Field of invention

The present invention relates to a process for the preparation of Ixabepilone and also to novel intermediates useful in the process according to the present invention.

Ixabepilone is an anti cancer agent acting as a microtubule inhibitor, and which in particular are efficient in the treatment of cancer not reacting to other anti cancer agents, such as e.g. paclitaxel. Ixabepilone is marketed under the trade name Ixempra® and are approved for the treatment of aggressive metastatic or locally advanced breast cancer which not responding to the current prevailing chemotherapies.

Ixabepilone known under the CAS no. 219989-84-1 has the following structure:

Ixabepilone

Ixabepilone may be prepared from a starting material named Epothilone B having the structural formula:

Epothilone B Ixabepilone as a compound and different aspects regarding the compound are described in the USRE4191 1 , US 6,365,749 and USRE39356. Summary of invention

The present provides a process for the preparation of Ixabepilone having the formula 5 :

comprising the step of:

reducing a compound of formula

4

wherein X is N, Ri and R ₂ are independently selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group to form Ixabepilone.

According to one aspect of the present invention, a process is provided for the preparation of Ixabepilone comprising the steps of reducing a compound of formula 4, wherein X is N; and Ri is H and R ₂ is Ci_s alkyl or Ci_s acyl. According to another aspect, X is N; and Ri is H and R ₂ is Ci_ ₃ alkyl or Ci_ ₃ acyl. According to yet another aspect, X is N; and Ri is H and R ₂ is acetyl.

According to another aspect of the present invention, a process is provided, wherein the starting material of formula 4 of step a) is prepared by: i) reacting Epothilone B with a nucleophile of formula NH ₂ -XRiR ₂ , wherein X, Ri and R ₂ is as defined above, in order to form a compound of formula 2

2

ii) ringclosure of the compound of formula II in order to form the compound of formula 4.

According to a further aspect of the above invention, the ring closure step ii) is performed by reacting the compound of formula 2 with an activation system or activation agent. According to yet a further aspect of the present invention, the said activation system or activation agent is selected from the group consisting of EDC/HOBt,

EDC/pentafluorophenol, pentafluorophenyl diphenylphosphinate, pentafluorophenyl diphenylphosphinate and diphenyl phosphoryl azide. According to another aspect of the present invention, the conversion of Epothilone B to the compound of formula 4 is performed in a one-pot step.

According to yet another aspect of the present invention, the reacting of Epothilone B with a nucleophile is performed in the presence of a palladium catalyst.

The present invention furthermore provides a novel compound of formula 4

wherein X is N, Ri and R ₂ are independently selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group. According to one embodiment of the present invention, a compound of formula 4 is provided wherein X is N; and Ri is H and R ₂ is Ci_s alkyl or Ci_s acyl. According to another embodiment, a compound of formula 4is provided, wherein X is N; and Ri is H and R ₂ is Ci_ ₃ alkyl or Ci_ ₃ acyl. According to yet another aspect of the present invention, a compound of formula 4 is provided, wherein X is N; and Ri is H and R ₂ is acetyl.

The present invention furthermore provides a process for the preparation of compound 4, wherein said process comprises the steps of activating the carboxylic acid of a compound of formula 2

2

wherein X is N, Ri and R ₂ are independently selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group with an activation system or activation agent, such as e.g. EDC/HOBt, EDC/ pentafluorophenyl, pentafluorophenyl diphenylphosphinate and pentafluorophenyl diphenylphosphinate, diphenyl phosphoryl azide followed by ringclosure.

Finally, a process is provided for the preparation of Ixabepilone of the formula 5:

2 wherein X is N, Ri and R ₂ are independently selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group by a one pot ring closure and reduction step.

According to aspect, the ring closure of the above process is performed by activating the carboxylic acid group of the compound of formula 2 using diphenylphosphoryl azide as an activation agent.

Detailed description of the invention.

The present method is further illustrated by the following detailed description of the present invention.

According to the present invention, Ixabepilone may be prepared by reduction of a compound of formula 4

wherein is X is N, and Ri and R ₂ is independently selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group. The compound of formula 4, may be prepared by the ring opening of Epothilone B to form a compound of formula 2:

2

using a nitrogen nucleophile, followed by the conversion of the compound formula 2 to the compound of formula 4. According to one embodiment of the present invention, Ixabepilone is prepared according to the steps of reactions illustrated in Scheme 1.

Activation

of acid

Reduction

Ixabepilone

Scheme 1

The compound H ₂ N-XRiR ₂ includes nitrogen nucleophiles wherein X denotes the heteroatom N. The heteroatom X carries two entities Ri and R ₂ according to its preferred valency. Ri or R ₂ may independently be selected from the group consisting of H, a straight or branched alkyl group or a straight or branched acyl group, wherein said alkyl or acyl may be substituted or un- substituted. Other examples of nitrogen nucleophiles that may be used according to the present invention are the free forms of hydrazine, which optionally may be substituted, e.g. N-alkylhydrazines, and N-acylhydrazines, e.g. acethydrazide (CH3CONH-NH ₂ ).

According to one embodiment, Ri or R ₂ are selected from the group consisting of Ci_8 alkyl or Ci_s acyl. According to anther embodiment, Ri and R ₂ is independently selected from the group consisting of Ci_ ₃ alkyl or Ci_ ₃ acyl. According to a preferred embodiment, Ri is H and R ₂ is acetyl.

