, Kyowa Pharmaceutical Industry Co. Ltd. (Kyowa), a company Lupin acquired in 2007,^[29]^[30] and I’rom, Pharmaceutical Co. Ltd (IP), acquired in 2011.^[31]^[32] Kyowa has an active presence in Neurology, Cardiovascular, Gastroenterology and the Respiratory therapy segments. I’rom is a niche injectables company with significant presence in the DPC hospital segment.

South Africa
Lupin’s South African subsidiary, Pharma Dynamics (PD)^[33] is the fastest growing and the 5th largest generic company in the South African market (IMS). The company is a market leader in the Cardiovascular segment and has a growing presence in Neurology, Gastroenterology and the Over the Counter (OTC) segments.

Australia
Lupin entered the Australian market through its subsidiary, Generic Health Pte. Ltd. (GH).^[34] It subsequently acquired the worldwide marketing rights to the over 100 year old Australian brand Goanna,^[35] used for pain management.

Philippines
Lupin’s Philippines subsidiary Multicare Pharmaceuticals (Multicare),^[36] is a branded generic company focused on Women’s Health, Pediatrics, Gastro-Intestinal and Diabetes care. FY 2012 also marked its foray into the Neurology segment when it entered into a strategic marketing partnership with Sanofi.^[37]

API and Global TB

Lupin is one of the most vertically integrated global generic majors and a global leader in Cephalosporins, Cardiovasculars and the anti-TB space. The company is also a strategic supplier of anti-TB products to the Global Drug Facility (GDF), with its formulations being supplied to more than 50 countries through GDF procurement.

Lupin is also a global leader in anti-TB APIs, and is associated with the Revised National TB Program of the Government of India, thereby partnering the Government in its fight against TB in the country. It also supplies to various Government agencies, the Stop TB Partnership and various other international agencies like Pan America Health Organisation (PAHO), Médecins Sans Frontières (MSF) and the Damien Foundation. Ethambutol, Rifampicin and Pyrazinamide are the company’s top selling TB molecules.

Biotechnology Research
The Lupin Biotechnology Research Group, based out of Wakad, near Pune is focussed on developing affordable, high quality biopharmaceuticals with an emphasis on biosimilars. As of May 2013, it has a pipeline of 10 biosimilar products under development, and is close to getting marketing authorization for 2 of its oncology products for the Indian market. Lupin has competencies for the complete development and manufacture of recombinant protein therapeutic products from high yielding and proprietary microbial and mammalian cell culture platforms. The Biotech R&D infrastructure offers a range of product development capabilities ranging from clone development, process optimization, analytical method development, bioassay, formulation, stability studies, non-clinical and clinical studies backed by a sound understanding of regulatory and IP aspects. The company’s biotech development programs are in compliance of and follow ICH, EMEA and Indian Regulatory guidelines.

Corporate Social Responsibility

To further its social responsibility objectives, Lupin established the Lupin Human Welfare & Research Foundation (LHWRF) on 2 October 1988. Its chief objective was to provide an alternative sustainable, replicable and ever evolving model of holistic rural development. LHWRF started with a few small rural development projects covering around 35 villages in Bharatpur District,Rajasthan. Its efforts have touched the lives of over a million people across 2,200 villages in the states of Rajasthan, Madhya Pradesh, Maharashtra and Uttarakhand.

Lupin gets USFDA nod to market HIV drug in US market

ANKLESHWAR GUJARAT PLANT INDIA

LUPIN TARAPUR INDIA

LUPIN GOA INDIA

Oncologyand inflamation

Lupin is building a discovery pipeline with over seven to eight molecules targeting the oncology and inflammation segments.

Lupin is building a discovery pipeline with over seven to eight molecules targeting the oncology and inflammation segments. The company, producing both branded and generic drugs, is also planning an investment of $20 million to expand and build additional facilities and capacities exclusively for biologics in Pune in next two to three years.

“We have over seven to eight molecules in the pipeline. Of these, three are already undergoing clinical trials, while two are in pre-clinical stage. One more molecule will be entering pre-clinical stage soon,” Cyrus Karkaria, Lupin president said, adding that the company was gearing up to launch a new product this year. He also indicated that some of these lead molecules could be potential out-licensing targets at some point of time.

