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Afatinib

April 19, 2014 2:55 am / Leave a comment

Afatinib

439081-18-2

850140-73-7 dimaleate

Tovok, BIBW2992, Tomtovok

Boehringer Ingelheim Int,

An irreversible EGFR/HER2 inhibitor

Molecular Weight:	485.94
Molecular Formula:	C₂₄H₂₅ClFN₅O₃

N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide

4 – [(3-chloro-4-fluorophenyl) amino] -6 – {[4 – (N, N-dimethylamino)-1-oxo-2-buten-1-yl] – amino} -7 – ((S )-tetrahydrofuran-3-yloxy)-quinazoline

(E)-4-Dimethylamino-but-2-enoic acid {4-(3-chloro-4-fluoro- phenylanimo)-7-[(S)-(tetrahydro-furan-3-yl) oxy]-quinazolin-6-yl} -amide

4 – [(3_ chloro-4 – fluorophenyl) amino] -6 – {[4_ (N, N-dimethylamino)-buten-1-oxo-_2_ – yl] amino}-7 – ((S) – tetrahydrofuran-3 – yloxy) – quinazoline

EMA:Link, US FDA:link

The endorsement for Giotrif (afatinib) covers the drug’s use in the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) who have the epidermal growth factor receptor (EGFR) gene mutation, which is present in about 10 per cent of people with NSCLC.

It caps a good month for Boehringer, which won US approval for the drug under the brand name Gilotrif two weeks ago, adding to the company’s list of therapy areas, which so far include chronic obstructive pulmonary disease (COPD), anticoagulation, HIV, Parkinson’s disease and diabetes.

In the US, the drug is approved alongside a companion diagnostic to help determine if a patient’s lung cancer cells express the EGFR mutations, whereas the EMA recommendation just includes the requirement that Giotrif be initiated and supervised by a physician experienced in the use of anti-cancer therapies.

http://www.pmlive.com/pharma_news/boehringers_first_cancer_drug_leads_ema_recommendations_493051

GILOTRIF tablets contain afatinib, a tyrosine kinase inhibitor which is a 4-anilinoquinazoline. Afatinib is presented as the dimaleate salt, with the chemical name 2-butenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-,(2E)-, (2Z)-2-butenedioate (1:2). Its structural formula is:

GILOTRIF™ (afatinib) Structural Formula Illustration

Afatinib dimaleate is a white to brownish yellow powder, water soluble and hygroscopic, with an empirical formula of C₃₂H₃₃ClFN₅O₁₁, and a molecular weight of 718.1 g/mol.

GILOTRIF tablets for oral administration are available in 40 mg, 30 mg, or 20 mg of afatinib (equivalent to 59.12 mg, 44.34 mg, or 29.56 mg afatinib dimaleate, respectively). The inactive ingredients of GILOTRIF are the following: Tablet Core: lactose monohydrate, microcrystalline cellulose, crospovidone, colloidal silicon dioxide, magnesium stearate. Coating: hypromellose, polyethylene glycol, titanium dioxide, talc, polysorbate 80, FD&C Blue No. 2 (40 mg and 30 mg tablets only).

Afatinib (BIBW2992) is an irreversible EGFR/Neu inhibitor with an IC₅₀ of 14 nM. Afatinib is a potent inhibitor of EGFR phosphorylation. Afatinib showed positive results in assays against a variety of human cancer cell lines, including A431, murine NIH-3T3 cells, and breast cancer cell line BT-474.

Afatinib^[2] (INN; trade name Gilotrif in the US and Giotrif in Europe, previously Tomtovok and Tovok^[3]) is a drug approved inmuch of the world (including the United States, Canada, the United Kingdom and Australia) for the treatment of metastatic non-small cell lung carcinoma (NSCLC), developed by Boehringer Ingelheim.^[4]^[5]^[6] It acts as an angiokinase inhibitor.

Quinazoline derivatives, such as afatinib, are described in WO2002050043. This document also describes certain favourable pharmacological properties of this compound. The dimaleate salt and its crystalline form are described in WO2005037824.

It is known in the W002/50043, which describes the pharmacological properties has important compounds include in particular their pharmacological properties mediated by the tyrosine kinase inhibitory effect and the signal transmission through the skin growth factor receptor (EGF-R) signal transduction mediated inhibitory effect. Therefore, this type of compounds are useful in the treatment of diseases, in particular for the treatment of tumor diseases, lung and gastrointestinal and respiratory tract and gall bladder and bile duct disease.

W002/50043 discloses a method for preparing a compound wherein the amino crotonic group (IV), such as 4_ [(3 – chloro-4 – fluorophenyl) amino] -6 – {[4 – (N, N-two methyl-amino)-oxo-2-1_ – buten-1 – yl] amino} -7 – ((S) – tetrahydrofuran-3 – yloxy) – quinazoline in the one-pot reaction from the corresponding aniline component (II), bromo crotonic acid (III), oxalyl chloride and a secondary amine prepared (see Figure 1).

Figure 1:

In the method, the yield was 50% at most. In addition, the implementation typically purified by column chromatography. Therefore Preparation of 4 – [(3_ chloro-4 – fluorophenyl) amino] -6 – {[4 – (N, N-dimethylamino)-l-oxo-2 – buten-1 – yl] amino} -7 – ((S) – tetrahydrofuran-3 – yloxy) – quinazoline of the method is not for large-scale industrial production. Moreover, the method is not drawback bromo crotonate purchased by a large number of commercial sources, and the corresponding bromo-methyl crotonate only be obtained in a purity of about 80%.These methods are used in this case is also 4 – [(3 – chloro-4 – fluorophenyl) amino] -6 – {[4 – (N, N-dimethylamino) -1 – oxo – butene-1 – yl] amino} -7 – (⑶ – tetrahydrofuran-3 – yloxy) – quinazoline industrialized production adversely affect the applicability.

In the above-mentioned drawbacks of known production methods, the present invention is to provide a produce aminocrotonate aryl amides, in particular 4 – [(3 – chloro-4 – fluorophenyl) amino] -6 – {[4 – (N, N-dimethylamino)-buten-1-oxo-_2_ – yl] amino} -7 – ((S) – tetrahydrofuran-3 – yloxy) – quinazoline The method of the method can be easily obtained using high purity starting materials and does not require the use of any material technology. Thus, the new method should be applicable on an industrial scale synthesis grade and therefore suitable for commercial applications.

This task is according to the present invention for preparing 4 – [(3 – chloro-4 – fluorophenyl) amino] -6 – {[4 – (N, N-dimethylamino) -1 – oxo-2 – buten-1 – yl] amino} -7 – (⑶ – tetrahydrofuran-3 – yloxy) – quinazoline, and other amino crotonic method based compound. In addition to high yield industrially embodiment, the synthesis method according to the present invention also has a very good purity and less than 0.1 of the advantages of a low cis content.

According to Figure 2, in the method according to the present invention, an aryl group corresponding amino compound (V) with two – (Ch-ware yl) _ phosphono acetic acid, preferably with diethyl phosphonoacetate, by After appropriate activation, in a suitable reaction solvent, preferably for the use of the active 1,1 – carbonyldiimidazole, 1,1 – carbonyldiimidazole – triazole or propane phosphonic acid anhydride, is preferred for the use of 1, 1 – carbonyl diimidazole. The solvent used may be, for example, tetrahydrofuran (THF), dimethylformamide (DMF) or ethyl acetate.

The amide may be connected through any possible approach for activation, i.e., for example, 1,1 _ carbonyldiimidazole, 1,1 – carbonyldiimidazole – triazole, DCC (N, N-dicyclohexyl carbodiimide ), EDC (N ‘_ (dimethylaminopropyl)-N-ethylcarbodiimide), TBTU (0 – (benzotriazol-1 – yl)-N, N, N’, N ‘ – pan tetramethyluronium tetrafluoroborate), thiazolidine-2 – thione, or through the use of thionyl chloride may be converted to the corresponding acyl chloride. If desired, activation may be used an organic base such as triethylamine or pyridine embodiment, and can additionally added DMAP (dimethylaminopyridine). Suitable solvents include DMF, THF, ethyl acetate, toluene, chlorinated hydrocarbons or mixtures thereof.

Figure imgf000002_0001

http://www.google.com/patents/CN1867564B?cl=en

Example 1

{[4 – (3 – chloro-4 – fluoro – phenylamino) -7 – (⑶ – tetrahydrofuran _3_-yloxy) – quinazoline _6_ yl carbamoyl] methyl}-_ _ Diethyl

A 3. 58kg of 1,1 _ carbonyldiimidazole (22.16 mol) was placed in 12.8 l of tetrahydrofuran, and at a temperature of 40 ° C was dissolved in it with 6.5 l of tetrahydrofuran, 4. 52kg (22. 16 mol) of diethyl phosphono acetic acid mixture. Temperature at 40 ° C the mixture was stirred for 30 minutes. The resulting solution was referred to as Solution A.

A 6. 39kg (17. 05 moles) of N4-(3_ _4_ chloro fluoro – phenyl) _7_ (tetrahydrofuran _3_ yloxy) quinazoline-4, 6 – diamine Add 26 5 of tetrahydrofuran at 40 ° C and the solution A were mixed and stirred at a temperature 30 ° C for 2 hours.To the suspension was added 64 l tert-butyl methyl ether and, after cooling to 20 ° C, the precipitate was removed by centrifugation. Using 16 liters of tetrahydrofuran and 16 l of a mixture of tert-butyl methyl ether, washed, and then washed with 32 liters of water and dried at 50 ° C.

Yield: 6. 58kg (69. 8%) of white crystals, the content = HPLC 99. IFl%

Example 2

(E) -4 – dimethylamino – D -2 – acid – [4 – (chloro-3_ _4_ fluoro – phenylamino) _7_ (⑶ – tetrahydrofuran-3 – yloxy) – quinoline yl-6 – yl] – amide

A 5.6 l of 30% hydrochloric acid (53.17 mol) was added to 4.4 liters of water. Then the temperature is under 30 ° C was added dropwise over 20 minutes 4. 28kg 95% of (dimethylamino) _ acetaldehyde – diethyl acetal (26.59 mol).Temperature at 35 ° C the reaction solution was stirred for 8 hours was cooled to 5 ° C and kept under argon. This solution is called Solution B.

A 4. 55kg (68. 06 mol) of potassium hydroxide dissolved in 23.5 liters of water and cooled to _5 ° C. This solution is called Solution C.

A 5. 88kg (10. 63 mol) ((4_ (3_ _4_ chloro fluoro – phenylamino) _7_ (tetrahydrofuran _3_-yloxy) – quinazolin-6 – yl carbamoyl) – methyl)-phosphonic acid diethyl ester and 0.45kg _ lithium chloride (10.63 moles) was placed in 23.5 l of tetrahydrofuran and cooled to -7 ° C. Was added over 10 minutes a cold solution of C. Then _7 ° C temperature of the solution was added over 1 hour B. At _5 ° C temperature for 1 hour under stirring the reaction mixture was heated to 20 ° C and mixed with 15 liters of water. After cooling to; TC temperature, the suspension was suction filtered, the precipitate was washed with water and dried. Yield: 5.21kg The crude product, 100%, water content: 6.7%.

Using Titanium Dioxide / methyl cyclohexane embodiment the crystallization of the crude product.

Yield: 78%, purity: HPLC99. 4F1%, water content: 5.4%

Example 3

(E) -4 – dimethylamino – D -2 – acid – (4 – (chloro-3_ _4_ fluoro – phenylamino) ~ 7 ~ ((S) – tetrahydrofuran-3 – yl oxy) – quinazolin-6 – yl) – amide dimaleate

A 6. Okg (12. 35 mol) of (E_) _4_ _2_ dimethylamino acid _ D – (4_ (3_ _4_ chloro fluoro – phenylamino) -7 – ((S) – tetrahydrofuran-3 – yloxy) – quinazolin-6 – yl) – amide into 84 liters of ethanol and heated to 70 ° C, and dissolved in 36 l of ethanol and 2.94kg (25.31 moles) of maleic acid was mixed . At the beginning of crystallization, the first mixture was cooled to 20 ° C and stirred for 2 hours and then at 0 ° C temperature for 3 hours. Precipitate was suction filtered, washed with 19 l of ethanol at a temperature of 40 ° C in vacuo.

Yield: 8. Ilkg (91. 5%)

Melting point: 178 ° C

[0096] 1H-NMR (CD3OD): δ = 2. 47 + 2. 27 (m + m, 2H), 2. 96 (s, 6H), 4. 03 (m, 2Η), 4. 07 +3 . 92 (m + m, 2Η), 4. 18 +4. 03 (m + m, 2Η), 5. 32 (m, 1Η), 6. 26 (s, 4H), 6. 80 (m, 1H ), 6. 99 (m, 1H), 7 · 27 (s, 1Η), 7 · 30 (t, 1Η), 7 · 66 (m, 1Η), 7 · 96 (dd, 1Η), 8 · 62 (s, 1Η), 9 · 07 (s, 1Η) ppm

PATENT

Examples:

Example 1

{[4 – (3-chloro-4-fluoro-phenylamino) -7 – ((S)-tetrahydrofuran-3-yloxy)-quinazolin-6-ylcarbamoyl]-methyl)-phosphonic acid diethyl ester

3.58 kg 1 ,1-carbonyldiimidazole (22.16 mole) were placed in 12.8 liters of tetrahydrofuran at 40 ° C with 4.52 kg (22.16 mol) diethylphosphonoacetic acid, dissolved in 6.5 liters of tetrahydrofuran, . The mixture is stirred for 30 minutes at 40 ° C. The solution thus obtained is referred to as solution A.

6.39 kg (17.05 mol) of N ⁴ – (3-chloro-4-fluoro-phenyl) -7 – (tetrahydrofuran-3-yloxy) quinazolin-4,6-diamine in 26.5 liters of tetrahydrofuran and submitted to 40 ° C and mixed with the solution A and stirred at 30 ° C for 2 hours. To 64 liters of suspension of tert -. Added butyl methyl ether and, after cooling to 20 ° C., the precipitate is removed by centrifugation. It is dried with a mixture of 16 liters and 16 liters of tetrahydrofuran tert-butyl methyl ether and then washed with 32 liters of water at 50 ° C. Yield: 6.58 kg (69.8%) of white crystals Assay: HPLC 99.1 area% Example 2

(E)-4-dimethylamino-but-2-enoic acid [4 – (3-chloro-4-fluoro-phenylamino) -7 – ((S) – tetrahvdrofuran-3-yloxy)-quinazolin-6yl1 amide

5.6 liters to 4.4 liters of water are added 30% hydrochloric acid (53.17 mol). Then 4.28 kg 95% pure (dimethylamino) acetaldehyde diethyl acetal (26.59 mol) at 30 ° C was added dropwise over 20 minutes. The reaction solution is stirred for 8 hours at 35 ° C, cooled to 5 ° C and kept under argon. This solution is referred to as solution B.

4.55 kg (68.06 mol) of potassium hydroxide are dissolved in 23.5 liters of water and cooled to -5 ° C. This solution is called solution C..

5.88 kg (10.63 mol) of ((4 – (3-chloro-4-fluoro-phenylamino) -7 – (tetrahydrofuran-3-yloxy) – quinazolin-6-ylcarbamoyl)-methyl)-phosphonic acid diethyl ester, and 0.45 kg lithium chloride (10.63 mole) were placed in 23.5 liters of tetrahydrofuran and cooled to -7 ° C. The cold solution C is added within 10 minutes. The solution B is added at -7 ° C over 1 hour. After stirring for one hour at -5 ° C, the reaction mixture is heated to 20 ° C and mixed with 15 liters of water. After cooling to 3 ° C, the suspension is filtered with suction, the precipitate washed with water and dried. Yield: 5.21 kg raw 100% Water content: 6.7%

The crystallization of the raw product is butyl acetate / methylcyclohexane yield: 78% HPLC purity 99.4 area%, water content 5.4% Example 3

(E)-4-dimethylamino-but-2-enoic acid (4 – (3-chloro-4-fluoro-pheny hvdrofuran-3-yloxy)-quinazolin-6YL) amide dimaleate

6.0 kg (12.35 mol) of (E)-4-dimethylamino-but-2-enoic acid (4 – (3-chloro-4-fluoro-phenyl-amino) -7 – ((S)-tetrahydrofuran- 3-yloxy) quinazolin-6YL)-amide are in 84 liters

Submitted ethanol and heated to 70 ° C and a solution of 2.94 kg (25.31 mol) of maleic acid in 36 liters of ethanol added.Following the onset of crystallization is first cooled to 20 ° C. and stirred for 2 hours, then 3 hours at 0 ° C. The precipitate is filtered off, washed with 19 liters of ethanol and dried in vacuum at 40 ° C.

Yield: 8.11 kg (91, 5%)

Mp: 178 ° C.

¹ H NMR (CD ₃ OD): δ = 2.47 + 2.27 (m + m, 2H), 2.96 (s, 6H), 4.03 (m, 2H), 4.07 + 3 , 92

(M + m, 2H), 4.18 + 4.03 (m + m, 2H), 5.32 (m, 1 H), 6.26 (s, 4H), 6.80 (m, 1 H ), 6.99 (m, 1 H), 7.27 (s, 1 H), 7.30 (t, 1 H), 7.66 (m, 1 H), 7.96 (dd, 1 H ), 8.62 (s, 1 H), 9.07 (s, 1H) ppm

…………..

Chemical Synthesis for the Preparation of Gilotrif - Afatinib-Gilotrif-Covalent Lung Cancer Drug lung cancer drug erlotinib A method of preparation of

U.S. Patent No. : 8,426,586 patent expires : October 10, 2029

WO200250043A1 (compound);

WO2003094921A2 (anticancer purposes);

WO2003066060A2 (anti-inflammatory purposes);

US2005085495A1 (process);

WO2005037824A2 (process);

WO2007085638A1 (process);

US2011207932A1 (process);

WO2011084796A2 (deuterated);

WO2012121764A1 (crystalline);

WO2013052157A1 (crystalline)

Chinese patents : CN1867564

CN101402631

UPDATE…………………

(WO2015186065) PROCESS FOR THE PREPARATION OF 4-DIMETHYLAMINOCROTONIC ACID

SUN PHARMACEUTICAL INDUSTRIES LIMITED [IN/IN]; Sun House, Plot No. 201 B/1 Western Express Highway Goregaon (E) Mumbai, Maharashtra 400 063 (IN)

VERMA, Shyam Sunder; (IN).
SINGH, Shravan Kumar; (IN).
SINGH, Kaptan; (IN).
PRASAD, Mohan; (IN)

Afatinib is a tyrosine kinase inhibitor disclosed in U.S. Patent Nos. RE43,431 and

6,251,912. Afatinib is depicted by Formula la:

Formula la

Afatinib is presented as the dimaleate salt and is chemically designated as 2-butenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(35)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-,(2£)-,(2Z)-2-butenedioate (1 :2) having the structure depicted by Formula I:

Formula I

Processes for the preparation of 4-dimethylaminocrotonic acid or its salts are disclosed in U.S. Patent No. 7,126,025 and U.S. Publication No. 2012/0046494.

U.S. Patent No. 7,126,025 discloses a process for the preparation of 4-dimethylaminocrotonic acid or its salts by reacting but-2-enoic acid with

chlorotrimethylsilane in pyridine to obtain trimethylsilylcrotonate, which is brominated with a brominating agent under free radical conditions and in the presence of methylene chloride, acetonitrile, 1,2-dichloroethane, carbon tetrachloride, or ethyl acetate to give trimethylsilyl-4-bromocrotonate. The bromocrotonate compound is treated with dimethylamine in tetrahydrofuran to provide the 4-dimethylaminocrotonic acid.

U.S. Patent No. 7,126,025 also discloses a process for the preparation of 4-dimethylaminocrotonic acid by treating methyl or ethyl 4-bromocrotonate with dimethylamine to provide methyl or ethyl 4-dimethylaminocrotonate, which is hydrolyzed to provide the 4-dimethylaminocrotonic acid.