Biosimilar products include recombinant erythropoietin, recombinant granulocyte colony stimulating factor (G-CSF), interferon alpha and beta, human insulin, monoclonal antibodies and human growth hormone. These are used oncology, infectious diseases, chronic autoimmune diseases, growth-related deficiencies and haematology.

As part of its expansion, Lupin will be expanding facilities in Pune, which will be operational in next two to three years. “We currently have a production facility near the lab. We will be building additional facilities with about $20 million investment in next two to three years,” Cyrus explained. Last year, the company announced plans to invest over R450 crore towards capacity expansion and strengthening sales force. Lupin is also gearing up to launch its first biosimilar product in India by early next year besides targeting 5-7% of its business from biosimilar business.

The company had entered into a licensing agreement with Sydney-based NeuClone for cell-line technology which will provide exclusive proprietary cell-line technology to be developed into biosimilar drugs targeting cancer.

Several drug companies, including Dr Reddy’s, Cipla and Biocon, among others, are eying the opportunity in biosimilars. Industry estimates global market for biosimilars or follow-on biologic drugs is about $100 billion and the Indian market is about R2,500 crore.

Meanwhile, the US Food and Drug Administration has issued three guidances on biosimilar product development to assist industry in developing such products in the US. These draft documents are designed to help industry develop

About Lupin Limited
Lupin is an innovation led transnational pharmaceutical company producing and developing a wide range of branded and generic formulations and APIs globally. The Company is a significant player in the Cardiovascular, Diabetology, Asthma, Pediatric, CNS, GI, Anti-Infective and NSAID space and holds global leadership positions in the Anti-TB and Cephalosporin segment.

Lupin is the 5th largest and fastest growing top 5 generics player in the US (5.3% market share by prescriptions, IMS Health) and the 3rd largest Indian pharmaceutical company by revenues. The Company is also the fastest growing top 10 generic pharmaceutical players in Japan and South Africa (IMS).

For the financial year ended March 2013, Lupin’s Consolidated turnover and Profit after Tax were Rs. 94,616 million (USD 1.74 billion) and Rs. 13,142 million (USD 242 million) respectively. Please visithttp://www.lupinpharmaceuticals.com for more information.

Mumbai, February 03, 2014: Pharmaceutical major Lupin Limited announced the acquisition of Nanomi B.V. in the Netherlands today. With this acquisition, Lupin has made its foray into the technology intensive complex injectables space.

Nanomi has patented technology platforms to develop complex injectable products. Nanomi has a rich talent pool of scientists who would be backed by Lupin’s global R&D and manufacturing teams.

Commenting on the acquisition Ms. Vinita Gupta, Chief Executive Officer, Lupin Limited said “We are very pleased with the acquisition of Nanomi. With the use of Nanomi’s proprietary technology platform, Lupin would be able to make significant in-roads into the niche area of complex injectables.”

Mumbai, Baltimore, December 18, 2013: Pharma Major Lupin Limited (Lupin) announced today that its US subsidiary Lupin Pharmaceuticals Inc. has launched its Abacavir Sulfate, Lamivudine, and Zidovudine Tablets, 300 mg (base) / 150 mg / 300 mg in the US after the US District Court for the District of Delaware ruled that the Lupin’s generic version of Trizivir® did not infringe on patents. Lupin had earlier received approval for the same.

Lupin’s Abacavir Sulfate, Lamivudine Zidovudine 300mg (Base)/150mg/300mg Tablets are the AB-rated generic equivalent of ViiV Healthcare’s (ViiV) Trizivir® Tablets, 300 mg (base) / 150 mg / 300mg and are indicated in combination with other antiretrovirals or alone for the treatment of HIV-1 infection.

Lupin is the first applicant to file an ANDA for Trizivir® Tablets and as such is entitled to 180 days of marketing exclusivity.

Trizivir® Tablets, 300 mg (base) / 150 mg / 300mg had annual U.S sales of approximately US$ 111.6 million (IMS MAT Sep, 2013).

Mumbai, Baltimore, December 12, 2013: Pharma Major Lupin Limited (Lupin) announced today that its US subsidiary, Lupin Pharmaceuticals, Inc. (LPI) has launched its Duloxetine Hydrochloride Delayed-release (HCl DR) Capsules 20 mg, 30 mg and 60 mg strengths. The Company received final approval to market its Duloxetine HCl DR Capsules USP, 20 mg, 30 mg, 40 mg and 60 mg strengths from the United States Food and Drugs Administration (FDA) yesterday.

Lupin’s Duloxetine HCl DR Capsules 20 mg, 30 mg and 60 mg strengths are the generic equivalent of Eli Lilly & Company’s (Lilly) Cymbalta® Delayed-release Capsules 20 mg, 30 mg and 60 mg.

Duloxetine HCl DR Capsules are indicated for the treatment of major depressive disorder (MDD), generalized anxiety disorder (GAD) and management of neuropathic pain (DPNP) associated with diabetic peripheral neuropathy.

Cymbalta® Delayed-Release Capsules 20 mg, 30 mg and 60 mg strengths had annual U.S sales of approximately USD 5.43 billion (IMS MAT Sep, 2013).

Lupin launches Generic Trilipix® Delayed – Release Capsules 45 mg & 135 mg in the US

Mumbai, Baltimore, December 06, 2013: Pharma Major Lupin Limited (Lupin) announced today that its US subsidiary, Lupin Pharmaceuticals, Inc. (LPI) has launched its generic Fenofibric Acid Delayed‐Release Capsules 45 mg and 135 mg. Lupin had earlier received final approval from the US FDA for the same.

Lupin�s Fenofibric Acid Delayed‐Release Capsules 45 mg and 135 mg are the generic equivalent of AbbVie Inc.�s Trilipix® Delayed‐Release Capsules 45 mg & 135 mg strengths are indicated as co‐administration therapy with statins for the treatment of mixed dyslipidemia, treatment of severe hypertriglyceridemia and primary hypercholesterolemia or mixed dyslipidemia.

Trilipix® Delayed‐Release Capsules 45 mg & 135 mg strengths had annual U.S sales of approximately US$ 449.5 million (IMS MAT Sep, 2013).

References

PEMETREXED

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Mar 022014

Pemetrexed

US 5,344,932 .

(2S)-2-{[4-[2-(2-amino-4-oxo-1,7-dihydro
pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino}
pentanedioic acid

N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl] benzoyl]-L-glutamic acid or N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1 H-pyrrolo [2,3-d]-pyrimidin-5-yl)ethyl] benzoyl]-L-glutamic acid

GENERIC LICENSING NEWSLETTER TODAY 23 APRIL 2013 REPORTED, SEE LINK BELOW

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PEMETREXED

Pemetrexed is a chemotherapy drug used in the treatment of pleural mesothelioma as well as non-small cell lung cancer.Used in combination with cisplatin for the treatment of malignant pleural mesothelioma in adults whose disease is unresectable or who otherwise are not candidates for potentially curative surgery. Also used as a monotherapy for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) after prior chemotherapy.Click here to contact Logenex about this product.

Pemetrexed (brand name Alimta) is a chemotherapy drug manufactured and marketed by Eli Lilly and Company. Its indications are the treatment of pleural mesothelioma andnon-small cell lung cancer.

The molecular structure of pemetrexed was developed by Edward C. Taylor at Princeton University and clinically developed by Indianapolis based drug maker, Eli Lilly and Company in 2004.

PEMETREXED

Pemetrexed is chemically similar to folic acid and is in the class of chemotherapy drugs called folate antimetabolites. It works by inhibiting three enzymes used in purine andpyrimidine synthesis—thymidylate synthase (TS), dihydrofolate reductase (DHFR), andglycinamide ribonucleotide formyltransferase^[1]^[2] (GARFT). By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNAand RNA, which are required for the growth and survival of both normal cells and cancer cells.

Pemetrexed disodium is chemically described as L-Glutamic acid, N-[4-[2- (2-amino-4,7-dihydro-4-oxo-1 H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]- disodium salt heptahydrate, represented by the chemical structure of Formula (I).

Formula I

Pemetrexed is an anti-folate anti-neoplastic agent that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication. It is believed to work by inhibiting three enzymes that are required in purine and pyrimidine biosynthesis — thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyl transferase (GARFT). Pemetrexed is available in the market under the brand name ALIMTA^®.