U.S. Publication No. 2012/0046494 discloses a process for the preparation of 4-dimethylaminocrotonic acid or its salts by converting alkyl 4-chloro-3 -hydroxy butyrate to alkyl 4-hydroxy crotonate, which is brominated to obtain alkyl 4-bromo crotonate. The alkyl 4-bromo crotonate is treated with dimethyl amine to provide alkyl 4-dimethylaminocrotonate, which is hydrolyzed to get the 4-dimethylaminocrotonic acid.

The use of pyridine or carbon tetrachloride is toxic to humans and therefore their use for the manufacture of a drug substance is not advisable. The bromocrotonate compounds, being lachrymatory in nature, are difficult to handle on an industrial scale.

The present invention provides a faster, more efficient, and industrially feasible process for the preparation of 4-dimethylaminocrotonic acid of Formula II, which is used as an intermediate for the preparation of afatinib or its salts.

A first aspect of the present invention provides a process for the preparation of 4-dimethylaminocrotonic acid of Formula II or its salts,

Formula II

comprising the steps of:

i) converting 2,2-diethoxy-N,N-dimethylethanamine of Formula III

Formula III

to ethyl-4-(dimethylamino)crotonate of Formula IV; and

Formula IV

ii) hydrolyzing the ethyl-4-(dimethylamino)crotonate of Formula IV.

A second aspect of the present invention provides a process for the preparation of afatinib of Formula la or its salts,

Formula la

comprising the steps of:

i) converting 2,2-diethoxy-N,N-dimethylethanamine of Formula III

Formula III

to ethyl-4-(dimethylamino)crotonate of Formula IV;

Formula IV

ii) hydrolyzing the ethyl -4-(dimethylamino)crotonate of Formula IV to obtain 4- dimethylaminocrotonic acid of Formula II or its salts; and

Formula II

iii) converting the 4-dimethylaminocrotonic acid of Formula II or its salts to afatinib of Formula la or its salts.

EXAMPLES

Example 1 : Preparation of ethyl-4-(dimethylamino)crotonate (Formula IV)

In a round bottom flask, 2,2-diethoxy-N,N-dimethylethanamine (Formula III, 200 g) and deionized water (100 mL) were added at about 20°C to about 25°C. To the solution, concentrated hydrochloric acid (240 mL) was added at about 25°C to about 50°C. The temperature of the reaction mixture was raised to about 70°C. The reaction mixture was stirred at about 60°C to about 70°C for about 12 hours. The reaction mixture was cooled to about 0°C. To the reaction mixture, about 200 mL of aqueous potassium hydroxide (240 g in 250 mL water) was added at about 0°C to about 10°C to attain a pH of 9.0. To the reaction mixture, ethyl(diethoxyphosphoryl) acetate (200 g) and 2-methyltetrahydrofuran (600 mL) were added at about 0°C to about 5°C. Further, 50 mL of aqueous potassium hydroxide was added to the reaction mixture at about -5°C to about 0°C to attain a pH of about 13.5. The reaction mixture was stirred at about -5°C to about 0°C for about 1 hour. The reaction mixture was filtered, and then the filtrate was recovered under vacuum at about 45°C to about 50°C to obtain ethyl-4-(dimethylamino)crotonate as an oily mass.

Yield: 89%

Example 2: Preparation of 4-dimethylaminocrotonic acid hydrochloride (Formula ID

In a round bottom flask, ethyl -4-(dimethylamino)crotonate (Formula IV, 120 g) and ethanol (480 mL) were added at about 25°C to about 35°C. To the solution, aqueous sodium hydroxide (30.5 g in 60 mL water) was added at about 10°C to about 20°C. The temperature of the reaction mixture was raised to about 50°C. The reaction mixture was stirred at about 50°C to about 55°C for about 1 hour. The reaction mixture was cooled to about 5°C. To the reaction mixture, concentrated hydrochloric acid (120 mL) was added to attain a pH of 1.5. The reaction mixture was filtered on Celite^® and washed with ethanol (50 mL). The filtrate was recovered under vacuum at about 55°C to about 60°C to obtain a crude mass. Ethanol (240 mL) was added to the crude mass, and then the reaction mixture was stirred at about 55°C to about 60°C for about 15 minutes to obtain a solution. In the solution, sodium chloride was obtained as a byproduct. The solution was filtered to discard sodium chloride. The filtrate was recovered under vacuum at about 55°C to about 60°C to obtain a residue. To the residue, isopropanol (400 mL) was added, and then the reaction mixture was stirred at about 55°C to about 60°C to obtain a clear solution. The solution was gradually cooled to about 25°C to about 30°C. The solution was further stirred at the same temperature for about 2 hours. The solid obtained was filtered, and then washed with isopropanol (50 mL). The solid was dried under vacuum at about 55°C to about 60°C to provide 4-dimethylaminocrotonic acid hydrochloride.

Yield: 63%

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//////

Afatinib

- Synonyms:BIBW 2992
- ATC:L01XE13
Use:anticancer; tyrosine kinase inhibitor
Chemical name:N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide; N-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline
Formula:C₂₄H₂₅ClFN₅O₃

MW:485.9 g/mol
CAS-RN:439081-18-2; 850140-72-6

Derivatives

dimaleate

Formula:C₃₂H₃₃ClFN₅O₁₁
MW:718.1 g/mol
CAS-RN:850140-73-7

Synthesis Path

Substances Referenced in Synthesis Path

CAS-RN	Formula	Chemical Name
446-32-2	C₇H₆FNO₂	4-fluoro-anthranilic acid
162012-70-6	C₈H₃ClFN₃O₂	4-chloro-7-fluoro-6-nitroquinazoline
367-21-5	C₆H₅ClFN	3-chloro-4-fluoroaniline
86087-23-2	C₄H₈O₂	(S)-(+)-3-hydroxytetrahydrofuran
314771-76-1	C₁₈H₁₆ClFN₄O₂	N-(3-chloro-4-fluorophenyl)-7-((tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine
13991-36-1	C₄H₅BrO₂	bromocrotonic acid
3095-95-2	C₆H₁₃O₅P	diethylphophonoacetic acid
618061-76-0	C₂₄H₂₇ClFN₄O₆P	Diethyl-{[4-((3-chloro-4-fluorophenyl)amino)-7-(((S)-tetrahydro- furan-3-yloxy)quinazolin-6-yl)carbamoyl]-methyl}phosphonate
3616-56-6	C₈H₁₉NO₂	(dimethylamino)-acetaldehyde diethylacetate

Trade Names

Country	Trade Name	Vendor	Annotation
USA	Gilotrif	Boehringer Ingelheim, 2013
EU	Giotrif	Boehringer Ingelheim, 2013

Formulations

tabs.; 20, 30 and 40 mg

References

- a US 6 251 912 (American Cyanamid; 26.6.2001; appl. 29.7.1998; USA-prior. 1.8.1997).
- WO 0 250 043 (Boehringer Ingelheim; 27.6.2002; appl. 12.12.2001; DE-prior. 20.12.2000).
- US RE 43431 (Boehringer Ingelheim; 29.5.2012; appl. 18.8.2009; DE-prior. 20.12.2000).
- b US 8 426 586 (Boehringer Ingelheim; 1.2.2007; appl. 14.7.2006; DE-prior. 17.10.2003).

crystalline forms of Afatinib di-maleate:
- Solca, F. et al., J. Pharmacol. Exp. Ther., (2012) 343(2), 342-350.
- WO 2013 052157 (Ratiopharm/Teva; 11.4.2013; appl. 25.4.2012; USA-prior. 6.10.2011).

Teva Pharmaceutical has been given a green light by the European Commission (EC) for Lonquex, a rival to Amgen’s blockbuster Neulasta.

April 18, 2014 9:47 am / 2 Comments on Teva Pharmaceutical has been given a green light by the European Commission (EC) for Lonquex, a rival to Amgen’s blockbuster Neulasta.

lipegfilgrastim

C ₈₆₄ H ₁₃₆₉ N ₂₂₅ O ₂₅₈ S ₉ [C ₂ H ₄ O] _N

117844-87-7

pegylated granulocyte colony stimulating factor; O3.133-[N5-(N-{[ω-methoxypoly (oxyethylene)] carbonyl} glycyl)-α-neuraminyl-(2 → 6)-α-D-galactopyranosyl]-L-methionyl -des-1-L-alanine-des-37-L-valine-des-38-L-serine-des-39-L-glutamic acid-human granulocyte colony-stimulating factor (G-CSF, pluripoietin)

Lonquex (lipegfilgrastim) has been approved to reduce the duration of neutropaenia (low white blood cell counts) and febrile neutropaenia in patients undergoing cytotoxic chemotherapy for cancer, and is given as a single subcutaneous dose per cycle of chemotherapy.

Like Neulasta (pegfilgrastim), Lonquex is a long-acting recombinant granulocyte colony-stimulating factor (G-CSF) and is dosed at the same frequency as Amgen’s drug.

http://www.pmlive.com/pharma_news/neulasta_rival_from_teva_cleared_in_eu_495953

OMARIGLIPTIN. MK 3102 IN PHASE 3 FOR TYPE 2 DIABETES

April 18, 2014 2:21 am / 1 Comment on OMARIGLIPTIN. MK 3102 IN PHASE 3 FOR TYPE 2 DIABETES

OMARIGLIPTIN. MK 3102

cas 1226781-44-7

Approved in japan SEPT 28 2015

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(2-methylsulfonyl-4,6-dihydropyrrolo[3,4-c]pyrazol-5-yl)oxan-3-amine

(2R,3S,5R)-2-(2,5-Difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine

1226781-44-7, Omarigliptin [USAN], UNII-CVP59Q4JE1, CHEMBL2105762, MK-3102, PB39113

Molecular Formula: C₁₇H₂₀F₂N₄O₃S Molecular Weight: 398.427506

IN PHASE 3

omarigliptin

CLICK ON IMAGES FOR CLARITY VIEW

PAPER

J. Med. Chem., 2014, 57 (8), pp 3205–3212

DOI: 10.1021/jm401992e

http://pubs.acs.org/doi/abs/10.1021/jm401992e

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

Omarigliptin (MK-3102) (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine,is a 2,3,5-substituted tetrahydropyran analogue currently in phase 3 clinical trial for type 2 diabetes mellitus (T2DM).

(2R,3S,5R)-2-(2,5-Difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine, Omarigliptin

Crystallization from ethyl acetate gave a compound with greater than 99% purity.

Optical rotation [α]_D²⁰ −12.0° (c 1.0, CH₃OH).

¹H NMR (CD₃OD, 500 MHz) δ = 1.71 (q, 1H, J = 12 Hz), 2.56–2.61 (m, 1H), 3.11–3.18 (m, 1H), 3.36–3.40 (m, 1H), 3.48 (t, 1H, J = 12 Hz), 3.88–3.94 (m, 4H), 4.30–4.35 (m, 1H), 4.53 (d, 1H, J = 12 Hz), 7.14–7.23 (m, 2H), 7.26–7.30 (m, 1H), 7.88 (s, 1H).

LC–MS: 399.04 (M + 1).

PATENT

http://www.google.com.tr/patents/US20100120863?hl=tr&cl=ja

Example 1

(2R,3S,5R)-2-(2,5-Difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amineStep A: tert-Butyl {(2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5 (4H)-yl]tetrahydro-2H-pyran-3-yl}carbamate

A mixture of Intermediate 2 (26.3 g, 80 mmol) and 2-(methylsulfonyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole (Intermediate 5) (15.07 g, 80 mmol) in anhydrous methanol (1.5 L) was stirred at room temperature for 2 h. To the resulting white suspension was added decaborane (2.95 g, 24.15 mmol) and the mixture was stirred at room temperature overnight. Methanol was removed and the residue was purified on two 65i Biotage™ columns eluting with 5-50% ethyl acetate in dichloromethane to afford the title compound as a white solid. LC-MS: 499.10 (M+1).

Step B: (2R,3S,5R)-2-(2,5-Difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine

Removal of the BOC group in the product from Step A (13.78 g, 27.67 mmol) was accomplished with trifluoroacetic acid (100 ml) in dichloromethane (200 mL) at room temperature. After stirring for 2 h, the reaction was concentrated and neutralized with 25% MeOH and 2.5% ammonium hydroxide in dichloromethane. Solvents were removed under reduced pressure and the resulting crude material was purified on a 65i Biotage™ column eluting with 1.25-5% MeOH and 0.125-0.5% ammonium hydroxide in dichloromethane. The isolated material was further purified by recrystallization from 5:1 EtOAc/CH₂Cl₂at 60° C. The crystalline product was washed with cold 2:1EtOAc/hexanes to give the title compound as a light brown solid. ¹H NMR (500 MHz, CD₃OD): 1.71 (q, 1H, J=12 Hz), 2.56-2.61 (m, 1H), 3.11-3.18 (m, 1H), 3.36-3.40 (m, 1H), 3.48 (t, 1H, J=12 Hz), 3.88-3.94 (m, 4H), 4.30-4.35 (m, 1H), 4.53 (d, 1H, J=12 Hz), 7.14-7.23 (m, 2H), 7.26-7.30 (m, 1H), 7.88 (s, 1H). LC-MS: 399.04 (M+1).

Intermediate 2

tert-Butyl[(2R,3S)-5-oxo-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate Step A: 1-(2,5-Difluorophenyl)-2-nitroethanol

To sodium hydroxide (1N, 3L) and methanol (1500 mL) at 5° C. was added a solution of 2,5-difluorobenzaldehyde (350 g, 2.46 mol) and nitromethane (157 mL, 2.9 mol) in methanol (350 mL) dropwise over a period of 1 h. The reaction mixture was then neutralized with glacial acetic acid (165 mL). Diethyl ether (1500 mL) was added and the layers separated. The organic layer was washed successively with saturated aqueous sodium carbonate solution (1000 mL), and saturated aqueous brine (1000 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to afford 1-(2,5-difluorophenyl)-2-nitroethanol that was used without further purification in Step B.

Step B: 2-Nitro-1-(2,5-difluorophenyl)ethanone

A solution of Dess-Martin periodinane (125 g) in dichloromethane (600 mL) was added to a solution of the nitroalcohol made in Step A (46.3 g) at 10° C. over a period of 30 min. Stirring was continued for 2 h, and the reaction mixture was then poured onto a mixture of sodium bicarbonate (300 g) and sodium thiosulfate (333 g) in water (3 L). The desired product was extracted with methyl t-butyl ether (MTBE) (2 L). The aqueous layer was neutralized with HCl (2N, 1.5 L) and extracted with MTBE (3 L). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, evaporated and the residue was purified by chromatography (silica gel, eluting with dichloromethane) to yield the desired nitroketone.

Step C: 3-Iodo-2-(iodomethyl)prop-1-ene

A mixture of 3-chloro-2-(chloromethyl)prop-1-ene (1.0 g, 8 mmol) and sodium iodide (6.6 g, 44 mmol) in acetone (60 mL) was stirred at room temperature for 20 h, evaporated under reduced pressure and partitioned between dichloromethane (150 mL) and water (50 mL). The organic layer was dried over sodium sulfate, filtered and evaporated to yield 3-iodo-2-(iodomethyl)prop-1-ene as a reddish oil.

Step D: 3-Methylene-5-nitro-6-(2,5-difluorophenyl)-3,4-dihydro-2H-pyran

N,N-diisopropylethylamine (184 mL) was added to a solution of 2-nitro-1-(2,5-difluorophenyl)ethanone (92.7 g, 461 mmol) in N,N-dimethylformamide (1000 mL) and 3-iodo-2-(iodomethyl)prop-1-ene (156 g, 507 mmol). The mixture was heated at 60° C. for 2 h, evaporated and purified by chromatography (silica gel, gradient 0-30% dichloromethane in hexane) to yield 3-methylene-5-nitro-6-(2,5-difluorophenyl)-3,4-dihydro-2H-pyran.

Step E: (2R,3S)-5-Methylene-3-nitro-2-(2,5-difluorophenyl)tetrahydro-2H-pyran

This compound was made by following the same method described in Intermediate 1, Step D by using 3-methylene-5-nitro-6-(2,5-trifluorophenyl)-3,4-dihydro-2H-pyran.

Step F: (2R,3S)-5-Methylene-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-amine

This compound was made by following the same method described in Intermediate 1, Step E by using (2R,3S)-5-Methylene-3-nitro-2-(2,5-difluorophenyl)tetrahydro-2H-pyran.

Step G: tert-Butyl[(2R,3S)-5-methylene-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate

This compound was made by following the same method described in Intermediate 1, Step F by using (2R,35)-5-methylene-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-amine.

Step H: tert-Butyl[(2R,3S)-5-hydroxy-5-(hydroxymethyl)-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate

This compound was made by following the same method described in Intermediate 1, Step G by using tert-butyl[(2R,35)-5-methylene-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate.

Step I: tert-Butyl[(2R,3S)-5-oxo-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate

To a solution of tert-butyl[(2R,3S)-5-hydroxy-5-(hydroxymethyl)-2-(2,5-trifluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate (10.5 g) in methanol (100 mL) at 0° C. was added pyridine (7.8 mL) and lead tetraacetate (21.7 g). The reaction mixture was stirred for 20 min. Aqueous work-up with ethyl acetate gave crude product which was purified by chromatography (silica, 0-50% ethyl acetate/heptane) to yield tert-butyl[(2R,35)-5-oxo-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-yl]carbamate as white solid.

Intermediate 3

Step A: tert-Butyl (3Z)-3-[(dimethylamino)methylene]-4-oxopyrrolidine-1-carboxylate

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (40 g, 216 mmol) was treated with DMF-DMA (267 g, 2241 mmol) and heated at 105° C. for 40 min. The solution was cooled and evaporated under reduced pressure and the resulting orange solid was treated with hexane (200 mL) and cooled in a refrigerator for 3 days. The resulting brownish-yellow solid obtained as such was collected by filtration, dried and used in the next step without further purification.

Step B: 1,4,5,6-Tetrahydropyrrolo[3,4-c]pyrazole

A solution of hydrazine (3 mL) and tert-butyl (3Z)-3-[(dimethylamino)methylene]-4-oxopyrrolidine-1-carboxylate (19.22 g) in ethanol (40 mL) was heated at 85° C. in a sealed tube for 4 h. Solvent was removed under reduced pressure, and the residue was triturated with dichloromethane (160 mL) and ethyl acetate (15 mL). The resulting solid was filtered. The filtrate was concentrated and the resulting solid was triturated again and filtered. The combined solids were treated with 4N hydrochloric acid (250 mL) in methanol and stirred for 6 h. The reaction mixture was concentrated and dried. The resulting solid was treated again for 6 h with 4N hydrochloric acid (250 mL) in methanol. After concentration and drying, the resulting hydrochloride salt was treated with ammonia in methanol (2N, 300 mL) and ammonium hydroxide solution in water (28%, 30 mL) and concentrated to dryness. The solid obtained was treated with methanol (70 mL) and water (5 mL) and purified in three batches on Biotage Horizon® system (silica, gradient 5-17% methanol containing 10% concentrated ammonium hydroxide in ethyl acetate) to yield 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole. ¹H NMR (500 MHz, CD₃OD): δ 4.04 (d, 4H); 7.39 (s, 1H).