Taylor et al., in describe pemetrexed, its related compounds and pharmaceutically acceptable cation. Chelius et al., in WO 01/14379 A2 disclose pemetrexed disodium crystalline hydrate Form I and process for preparation thereof.

Chelius et al., in WO 01/62760 disclose pemetrexed disodium heptahydrate crystalline Form Il and process for the preparation thereof.

Journal of Organic Process Research & Development, Volume 3, 1999, page 184 describes a process for the preparation of pemetrexed diacid. Busolli et al., in WO200802141 1 disclose process for preparation of pharmaceutically acceptable salt of pemetrexed diacid.

Busolli et al., in WO2008021405A1 disclose seven crystalline forms of pemetrexed diacid designated as Form A, B, C, D, E, F, & G and processes for preparation thereof.

In February 2004, the Food and Drug Administration approved pemetrexed for treatment of malignant Pleural Mesothelioma, a type of tumor of the lining of the lung, in combination with cisplatin^[3] for patients whose disease is either unresectable or who are not otherwise candidates for curative surgery.^[4] In September 2008, the FDA granted approval as a first-line treatment, in combination with cisplatin, against locally-advanced and metastatic non-small cell lung cancer (NSCLC) in patients with non-squamous histology. A Phase III study showed benefits of maintenance use of pemetrexed for non-squamous NSCLC.Activity has been shown in malignant peritoneal mesothelioma.Trials are currently testing it against esophagus and other cancers.

MECHANISM

Pemetrexed is also recommended in combination with carboplatin for the first-line treatment of advanced non-small cell lung cancer.However, the relative efficacy or toxicity of pemetrexed-cisplatin versus pemetrexed-carboplatin has not been established beyond what is generally thought about cisplatin or carboplatin doublet drug therapy

In addition to the brand name Alimta, this drug is also marketed in India by Abbott Healthcare as Pleumet and by Cadila Healthcare asPemecad.

Pemetrexed disodium is a multitargeted antifolate agent approved as a single agent for the treatment of non-small cell lung cancer, and in combination with cisplatin for the treatment of patient with malignant pleural mesothelioma, under the trade name Alimta^®.
Pemetrexed disodium is available in a number of crystalline forms.
Barnett et al, Organic Process Research & Development, 1999, 3, 184-188 discloses synthesis and crystallization of pemetrexed disodium from water-ethanol. The product obtained by the process disclosed herein is the 2.5 hydrate of pemetrexed disodium.
United States patent number 7,138,521 discloses a crystalline heptahydrate form of pemetrexed disodium, which has enhanced stability when compared to the known 2.5 hydrate.
To date workers have concentrated on producing stable crystalline forms of pemetrexed disodium and there has been no disclosure of any non-crystalline form of this active.
We have now found a new form of pemetrexed disodium, which is an amorphous form, as characterized by powder X-ray diffraction. Surprisingly, we have found that it is possible to prepare an amorphous form of pemetrexed disodium and that this form is stable. The amorphous form of the invention is stable contrary to expectations. The amorphous form of pemetrexed disodium of the present invention is stable as it retains it’s amorphous character under a variety of storage conditions. The amorphous form of the present invention is particularly advantageously characterized by a bulk density in the range of 0.15 to 0.35 gm/ml.

“Pemetrexed”)

R = H: Pemetrexed; I

R = Na: Pemetrexed Disodium; II is a known compound. Pemetrexed Disodium is an known anticancer agent. It is clinically active in several solid tumors and approved for treatment of malignant pleural mesothelioma (MPM) and metastatic non-small cell lung cancer (NSCLC). Pemetrexed Disodium is supplied as a sterile lyophilized powder for intravenous administration.

The compound of formula I including pharmaceutically salts thereof as well as a process for its preparation is at first and specifically disclosed in EP patent no. 0432677 B1. The preparation and isolation of Pemetrexed (compound of formula I) as its Disodium salt (compound of formula II) was described for the first time in WO patent no. 9916742 A1 and in Drugs of the future 1998, 23(5), 498-507 as well as by Charles J. Barnett et al. in Organic Process Research & Development, 1999, 3, 184-188 and by Peter Norman in Current Opinion in Investigational Drugs 2001 , 2(11 ), 1611-1622.

Detailed information about the crystalline form of Pemetrexed Disodium prepared according to the process as described above were not provided but it is reported by Charles J. Barnett et al. in Organic Process Research & Development, 1999, 3, 184-188 that the disodium salt II was obtained as a hygroscopic solid.

The first crystalline form of Pemetrexed Disodium has been described in WO patent no. 0114379 designated Disodium MTA Hydrate Form I (MTA = multi- targeted antifolate, disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H- pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt). The Disodium MTA Hydrate Form I obtained according to the Examples 2, 3 and 4 contained different amounts of water (Example 2: water = 9.1%; Example 3: water = 17.7%; Example 4: water = 11.7%). The Disodium MTA Hydrate Form I has a typical XRD pattern as shown in Figure 4 (the corresponding 2theta values have been calculated from the provided d-spacing values).

An improved crystalline form of Pemetrexed Disodium has been disclosed in WO patent no. 0162760. It is teached that Pemetrexed Disodium can exist in the form of a heptahydrate (Form II; theoretical amount of water: approx 21%) which is much more stable than the previously known 2.5 hydrate (Form I; theoretical amount of water: 8.7%). The Pemetrexed Disodium Heptahydrate Form (Form II) has a typical XRD pattern as shown in Figure 5 (the corresponding 2theta values have been calculated from the provided d- spacing values).

The Chinese patent no. 1778802 describes a hydrate or trihydrate form of Pemetrexed Disodium. The preparation of Pemetrexed Disodium hydrate or trihydrate includes crystallization from water and water soluble solvent. An overview of the X ray powder diffraction data for Pemetrexed Disodium Hydrate provided in Chinese patent no. 1778802 is shown in Figure 6.

The WO patent no. 2008124485 disclose besides crystalline Forms of the diacid Pemetrexed also amorphous Pemetrexed Disodium as well as a crystalline Form III thereof including a composition containing a major amount of amorphous Form and a minor amount of crystalline Form III of Pemetrexed Disodium. An overview of the X ray powder diffraction data for Pemetrexed Disodium crystalline Form 3 is shown in Figure 7.

EP patent application no. 2072518 disclose a stable amorphous form of Pemetrexed Disodium.

According to the more recent US 5,416,211 , which is incorporated herein by reference, pemetrexed can be synthesized from 4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid of formula 1, obtained from simple precursors, in accordance with the following Scheme 1:
This second method seems to be used also for the industrial preparation of the active ingredient. In fact, the same type of synthesis scheme is also described in C. J. Barnett, T. W. Wilson and M. E. Kobierski, Org. Proc. Res. & Develop., 1999, 3, 184-188, in which the experimental examples refer to a scale of the order of tens of kgs.

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WO2012134392A1

Example 1 Preparation of crude pemetrexed disodium

[0023] N-[4-2-(2-Amino-4, 7-dihydro-4-oxo-

1 H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic Acid Diethyl Ester

4-Methylbenzenesulfonic Acid Salt and purified process water (PPW) (about 10 kg) are charged to a suitable vessel under nitrogen. The reactor is cooled to NMT 10Ό under nitrogen. Pre-cooled sodium hydroxide solution (about 1.5 kg )/PPW (about 11.4 kg) are added and the temperature is maintained at NMT 10Ό. The mixture is stirred at NMT 0 until the solid is dissolved. Pre-cooled isopropanol (about 62.8 kg) is added and the mixture temperature is maintained at NMT 5 . Pre-cooled 1 N hydrochloric acid in isopropanol is added to adjust the pH to 6.5 to 9.5, preferably between pH 7.5 to pH 8.5, at NMT 5 . The mixture is warmed to a room temperature (i.e., 15-30Ό, preferably 20-25″C) and stirred. The solids are filtered and washed with isopropanol/PPW. The wet cake is vacuum dried to provide crude pemetrexed disodium (about 2.30 kg).