Intermediate 5

2-(Methylsulfonyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole Step A: tert-Butyl 1-(methylsulfonyl)]-4,6-dihydropyrrolo[3,4-c]pyrazole-5(1H)-carboxylate (A) and tent-butyl 2-(methylsulfonyl)]-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate (B)

A suspension of N-Boc-pyrazolopyrrolidine (Intermediate 3, Step B) (27.16 g, 130 mmol) in anhydrous acetonitrile (1.0 L) was charged in a 2.0 L three-neck flask fitted with a thermometer and an addition funnel and then treated with sodium hydride (60% dispersion in oil, 6.23 g, 156 mmol) while under nitrogen atmosphere in one portion. The reaction mixture was stirred at room temperature for 2 h. The resulting white suspension was then cooled in an ice bath and methanesulfonyl chloride (25.2 mL, 324 mmol) was slowly added via addition funnel The ice bath was then removed and the mixture was stirred 1 h at room temperature. The reaction mixture was quenched with water (500 mL) and the layers were separated. The aqueous layer was then extracted with 2×500 mL of dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give a mixture of products A and B as colorless syrups. NMR in CD₃OD indicated a 1:1 mixture of two products, in which the proton on the pyrazole ring in product A appeared at 7.70 ppm while the proton in product B appeared at 7.95 pm. LC-MS: 288.08 (M+1).

Step B: 2-(Methylsulfonyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole

Trifluoroacetic acid (200 mL) was added slowly to a solution containing intermediates A and B prepared in the previous step (48.4 g, 168 mmol) in dichloromethane (400 mL) at 0° C. After addition, the cooling bath was removed and the reaction was allowed to stir at room temperature for 2 h. Solvent was removed under reduced pressure and the resulting trifluoroacetate salt was then neutralized with 500 mL of 25% methanol and 2.5% ammonium hydroxide in dichloromethane. After removal of solvent, the desired Intermediate 5 was obtained after chromatography on a Biotage™ column (2×340 g) eluting with 2.5-12.5% methanol and 0.25-1.25% ammonium hydroxide in dichloromethane. LC-MS: 109.85 (M+1).

PATENT

below patent caution…………….similar not same….examples below will help you in synthesis similarities

http://www.google.com/patents/WO2014018355A1?cl=en

Step 1 ² Step 2

Example 1

Synthesis of 3: (Step 1 & 2)

Dimethyldisulfide 1 (5 g, 53 mmol) and acetic acid (6 mL, 106 mmol) were mixed under nitrogen atmosphere and cooled to – 20 °C. Sulfuryl chloride (13 mL, 159 mmol) was added dropwise with stirring. The mixture was then stirred for 1 hour at -20 °C and afterwards allowed to come to room temperature and continued for another two hours. Acetyl chloride was distilled off from the reaction mixture. Crude methanesulfinyl chloride 2 obtained was used in the next step without further purification.

To a solution of chloramine T (14.95 g, 53 mmol) in dry toluene (220 mL) was added a solution of methanesulfinyl chloride 2 (5.2 g, 53 mmol) in dry toluene (10 mL) at 0 °C. The resulting suspension was heated at 80 °C for 2 hours with stirring. After cooling, the solid was filtered off and washed with dry toluene (100 mL). The filtrate was evaporated in vacuo and the crude mixture was purified through silica gel chromatography to obtain 3 as off white solid. ^XH NMR (300 MHz, CDC1₃): δ 7.85 – 7.91 (m, J= 8.42 Hz, 2H), 7.31 – 7.38 (m, J= 8.23 Hz, 2H), 3.78 (s, 3H), 2.45 (s, 3H).

Synthesis of 4: (Step 3)

To a solution of Ml (1.0 g, 2.2 mmol) in THF (10 mL) and DMF (10 mL) under nitrogen atmosphere at 0 °C was added Et₃N (0.92 mL, 6.6 mmol) followed by B0C₂O (0.48 g, 2.2 mmol). The reaction mixture was allowed to come to room temperature and continued the stirring for over night. The reaction mixture was diluted with water (100 mL) and extracted with CH₂CI₂ (3 x 100 mL). Combined organics were dried over Na₂S0₄, filtered, concentrated under vacuum and purified by silica gel chromatography afforded 4 as a off white solid.

^XH NMR (400 MHz, CDC1₃): δ 7.27 – 7.35 (m, 1H), 4.44 – 4.54 (m, 4H), 1.52 (s, 9H).

Synthesis of 5: (Step 4)

To a suspension of NaH (0.30 g, 7.5 mmol) in dry THF (5 mL) under nitrogen atmosphere at 0 °C was added a solution of 4 (0.78 g, 3.7 mmol) in dry THF (30 mL). The reaction mixture was allowed to come to room temperature and continued the stirring for 2 hours. Reaction mixture was again cooled to 0 °C. A solution of 3 (2.0 g, 7.4 mmol) in THF (25 mL) was added to the reaction mixture and continued the stirring for another 1 hour. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (3 x 200 mL). Combined organics were dried over Na₂S0₄, filtered, concentrated under vacuum and purified by silica gel chromatography afforded 5 as an off-white solid.

^XH NMR (400 MHz, CDC1₃): δ 7.84 – 7.88 (m, 1H), 7.78 (t, J= 8.27 Hz, 2H), 7.23 – 7.30 (m, 2H), 4.39 – 4.49 (m, 4H), 3.53 (d, J= 2.40 Hz, 3H), 2.42 (s, 3H), 1.53 (s, 9H).; Molecular Formula: Ci₈H24N₄0₅S₂; LCMS purity: 98.18%; Expected: 440.1 ; Observed: 341.0 (M-99).

Synthesis of 6: (Step 5)

To a solution of 5 (0.47 g, 1.06 mmol) in dry CH₂CI₂ (1 1 mL) under nitrogen atmosphere at 0 °C was added TFA (3 mL). The reaction mixture was allowed to come to room temperature and continued the stirring for 2 hours. Solvent was removed under vacuum and solid mass was washed with Et₂0 (3 x 10 mL) to get amine TFA salt as white solid.

^XH NMR (300 MHz, CD₃OD): δ 7.78 (s, 1H), 7.63 – 7.70 (m, J= 8.11 Hz, 2H), 7.26 – 7.35 (m, J = 8.33 Hz, 2H), 3.93 (s, 2H), 3.86 (s, 2H), 3.34 (s, 3H), 2.42 (s, 3H).

The amine TFA salt was dissolved in minimum volume of MeOH:CHCi₃ (1 : 1) and passed through a column [Orochem 5 g, 10 ml, Amino (N¾)] using MeOH as eluent.

Organics were concentrated under vacuum to get free 6.

Synthesis of 7: (Step 6) To a stirred solution of 6 (0.34 g, 0.95 mmol) and M2 (0.26 g, 0.79 mmol) in DMAc (6.78 mL) under nitrogen atmosphere for 10 minutes was added AcOH (0.067 mL, 1.19 mmol). The reaction mixture was stirred for further 5 minutes and cooled to 0 °C. NaBH(OAc)3 (0.20 g, 0.95 mmol) was added to the reaction mixture and allowed to stirrer at room temperature for overnight. NH₄OH (2 mL) was added to the reaction mixture and heated at 50 °C for 1 hour followed by water (3.39 mL) and again heated at 50 °C for another hour. Reaction mixture was cooled to room temperature and filtered. The solid residue was washed with water (4 x 100 mL) and the crude residue was purified by silica gel chromatography to afford 7.

XH NMR (300 MHz, CDC1₃): δ 7.80 (d, J= 6.95 Hz, 3H), 7.25 – 7.29 (m, 2H), 7.22 (br. s., 1H), 6.92 – 7.02 (m, 2H), 4.52 (d, J= 9.33 Hz, 1H), 4.24 – 4.40 (m, 2H), 3.85 (br. s., 5H), 3.48 (s, 3H), 3.39 – 3.47 (m, 1H), 3.07 (br. s., 1H), 2.52 (d, J= 10.25 Hz, 1H), 2.44 (s, 3H), 1.61 (br. s., 1H), 1.28 (s, 9H).; Molecular Formula: C₂9H35F₂N₅0₆S₂; LCMS purity: 99.08%; Expected: 651.2; Observed: 652.0 (M+l). Synthesis of Example 1: (Step 7)

To a solution of 7 (20 mg, 0.03 mmol) in dry CH2CI2 (2 mL) under nitrogen atmosphere at 0 °C was added TFA (0.5 mL). The reaction mixture was allowed to come to room temperature and continued the stirring for 2 hours. Solvent was removed under vacuum and solid mass was washed with Et₂0 to get amine di-TFA salt Example 1 as white solid. Unless otherwise noted the IC50 values were determined using the assay discussed earlier.

^XH NMR (400 MHz, CD₃OD): δ 8.05 (s, 1H), 7.73 (d, J= 8.03 Hz, 2H), 7.36 (d, J= 8.28 Hz, 2H), 7.29 – 7.34 (m, 1H), 7.20 – 7.27 (m, 2H), 4.71 (d, J= 10.04 Hz, 1H), 4.40 – 4.53 (m, 5H), 3.72 – 3.82 (m, 2H), 3.68 (s, 3H), 3.59 – 3.65 (m, 1H), 2.77 – 2.85 (m, 1H), 2.44 (s, 3H), 2.00 – 2.14 (m, 1H).; Molecular Formula: C₂4H₂7F_{2 5}0₄S₂; HPLC purity: 99.74%; LCMS Expected: 551.2; Observed: 552.2 (M+l).

SCHEME 2

Example 2: Synthesis of Compound 1 & 2 (Step 1):

To a suspension of M2 (0.95 g, 2.8 mmol) in water (8.67 mL) was added sodium metabisulfite (0.55 g, 2.8 mmol) and stirred a room temperature for lhour. A solution of M3* (0.52 g, 2.8 mmol) in ethanol (8.67 mL) was added to the above reaction mixture and continued the stirring for further 4 hours. Neat aCN (0.14 g, 2.8 mmol) was added to the above reaction mixture in one portion and heated the reaction mixture at 50 °C for 2 days. Reaction mixture was concentrated under vacuum to remove most of the ethanol. The crude mixture was extracted with CHCI₃ (50 x 3 mL). The combined organic layer was washed with water, dried over a₂S04, filtered, concentrated and purified by flash chromatography to obtain 1 and 2 as solids.

Compound 1: ‘H NMR (300 MHz, CDC1₃): δ 7.77 (s, 1H), 7.26 – 7.35 (m, 1H), 7.00 (t, J= 5.76 Hz, 2H), 4.57 (t, J= 9.88 Hz, 2H), 4.32 – 4.39 (m, 1H), 3.85 – 4.09 (m, 5H), 3.60 (d, J= 11.34 Hz, 1H), 3.34 (s, 3H), 2.63 – 2.74 (m, 1H), 2.02 – 2.15 (m, 1H), 1.31 (s, 9H).

Compound 2: ^XH NMR (300 MHz, CDC1₃): δ 7.28 – 7.36 (m, 2H), 7.00 (t, J= 5.85 Hz, 2H), 4.55 (d, J= 8.97 Hz, 2H), 4.37 (dd, J= 2.65, 11.25 Hz, 1H), 3.88 – 4.07 (m, 5H), 3.60 (d, J = 1 1.34 Hz, 1H), 2.71 (td, J= 3.45, 12.49 Hz, 1H), 1.97 – 2.12 (m, 1H), 1.31 (s, 9H).; Molecular Formula: C₂2H₂5F_{2 5}0₃; LCMS purity: 94.48%; Expected: 445.2; Observed: 446.0 (M+l). (*Preparation of M3: M3.PI1SO₃H (1.0 g, 2.8 mmol) was dissolved in minimum volume of MeOH:CHCl₃ (1 : 1) and passed through a column [Orochem 5 g, 10 ml, Amino (NH₂)] using MeOH as eluent. Organics were concentrated under vacuum to get free M3, which was used directly without further purification.) Synthesis of compound 3 (Step 2):

To a solution of compound 2 (0.40 g, 0.89 mmol) in THF (5 mL) under ₂ atmosphere at -78 °C was added a solution of MeMgBr (0.89 mL, 2.6 mmol, 3M in Et₂0). The reaction mixture was allowed to attain room temperature over 1 hour. TLC shows complete conversion. The reaction mixture was again cooled to -10 °C and quenched with saturated aq. NH4CI solution (10 mL). The reaction mixture was extracted with CH₂CI₂ (50 x 3 mL).

Combined organics were dried over Na₂S0₄, filtered, concentrated and purified by reversed phase chromatography to obtain 3 as di-TFA salt.

Molecular Formula: C22H2₈F2 4O₃; LCMS purity: 88.82%; Expected: 434.2; Observed: 435.2 (M+l).

Synthesis of Example 2 (Step 3):

To a solution of compound 3 (35 mg, 0.053 mmol) in CH₂CI₂ (2 mL) was added TFA (0.5 mL) dropwise at 0 °C. Reaction mixture was allowed to attain room temperature over 2 hours time. TLC shows complete conversion. Reaction mixture was concentrated to dryness. The solid residue was washed with Et₂0 (10 x 3 mL) and dried under vacuum to obtain Example 2 as tri-TFA salt.

^XH NMR (400 MHz, CD₃OD): δ 7.60 (s, 1H), 7.37 (dd, J= 5.02, 8.03 Hz, 1H), 7.22 – 7.31 (m, 2H), 4.70 (d, J= 10.04 Hz, 1H), 4.48 – 4.61 (m, 4H), 4.17 (dd, J= 2.26, 11.29 Hz, 1H), 3.91 (d, J = 11.04 Hz, 1H), 3.73 – 3.83 (m, 1H), 2.54 – 2.62 (m, 1H), 2.22 (t, J= 12.05 Hz, 1H), 1.71 (s, 3H).; Molecular Formula: C17H2₀F2 4O; HPLC purity: 94.98%; Expected: 334.2; Observed: 335.2 (M+l).

SCHEME 3

Example 3

Synthesis of 1 & 2: (Step 1)

Compound 1: ‘H NMR (300 MHz, CDC1₃): δ 7.77 (s, 1H), 7.35 – 7.26 (m, 1H), 7.00 (t, J= 5.76 Hz, 2H), 4.57 (t, J= 9.88 Hz, 2H), 4.39 – 4.32 (m, 1H), 4.09 – 3.85 (m, 5H), 3.60 (d, J= 1 1.34 Hz, 1H), 3.34 (s, 3H), 2.74 – 2.63 (m, 1H), 2.15 – 2.02 (m, 1H), 1.31 (s, 9H).

Compound 2: ^XH NMR (300 MHz, CDC1₃): δ 7.36 – 7.28 (m, 2H), 7.00 (t, J= 5.85 Hz, 2H), 4.55 (d, J= 8.97 Hz, 2H), 4.37 (dd, J= 2.65, 11.25 Hz, 1H), 4.07 – 3.88 (m, 5H), 3.60 (d, J= 1 1.34 Hz, 1H), 2.71 (td, J= 3.45, 12.49 Hz, 1H), 2.12 – 1.97 (m, 1H), 1.31 (s, 9H).; Molecular Formula: C₂2H₂5F_{2 5}0₃; LCMS purity: 94.48%; Expected: 445.2; Observed: 446.0 (M+l).

(*Preparation of M3: M3.PI1SO₃H (1.0 g, 2.8 mmol) was dissolved in minimum volume of MeOH:CHCl₃ (1 : 1) and passed through a column [Orochem 5 g, 10 ml, Amino (NH₂)] using MeOH as eluent. Organics were concentrated under vacuum to get free M3, which was used directly without further purification.) Synthesis of compound 3 (Step 2):

To a solution of 2 (0.40 g, 0.89 mmol) in THF (5 niL) under 2 atmosphere at -78 °C was added a solution of MeMgBr (0.89 mL, 2.6 mmol, 3M in Et20). The reaction mixture was allowed to attain room temperature over 1 hour. TLC shows complete conversion. The reaction mixture was again cooled to -10 °C and quenched with saturated aq. NH4CI solution (10 mL). The reaction mixture was extracted with CH2CI2 (50 x 3 mL). Combined organics were dried over Na₂S0₄, filtered, concentrated and purified by reversed phase chromatography to obtain 3 (0.05 g, 8.4%) as di-TFA salt.

Molecular Formula: C22H2₈F2 4O₃; LCMS purity: 88.82%; Expected: 434.2; Observed: 435.2 (M+l).

Synthesis of compound 4 (Step 3):

To a suspension of NaH (22 mg, 0.55 mmol) in dry THF (0.1 mL) under nitrogen atmosphere at 0 °C was added a solution of 3 (120 mg, 0.27 mmol) in dry THF (4.8 mL). The reaction mixture was allowed to come to room temperature and continued the stirring for 2 hours. Reaction mixture was again cooled to 0 °C. Methanesulfonyl chloride (0.42 mL, 0.55 mmol) was added to the reaction mixture and continued the stirring for another 1 hour. The reaction mixture was quenched with water and extracted with EtOAc (3 x 50 mL). Combined organics were dried over Na₂S0₄, filtered, concentrated under vacuum and purified by silica gel chromatography afforded 4 as off white solid.

Molecular Formula: C2₃H₃₀F2N4O5S; LCMS purity: 95.64%; Expected: 512.2; Observed: 513.2 (M+l). Synthesis of Example 3: (Step 4)

To a stirred solution of compound 4 (9.0 mg, 0.017 mmol) in CH2CI2 (2.0 mL) was added TFA (0.2 mL) dropwise at 0 °C. Reaction mixture was allowed to attain room temperature over 2 hours time. TLC shows complete conversion. Reaction mixture was concentrated to dryness. The solid residue was washed with Et₂0 (2 x 10 mL) and dried under vacuum. The solids were once again washed with a mixture of CH2CI2 (0.1 mL) and Et₂0 (5.0 mL) to obtain Example 3 (8.0 mg, 72.7%) as di-TFA salt. The IC50 value of Example 3 is 4nM. ¾ NMR (400MHz ,CD₃OD): δ 7.96 (s, 1 H), 7.41 – 7.31 (m, 1 H), 7.30 – 7.19 (m, 2 H), 4.68 – 4.60 (m, 1 H), 4.22 – 4.07 (m, 4 H), 4.01 (d, J= 11.0 Hz, 1 H), 3.77 (d, J= 11.0 Hz, 1 H), 3.74 – 3.63 (m, 1 H), 3.39 (s, 3 H), 2.43 (d, J= 10.8 Hz, 1 H), 2.04 (t, J= 11.9 Hz, 1 H), 1.51 (s, 3 H).; Molecular Formula: C1₈H22F2 4O₃S; HPLC purity: 95.01%; LCMS mass Expected: 412.2;

Observed: 413.0 (M+l).

Synthesis of Omarigliptin (MK-3102), a once-weekly DPP-4 drug for the treatment of type 2 diabetes_Merck 默沙东超长效2型糖尿病药物Omarigliptin的制备方法

PAPER

http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00267

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00267

Development of a convergent synthesis of omarigliptin (MK-3102) suitable for commercial manufacture is described. The target molecule is assembled through a diastereoselective reductive amination of a highly functionalized pyranone with a mesylated pyrazole followed by deprotection of a Boc group. The synthesis of the pyranone relies on three Ru-catalyzed reactions: (1) a DKR reduction of a rac-α-aminoketone to set the two contiguous stereogenic centers, (2) a cycloisomerization of a bis-homopropargylic alcohol to a dihydropyran, and, finally, (3) a Ru-catalyzed oxidation of a pyranol to the desired pyranone. The regioselective synthesis of a N-Boc-1-mesyl pyrazole fragment was achieved via base-promoted mesyl group isomerization to afford 30:1 selectivity. A highlight of the endgame process development is telescoping a Boc deprotection and reductive amination followed by direct crystallization of the penultimate from the reaction mixture. This avoids handling of an unstable, mutagenic 1-mesylpyrazole BSA salt used in the earlier multikilogram deliveries and improves the overall diastereoselectivity and efficiency of the route.