Example 2 Purification of crude pemetrexed disodium to pemetrexed disodium

[0024] Crude pemetrexed disodium (about 2.1 kg) and PPW (about 23.3 kg) are charged under nitrogen to a suitable vessel at 15 to 30 . Isopropanol (about 28.3 kg) is added slowly to cloud point and stirred. Isopropanol (up to about 55 kg) is charged and stirred. The solids are filtered arid washed with isopropanol/PPW. The wet cake is vacuum dried to provide pemetrexed disodium (about 1.9 kg) (90% Yiled). 1 H NMR (D₂0): δ 7.51 (2H, d, J=8.0 Hz), 6.98 (2H, d, J=8.0 Hz), 6.12 (1 H, s), 4.26-4.23 (H, m), 3.60-3.54 (4H, m), 2.27-2.23 (2H, m), 2.13-2.08 (1 H, m), 2.00-1.94 (1 H, m)

HPLC

EP2072518A1

Example 7

HPLC Analysis method

Reagent: Water :milliQ,
Sodium perchlorate :AR Grade
Perchloric acid :AR Grade
Acetonitrile :J.T.Baker gradient
Trifluroacetic acid :AR Grade
Buffer solution: 6.1 g of sodium perchlorate into a 1000ml water. Adjust the pH to 3.0 (± 0.1) with perchloric acid.
Mobile phase A:
mixture of buffer and acetonitrile in the proportion of (90:10).
Mobile phase B:
mixture of buffer and acetonitrile in the proportion of (10 : 90).
Diluent -1 : mixture of water and acetonitrile in the ratio of 50 : 50.
Diluent -2: mixture of water and acetonitrile in the ratio of 90 : 10.
Standard Stock Solution:
Transfer accurately weighed 1.5 mg impurity-E RS and into a 200 ml volumetric flask. Dissolve in and dilute upto mark with diluent-1.

Blank solution

Add 10 ml diluent-2 and 50µl of 3% trifluro acetic acid to a 50 ml volumetric flask, and dilute upto mark with diluent-2.

System suitability solution

Transfer about 25 mg accurately weighed pemetrexed disodium sample in to a 50 ml volumetric flask. First add 10ml of diluent-2 and sonicate to dissolve the contents.Then add 50µl of 3% trifluro acetic acid (prepared in water) and add 5 ml of standard stock solution and dilute up to mark with diluent-2.

Sample preparation

Transfer about 25 mg accurately weighed pemetrexed disodium sample in to a 50 ml volumetric flask. First add 10ml of diluent-2 and sonicate to dissolve the contents.Then add 50µl of 3% trifluro acetic acid (prepared in water) and dilute up to mark with diluent-2 (500 µg/ml).

Chromatographic system :

Use a suitable high pressure liquid chromatography system equipped with Column: 250 mm x 4.6mm containing 5µ packing material (suggested column – Inertsil ODS 3V)
Detector: UV detector set to 240 nm
Cooler temp: 5°C.
Flow rate: about 1.5 ml/min.
The system is also equipped to deliver the two phases in a programmed manner as shown in the following table :

Gradient programme :

[0082]

0 92 8

15 85 15

30 65 35

35 65 35

36 92 8

40 92 8

Procedure:

Inject 20µl of blank and system suitability solution into the chromatograph set to above conditions and record the chromatograms up to 40 min.
Calculate the resolution between pemetrexed disodium and impurity-E. The resolution should not be less than 3.0. Calculate the Number of theoretical plate and tailing factor for pemetrexed peak. Number of theoretical plate is NLT 4000 and tailing factor is NMT 2.0.
Inject 20µl of test solution and calculate the chromatographic purity by area normalisation method.

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US20120329819

Synthetic Route for the Preparation of Pemetrexed Disodium

Starting from commercially available materials a novel synthetic route for the synthesis of Pemetrexed-IM8 (the dimethyl ester of Pemetrexed) was developed which was then used for the preparation of Pemetrexed Disodium (Scheme 1).