Tesfaye Biftu et al, Omarigliptin (MK-3102): A Novel Long-Acting DPP-4 Inhibitor for Once-Weekly Treatment of Type 2 Diabetes;Journal of Medicinal Chemistry, Articles ASAP, March 24, 2014,DOI: 10.1021/jm401992e

Zacuto, Michael J. et al, Process for preparing chiral dipeptidyl peptidase-IV inhibitors;PCT Int. Appl., WO2013003250

Biftu, Tesfaye et al, Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine, Bioorganic & Medicinal Chemistry Letters, 23(19), 5361-5366; 2013

Biftu, Tesfaye et al, Preparation of aminotetrahydropyrans as dipeptidyl peptidase IV inhibitors for the treatment or prevention of diabetes,PCT Int. Appl., WO2011028455

Biftu, Tesfaye et al, Preparation of aminotetrahydropyrans as dipeptidyl peptidase IV inhibitors for treatment or prevention of diabetes,U.S. Pat. Appl. Publ., US20100120863

Xu, Feng et al, Process for preparation of chiral trans-2,3-disubstituted 5-oxotetrahydropyrans from ethyl N-(diphenylmethylene)glycinate and propargyl besylate, U.S. Pat. Appl. Publ., US20090187028

Ru(p-cymene)-N-sulfonyl-l,2-diphenylethylenediamine (DPEN) catalyst
R. Noyori, et al., J. Org. Chem., 66: 7931-7944 (2001)
B. Mohar, et al., Chem. Commun., 2572-2573 (2001)

The rhodium-catalyzed cycloisomerization
B. Trost etal., J.Amer. Chem.Soc., 125:7482-7483 (2003).

The ruthenium-catalyzed cycloisomerization
B. Trost, et al., J. Amer. Chem. Soc., 124: 2528-2533 (2002)

Gantz, I.; Chen, M.; Mirza, A.; Suryawanshi, S.; Davies, M. J.; Goldstein, B. J. Eﬀect of MK-3102, a novel once-weekly DPP-4 inhibitor, over 12 weeks in patients with type 2 diabetes mellitus. Presented at the 48th Annual Meeting of the European Association for the Study of Diabetes (EASD), Berlin, Germany, October 2012; Abstract 101 (Clinical Research, Metabolism, Merck Research Laboratories).

Enzyme revealed as promising target to treat asthma and cancer

April 18, 2014 1:13 am / Leave a comment

Lyra Nara Blog

In experiments with mice, Johns Hopkins Kimmel Cancer Center scientists have identified an enzyme involved in the regulation of immune system T cells that could be a useful target in treating asthma and boosting the effects of certain cancer therapies.

In research described online April 6 in Nature Immunology, the investigators show that mice without the enzyme SKG1 were resistant to dust mite-induced asthma. And mice with melanoma and missing the enzyme, developed far fewer lung tumors—less than half as many—than mice with SKG1.

“If we can develop a drug that blocks the enzyme in a way that mimics what happens when the enzyme is missing, we would not only have a treatment to inhibit asthma, but also a drug that could be used in conjunction with other experimental therapies aimed at helping the immune system fight cancer,” said Jonathan D. Powell, M.D., professor of oncology at the…

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New pain relief targets discovered

April 18, 2014 1:11 am / Leave a comment

Lyra Nara Blog

The immune cell enters the nerve. Credit: Dr. Marzia Malcangio, King’s College London

Scientists have identified new pain relief targets that could be used to provide relief from chemotherapy-induced pain. BBSRC-funded researchers at King’s College London made the discovery when researching how pain occurs in nerves in the periphery of the body.

Dr Marzia Malcangio said: “We have been investigating and identifying mechanisms underlying pain generation and our findings could help chemotherapy patients who suffer pain related side effects.”

One potential side effect of some chemotherapy drugs (such as vincristine) is damage to nerves. This is particularly prominent in hands and feet as the drugs affect nerves in the periphery of the body. This causes pain which doctors treat with painkillers. However, some people find that the pain persists.

Dr Malcangio’s team investigated why the chemotherapy drugs were causing pain in hope to solve the problem. The used mice in…

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lei gong teng ,:雷公藤, Tripterygium wilfordii Hook F. for Rheumatoid Arthritis

April 17, 2014 10:01 am / 2 Comments on lei gong teng ,:雷公藤, Tripterygium wilfordii Hook F. for Rheumatoid Arthritis

A Chinese herb called thunder god vine works better than a widely-prescribed pharmaceutical drug at easing rheumatoid arthritis, a new study has found.

Tripterygium regelii, Aizu area, Fukushima pref, Japan

The herb has long been used in China to treat this potentially crippling autoimmune disease, which typically strikes hand and foot joints. It is known in Mandarin as ‘lei gong teng’ and to botanists as Tripterygium wilfordii Hook F.

Extracts of the herb have already fired the interest of drug laboratories as they contain hundreds of compounds, including intriguing molecules called diterpenoids which are believed to ease inflammation and immune response.

read at

http://lyranara.me/2014/04/16/chinese-herb-beats-drug-at-treating-rheumatoid-arthritis/

http://www.scmp.com/lifestyle/health/article/1482780/chinese-herb-beats-drug-rheumatoid-arthritis-study

Researchers at the Johns Hopkins School of Medicine have discovered that a natural constituent isolated from a traditional Chinese medicinal herb, Triptergium wilfordii Hook F. (雷公藤, Lei Gong Teng, Thunder God Vine), used for hundreds of years to treat many conditions, works well by blocking gene control machinery in the cell. Thunder God Vine (Lei Gong Teng) is regarded as toxic and used externally to treat rheumatoid arthritis and sciatica. This report, published as a cover story of the March issue of Nature Chemical Biology, suggests that the natural constituent could be a starting point for developing new anti-cancer drugs.

The extracts of Triptergium wilfordii have been used to treat a whole host of conditions and highly lauded for anti-inflammatory, immunosuppressive, contraceptive and anti-tumor activities. The researchers have known about the active compound, triptolide, which can stop cell growth, since 1972, but only now have they figured out what it does.

Triptolide, the active ingredient purified from Tripterygium wilfordii, has been shown in animal models to be effective against cancer, arthritis, and skin graft rejection. In fact, triptolide has been shown to block the growth of all 60 U.S. National Cancer Institute cell lines at very low doses, and even causes some of those cell lines to die. Other experiments have suggested that triptolide interferes with proteins known to activate genes, which gives the researchers an entry point into their research. Using information already known about these proteins and testing the rest to see if triptolide would alter their behaviors, the research team finally found that triptolide directly binds to and blocks the enzymatic activity of a protein.

Triptolide’s general ability to stop enzymatic activity explains its anti-inflammatory and anticancer effects. And its behavior has important additional implications for circumventing the resistance that some cancer cells develop to certain anti-cancer drugs. The researchers are eager to study it further to see what it can do for future cancer therapy.

Source:

http://www.physorg.com/news/2011-03-traditional-chinese-medicine-mystery.html

Tripterygium wilfordii, or léi gōng téng (Mandarin) (Chinese:雷公藤, Japanese: raikōtō), sometimes called thunder god vine but more properly translated thunder duke vine, is a vine used in traditional Chinese medicine for treatment of fever, chills, edema and carbuncle.

Tripterygium wilfordii recently has been investigated as a treatment for a variety of disorders including rheumatoid arthritis, cancer, chronichepatitis, chronic nephritis, ankylosing spondylitis, polycystic kidney disease as well as several skin disorders. It is also under investigation for its apparent antifertility effects, which it is speculated, may provide a basis for a Male oral contraceptive.^[1]

Triptolide, a diterpene triepoxide, is a major active component of extracts derived from Tripterygium wilfordii. Triptolide has multiple pharmacological activities including anti-inflammatory, immune modulation, antiproliferative and proapoptotic activity.^[2]

The Chinese herb, Lei Gong Teng, comes from the roots, leaves and flowers of the tripterygium wilfordii Hook. f. It is collected during summer and autumn. Tripterygium wilfordii Hook is a deciduous climbing vine growing to 12 meters, with brown, angular, downy twigs. The leaves are light green, smooth on top, and pale gray with light hairs underneath. They have crenate margins and pointed apexes, and are ovate to elliptic, 5-15 cm long, 2.5 – 7 cm wide. The scented hermaphroditic (having male and female organs) flowers, which bloom in September, are small and whitish with five petals and are about 9 mm across, in terminal panicles in July. The fruit is 3-winged, and brownish red, about 1.5 cm long. The plant can grow in light (sandy), medium (loamy) and heavy (clay) soils. It can survive in acid, neutral and basic (alkaline) soil. It can grow in semi-shade (light woodland) or no shade. It requires moist soil.

Source: The whole plant of Triptergium wilfordil Hook. f., family Celastraceae.

Reduction of male fertility

The plant contains many active compounds, at least six of which have male anti-fertility effect (triptolide, tripdiolide, triptolidenol, tripchlorolide, 16-hydroxytriplide and a compound known as T7/19, whose structure is unpublished). The mechanism by which they affect fertility is not yet understood. What is known is that daily doses of these compounds reduce sperm counts and also severely affect the formation and maturation of sperm, causing them to be immotile.

Scientific research into medical effects

Contraception

Certain extracts from Tripterygium wilfordii, as well as from Tripterygium hypoglaucum (now considered identical to T. regelii) and Tripterygium regelii, were discovered in the 1980s to have temporary antifertility effects, which has led to research on its potential as a contraceptive.

“Tripterygium wilfordii Hook.f., known as Leigongteng (Thunder God Vine) in traditional Chinese medicine, has attracted much attention for its applications in relievingautoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, and for treating cancer. Molecular analyses of the ITS and 5S rDNA sequences indicate that T. hypoglaucum and T. doianum are not distinct from T. wilfordii, while T. regelii should be recognized as a separate species. The results also demonstrate potential value of rDNA sequence data in forensic detection of adulterants derived from Celastrus angulatus in commercial samples of Leigongteng.”^[3]

Not enough is known about T. wilfordii to actually test it as a contraceptive. Research thus far has dealt with establishing the mechanism by which the plant affects fertility, and investigating toxicity and side effects. What has been learned is encouraging, however: in both animals and humans, low doses of various Tripterygium extractscan produce significantly lowered sperm density and motility indices without major side effects. When the treatment was ended in the various trials, all indices returned to normal within months.

T. wilfordii could be an effective pharmaceutical alternative to contraceptives based on hormonal manipulation.

Kidney function

As of 2012 The Nanjing University School of Medicine is conducting a clinical trial of Tripterygium wilfordii to determine its possible beneficial effects on kidney volume and kidney function for polycystic kidney disease (PKD) patients.^[4] It should report in late 2013.^{[dated info]}

Immunosuppression

A small molecule Triptolide derived from T. wilfordii has been shown to disrupt mitochondrial function in cells and is under investigation as an anti-tumor agent or to suppress auto-immune disorders.

Rheumatoid arthritis

In China Tripterygium wilfordii has an established history of use in the treatment of rheumatoid arthritis. The herb shows immunosuppressive, cartilage protective, and anti-inflammatory effects.^[5]^[6] The National Center for Complementary and Alternative Medicine has noted that one systematic review of the literature found that Tripterygium wilfordii may improve some RA symptoms, though another systematic review has stated that the serious side effects occur frequently enough to make the risks of taking this herbal supplement too high for the possible benefits.^[7]

Pancreatic cancer

Two compounds, the diterpenoid epoxide triptolide and the quinone triterpene celastrol found in the plant may have potential as antitumor drugs.^[8]

Drugs derived from the plant also show potential for reduction and elimination of pancreatic tumors in mice. Clinical trials may soon begin for the development of a drug for use in humans.^[9]

At medicinal doses, T. wilfordii extract does have significant side effects, including immunosuppression. However, this may not apply to contraceptive use. Many of the side effects are caused by the other active compounds found in the plant, and do not appear when a pure extraction of its compounds with anti-fertility effect is used. In addition, the dose required to lower fertility is significantly lower than the standard medicinal dose.

In August 2011, the UK Medicines and Healthcare products Regulatory Agency (MHRA) published a drug safety bulletin advising consumers not to use medicines containing Lei Gong Teng. This was due to concerns over potentially serious side effects.

Baidu Baike cautions do not take internally; China State Food and Drug Administration issued a warning in April 2012 about this medicine, urging caution.^[10]

However, a recent review stated that although Tripterygium wilfordii has toxic potential, careful extraction gives an acceptable frequency of adverse reactions, which are largely related to the gastrointestinal tract and amenorrhea. The review found that T. wilfordii extract is useful remedy for postmenopausal rheumatoid arthritis.^[11]

The Beijing TV series of China Medicine has shown people being treated successfully with the herb in a formula for rheumatoid arthritis. and outlined some practice to alleviate problems of using the herb. As often the case of TCM, formulations need to to be adjusted for individual’s physiology for best result.

Composition:

1. Saponins

(1). Wilforgine, wilforgine-B,wilfordine, wilfornine, wilfortrine, wilfortrine-D, wilforzine, wilformine, wilfordinic acid, hydroxywilfordii acid ,wilfornine , neowilforine.

(2). Celacinnine, celafurine, celabenzine, celallocinnine.

(3). Triptofordinine A-1, A-2, triptofordin D-1, D-2, E , triptofordin A, B, C-1 C-2 , triptofordin F-1, F-2, F-3, F-4.

2. Diterpene group

(1). Triptolide, tripdiolide, triptonide,tripterolide.

(2.). Triptolidenol, tripnolide, neotriptophenolide, triptophenolide methyl ether , isoneotrip-tophenolide, hypolide methyl ether.

(3). Triptonoterpene, triptonoterpene methyl ether, triptonoterpenol 12-ydroxy-abieta-8, 11, 13 -trien-3-one, 11-hydroxy-14-methoxy-abieta-8, 11-hydroxy-14-methoxy-abieta-8, 11, 13-trien-3-one.

3. Tetra-triterpene group

(1). Wilforlide A, wilforlide B.

(2). Tritotriterpenoid lactone, tretotriterpenic acid A, tritotriterpenic acid B, tritotriterpenic acid C, 3-epikatonic acid, polpunonic acid, triptodihydroxy acid methyl ester, tripterine.

(3). 3,24-dioxofridelan-29-oic acid, salaspermic acid.

4. Wilfornide

5. 1,8-dihydroxy-4-hydroxymethyl anthraquinone

6. Syringareisno

7 Other Chemicals: dulcitol, glucose, tannin.

8. Trace mineral: iron, manganese, zinc, copper, selenium etc.

Pharmacology

PG490-88 (14-succinyl triptolide sodium salt) is a semisynthetic compound derived from the diterpene triepoxide, triptolide (PG490). PG490 was first isolated and structurally characterized in 1972 when it was extracted from the Chinese medicinal herb, Tripterygium wilfordii Hook F (TWHF), a member of the Celastraceae family. Historically, extracts of TWHF have been used for centuries in traditional Chinese medicine but in the 1970s, they were identified as being effective in the treatment of inflammatory/autoimmune disorders such as rheumatoid arthritis. Since then, more rigorous attempts were made to better identify biologically active constituents of TWHF responsible for its various clinical properties. We now know, for example, that diterpenoid components of TWHF, especially PG490, exert their anti-inflammatory and immunosuppressant effects by inhibition of cytokine production (e.g. , IL-2, IL-4, IFN) by T lymphocytes. These effects of PG490 have also been explored in mouse models where it was shown that PG490 prevents graft versus host disease (GVHD) and prolongs skin, heart, and kidney allograft survival.

The isolation of PG490 has also led to studies supporting its potential development as an antineoplastic agent. Shamon et al., for example, showed that PG490 inhibited growth of several human cancer-derived cell lines (including breast, prostate, and lung) grown in culture. PG490 was also shown to induce apoptosis of human promyelocytic leukemia, T-cell lymphoma, and hepatocellular carcinoma cell lines grown in culture. Interestingly, the inhibitory effects of PG490 on the growth of tumor cells in culture were enhanced in the presence of other inducers of apoptosis such as tumor necrosis factor- (TNF) and chemotherapeutic agents. When combined with chemotherapeutic drugs, PG490 enhanced apoptosis through signaling pathways involving both p53 and p21.

Data on the effects of PG490 on tumor cell growth in vivo , however, are limited. Previous reports have shown that PG490 inhibits tumor development in a hamster model of cholangiocarcinoma and in a murine breast cancer model. These beneficial effects of PG490, however, were counterbalanced by toxicity that was observed at high doses. In the present studies, we further examined the role of PG490 in inhibition of tumor cell growth both in vitroand in a tumor xenograft model. We show that PG490-88, a water-soluble prodrug of PG490, suppresses tumor cell growth in vivo without toxicity. We also show that PG490 acts in synergy with chemotherapy. Our results suggest a potential role of PG490-88 alone and in combination with chemotherapy as a novel antineoplastic regimen for the treatment of patients with solid tumors

The molecular target(s) for PG490 is currently unknown. Clues to the cellular target, however, are emerging from its effect on transcriptional activity. For example, we have shown along with Qiu et al. , that PG490 blocks transcriptional activation of NF- B by blocking transcriptional activation of p65 but without affecting DNA binding by p65. Additionally, we have found that PG490 blocks transcriptional activation by AP-1 and p53 without affecting DNA binding by Jun/Fos or p53. Recent studies show that the transcriptional activity of AP-1, NF-B, and p53 is regulated by a chromatin structure that is controlled, in part, by histone acetylation. In support of this, a recent study showed that p65 interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate NF- B transcriptional activity. Also, silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) was shown to inhibit transactivation of AP-1, NF-B, and serum response factor (SRF) by binding to their cognate transcription factors. Recent studies also show that p53-mediated transcriptional activity is regulated by histone acetylation. However, we have not observed an effect of PG490 on histone acetyltransferase (HAT) activity or histone acetylation.

PG490 at doses of 5–10 ng/ml does not repress basal transcriptional activity mediated by AP-1, NF-B, and p53 but it does block induction of NF-B by TNF and p53 transcriptional activity induced by chemotherapy. Also, PG490 does not affect topoisomerase I or II activity or increase topoisomerase cleavage complexes. Therefore, its synergy with chemotherapy may in large part be due to its inhibition of p21 mediated growth arrest, which activates an apoptotic pathway.

The treatment of solid tumors is evolving to more targeted treatments that may be helped by genetic profiling of tumors and targeting tumor-specific angiogenic and growth factor pathways. Also, several recent studies have shown that disrupting checkpoints in tumors drives tumor cells into apoptosis by abrogating checkpoint arrest. Here we show that PG490-88, a water-soluble derivative of PG490, reduces tumor growth, induces marked regression, or completely eradicates human tumor xenografts. Moreover, PG490-88 is a potent and well-tolerated antitumor agent that acts in synergy with DNA damaging agents and is effective in a clinically relevant dosing schedule. PG490-88 is now in phase I clinical trials for patients with solid tumors. A recent study showing that PG490 inhibits metastasis of solid tumors coupled with our findings that PG490-88 markedly enhances the cytotoxicity of DNA damaging agents suggests that PG490 or PG490-88 alone or in combination with chemotherapy may become an effective therapy for patients with solid tumors. Also, our finding that PG490 sensitizes tumor cells to TNF by blocking NF-B suggests a role for the combination in treating patients with TNF sensitive tumors such as melanoma. Identification of the target of PG490 and its mechanism of action will complement the ongoing clinical trials, and will provide insight into potential mechanisms of toxicity and the design of compounds that may be more selective and more potent.

Zhen QS, Ye X, Wei ZJ (February 1995). “Recent progress in research on Tripterygium: a male antifertility plant”. Contraception 51 (2): 121–9. doi:10.1016/0010-7824(94)00018-R.PMID 7750290.
Liu Q. (2011). “Triptolide and its expanding multiple pharmacological functions”. International Immunopharmacology 11 (3): 377–383. doi:10.1016/j.intimp.2011.01.012.PMID 21255694.
Law et al (2010), p. 21. Source undefined
Randomized Clinical Trial of Triptolide Woldifii for Autosomal Dominant Polycystic Kidney Disease
Bao J., Dai S.-M. “A Chinese herb Tripterygium wilfordii Hook F in the treatment of rheumatoid arthritis: mechanism, efficacy, and safety” Rheumatology International 2011 (1-7)
Moudgil K.D., Venkatesha S.H., Rajaiah R., Berman B.M. “Immunomodulation of autoimmune arthritis by herbal CAM” Evidence-based Complementary and Alternative Medicine 2011 2011 Article Number 986797
“Rheumatoid Arthritis and Complementary Health Approaches”. National Center for Complementary and Alternative Medicine. Retrieved 21 April 2013.
Liu Z, Ma L, Zhou GB. (2011). “The main anticancer bullets of the Chinese medicinal herb, thunder god vine.”. Molecules 16 (6): 5283–97. doi:10.3390/molecules16065283.PMID 21701438.
Drug From Chinese ‘Thunder God Vine’ Slays Tumors in Mice. 17 Oct 2012
中医・我が愛しの上海へ／理想の中医学・漢方を求めて-
Bao J., Dai S.-M., (September 2011). “A Chinese herb Tripterygium wilfordii Hook F in the treatment of rheumatoid arthritis: Mechanism, efficacy, and safety.”. Rheumatology International 31 (9): 1123–1129. doi:10.1007/s00296-011-1841-y. PMID 21365177.

References

Downloadable PDF – “Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.)” (2010). Sue Ka-Yee Law et al. Phytochemistry 72 (2011) 21–26, Elsevier.[1]
Adv Exp Med Biol. 2007;599:139-46.
Journal of Andrology 1998; vol 19 no 4, pp 479–486.
Contraception 1995; vol 51, pp 121–129.
Contraception 1986; vol 36 no 3, pp 335–345.
MHRA safety bulletin

Drug From Chinese ‘Thunder God Vine’ Slays Tumors in Mice

Cerecor’s selective NMDA receptor subunit 2B antagonist CERC-301 (MK-0657) for depression in phase 2

April 17, 2014 9:33 am / 1 Comment on Cerecor’s selective NMDA receptor subunit 2B antagonist CERC-301 (MK-0657) for depression in phase 2

CERC-301 (MK-0657) MK-657, c-6161, AGN-PC-00887R

structure source….http://www.google.com/patents/WO2013156614A1?cl=en my id is amcrasto@gmail.com

Treat depression; Treat major depressive disorder (MDD); Treat suicidality

808732-98-1 free form, C₁₉ H₂₃ F N₄ O₂

(-) (3S,4R) – 1-Piperidinecarboxylic acid, 3-fluoro-4-[(2-pyrimidinylamino)methyl]-, (4-methylphenyl)methyl ester,

AND

1-Piperidinecarboxylic acid, 3-fluoro-4-[(2-pyrimidinylamino)methyl]-, (4-methylphenyl)methyl ester, (3S,4R)-

(-)-(3S,4R)-4-Methylbenzyl 3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

(3S,4R)-4-methylbenzyl 3-fluor-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate

cas no of hydrochloride 808733-06-4

read at

http://www.allfordrugs.com/2014/04/17/cerecors-selective-nmda-receptor-subunit-2b-antagonist-cerc-301-mk-0657-for-depression-in-phase-2/

NICE nod for Firmagon’s prostate cancer drug degarelix for the management of advanced prostate cancer.

April 17, 2014 3:36 am / Leave a comment

Degarelix

214766-78-6 CAS

EMA:Link,

US FDA:link

Degarelix is used for the treatment of advanced prostate cancer. Degarelix is a synthetic peptide derivative drug which binds to gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland and blocks interaction with GnRH. This antagonism reduces luteinising hormone (LH) and follicle-stimulating hormone (FSH) which ultimately causes testosterone suppression. Reduction in testosterone is important in treating men with advanced prostate cancer. Chemically, it is a synthetic linear decapeptide amide with seven unnatural amino acids, five of which are D-amino acids. FDA approved on December 24, 2008.

A subgroup of patients with advanced prostate cancer could now get access to a new treatment option in England and Wales after cost regulators for the NHS issued a green light for Ferring’s Firmagon (degarelix).

Clinical effectiveness

A Phase III, randomised, 12 month clinical trial (CS21) in prostate cancer^[4] compared androgen deprivation with one of two doses of degarelix or the GnRH agonist, leuprolide. Both degarelix doses were at least as effective as leuprolide at suppressing testosterone to castration levels (≤0.5 ng/mL) from Day 28 to study end (Day 364). Testosterone levels were suppressed significantly faster with degarelix than with leuprolide, with degarelix uniformly achieving castration levels by Day 3 of treatment which was not seen in the leuprolide group. There were no testosterone surges with degarelix compared with surges in 81% of those who received leuprolide. Degarelix resulted in a faster reduction in PSA levels compared with leuprolide indicating faster control of the prostate cancer. Recent results also suggest that degarelix therapy may result in longer control of prostate cancer compared with leuprolide.^[5]

Side effects

As with all hormonal therapies, degarelix is commonly associated with hormonal side effects such as hot flashes and weight gain.^[4]^[6]^[7] Due to its mode of administration (subcutaneous injection), degarelix is also associated with injection-site reactions such as injection-site pain, erythema or swelling. Injection-site reactions are usually mild or moderate in intensity and occur predominantly after the first dose, decreasing in frequency thereafter.^[4]

FSH receptors in other solid tumors

FSH receptors are selectively expressed on the luminal surface of the blood vessels of a wide range of tumors.^[8] There may be a potential role for suppression of FSH or FSH receptors. This work is in early stages. It is thought that FSH receptors are important in tumor angiogenesis by signalling via two pathways, one involving VEGF, and a Gq/11mechanism that activates VEGFR-2 independently of VEGF.^[8]

N-Boc-D-alanine (I) was coupled to the resin using diisopropyl carbodiimide and 1-hydroxybenzotriazole to afford resin (II). Subsequent cleavage of the Boc protecting group by means of trifluoroacetic acid provided the D-alanine-bound resin ( III) Sequential coupling and deprotection cycles were carried out with the following protected amino acids:. N-Boc-L-proline (IV), N-alpha-Boc-N6-isopropyl-N6-carbobenzoxy-L-lysine (VI) and N-Boc-L-leucine (VIII) to afford the respective peptide resins (V), (VII) and (IX). N-alpha-Boc-D-4-(Fmoc-amino) phenylalanine (X) was coupled to (IX), yielding resin (XI). Cleavage of the side-chain Fmoc protecting group with piperidine in DMF gave the aniline derivative (XII). After conversion to the corresponding urea by treatment with tert-butyl isocyanate, the Boc group was cleaved with trifluoroacetic acid to produce resin (XIII).

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Bioorganic & Medicinal Chemistry

doc.sciencenet.cn/upload/file/2011531154034454.pdf‎

by KCL Kevin – ‎2011 – ‎Cited by 10 – ‎Related articles

Keywords: Synthesis. New drug molecules. New chemical entities. Medicine …Degarelix acetate (Firmagon®) . ….. Scheme 5. Synthesis of degarelix acetate (V).

http://doc.sciencenet.cn/upload/file/2011531154034454.pdf IS THE LINK

SEE SCHEME IN THIS PUBLICATION

………………………………

http://www.google.com/patents/US20120041172
Example 1

Hydantoin formation in the synthesis of degarelix. The rearrangement of the hydroorotic group to a hydantoinacetyl group in the production of degarelix has been seen at two stages and two sets of basic conditions.

The first rearrangement appeared during basic extractions of the segment Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-ILys(Boc)-Pro-D-Ala-NH₂. The pH was adjusted to 9.1 in the organic/aqueous two-phase system using conc. NaOH solution, resulting in the formation of 4.5% by weight of the hydantoin analogue. The mechanism appeared to comprise two steps: (a) hydrolysis of the 6-membered hydroorotic moiety under basic conditions followed by ring closure to the 5-membered hydantoin analogue under acidic conditions.

The second rearrangement was observed during evaporation of the segment Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-OH.DCHA. After the preceding extractions, Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-OH was dissolved in a mixture of ethyl acetate and 2-butanol. DCHA (2.5 eq.) was added because the segment is isolated as the DCHA salt after evaporation of the solvent followed by a precipitation step. In the particular batch both the hydantoin analogue and the hydrolysed form (mentioned above) were identified. Quantification of the hydantoin was not possible because poor separation by HPLC from other products; the hydrolyzed form was formed in an amount of 1.34% by weight of the combined products. Experimental evidence showed that the amount of rearrangement/hydrolysis was related to the amount of DCHA used in the method.

The following experiment provided further proof of the instability of the hydrooroic moiety under basic conditions. Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-OH.DCHA (67 mM) was dissolved in wet 2-BuOH with 167 mM (2.5 eq) DCHA at 31° C. After 25 h, 1.3% of the hydantoin analogue and 0.3% of the hydrolysed intermediate had been formed.

Example 2

Stability of degarelix in DBU/DMF and piperidine/DMF. The stability of degarelix was tested under conditions corresponding to those used for removal of the Fmoc-group during SPPS. The hydroorotic group in the side chain of 4Aph(Hor), amino acid residue no. 5 in the sequence of degarelix, is known to be sensitive to base and rearrange to a hydantoinacetyl group. All SPPS procedures known to the inventors had been based on Boc-chemistry.

Samples of degarelix were dissolved in 20% piperidine/DMF; 2% DBU in DMF, and 2% DBU+5% water in DMF; respectively. The samples were analysed by HPLC after 20 h and the amount of the hydantoin analogue determined.

2% DBU/DMF resulted in the formation of 1.8% hydantoin. If 5% water was present, too (simulating wet DMF), the amount was increased to 7%. Surprisingly, the use of 20% piperidine in DMF did not result in any formation of the hydantoin analogue, indicating that this mixture might be useful for Fmoc-based SPPS of Degarelix.

Example 3 Synthesis and Purification of Degarelix Using Fmo-/Rink Amide AM Resin

Step 1. Fmoc-Rink amide AM resin (64 g; substitution 0.67 mmol/g) was placed in a reactor and washed with 1.9 L DMF. To the swollen resin 250 ml of 20% piperidine in DMF is added and stirred for 20 min. The reactor is emptied through the filter in the bottom by applying vacuum to the reactor and a second treatment with 250 ml 20% piperidine in DMF is performed for 20 min. The reactor is once again emptied by applying vacuum to it followed by a wash of the peptide resin using 2 L of DMF. The reactor is then emptied by applying vacuum. The peptide resin is now ready for step 2.

Step 2. A solution of 27.0 g Fmoc-D-Ala-OH (2 eq.), 14.3 g HOBt and 13.2 ml DIC is dissolved in 250 ml of DMF and allowed to activate for 15 min, after which it is poured into the reactor containing the peptide resin. After 1 h of reaction time, 2.2 ml of NMM is added to the solution and the reaction is allowed to proceed for another hour. Then 30 ml acetic acid anhydride and 2 ml NMM is added to the mixture, which is allowed to stand under stirring for 15 min. Then the reactor is emptied by using vacuum. The peptide resin is washed with 2 L DMF. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 250 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second treatment of 250 ml 20% piperidine in DMF for 20 min is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 2 L of DMF. It is now ready for step 3.

Step 3. A solution of 29 g Fmoc-L-Pro-OH (2 eq), 14.3 g HOBt and 13.2 ml DIC is dissolved in 250 ml DMF and allowed to activate for 25 min, after which it is poured into the reactor containing the peptide resin. After 75 min of reaction, 2.2 ml NMM is added to the solution, and the reaction is allowed to proceed for another hour. Then 30 ml acetic acid anhydride and 2 ml NMM is added to the mixture, which is allowed to stand under stirring for 15 min, The reactor is then emptied by using vacuum. DMF (2.6 L) is used for washing the peptide resin. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 250 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum, and a second treatment with 250 ml 20% piperidine in DMF for 20 min is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 2 L of DMF. It is now ready for step 4.

Step 4. A solution of 33 g Fmoc-L-ILys(Boc)-OH (1.5 eq), 10.7 g HOBt and 10.1 ml DIC is dissolved in 250 ml of DMF and allowed to activate for 0.5 h, after which it is poured into the reactor containing the peptide resin. After 2 h of reaction, 2.2 ml NMM is added to the solution and the reaction is allowed to proceed for another hour. Then 30 ml acetic acid anhydride and 2.2 ml NMM is added to the mixture, which is allowed to stand under stirring for 15 min, whereupon the reactor is emptied by using vacuum. The peptide resin is washed with DMF (3 L). After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 250 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second treatment of 250 ml 20% piperidine in DMF for 20 min is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 3.5 L DMF. It is now ready for step 5.

Step 5. A solution of 38 g Fmoc-L-Leu-OH (2.5 eq), 18 g of HOBt and 16.8 ml of DIC is dissolved in 250 ml of DMF and allowed to activate for 0.5 h, after which it is poured into the reactor containing the peptide resin. After 2 h of reaction, 2.2 ml NMM is added to the solution, and the reaction is allowed to proceed for another 50 min. Then 30 ml acetic acid anhydride and 2 ml NMM is added to the mixture, which is allowed to stand under stirring for 15 min. Then the reactor is emptied by using vacuum. DMF (2.6 L) is used for washing the peptide resin. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 250 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second treatment with 250 ml 20% piperidine in DMF for 20 min is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 2.5 L of DMF. It is now ready for step 6.

Step 6. A solution of 32 g of Fmoc-D-4Aph(tBu-Cbm)-OH (1.5 eq), 10.7 g HOBt and 10.1 ml DIC is dissolved in 250 ml of DMF and allowed to activate for 1 hour, after which it is poured into the reactor containing the peptide resin. After 20 min of reaction, 22 ml NMM is added to the solution and the reaction is allowed to proceed for another 20 h. Then 30 ml acetic acid anhydride and 2 ml NMM is added to the mixture, which is allowed to stand under stirring for 15 min. Then the reactor is emptied by using vacuum. The peptide resin is washed with 4 L DMF. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 250 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment with 250 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 3.4 L DMF. It is now ready for step 7.

Step 7. A solution of 35 g Fmoc-L-4Aph(L-Hor)-OH (1.5 eq), 11 g HOBt and 10.1 ml DIC is dissolved in 350 ml DMF and allowed to activate for 1 h, after which it is poured into the reactor containing the peptide resin. After 50 min of reaction, 2.2 ml NMM is added to the solution and the reaction is allowed to proceed for another 21.5 h. The reactor is emptied by using vacuum. The peptide resin is washed with 4.4 L DMF. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 350 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment with 350 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin is washed with 4.4 L DMF. It is now ready for step 8.

Step 8. Fmoc-L-Ser(tBu)-OH (2.5 eq) (41 g), 17.9 g HOBt, 16.8 ml DIC and 4.9 ml of NMM is dissolved in 500 ml of DMF and poured into the reactor containing the peptide resin. The reaction is allowed to proceed for 3.5 h. The reactor is then emptied by using vacuum. The peptide resin is washed with 4.2 L DMF. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 375 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment of 375 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin washed with 4.2 L of DMF. It is now ready for step 9.

Step 9. A solution of 25 g Fmoc-D-3 Pal-OH (1.5 eq), 10.7 g HOBt, 10.1 ml DIC and 4.9 ml NMM is dissolved in 400 ml of DMF and poured into the reactor containing the peptide resin. The reaction is allowed to proceed for 4.5 h. Then the reactor is emptied by using vacuum. The peptide resin is washed with 4.2 L DMF. After applying vacuum to the reactor, removing the DMF, the peptide resin is treated with 375 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment with 375 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin washed with 4.2 L of DMF. It is now ready for step 10.

Step 10. A solution of 27 g Fmoc-D-Phe(4Cl)—OH (1.5 eq), 10.7 g HOBt, 10.1 ml DIC and 4.9 ml NMM is dissolved in 400 ml of DMF and is poured into the reactor containing the peptide resin. The reaction is allowed to proceed for 10 h. The reactor is emptied by using vacuum. The resin is washed with 5.5 L DMF. After applying vacuum to the reactor and removing the DMF, the peptide resin is treated with 375 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment with 375 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin washed with 5 L DMF. It is now ready for step 11.

Step 11. A solution of 28 g Fmoc-D-2Nal-OH (1.5 eq), 10.7 g HOBt, 10.1 ml DIC and 4.9 ml NMM is dissolved in 400 ml DMF and poured into the reactor containing the peptide resin. The reaction is allowed to proceed for 2.5 h. The reactor is emptied by using vacuum. The peptide resin is washed with 5.2 L DMF. After applying vacuum to the reactor and removing the DMF, the peptide resin is treated with 375 ml of 20% piperidine in DMF for 20 min. The reactor is emptied by applying vacuum and a second 20 min treatment of 375 ml 20% piperidine in DMF is performed. The reactor is once again emptied by applying vacuum and the peptide resin washed with 5 L DMF. It is now ready for and is ready for step 12.

Step 12. Acetylimidazole (3 eq) (14.5 g) and 4.9 ml NMM is dissolved in 400 ml DMF and poured into the reactor. After 1.5 h, the reactor is emptied by applying vacuum to the reactor. The peptide resin is washed with 5 L DMF and the reactor emptied using vacuum.

Step 13. The peptide resin is washed with WA and dried under vacuum. Peptide resin (129.8 g; yield 96%) was isolated.

Step 14. Dry peptide resin (60 g) is suspended in 600 ml TFA for 25 h at room temperature. It was then poured into a mixture of 2.4 L water, 620 g ammonium acetate, 600 ml ethanol and 600 ml acetic acid. The mixture is adjusted to a pH between 3 and 4 using TFA and filtered.

Step 15. The product is purified using a two step purification protocol. In the first step a column (2.5 cm×34 cm) packed with reversed phase C-18 material is used with a buffer system consisting of buffer A (0.12% aqueous TFA) and buffer B (99.9% ethanol) A volume from the filtered solution from step 14 corresponding to 1.6 g of the product is applied to the column. Purification is executed using a step gradient starting with 10% B for 2-3 column volumes, 29% B for 5-7 column volumes and a gradient from 29% B to 50% B over 3 column volumes at a flow rate of 70 ml/min. This procedure is followed until all the filtered solution from step 14 has been processed. All fractions collected are analyzed by analytical HPLC. Fractions containing product with a purity higher than 94% are pooled. The second purification step is performed using a column (2.5 cm×34 cm) packed with reverse phase C-18 material and a buffer system consisting of a buffer A (1% aqueous acetic acid), buffer B (99.9% ethanol), and buffer C (0.5 M aqueous ammonium acetate). From the pooled fractions containing the product an amount equivalent to 1.3 g of the product is applied to the column and purification performed by applying a step gradient starting with 10% B+90% C for 2-3 column volumes followed by 90% A+10% B for 2-3 column volumes. The product is eluted by 24% B+76% A. The fractions containing product with the acceptable purity are pooled and desalted using the same column. Desalting is performed using buffer A (1% aqueous acetic acid) and buffer B (99.9% ethanol). A volume from the pooled purified fraction corresponding to 1.6 g of product is applied to the column, 2-3 column volumes buffer A being used to wash out any ammonium acetate in the product. Then the product is eluted using 50% buffer A+50% buffer B. The solution of the purified product containing 50% ethanol is concentrated on a rotary evaporator. When all the ethanol has been removed the remaining solution containing the product is lyophilized. A total of 11.8 g (overall yield 37%) of degarelix is obtained as a fluffy solid. 4-([2-(5-Hydantoyl)]acetylamino)-phenylalanine could not be detected in the product (HPLC).

Example 4 Synthesis and Purification of Degarelix Using Fmoc-Rink Amide MBHA

Performed substantially as the synthesis and purification of Example 1. Deviations from the method of Example 1:

- a) Fmoc-D-Aph(Cbm)-OH was used instead of Fmoc-D-Aph(tBu-Cbm)-OH;
- b) Acetylation of the N-terminal of H-D-2-Nal-peptide-resin was performed using acetic acid anhydride instead of acetylimidazole;
- c) Acetonitrile was used in purification instead of ethanol.

4-([2-(5-Hydantoyl)]acetylamino)-phenylalanine could not be detected in the product by HPLC.