The current synthetic route for the preparation of Pemetrexed IM8 starts with an aldol-condensation reaction of Methyl-4-formylbenzoate (SM1) with 1,1-Dimethoxyacetone (SM2) to give Pemetrexed IM1a. As Pemetrexed IM1a irreversibly converts to its aldol-addition product Pemetrexed IM1b under reaction conditions the reaction mixture is directly submitted to hydrogenation (i.e. without isolation of Pemetrexed IM1a) over Pd/C to give Pemetrexed IM2. As under the hydrogenation conditions not only the double-bond of IM1a is hydrogenated but also some amount of Pemetrexed IM2 is converted to Pemetrexed IM3 (hydrogenation of the carbonyl function to the corresponding secondary alcohol) a solution of NaBH4 is added to the reaction mixture to ensure complete conversion to Pemetrexed IM3. The Pd-catalyst is removed by filtration and the reaction mixture is extracted with toluene. The combined organic layers are evaporated to give crude Pemetrexed IM3 as oil. This oil is dissolved in THF and the alcohol functionality is converted to a mesylate using MsCl and NEt3. The salts are removed by filtration, glacial acetic acid is added and THF is removed by distillation. Upon addition of water Pemetrexed IM4 crystallizes and is isolated by filtration. The dried Pemetrexed IM4 is dissolved in glacial acetic acid and gaseous HCl is added to cleave the dimethoxy acetale and liberate the aldehyde functionality of Pemetrexed IM5. Upon complete deprotection a solution of 2,6-diamino-4-hydroxypyrimidine in aq. NaOH and acetonitrile is added. Upon complete conversion the crystallized Pemetrexed IM6 is isolated by filtration. The saponification of the methyl ester of Pemetrexed IM6 to Pemetrexed IM7 is done using aqueous NaOH. Upon addition of aq. HCl first the Na-salt of Pemetrexed IM7 crystallizes from the reaction mixture. The salt is isolated by filtration, purified by slurry in a mixture of MeOH and water and then converted to Pemetrexed IM7 by pH adjustment in water using aq. HCl. Dried Pemetrexed IM7 (water content not more than 6.0%) is dissolved in DMF, activated using 1,1-carbonyldiimidazolide (CDI) and then reacted with dimethyl-L-glutamate hydrochlorid to give, upon addition of water and filtration, crude Pemetrexed IM8. This intermediate is purified by tosylate salt formation, followed by recrystallization and liberation to give pure Pemetrexed IM8. Starting with the saponification of Pemetrexed IM8 the preparation of different solid forms of Pemetrexed Disodium can be achieved.

Methods For Preparing Pemetrexed Disodium Form IV and Investigation of its Stability

An overview on the possible transformations of Pemetrexed IM8 to Pemetrexed Disodium Form IV is shown in FIG. 20.

Description of Possible Routes for the Preparation of Pemetrexed Disodium Form IV Starting from Pemetrexed IM8

All routes start with saponification of Pemetrexed IM8 in water at IT=20° C. to 30° C. using 3.25 eq of NaOH. Upon complete conversion an aqueous solution of Pemetrexed Disodium with a pH of 13.0 to 13.5 is obtained. Starting from this mixture the desired route can be accessed by addition of HCl to adjust the pH to a certain value (depending on the route, FIG. 20).

Structures of Pemetrexed (Compound I), Pemetrexed Disodium (Compound II) and Pemetrexed Monosodium (Compound IV)

Surprisingly we found that the crucial feature of all successful transformations to Pemetrexed Disodium Form IV is the presence of Pemetrexed Monosodium during the transformation. Routes starting from pure Pemetrexed Disodium Heptahydrate, Pemetrexed Disodium 2.5 hydrate or Pemetrexed Disodium Form A in the presence of seeding crystals of Pemetrexed Disodium Form IV were not successful and resulted in isolation of Pemetrexed Disodium Form A. The same transformations, if carried out in the presence of 0.15 eq of Pemetrexed Monosodium were successful and after addition of 0.15 eq NaOH allowed the isolation of pure Pemetrexed Disodium Form IV. The use of 0.15 eq HCl instead of 0.15 eq Pemetrexed Monosodium under the same conditions resulted in isolation of Pemetrexed Disodium Form A without any Pemetrexed Disodium Form IV. Transformations via isolated Pemetrexed Monosodium gave complete transformation to Pemetrexed Disodium Form IV if either 1.0 eq NaOH were added slowly (over a period of several hours) to Pemetrexed Monosodium or if initially only 0.85 eq of NaOH (based on Pemetrexed Monosodium) were added, followed by 0.15 eq once the transformation to Pemetrexed Disodium Form IV was complete. Very fast addition (<1 min) of 1.0 eq NaOH resulted in formation of Pemetrexed Disodium Form A containing traces of Pemetrexed Disodium Heptahydrate.

Starting from Pemetrexed (compound I) the transformation to Pemetrexed Disodium Form IV would be possible if initially 1.85 eq of NaOH were added followed by 0.15 eq once the transformation was complete. Alternatively, 2.0 eq of NaOH could be added over a long period of time (i.e several hours) to achieve formation of Pemetrexed Form IV. Fast addition (<1 min) of 2.0 eq of NaOH is assumed to result in formation of Pemetrexed Disodium Form A. All these experiments show the presence of Pemetrexed Monosodium to be crucial during the transformations. This presence can be achieved by either addition of catalytic amounts of Pemetrexed Monosodium to Pemetrexed Disodium, by slow addition over several hours of NaOH to Pemetrexed Monosodium or by portionwise addition of NaOH to Pemetrexed Monosodium. Addition of catalytic amounts of HCl to Pemetrexed Disodium (in situ preparation of Pemetrexed Monosodium) failed to give Pemetrexed Disodium Form IV.

Fast addition of NaOH to Pemetrexed Monosodium results in fast formation of Pemetrexed Disodium, thereby lacking the necessary catalytic amounts of Pemetrexed Monosodium to catalyze the transformation to Pemetrexed Disodium Form IV. EtOH as solvent and water content of EtOH were found to be crucial parameters for the transformation to Pemetrexed Disodium Form IV. So far the transformation has only been observed in EtOH containing 0-4% water (v/v). A water content>4% (v/v) results in formation of Pemetrexed Disodium Heptahydrate. Under the conditions used (EtOH containing 0-4% water (v/v)) both Pemetrexed Disodium Heptahydrate and Pemetrexed Disodium 2.5 hydrate are transformed to Pemetrexed Form A. Therefore the mechanism of the transformation to Pemetrexed Disodium Form IV is assumed to proceed via Pemetrexed Disodium Form A with Pemetrexed Monosodium acting as catalyst for the transformation.

Preparation of Pemetrexed Disodium Heptahydrate

a) Preparation of Pemetrexed Disodium Heptahydrate Starting from Pemetrexed IM8

Pemetrexed Disodium Heptahydrate was prepared by adjustment of the pH of the Pemetrexed Disodium solution after saponification from pH=13 to pH=8 using HCl followed by addition of EtOH (3 times the volume of water) to achieve crystallization. Precipitated Pemetrexed Disodium Heptahydrate was isolated by filtration, washed with a mixture of EtOH and water (4:1 v/v) followed by EtOH. The wet product was dried in vacuo at 200 mbar at 20° C. to 30° C. until water content of the dried product was 20.1% to 22.1%.

b) Conversion of Pemetrexed Disodium Form A to Pemetrexed Disodium Heptahydrate

To a suspension of Pemetrexed Disodium Form A in EtOH was added water until a mixture of EtOH containing 25% water (v/v) was obtained. The resulting suspension was stirred at 20° C. to 30° C. until conversion was complete according to PXRD. Pemetrexed Disodium Heptahydrate was isolated by filtration, washed with EtOH and dried in vacuo at 200 mbar at 20° C. to 30° C. until water content of the dried product was 20.1% to 22.1%.

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EP2504341A1

REFERENCES

McLeod, Howard L.; James Cassidy, Robert H. Powrie, David G. Priest, Mark A. Zorbas, Timothy W. Synold, Stephen Shibata, Darcy Spicer, Donald Bissett, Yazdi K. Pithavala, Mary A. Collier, Linda J. Paradiso, John D. Roberts (Jul-2000).“Pharmacokinetic and Pharmacodynamic Evaluation of the Glycinamide Ribonucleotide Formyltransferase Inhibitor AG2034”. Clinical Cancer Research 6 (7): 2677–84.PMID 10914709. More than one of |work= and |journal=specified (help)
Avendano, Carmen; Menendez, J. Carlos (April 2008).Medicinal Chemistry of Anticancer Drugs. Amsterdam:Elsevier. p. 37. ISBN 0-444-52824-5.
Manegold C (August 2003). “Pemetrexed (Alimta, MTA, multitargeted antifolate, LY231514) for malignant pleural mesothelioma”. Semin. Oncol. 30 (4 Suppl 10): 32–6.doi:10.1016/S0093-7754(03)00283-5. PMID 12917819.
National Cancer Institute: FDA Approval for Pemetrexed Disodium

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