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http://www.google.com/patents/EP2447276A1?cl=en

Prostate cancer is a leading cause of morbidity and mortality for men in the industrialised world. Degarelix, also known as FE200486, is a third generation gonadotropin releasing hormone (GnRH) receptor antagonist (a GnRH blocker) that has been developed and recently approved for prostate cancer patients in need of androgen ablation therapy (Doehn et al., Drugs 2006, vol. 9, No. 8, pp. 565-571; WO 09846634 ). Degarelix acts by immediate and competitive blockade of GnRH receptors in the pituitary and, like other GnRH antagonists, does not cause an initial stimulation of luteinizing hormone production via the hypothalamic-pituitary-gonadal axis, and therefore does not cause testosterone surge or clinical flare (Van Poppel, Cancer Management and Research, 2010:2 39-52; Van Poppel et al., Urology, 2008, 71(6), 1001-1006); James, E.F. et al., Drugs, 2009, 69(14), 1967-1976).
[0003]

Degarelix is a synthetic linear decapeptide containing seven unnatural amino acids, five of which are D-amino acids. It has ten chiral centers in the back bone of the decapeptide. The amino acid residue at position 5 in the sequence has an additional chiral center in the side-chain substitution giving eleven chiral centers in total. Its CAS registry number is 214766-78-6 (of free base) and it is commercially available under the Trademark Firmagon™. The drug substance is chemically designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-dioxo-4-pyriminyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl- and is represented by the chemical structure below (in the following also referred to as Formula I):
[0004]

The structure of Degarelix can also be represented as:Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂

where Ac is acetyl, 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Ser is serine, 4Aph is 4-aminophenylalanine, Hor is hydroorotyl, Cbm is carbamoyl, Leu is leucine, Lys(iPr) is N6-isopropyllysine, Pro is proline and Ala is alanine.
[0005]

For the purposes of describing this invention, each amino acid in Degarelix will be given the shorthand notation as follows:AA₁ is D-2Nal, AA₂ is D-4Cpa, AA₃ is D-3Pal, AA₄ is Ser, AA₅ is 4Aph(L-Hor), AA₆ is D-Aph(Cbm), AA₇ is Leu, AA₈ is Lys(iPr), AA₉ is Pro and AA₁₀ is D-Ala.
[0006]

Thus, as an example, Degarelix can be represented as Ac-AA_1-AA₁₀-NH₂, the tetrapeptide Ac-D-2Nal-D-4Cpa-D-3Pal-Ser can be represented as Ac-AA₁-AA₄ and the hexapeptide 4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂ as AA₅-AA₁₀-NH₂.
[0007]

Degarelix has previously been prepared using Boc-solid phase peptide synthesis (SPPS) methodology as reported in WO 98/46634 and Jiang et al., J. Med. Chem. 2001, 44, 453-467. Basically, Boc-protected D-Ala is first coupled to MBHA resin in dimethylformamide (DMF)/CH₂Cl₂using diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) as activating or coupling agents. Once D-Ala is coupled to the resin, synthesis proceeds by washing, deblocking and then coupling the next amino acid residue until the decapeptide has been completed. The side chain primary amino groups of 4Aph in the 5-position and of D-4Aph in the 6-position are protected by Fmoc when they are added and modified with L-Hor and Cbm respectively before the next amino acid in the chain is added. This requires the additional steps of first removing the side-chain protection with piperdine, reacting the newly freed amino group on the peptidoresin with tert-butyl isocyanate or L-hydroorotic acid, ensuring that the reaction is complete with a ninhydrin test and then washing the peptidoresin before adding the next amino acid residue (see also Sorbera et al., Drugs of the Future 2006, Vol. 31, No. 9, pp 755-766).
[0008]

While Boc-SPPS methodology has afforded sufficient quantities of Degarelix until now, the growing demand for this polypeptide means that ever larger quantities are required. Boc-SPPS, which requires HF cleavage, is not suited to large scale industrial synthesis. Indeed, WO 98/46634 mentions that SPPS is only suitable for limited quantities of up to 1 kg while classical peptide solution synthesis, or liquid phase peptide synthesis (LPPS), is preferred for larger quantities of product.WO 98/46634 does not specify how such synthesis should be performed. While the existence of a liquid phase peptide synthesis of Degarelix has been reported [EMEA Report: Assessment Report for Firmagon™ (Degarelix): Doc. Ref. EMEA/CHMP/635761/2008], as of now no details of such a process have been publically disclosed.
[0009]

WO 97/34923 and WO 99/26964 are documents concerned with liquid phase processes for the preparation of biologically active peptides. WO 99/26964 is particularly concerned with the liquid phase synthesis of decapeptides having activity as GnRH antagonists. WO 99/26964 lists a number of inherent limitations of the SPPS methodology for producing GnRH antagonists including the limited capacity of the resin, the large excess of reagents and amino acids required, as well as the need to protect all reactive side chains such as the hydroxy group in Ser, the aromatic amino groups in Aph and D-Aph, the ε-i-propylamino group in Lys(i-Pr).
[0010]

WO 99/26964 proposes a liquid phase process which involves first preparing the central peptide fragments of the 5 and 6 positions of a decapeptide with the side chains fully elaborated and then assembling the peptide through a “4-2-4”, “3-3-4” or “3-4-3” fragment assembly pattern. For example, in the preparation of the GnRH antagonist Azaline B, a tetrapeptide is coupled with a hexapeptide to form the desired decapeptide. When the same fragment assembly pattern is attempted for Degarelix, racemisation of the Ser amino acid (AA₄) occurs resulting in about 20% impurity of L-Ser. This impurity carries over into the final decapeptide and is difficult to remove. Furthermore, when preparing the tetrapeptide AA₁-AA₄ by adding the Ser unit to the tripeptide AA₁-AA₃following the procedure described in WO 99/26964 , tetrabutylammonium ions from the hydrolysis of the benzyl ester group could not be removed completely during the subsequent operations and were carried through to the final product. It was further found that in the Degarelix synthesis, the L-hydroorotyl group rearranges to its hydantoinacetyl analogue when L-dihydroorotic acid is coupled with 4Amp to prepare AA₅. These and other problems with the solution-phase synthesis of Degarelix have now been overcome and a new solution-phase polypeptide synthesis of this decapeptide is disclosed herein for the first time.

Starting materials:

[0073]

N-t-Butyloxycarbonyl-D-4-chlorophenylalanine	Boc-D-4Cpa-OH C₁₄H₁₈NO₄
N-t-Butyloxycarbonyl-D-2-naphtylalanine	Boc-D-2Nal-OH C₁₈H₂₁N0₄
D-3-Pyridylalanine hydrochloride	H-D-3Pal-OH x 2HCl C₈H₁₂Cl₂N₂O₂
N-α-t-Butyloxycarbonyl-N-4-(t-Butylcarbamoyl)-D-4-Aminophenylalanine	Boc-D-4Aph(tBuCbm)-OH C₁₉H₂₉N₃O₅
N-α-t-Butyloxycarbonyl-N-4-(L-Hydroorotyl)-4-Aminophenylalanine	Boc-4Aph(L-Hor)-OH C₁₉H₂₄N₄O₇
Leucine benzyl ester p-tosylate	H-Leu-OBzl x TOS C₂₀H₂₇NO₅
N-Benzyloxycarbonyl-O-t-butyl-serine	Z-Ser(tBu)-OH C₈H₁₅NO₅
N-t-Butyloxycarbonyl-proline	Boc-Pro-OH C₁₀H₁₇NO₄
D-Alaninamide hydrochloride	H-D-Ala-NH₂ x HCl C₃H₈ClNO₂
N-α-Benzyloxycarbonyl-N-ε-t-butyloxycarbonyl-N-ε-isopropyl-lysine, dicyclohexylamine salt	Z-Lys(iPr,Boc)-OH x DCHA C₃₄H₅₇N₃O₆

Example 1: Synthesis of Intermediate Ac(1-3)ONa: Ac-D-2Nal-D-4Cpa-D-3Pal-ONa[7]Activation of Boc-D-4Cpa-OH and isolationStep 1 (Reaction step)

[0074]

Boc-D-4Cpa-OH ( 299.75 g) is dissolved in iPrOH (3.53 kg), the mixture is stirred and HOSu (0-184 kg) and DIC (0.164 kg) are added, and stirred at 0°C for 1 hour. The precipitate is filtered off and washed with iPrOH. The solid is dried under reduced pressure to yield Boc-D-4Cpa-OSu[1].

Activation of Boc-D-2Nal-OH and isolationStep 2 (reaction step)

[0075]

Boc-D-2Nal-OH (315.38 g) is dissolved in iPrOH (5.35 kg) and IBC (157.07 g) and NMM (116.7 g) are added. A mixture of water (42 mL), iPrOH (1.1 kg) and HOSu (230.14 g) is added after cooling to -10°C together with additional NMM (10.11g), and the mixture stirred 30 min. Water (0.82 L) is added and the precipitate is filtered off, and washed with iPrOH and dried under reduced pressure to yield Boc-D-2Nal-OSu[2].

Synthesis of Boc(2-3)OH: Boc-D-4Cpa-D-3Pal-OHStep 3 (Reaction step)

[0076]

H-D-3Pal-OH x 2 HCl (0.251 kg) and Boc-D-4Cpa-OSu [1] (0.397 kg) from step 1 are dissolved in DMSO (3.33 L) and NMM (318.8 g) is added. The mixture is stirred at 20°C for 6 hours. Water (17 L) is added and pH is adjusted by adding HCl to pH 4.25. The precipitate is filtered off, and dispersed in water. The obtained slurry is then filtered and washed with water. The solid is dried under reduced pressure to yield Boc-D-4Cpa-D-3Pal-OH[3].

Synthesis of Intermediate Ac(1-3)ONa: Ac-D-2Nal-D-4Cpa-D-3Pal-ONa[7] (Compound of formula IIIa)Step 4 (Reaction step)

[0077]

Boc-D-4Cpa-D-3Pal-OH [3] (447.93 g) from step 3 is dissolved in a mixture of AcOEt (3.4 L) and AcOH (675 mL), the mixture is cooled at 5°C where after MSA (672.77 g) is added. The reaction continues at 10°C for 2 hours and to the solution is added TEA (1214.28 g) to yield H-D-4Cpa-D-3Pal-OH [4].
[0078]

Boc-D-2Nal-OSu[2] (412.44 g) from step 2 is added to H-D-4Cpa-D-3Pal-OH [4], stirred for 24 hours at 20°C. 25% aqueous NH₃ (0.154L)and n-butanol (4.5L) are added, and the mixture is stirred at 45°C for 1 hour.
[0079]
The solution is washed with:
- Water
- Water at pH 9.5 (pH is adjusted while stirring with aq. NaOH)
- Water
[0080]

AcOH (4.5 L) is added to the organic phase and the solution is concentrated to an oil under reduced pressure. The oil is re-dissolved in AcOH (4.5 L) and re-concentrated under reduced pressure to yield Boc-D-2Nal-D-4Cpa-D-3Pal-OH[5] as an oil.
[0081]

Boc-D-2Nal-D-4Cpa-D-3Pal-OH [5] is dissolved in water (0.09 L) and AcOH (1.8L). MSA (672.77 g) is added and the mixture is stirred at below 35°C for 2 hours. The solution is neutralised with TEA (779.16 g). The solution is concentrated under reduced pressure to an oil. The oil is re-dissolved in toluene (2.5 L) and re-concentrated under reduced pressure to an oil. The last step is repeated to yield H-D-2Nal-D-4Cpa-D-3Pal-OH[6].
[0082]

H-D-2Nal-D-4Cpa-D-3Pal-OH [6] is dissolved in toluene (2.0 L) and a solution of acetyl imidazole (132.14 g) in toluene (0.25 L) is added. The solution is stirred at 20°C for 2 hours, and water (0.1 L) is added.
[0083]
n-Butanol (4.5 L) is added and the organic mixture is washed at 35° C with:
- 5% aqueous NaCl
- Methanol and water at acidic pH 5.5 (pH is adjusted while stirring with aq. NaOH)
- Methanol and water at pH 11 (pH is adjusted while stirring with aqueous NaOH)
- Methanol and 10% aqueous NaCl
[0084]

To the stirred organic phase from the extractions, heptane (15 L) is added at 20°C for 1 hour, and the resulting suspension is left with stirring at 20°C for 1 hour. The precipitate is isolated by filtration, and suspended in heptane (3.5 L). The suspension is filtered again. The last washing step with heptane and the filtration is repeated. The solid is then dried under reduced pressure to yield Ac-D-2Nal-D-4cpa-D-3Pal-ONa[7]. Purity of intermediate Ac(1-3)ONa[7] is ≥90% (HPLC).

Example 2: Synthesis of Intermediate Z(4-7)OH x DCHA: Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OHxDCHA[15]Synthesis of intermediate Boc(6-7)OBzl: Boc-D-4Aph(tBucbm)-Leu-OBzl Step 5 (Reaction step)

[0085]

Boc-D-4Aph(tBuCbm)-OH (379.45 g) is dissolved in NMP (0.76 L) and AcOEt (4.4 kg). After cooling at – 4°C, IBC (150.2 g) and NMM (101.1 g) are added, and the solution stirred at -7°C for 0.5 hour to yield Boc-D-4Aph(tBuCbm)-OAct[8].
[0086]

H-Leu-OBzl x TOS (491.88 g) is dissolved in NMP (1.5 L) and AcOEt (2.7 kg) is added, followed by NMM (126.4 g). This solution is subsequently transferred to Boc-17-4Aph(tBuCbm)-OAct [8], and stirred at – -10°C for 1 hour. Then, water (0.5 L) is added.
[0087]
The reaction mixture is washed at 20°C with:
- Water at pH 8.5 (pH is adjusted while stirring with aq. NaOH)
- Water at pH 2.0 (pH is adjusted while stirring with aq. HCl)
- Water
[0088]

The organic phase is concentrated under reduced pressure to an oil. The oil is re-dissolved in AcOEt (0.6 kg) and re-concentrated under reduced pressue to an oil. The remaining oil is dissolved in AcOEt (0.6 kg). Heptane (15.5 L) is added while stirring at 20°C. The precipitate is isolated by filtration, and washed with heptane and subsequently dried under reduced pressure at to yield Boc-D-4Aph(tBuCbm)-Leu-OBzl[9].

Synthesis of Boc-(5-7)-OBzl: Boc-4Aph(L-Hor)-D-4Aph(tBucbm)-Leu-OBzlStep 6 (Reaction step)

[0089]

Boc-D-4Aph(tBuCbm)-Leu-OBzl [9] (582.7 g) from step 5 is dissolved in AcOEt (3.15 kg). MSA (481 g) is added, and stirred below 15°C for 5 hours, and TEA (406 g) is added. DMF (D.333 kg) is added followed by TEA (101 g) and NMM (51 g) to yield H-D-4Aph(tBuCbm)-Leu-OBzl[10].
[0090]

Boc-4Aph(L-Hor)-OH (462.46 g) is dissolved in DMF (2.09 kg) and AcOEt (1.44 kg). IBC (150.24 g) and NMP (111.27 g) are added, and stirred at -10°C for 0.5 h to yield Boc-4Aph(L-Hor)-OAct[11].
[0091]

H-D-4Aph(tBuCbm)-Leu-OBzl [10] is added to Boc-4Aph(L-Hor)-OAct [11] and stirred at -10°C for 1.5 hours. Then, AcOEt (5.4 kg) and n-butanol (6.0 L) are added.
[0092]
The organic phase is washed at 20°C with:
- 5% aqueous NaHCO₃ at pH 8 (pH is adjusted while stirring with aq NaHCO₃)
- 10% aqueous. NaCl at pH 2.5 (pH is adjusted while stirring with aq.H₃PO₄)
[0093]

DMF (0.9 L) is added to the organic phase, which is then concentrated under reduced pressure to an oil. The solution is poured into water (14 L) while stirring. The precipitate is isolated on a filter, and washed with water. The solid is dried under reduced pressure to yield Boc-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OBzl[12].

Synthesis of intermediate Z(4-7)OH x DCHA: Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA (Compound of formula Va)Step 7 (Reaction step)

[0094]

Boc-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OBzl [12] (885.02 g) from step 6 is added to a mixture of MSA (961.1g) and AcOEt (7.2 kg) and 2-butanol (2 L) is added, and the resulting mixture stirred at 0°C for 6 hours. MSA is then neutralised with TEA (909.0 g).
[0095]

5% Pd/C (88.5g) dispersed in 2-butanol (1L) is added and the mixture is hydrogenated under pressure at 20°C for 3 hours. Then, the Pd/C is filtered off, and washed with 2-butanol to yield the solution containing H-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH [13]. Z-Ser(tBu)-OH (413.5 g) is dissolved in MeCN (2.5 L) and the solution is cooled to -5°C. HONp (195 g) is added followed by DCC (278.5 g), and the mixture stirred at 20°C for 24 hours. The mixture is then filtered, and washed with MeCN to yield Z-Ser(tBu)-ONp [14]. NMM (354.2 g), DMF (4.75 kg) and Z-Ser(tBu)-ONp [14] is added to the solution of H-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH [13] and the mixture is left with stirring at 20°C for 3 days.
[0096]
The resulting mixture is washed with:
- 10% aqeuous NaCl at pH 2.5 (pH is adjusted while stirring with aqueous HCl)
- Water at acidic pH (pH 2.5) (pH is adjusted while stirring with aqueous HCl)
- 7.5% aqeuous NaHcO₃
- 5% aqeuous. NaCl at (pH 2.5) (pH is adjusted while stirring with aqueous HCl)
- 10% aqeuous NaCl
[0097]

To the final organic phase DCHA (181 g) is added and the organic phase is concentrated under reduced pressure to an oil. The oil is re-dissolved in iPrOH (3.14 kg) and re-concentrated under reduced pressure to an oil. The remaining oil is re-dissolved in iPrOH (3.14 kg) and while stirring the solution is poured into AcOEt (31.5 kg). Stirring is continued at 20°C for 1 hour until precipitation and the precipitate is then isolated by filtration, and washed with AcOEt. The solid is dried under reduced pressure at 30°C for 30 hours to yield Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA[15]. Purity of intermediate Z(4-7)OH x DCHA[15] is ≥80% (HPLC).

Example 3: Synthesis of Intermediate H(8-10)NH ₂:H-Lys(iPr,Boc)-Pro-D-Ala-NH ₂[21]Synthesis of Boc(9-10)NH₂: Boc-Pro-D-Ala-NH₂Step 8 (Reaction step)

[0098]

Boc-Pro-OH (226.02 g) is dissolved in iPrOH (1.73 kg). The reaction mixture is cooled to -5°C. IBC (143.4 g) and NMM (106.2 g) are added and the mixture is stirred at 5°C for 0.5 hour to yield Boe-Pro-OAct[16].
[0099]

H-D-Ala-NH₂ x HCl (124.57 g) is suspended in a mixture of iPrOH (1.57 kg) and NMM (106.2 g). The suspension is added to Boc-Pro-OAct [16]. The reaction mixture is left with stirring at 10°C for 3 hours. Then DMEDA (10.6 ml) is added. The mixture is filtered, and the filtrate is concentrated under reduced pressure to an oil. The oil is re-dissolved and re-concentrated with AcOEt (1.125 kg).
[0100]
The residual oil is dissolved in a mixture of AcOEt (1.8 kg) and n-butanol (0.6 L). The organic phase is washed with:
- 15% aqeuous. NaCl at pH 2.5 (pH is adjusted while stirring with aqeuous HCl)
- 15% aqeuous NaCl at pH 9.5 (pH is adjusted while stirring with aqeuous NaOH)
[0101]

The organic phase is concentrated under reduced pressur, re-dissolved in AcOEt (1.08 kg) and re-concentrated to an oil.
[0102]

A mixture of AcOEt (0.33 kg) and heptane (0.75 L) is added at 20°C and stirred for 16 hours. The resulting precipitate is filtered and washed with a mixture of AcOEt and heptane on the filter. The solid is then dried under reduced pressure to yield Boc-Pro-D-Ala-NH₂[17]

Synthesis of intermediate H(8-10)NH₂: H-Lys(iPr,Boc)-Pro-D-Ala-NH₂ (Compound of formulae Vla)Step 9 (Reaction step)

[0103]

Boc-Pro-D-Aia-NH₂ [17] (313.89 g) from Step 8 is dissolved in a mixture of MSA (528.61 g) and iPrOH (0.785 kg) and the solution is stirred at 45°C for 1 hour. The mixture then is neutralised with TEA (607.14 g) to yield H-Pro-D-Ala-NH₂[18].
[0104]
Z-Lys(iPr,Boc)-OH x DCHA (603.83 g) is suspended in AcOEt (1.17 kg) and washed with:
- 12% aqeuous NaHSO₄
- Water
- 15% aqeuous NaCl
[0105]
The organic phase of Z-Lys(iPr,Boc)-OH [19] from the extractions is added to H-Pro-D-Ala-NH₂ [18]. HOBt (183.79 g) and DCC (227.0 g) dissolved in AcOEt (0.135 kg) are added, and the mixture stirred at 20°C for 0.5 hours. Then, water (0.2 L) is added. The mixture is filtered and washed with AcOEt. The combined filtrates are concentrated under reduced pressure to an oil. The oil is dissolved in AcOEt (0.9 kg), filtered and the solution is washed with:
- Water at pH 2.5 (pH is adjusted while stirring with aqueous HCl)
- Water at pH 9 (pH is adjusted while stirring with aqueous NaOH)
- 10 % aqueous NaCl at pH 7 (pH is adjusted while stirring with aqueous HCl or aqeuous NaOH)
[0106]

The organic phase is concentrated under reduced pressure to yield Z-Lys(iPro,Boc)-Pro-D-Ala-NH₂[20].
[0107]

Z-Lys(iPro,Boc)-Pro-D-Ala-NH₂ [20] is dissolved in ethanol (0.04 kg) and water (0.5 L), and 5% Pd/C (50 g) is added. The slurry is acidified to pH 2.5 by addition of 6 M HCl and hydrogenated at 20°C. After completed reaction the catalyst is removed by filtration and pH is raised to pH 7.0 by addition of 32 % NaOH. The ethanol is subsequently removed by evaporation under reduced pressure. n-Butanol (1 L) is added to the resulting aqueous phase and the pH is adjusted to alkaline pH 9 with aqueous NaOH and the extraction starts. This step is repeated. The combined organic phases are concentrated under reduced pressure to an oil.
[0108]

The oil is dissolved in AcOBu (0.5 L), concentrated under reduced pressure at 20°C and re-dissolved in AcOBu (0.5 L). Then, heptane (2 L) is added at 50°C for 1 hour. The suspension is left with stirring at 0°C for 16 hours. The precipitate is isolated by filtration and washed with heptane. Finally, the solid is dried under reduced pressure at to yield H-Lys(iPr,Boc)-Pro-D-Ala-NH₂[21]. Purity of intermediate H(8-10)NH₂[21] is ≥95% (HPLC).

Example 4: Segment Condensations to Final Intermediate (compound of Formula II)intermediate Z(4-10)NH₂ : Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-leu-lys(iPr,Boc)-Pro-D-Ala-NH₂[22]

(Compound of formula lVg)

Step 10 (reaction step)

[0109]
Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA [15] (1153.41 g) from Step 7 is dissolved in DMF (2.1 kg). Then, HOBt (153.2 g) is added together with AcOEt (6.9 kg) and H-Lys(iPr,Boc)-Pro-D-Ala-NH₂ [21] (569.5 g) from step 9. When all solids are dissolved MSA (96.1 g) is added. The solution is cooled below 5°C and DCC (309.5 g) dissolved in AcOEt (0.810 kg) is added. The temperature is raised to 20°C and the reaction continues for 24 hours. Conversion of Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-OH x DCHA [15] is ≥96 % (HPLC). AcOEt (4.95 kg) and water (5.5 L) are added, and the mixture is stirred, and filtered. While stirring, 7.5.% NaHCO₃ (aq) (35L) is added to the filtrate. The phases are separated and the organic layer is further washed with:
- 7.5% NaHCO₃
- Water at pH 3 (pH is adjusted while stirring with aqueous HCl)
- Water
[0110]

The final organic phase is concentrated under reduced pressure to an oil. The oil is re-concentrated with EtOH (0.405 kg) and subsequently with AcOEt (0.45 kg). The remaining oil is dissolved in EtOH (0.405 kg), and AcOEt (0.45 kg) and AcOBu (4.6 L) are added. The solution is added to heptane (27.6 L) at 20°C for 1 hour. Then, the precipitate is filtered, and washed with heptane. The solid is dried under reduced pressure at maxiumum and it is checked by [Quality control 2] to meet the acceptance criteria to yield Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂[22]. Purity of Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂ [22] is ≥70 % (HPLC).

Final Intermediate Ac(1-10)NH₂: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-leu-Lys(iPr, Boc)-Pro-D-Ala-NH₂[24]Step 11 (Reaction step)

[0111]

Z-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂ [22] (1409.67 g) from Step 10 is added to a mixture of EtOH (10.98 kg) and water (3.2 L) and stirred until the solution is homogenous. 5 % Pd/C (211 g) is added. The mixture is hydrogenated at 20°C with pH-control at pH 2.5 ,with aqueous HCl.
[0112]

The catalyst is removed by filtration and the pH is adjusted to pH 3.8 ,with aqueous NaOH. The filtrate is concentrated under reduced pressure to an oil. EtOH (4.7 kg) is added to the oil and re-concentrated. Then, AcOEt (5.4 kg) is added to the oil and re-concentrated and this process is repeated again to yield H-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂[23]. H-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBucbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂ [23] is dispersed in AcOEt (1.125 kg), then HOBt (153.16 g) is added and the mixture is cooled to 0°C. Ac-D-2Nal-D-4Cpa-D-3Pal-ONa [7] (609.05 g) from Step 4 is dissolved in DMSO (2.5 L), this solution is mixed with the slurry containing H-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-PrO-D-Ala-NH₂ [23] and DCC (309.5 g) dissolved in AcOEt (0.45 kg) is added. The mixture is stirred at 5°C for 24 hours. Conversion of [23] is ≥96% (HPLC).
[0113]
Water (150 mL) and DMSO (0.5 L) are added and the stirring is continued at 20°C for more than 3 hours. The precipitate is filtered, and washed with a mixture of AcOEt and DMSO. The filtrates are combined, and n-butanol (17 L) is added. The organic solution is washed with:
- Water at pH 2.5 (pH is adjusted while stirring with aqueous HCl)
- 7% NaHCO₃ (aq)
- 10% aqueous NaCl (pH in the mixture is neutralised to pH 7.0, if necessary, while stirring with aqueous HCl)
[0114]

DMF (4.75 kg) is added and the organic phase is concentrated under reduced pressure to an oil. The oil is slowly added to water (50 L) at 20°C for 1 hour under vigorous stirring. The precipitate is isolated on a filter, and washed twice with water. The solid is subsequently dried under reduced pressure to yield Ac-D-2Nal-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-PrO-D-Ala-NH₂[24][Final intermediate]. Purity of [24] is ≥70% (HPLC)

Example 5: Deprotection of Final Intermediate Ac(1-10)NH ₂to Crude Degarelix[251]Step 12 (Reaction step)

[0115]

AC-D-2Nal-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-4Aph(tBuCbm)-Leu-Lys(iPr,Boc)-Pro-D-Ala-NH₂ [24] (Compound of formula IIb)(1844.59 g) from step 11 is dissolved in TFA (28.3 kg) at 20°C. The solution is stirred at 20°C (removal of 3 protection groups) for 24 hours. Conversion of [24] is ≥99 % (HPLC).
[0116]

The reaction mixture is then mixed with a cold solution (below 10°C) of water (74 L), AcONH₄ (19.1 kg), AcOH (18.4 L) and EtOH (14.52 kg). During mixing of the two solutions the temperature is kept below 25°C. The pH of the final solution is adjusted to pH 3 with TFA or AcONH₄, if necessary, to yield the solution of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH₂[25][Crude Degarelix].

Step 13 (purification and lyophilisation)

[0117]

The solution of crude degarelix is pumped through a reversed phase column. Degarelix is eluted from the column with a gradient of EtOH/ 0.12 % TFA in water. Fractions with a purity ≥95% are repurified on a reversed phase column using a gradient of EtOH/ 1% AcOH in water. Fractions of high purity are lyophilised.

…………….

The synthesis of Degarelix has been described by solid phase synthesis using Boc chemistry (2001 :880 SYNTHLINE):

N-Boc-D-alanine (I) was coupled to the MBHA resin using diisopropyl carbodiimide and 1-hydroxybenzotriazole to afford resin (II). Subsequent cleavage of the Boc protecting group by means of trifluoroacetic acid (TFA) provided the D-alanine-bound resin (III). Sequential coupling and deprotection cycles were carried out with the following protected amino acids: N-Boc-L-proline (IV), N-alpha-Boc-N6-isopropyl-N6-carbobenzoxy-L- lysine (VI) and N-Boc-L-leucine (VIII) to afford the respective peptide resins (V), (VII) and (IX). N-alpha-Boc-D-4-(Fmoc-amino)phenylalanine (X) was coupled to (IX), yielding resin (XI). Cleavage of the side-chain Fmoc protecting group with piperidine DMF gave the aniline derivative (XII). This portion of the synthesis route is shown below in Scheme 1.

Scheme 1

After conversion to the corresponding urea by treatment with tert-butyl isocyanate, the Boc group was cleaved with TFA to produce resin (XIII). Further coupling with N-alpha- Boc-L-4-(Fmoc-amino)phenylalanine (XIV), followed by Fmoc deprotection with piperidine, furnished (XV). The aniline derivative (XV) was acylated with L-hydroorotic acid (XVI) to yield, after Boc group cleavage, resin (XVII). Coupling of (XVII) with N- Boc-L-serine(O-benzyl) (XVIII) and subsequent deprotection gave (XIX), as shown in Scheme 2, below:

Sche

Peptide (XIX) was sequentially coupled with N-alpha-Boc-D-(3-pyridyl)alanine (XX) and N-Boc-D-(4-chlorophenyl)alanine (XXII) to furnish, after the corresponding deprotection cycles with TFA, the resins (XXI) and (XXIII), respectively, as shown in Scheme 3, below:

Scheme 3

The coupling of resin (XXIII) with N-Boc-D-(2-naphthyl)alanine (XXIV) as before gave, after the corresponding deprotection cycle with TFA, resin (XXV). The peptide resin (XXV) was acetylated with Ac20 and finally deprotected and cleaved from the resin by treatment with HF to provide the target peptide, as shown in Scheme 4 below:

Scheme 4

Alternatively, after coupling of the peptide resin (XIII) with alpha-Boc-L-4-(Fmoc- amino)-phenylalanine (XIV), the Fmoc protecting group was not removed, yielding resin (XXVI). Subsequent coupling cycles with amino acids (XVIII), (XX), (XXII) and (XXIV) as above finally produced resin (XXVII). The Fmoc group was then deprotected by treatment with piperidine, and the resulting aniline was acylated with L-hydroorotic acid (XVI) to provide resin (XXVIII), as shown in Scheme 5 below:

Scheme 5

Resin (XXVIII) was finally cleaved and deprotected by treatment with HF, as shown in Scheme 6 below:

US 6214798 Bl, which describes a similar synthetic approach, suggests that classical peptide solution synthesis would be preferable for producing large quantities of product. The purity of the final material obtained in US 6214798 Bl is estimated, therein, to be about 98%, yet J Med. Chem. 2005, 48, 4851-4860 describing the same preparation yields a purity as measured by capillary zone electrophoresis (CZE) of 98%, which corresponds to 96% according to HPLC analysis.

Fmoc chemistry generally employs milder reaction conditions than the Boc chemistry, and thus can sometimes be less likely to cause the formation of by-products..

In addition, Fmoc chemistry does not employ hydrofluoric acid or other very strong acid (such as TFMSA or HBr/AcOH) is needed. Therefore these processes are much safer and environmentally friendly; factors that are important when engineering such a synthesis process on the large production scale.

In stepwise SPPS at least two types of protection for reactive functionalities other than those involved in peptide bond formation are necessary: temporary protection of the alpha-amino group, removable after formation of each peptide bond; and, side chain protection removable after assembly of the complete peptide chain.

When using Fmoc chemistry, a tBu protecting group is commonly used for the protection of the hydroxyl groups on Ser, Tyr and Thr residues. The tBu group is easily removed together with other side-chain protecting groups at the end of the peptide synthesis by using TFA.

WO2010121835 (*W0’835′) describes the preparation of Degarelix using an Fmoc strategy and using tBu as the protecting group for the Ser residue. WO’835 also teaches that, unusually severe cleavage conditions such as long reaction time and 100% TFA as cleavage cocktail were required to successfully complete the final deprotection.. Such severe conditions are commonly known to result in increased side reactions, increased degradation of the peptide, and accordingly production of a lower quality of the resulting product.

One alternative to avoid using these severe deprotection conditions could be the use of different acid labile protecting group for Ser, such as Trt. However, it is well laiown in the art that the Trt group is very bulky and hydrophobic, and is frequently used to increase .the “steric hindrance” effect. See, e.g. Chem. Lett. 27, 1999 and J Pept. Sci. 2010; 16: 364-374, which describes that using the Trt protecting group in an Fmoc peptide synthesis could prevent formation of the target peptide due to “steric hindrance.” The Degarelix sequence is inherently very hydrophobic. Therefore coupling of a large hydrophobic residue to the growing peptide chain is expected to cause slowing of the reaction and thus increased liability for the formation of impurities associated with deletions in the sequence and racemization. Synthesis efficiency is an important consideration when determining which general coupling reaction to use in an SPPS process. This efficacy can be limited by a number of different variables. One major variable that must be considered is the steric hindrance. This steric hindrance is determined by the nature of the peptide side chains and of their protecting groups. It is an important variable because it will allow the rapid acylation of the amino function and have a limiting effect on possible side reactions. See, e.g., ChemPep®, Fmoc Solid Phase Peptide Synthesis; Coupling reaction). Moreover, for the Degarelix synthesis, Fmoc-

Ser(Trt)-OH would be added to extremely bulky 4Aph(L-Hor) [4-[[[(4S)-hexahydro-2,6- dioxo-4-pyrimidinyl]carbonyl] amino] -L-phenylalanyl] residue. Accordingly, the use of this protecting residue would likely be quite difficult due to “steric hindrance”. These “steric effects” are known to be of great importance when two molecules or two groups are in a close approach resulting in van der Waals forces repulsive effect. The repulsive potential can become quite large if the nonbonded distances are sufficiently short. This van der Walls repulsion can thus serve to slow a reaction, a situation that is typically refen-ed to as “steric hindrance” (Stereochemistry of Organic Compounds, E.L. Eliel et al, John Willey & Sons, 1994, 720 – 721 )patents
WO 2011004260
WO 2011066386
WO 2012013331
WO 2012055903
WO 2012055905
WO 2013104745
WO 2013178788

– See more at: http://worlddrugtracker.blogspot.in/2013/12/degarelix-nice-backs-ferrings-firmagon.html#sthash.x5FeHm6m.dpuf

References

Princivalle M, Broqua P, White R, et al (March 2007). Rapid suppression of plasma testosterone levels and tumor growth in the dunning rat model treated with degarelix, a new gonadotropin-releasing hormone antagonist. J. Pharmacol. Exp. Ther. 320: 1113-8.
PR Newswire. FDA approves Ferring Pharmaceuticals’ Degarelix (generic name) for the treatment of advanced prostate cancer. PR Newswire, Europe Ltd 2008 [cited 2009 Mar 2]; Available from here
Van Poppel H, Nilsson S (June 2008). Testosterone surge: rationale for gonadotropin-releasing hormone blockers? Urology 71: 1001-6.
Klotz L, Boccon-Gibod L, Shore ND, et al (December 2008). The efficacy and safety of degarelix: a 12-month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer. BJU Int. 102: 1531-8.
Schröder FH, Boccon-Gibod L, Tombal B, et al (March 2009) Degarelix versus leuprolide in patients with prostate cancer: effect in metastatic patients as assessed by serum alkaline phosphatase. European Association of Urology (EAU) Annual congress 17–21 March 2009, Stockholm, Sweden. Abstract 40.
Gittelman M, Pommerville PJ, Persson BE, et al (November 2008). A 1-year, open label, randomized phase II dose finding study of degarelix for the treatment of prostate cancer in North America. J. Urol. 180: 1986-92.
Van Poppel H, Tombal B, de la Rosette JJ, et al (October 2008). Degarelix: a novel gonadotropin-releasing hormone (GnRH) receptor blocker–results from a 1-yr, multicentre, randomised, phase 2 dosage-finding study in the treatment of prostate cancer. Eur. Urol. 54: 805-13.
Radu, A.; Pichon, C.; Camparo, P.; Antoine, M.; Allory, Y.; Couvelard, A.; Fromont, G. L.; Hai, M. T. V.; Ghinea, N. (2010). “Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels”. New England Journal of Medicine 363 (17): 1621–1630. doi:10.1056/NEJMoa1001283. PMID 20961245. edit

Degarelix Product website in the US
Steinberg, M. Clin. Ther. 2009, 31, 2312.
Jiang, G.; Stalewski, J.; Galyean, R.; Dykert, J.; Schteingart, C.; Broqua, P.; Aebi,
A.; Aubert, M. L.; Semple, G.; Robson, P.; Akinsanya, K.; Haigh, R.; Riviere, P.;
Trojnar, J.; Junien, J. L.; Rivier, J. E. J. Med. Chem. 2001, 44, 453.
Semple, G.; Jiang, G. WO 9846634 A1, 1998

Chinese Herb Beats Drug At Treating Rheumatoid Arthritis

April 17, 2014 1:15 am / Leave a comment

Lyra Nara Blog

A Chinese herb called thunder god vine works better than a widely-prescribed pharmaceutical drug at easing rheumatoid arthritis, a new study has found.

Tripterygium regelii, Aizu area, Fukushima pref, Japan

In a study published in the journal Annals of the Rheumatic Diseases, Chinese researchers recruited 207 patients with rheumatoid arthritis and gave them either the herb; the drug methotrexate; or a combination of the two.

After six months, the patients were given a doctor’s assessment and were also asked if they felt…

View original post 279 more words

Selumetinib.司美替尼 .. phase III trial in patients with KRAS mutation-positive NSCLC

April 16, 2014 7:50 am / Leave a comment

Selumetinib司美替尼

6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide

5-(4-Bromo-2-chlorophenylamino)-4-fluoro-1-methyl-1H-benzimidazole-6-carbohydroxamic acid 2-hydroxyethyl ester

6-(4-bromo-2-chloro- phenylamino)-7-fluoro-3 -methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy- ethoxy)-amide

606143-52-6

943332-08-9 (sulfate (1:1) salt) IS THE DRUG

Selumetinib, AZD6244, AZD-6244, ARRY-142886

Molecular Formula: C₁₇H₁₅BrClFN₄O₃ Molecular Weight: 457.681403

Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer. In October, AstraZeneca began a phase III trial of selumetinib in patients with KRAS mutation-positive NSCLC. AstraZeneca has also partnered with Roche Molecular Systems to develop a device to detect these mutations.

Selumetinib (AZD6244) is a drug being investigated for the treatment of various types of cancer, for example non-small cell lung cancer (NSCLC).

The gene BRAF is part of the MAPK/ERK pathway, a chain of proteins in cells that communicates input from growth factors. Activating mutations in the BRAF gene, primarily V600E (meaning that the amino acid valine in position 600 is replaced by glutamic acid), are associated with lower survival rates in patients with papillary thyroid cancer. Another type of mutation that leads to undue activation of this pathway occurs in the gene KRAS and is found in NSCLC. A possibility of reducing the activity of the MAPK/ERK pathway is to block the enzyme MAPK kinase (MEK), immediately downstream of BRAF, with the drug selumetinib. More specifically, selumetinib blocks the subtypes MEK1 and MEK2 of this enzyme.^[1]

Selumetinib is a novel, selective, non-ATP-competitive inhibitor of MEK1/2 currently in phase III clinical development at AstraZeneca for the oral treatment of non-small lung cancer with KRAS mutation. Additional phase II trials are under way at both AstraZeneca and Array BioPharma for the treatment of other oncological indications, including colorectal cancer, thyroid cancer and malignant melanoma. AstraZeneca is conducting phase I/II clinical trials for the treatment of Kaposi’s sarcoma (AIDS-related) in combination with highly active anti-retroviral therapy (HAART). Also, phase I trials are ongoing at the companies targeting several solid tumors, including skin, pancreatic, colon, lung and breast tumors. The National Cancer Institute (NCI) is also evaluating selumetinib for the treatment of thyroid cancer, ovary cancer, myeloid leukemia, glioma, multiple myeloma, metastatic uveal melanoma, sarcoma, pancreatic cancer, plexiform neurofibromas and for the treatment of recurrent or persistent endometrial cancer. Additional early clinical trials are under way at the Massachusetts General Hospital for the treatment of cancers with BRAF mutations. No recent development has been reported for phase II clinical trials for the treatment of metastatic pancreatic cancer.

In addition to thyroid cancer, BRAF-activating mutations are prevalent in melanoma (up to 59%), colorectal cancer (5–22%), serousovarian cancer (up to 30%), and several other tumor types.^[2]

KRAS mutations appear in 20 to 30% of NSCLC cases and about 40% of colorectal cancer.^[1]

. The National Cancer Institute (NCI) is also evaluating selumetinib for the treatment of thyroid cancer, ovary cancer, myeloid leukemia, glioma, multiple myeloma, metastatic uveal melanoma, sarcoma, pancreatic cancer, plexiform neurofibromas and for the treatment of recurrent or persistent endometrial cancer. Additional early clinical trials are under way at the Massachusetts General Hospital for the treatment of cancers with BRAF mutations. No recent development has been reported for phase II clinical trials for the treatment of metastatic pancreatic cancer.

A Phase II clinical trial about selumetinib in NSCLC has been completed in September 2011;^[3] one about cancers with BRAF mutations is ongoing as of June 2012.^[4]

Selumetinib appears to efficiently target cancers with overactivation of MEK and associated cell signaling pathways. According to laboratory studies, selumetinib has an effect on human tumors at nanomolar concentrations. Potential advantages of selumetinib over marketed therapies include improved efficacy linked to a novel mechanism and ease of use based on the drug candidate’s oral formulation.

In 2013, AstraZeneca acquired exclusive worldwide rights to selumetinib from Array BioPharma.

AZD6244 (Selumetinib)

6-(4-Bromo-2- chloro-ρhenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy- ethoxy)-amide, or “Compound 1”, is exemplified in WO 03/077914 and possesses the following structural formula:

Synthesis of Selumetinib_AZD6244_Non-small cell Lung Cancer_AstraZeneca 阿斯利康非小细胞肺癌药物司美替尼的化学合成

…………………………..

http://www.google.com/patents/US20030232869

Example 10

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide (29c)

Step A. 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid methyl ester 9a and 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-1-methyl-1H-benzoimidazole-5-carboxylic acid methyl ester

A solution of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-carboxylic acid methyl ester 8b (150 mg, 0.38 mmol), iodomethane (28 μL, 0.45 mmol) and potassium carbonate (78 mg, 0.56 mmol) in dimethylformamide (1.5 mL) is stirred at 75° C. for one hour. The reaction mixture is diluted with ethyl acetate, washed with saturated aqueous potassium carbonate (2×), brine, and dried (Na₂SO₄). Flash column chromatography (20:1 methylene chloride/ethyl acetate) provides 56 mg (36%) of the more mobile 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid methyl ester 9a as a white solid. ¹⁹F NMR (376 MHz, CD₃OD)-133.5 (s). MS APCI (+) m/z 412, 414 (M+, Br pattern) detected. Also isolated is 54 mg (35%) of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-1-methyl-1H-benzoimidazole-5-carboxylic acid methyl ester as a white solid. ¹⁹F NMR (376 MHz, CD₃OD)-139.9 (s). MS APCI (+) m/z 412, 414 (M+, Br pattern) detected.

Step B. 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid 10c

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid methyl ester 9a (56 mg, 0.14 mmol) is dissolved into 2:1 THF/water (3 mL) and NaOH (0.55 mL, 1.0 M aqueous solution, 0.55 mmol) is added. After stirring for two hours the reaction is reduced to one quarter initial volume via rotary evaporation and the remainder diluted to 50 mL with water. The aqueous solution is acidified to pH 2 by the addition of 1.0 M aqueous HCl and extracted with 1:1 tetrahydrofuran/ethyl acetate (3×), dried (Na₂SO₄) and concentrated under reduced pressure to provide 43 mg (79%) pure carboxylic acid as an off white solid. MS ESI (+) m/z 397, 398 (M+, Br pattern) detected.

Step C: 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-vinyloxy-ethoxy)-amide 29a

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid 10c (2.00 g, 5.0 mmol), O-(2-vinyloxy-ethyl)-hydroxylamine (0.776 g, 7.5 mmol), HOBt (0.88 g, 6.5 mmol), triethylamine (1.61 mL, 2.3 mmol) and EDCI (1.3 g, 6.5 mmol) are dissolved in dimethylformamide (52 mL) and stirred at room temperature for 48 hours. The reaction mixture is diluted with ethyl acetate, washed with water (3×), saturated potassium carbonate (2×), saturated ammonium chloride (2×), brine, dried (Na₂SO₄) and concentrated under reduced pressure to an off-white solid. Trituration of the solid with diethyl ether provides 2.18 g (90%) desired product as an off-white solid. MS ESI (+) m/z 483, 485 (M+ Br pattern) detected.

Step D: 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide 29c

Hydrochloric acid (14 mL, 1.0 M aqueous solution, 14 mmol) is added to a suspension of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-vinyloxy-ethoxy)-amide 29a (2.18 g, 4.50 mmol) in ethanol (50 mL) and the reaction mixture allowed to stir for 24 hours. The reaction mixture is concentrated to dryness by rotary evaporation and the solids partitioned between 3:1 ethyl acetate/tetrahydrofuran and saturated potassium carbonate. The aqueous phase is extracted with 3:1 ethyl acetate/tetrahydrofuran (3×), the combined organics dried (Na₂SO₄), and concentrated to provide 2.11 g (100%) 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide as an off-white solid. MS ESI (+) m/z 457, 459 (M+, Br pattern) detected. ¹H NMR (400 MHz, MeOH-d₄) δ8.26 (s, 1H), 7.78 (s, 1H), 7.57 (d, 1H), 7.24 (dd, 1H), 6.40 (dd, 1H), 3.86 (s, 3H), 3.79 (m, 2H), 3.49 (m, 2H). ¹⁹F NMR (376 MHz, MeOH-d₄)-133.68 (s).

…………

http://www.google.com/patents/WO2003077914A1?cl=en

Scheme 1

Scheme la

Scheme 2

Scheme 3

17 18

Scheme 4

Scheme 5

Example 1 and in this Example 9 by using the appropriate carboxylic acid and the appropriate hydroxylamine:

Example 10

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide (29c)Step A. 6-(4-Bromo-2-chloro-phenylamino)- 7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid methyl ester 9a and 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-l- methyl-lH-benzoimidazole-5-carboxylic acid methyl ester

A solution of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3H-benzoimidazole-5-

carboxylic acid methyl ester 8b (150 mg, 0.38 mmol), iodomethane (28 μL, 0.45 mmol)

and potassium carbonate (78 mg, 0.56 mmol) in dimethylformamide (1.5 mL) is stirred at

75 °C for one hour. The reaction mixture is diluted with ethyl acetate, washed with saturated aqueous potassium carbonate (2x), brine, and dried (Na SO ). Flash column chromatography (20:1 methylene chloride/ethyl acetate) provides 56 mg (36%) of the

more mobile 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3 -methyl-3H-benzoimidazole-

5-carboxylic acid methyl ester 9a as a white solid. ¹⁹F NMR (376 MHz, CD₃OD) -133.5

(s). MS APCI (+) m/z 412, 414 (M+, Br pattern) detected. Also isolated is 54 mg (35%)

of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-l-methyl-lH-benzoimidazole-5- carboxylic acid methyl ester as a white solid. ¹⁹F NMR (376 MHz, CD₃OD) -139.9 (s).

MS APCI (+) m/z 412, 414 (M+, Br pattern) detected.

Step B. 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid 10c

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid methyl ester 9a (56 mg, 0.14 mmol) is dissolved into 2:1 THF/water (3 mL ) and NaOH (0.55 mL, 1.0 M aqueous solution, 0.55 mmol) is added. After stirring for two hours the reaction is reduced to one quarter initial volume via rotary evaporation and the remainder diluted to 50 mL with water. The aqueous solution is acidified to pH 2 by the addition of 1.0 M aqueous HCl and extracted with 1 : 1 tetrahydrofuran/ethyl acetate (3x), dried (Na₂SO₄) and concentrated under reduced pressure to provide 43 mg (79%) pure carboxylic acid as an off white solid. MS ESI (+) m/z 397, 398 (M+, Br pattern) detected.

Step C: 6-(4-Bromo-2-chloro-phenylamino)~ 7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid (2-vinyloxy-ethoxy)-amide 29a

6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid 10c (2.00 g, 5.0 mmol), O-(2-vinyloxy-ethyl)-hydroxylamine (0.776 g, 7.5 mmol), HOBt (0.88 g, 6.5 mmol), triethylamine (1.61 mL, 2.3 mmol) and EDCI (1.3 g, 6.5 mmol) are dissolved in dimethylformamide (52 mL) and stirred at room temperature for 48 hours. The reaction mixture is diluted with ethyl acetate, washed with water (3x), saturated potassium carbonate (2x), saturated ammonium chloride (2x), brine, dried (Na₂SO₄) and concentrated under reduced pressure to an off-white solid. Trituration of the solid with diethyl ether provides 2.18 g (90%) desired product as an off- white solid. MS ESI (+) m/z 483, 485 (M+ Br pattern) detected.

Step D: 6-(4-Bromo-2-chloro-phenylamino)- 7-fluoro-3-methyl-3H-benzoimidazole-5- carboxylic acid (2-hydroxy-ethoxy) -amide 29c

Hydrochloric acid (14 mL, 1.0 M aqueous solution, 14 mmol) is added to a suspension of 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3 -methyl-3H-benzoimidazole -5-carboxylic acid (2-vinyloxy-ethoxy)-amide 29a (2.18 g, 4.50 mmol) in ethanol (50 mL) and the reaction mixture allowed to stir for 24 hours. The reaction mixture is concentrated to dryness by rotary evaporation and the solids partitioned between 3:1 ethyl acetate/tefrahydrofuran and saturated potassium carbonate. The aqueous phase is extracted with 3:1 ethyl acetate/tefrahydrofuran (3x), the combined organics dried (Na SO₄), and concentrated to provide 2.11 g (100%) 6-(4-bromo-2-chloro- phenylamino)-7-fluoro-3 -methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy- ethoxy)-amide as an off-white solid. MS ESI (+) m/z 457, 459 (M+, Br pattern) detected. 1H NMR (400 MHz, MeOH-c^) δ 8.26 (s, IH), 7.78 (s, IH), 7.57 (d, IH), 7.24 (dd, IH), 6.40 (dd, IH), 3.86 (s, 3H), 3.79 (m, 2H), 3.49 (m, 2H). ¹⁹F NMR (376 MHz, MeOH-d₄) -133.68 (s).

………………

http://www.google.com/patents/EP1968948A2?cl=en

Example 1

Preparation of the Hydrogen sulfate salt of Compound 1

[0076] To a stirred suspension of 6-(4-bromo-2-chloro-phenylamino)-7-fiuoro-3- methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide (100 g, 0.206 mol) (obtainable as described in Example 10 of WO 03/077914, which is incorporated herein by reference and as described below) in 2-butanone (680 mL) and water (115 mL) at 0-5 ⁰C was added sulfuric acid (12.3 mL, 0.226 mol) followed by water (5 mL) maintaining a temperature of 10 °C or lower. The stirred mixture was heated to 65 ⁰C and held for 30 minutes before filtering to remove any extraneous matter. The filter was washed with a mixture of 2-butanone (85 mL) and water (15 mL). The combined filtrates were heated to 72 ⁰C before adding 2-butanone (500 mL) maintaining a temperature of between 60-72 ⁰C. The resulting mixture was distilled at atmospheric pressure (approximate distillation temperature 73-74°C) until 500 mL of distillate had been collected.

[0077] A second aliquot of 2-butanone (500 mL) was added, maintaining the temperature of the mixture above 70 ⁰C. The resulting mixture was distilled again until 250 mL of distillate had collected. The mixture was cooled to 0-5 ⁰C over approximately 1 hour. The resulting slurry was filtered, washed with 2-butanone (240 mL) and dried under reduced pressure at 50 ⁰C, until a constant weight was achieved, to give 6-(4-bromo-2-chloro- phenylamino)-7-fiuoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)- amide hydrogen sulfate (103.5 g, 0.186 mol, 90% yield) as an off white crystalline solid.¹H NMR (400 MHz, D₆ DMSO) δ 3.58 (2H, t, CH₂OH), 3.89 (2H, t, CH₂ON), 3.99 (3H, s, CH₃), 6.47 (IH, dd, ArH), 7.29 (IH, dd, ArH), 7.63 (IH, d, ArH), 7.91 (IH, s, ArH), 7.96 (3H, br, ROH, NH, SOH), 8.10 (IH, br, ArNH), 8.94 (IH, s, NCHN), 11.79 (IH, s, ONH). ¹³C NMR (100 MHz, D₆ DMSO) δ 32.1 (CH₃), 58.5 (CH₂OH), 77.3 (CH₂ON), 108.2 (CH), 109.6 (CBr), 115.8 (CH), 120.6 (CCl), 122.0 (C), 125.0 (CC=O), 129.4 (C), 130.5 (CH), 131.1 (CH), 132.3 (C), 140.6 (C), 145.8 (CF), 146.5 (CH), 164.2 (C=O). [0078] The results of the infrared analysis are shown in Figure 2. Spectral assignments axe summarized in Table 1.

Table 1

Wavenumber (cm^“ ) Assignment 3,255 Includes the O-H stretching vibration of the primary alcohol group and the N-H stretching vibrations of the secondary aromatic amine and secondary amide groups.

3,200 – 2,700 Includes =C-H stretching vibrations of the aromatic ring and benzimidazole group and the aliphatic C-H stretching vibrations.

2,700 – 2,300 Includes the multiple NH⁺ stretching vibrations of the benzimidazole 1 : 1 sulfate salt group.

1,673 C=O stretching vibrations of the secondary amide group where

1,653 the carbonyl group is subject to different environmental effects such as hydrogen bonding.

1,640 – 1,370 Includes the C=C aromatic ring stretching vibrations, the C=C and C=N stretching vibrations of the benzimidazole group, the

O-H deformation vibration of the primary alcohol group and the aliphatic C-H deformation vibrations.

1,570 The CNH combination band of the secondary amide group.

1,506 Includes the CNH bending vibration of the secondary aromatic amine group.

1 ,213 The aryl C-F stretching vibration.

1,189 The asymmetric SO₃ ^“ stretching vibration of the benzimidazole

1 : 1 sulfate salt group. 1,100 – 1,000 Includes the C-O stretching vibration of the primary alcohol group and the aryl C-Br stretching vibration. 1,011 The symmetric SO₃ ^“ stretching vibration of the benzimidazole

1 :1 sulfate salt group. 920 – 600 Includes the C-H wag vibrations and C=C ring bending vibrations of the 1,2,4-trisubtituted aromatic ring and the benzimidazole group. 888 Includes the S-O(H) stretching vibration of the benzimidazole

1 : 1 sulfate salt group. Example IA

Preparation of the Hydrogen sulphate salt of Compound 1

[0079] Sulfuric acid (1.52 ml, 27.86 mmol) was added to a stirred suspension of 6-(4- bromo-2-chlorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2- hydroxyethoxy)-amide (1O g, 0.0214 mol) (obtainable as described in Example 10 of WO 03/077914, which is incorporated herein by reference and as described below) in tetrahydrofuran (THF) (62 ml) and water (8 ml) whilst maintaining a temperature of 10 ⁰C or lower. The stirred mixture was heated to 65 ⁰C and held for 30 minutes before filtering to remove any extraneous matter. THF (150 ml) was then added to the mixture maintaining the temperature above 60 ⁰C. The mixture was then cooled to 0-5 ⁰C over approximately 2 hour. The resulting slurry was filtered, washed with THF (30 ml) and dried under reduced pressure at 50 ⁰C until a constant weight was achieved, to give 6-(4-bromo-2-chlorophenylamino)-7- fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide hydrogen sulfate (9.81g, 0.17 mol, 82% yield) as an off white crystalline solid. The material was the same as that produced in Example 1 above.

References

Troiani, T.; Vecchione, L.; Martinelli, E.; Capasso, A.; Costantino, S.; Ciuffreda, L. P.; Morgillo, F.; Vitagliano, D.; d’Aiuto, E.; De Palma, R.; Tejpar, S.; Van Cutsem, E.; De Lorenzi, M.; Caraglia, M.; Berrino, L.; Ciardiello, F. (2012). “Intrinsic resistance to selumetinib, a selective inhibitor of MEK1/2, by cAMP-dependent protein kinase a activation in human lung and colorectal cancer cells”. British Journal of Cancer 106 (10): 1648–1659.doi:10.1038/bjc.2012.129. PMC 3349172. PMID 22569000. edit
Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J. (2002). “Mutations of the BRAF gene in human cancer”. Nature 417 (6892): 949–954. doi:10.1038/nature00766. PMID 12068308. edit
Jump up^ ClinicalTrials.gov NCT00890825 Comparison of AZD6244 in Combination With Docetaxel Versus Docetaxel Alone in KRAS Mutation Positive Non Small Cell Lung Cancer (NSCLC) Patients
Jump up^ ClinicalTrials.gov NCT00888134 AZD6244 in Cancers With BRAF Mutations
Journal of the American Chemical Society, 2013 , vol. 135, 35 p. 12994 – 12997
23787099

8-1-2013

Identification of potent Yes1 kinase inhibitors using a library screening approach.

Bioorganic & medicinal chemistry letters

WEDGE S R ET AL: “AZD2171: A HIGHLY POTENT, ORALLY BIOAVAILABLE, VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-2 TYROSINE KINASE INHIBITOR FOR THE TREATMENT OF CANCER“, CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 65, no. 10, 15 May 2005 (2005-05-15), pages 4389-4400, XP008066714, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-04-4409
52	*	WEDGE STEPHEN R ET AL: “ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration“, CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 62, no. 16, 15 August 2002 (2002-08-15), pages 4645-4655, XP002425560, ISSN: 0008-5472
53		WEDGE, S.R. ET AL.: ‘ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration‘ CANCER RES vol. 62, 2002, pages 4645 – 4655

Ho, Alan L.; Grewal, Ravinder K.; Leboeuf, Rebecca; Sherman, Eric J.; Pfister, David G.; Deandreis, Desiree; Pentlow, Keith S.; Zanzonico, Pat B. et al. (2013). “Selumetinib-Enhanced Radioiodine Uptake in Advanced Thyroid Cancer”. New England Journal of Medicine 368 (7): 623–32. doi:10.1056/NEJMoa1209288. PMC 3615415.PMID 23406027.

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Afatinib

Afatinib

Derivatives

dimaleate

Substance Classes

Synthesis Path

Substances Referenced in Synthesis Path

Trade Names

Formulations

References

crystalline forms of Afatinib di-maleate:

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PATENT

PATENT

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Reduction of male fertility

Scientific research into medical effects

Contraception

Kidney function

Immunosuppression

Rheumatoid arthritis

Pancreatic cancer

References

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Clinical effectiveness

Side effects

FSH receptors in other solid tumors

References

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