The starting material Epothilone B (1) is treated with the nitrogen nucleophile H ₂ N- XR ₁ R ₂ in the presence of a palladium catalyst. The substrate undergoes ring- opening and formation of the structure 2 as an intermediate.

Activation of the carboxylic acid group with a suitable activation system affords the activated compound 3. The term "Act" in Scheme 1 refers to any group that activates the carboxylic acid group for attack from a nucleophile, and is herein named "activation agents" or "activation systems". The skilled person will acknowledge that a number of well known activation agents or activation systems may be used in order to activate carboxylic acid group of the intermediate 2 in order to effect a ring closure. For example, one suitable activation system is the EDC/HOBt system where EDC stands for N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride and HOBt stands for 1 -hydro xybenzotriazole hydrate. Another suitable activation system is EDC/pentafluorophenol. Yet another example of a suitable activation agent is pentafluorophenyl diphenylphosphinate. Yet another example of a suitable activation agent is diphenyl phosphoryl azide.

Ring closing provides the intermediate structure 4, while reductive cleavage of the N-X bond releases the target Ixabepilone (5). The reductive cleavage may be obtained by various reduction agents. For example, the compound 4 may be treated with powdered Zn in acetic acid, resulting in cleavage of the N-N bond and formation of Ixabepilone (5). Examples

Example 1 - preparation of compound 2 according to Scheme 1

X = NHCOCH,

Nucleophile: NH ₂ NHAc;

250 mg (1.0 eq) of Epothilone B and 10 ml degassed THF was added to a 50 ml round bottom flask under nitrogen atmosphere and stirred for 10 min. The mixture was cooled to 0°C and added 0.1 eq of allyl palladium chloride dimmer and 0.1 eq of Xantphos. Then, 1.5 eq acetic hydrazide dissolved in degassed THF was added during 15 min. The reaction mixture was allowed to reach room temperature and stirred for 3 h. Complete conversion was observed by TLC. The reaction mixture was diluted with ethyl acetate and washed with water followed by brine solution. The solvents were subsequently removed under vacuum to furnish the crude Epothilone B - acetic hydrazide adduct 2.

HPLC - MS [Column: Xbridge C-18 (50x3.0mm, 3,5um), Mobil phase: A; 5.0mM NH ₄ OAc B; AcN (Gradient), Time/B%: 0.01/10 0.5/10 4/90 8/90, Flow: 0.8ml/Min, MS: ESI; positive mode] : Ret. time: 2.64 (82.5%), m/z: 582 (M+l , 100%).

Example 2: Activation of compound 2 and ring closure (I) in order to form compound 4 in accordance with scheme 1

In this example, a one-pot procedure starting from Epothilone B was used to form the compound 4 according to scheme 1 using EDC/HOBt as activation system:

To a 25 ml round bottom flask charged with 25 mg (1.0 eq) Epothilone B, 0.1 eq allyl palladium chloride dimmer, 0.1 eq Xantphos and 1.5 eq acetic hydrazide was added 1 ml degassed THF under nitrogen. The reaction mixture was stirred for 3 h at room temperature. Complete conversion was observed by TLC. Then 2.5 eq EDC and subsequently 1.1 eq HOBt was added and the reaction mixture stirred for 12 h at room temperature. The reaction mixture was diluted with ethyl acetate and washed with water followed by brine solution.

HPLC - MS [Column: Xbridge C-18 (50x3.0mm, 3,5um), Mobil phase: A; 5.0mM NH ₄ OAc B; AcN (Gradient), Time/B%: 0.01/10 0.5/10 4/90 8/90, Flow: 0.8ml/Min, MS: ESI; positive mode] : Ret. time: 2.51 (14%), m/z: 564 (M+l , 100%).

Example 3: Activation of compound 2 and ring closure (II) in order to form compound 4 in accordance with scheme 1.

In this example, a one-pot procedure starting from Epothilone B was used to form the compound 4 according to scheme 1 , using EDC and pentafluorophenol (C ₆ F ₅ OH) as activation system:

To a 25 ml round bottom flask charged with 25 mg (1.0 eq) Epothilone B, 0.1 eq allyl palladium chloride dimmer, 0.05 eq Xantphos and 1.5 eq acetic hydrazide was added 1 ml degassed THF under nitrogen. The reaction mixture was stirred for 3 h at room temperature. Complete conversion was observed by TLC. The reaction mixture was diluted with ethyl acetate and washed with water followed by brine solution. The solvents were subsequently removed under vacuum and the crude mass dissolved in 2 ml THF. To this was added 2.5 eq EDC followed by 1.5 eq pentafluorophenol. Then the reaction mixture was stirred under nitrogen at room temperature for 30 min. 2 eq DIPEA was added and the mixture stirred for an additional 30 min. before the addition of 0, 1 eq DMAP. The mixture was then stirred for 2 days, diluted with ethyl acetate and washed with water and then with brine solution.

HPLC - MS [Column: Xbridge C-18 (50x3.0mm, 3.5um), Mobil phase: A; 5.0mM NH ₄ OAc B; AcN (Gradient), Time/B%: 0.01/10 0.5/10 4/90 8/90, Flow: 0.8ml/Min, MS: ESI; positive mode] : Ret. time: 2.59 (72%), m/z: 564 (M+l , 100%).

Example 4 Activation of compound 2 and ring closure (III) in order to form compound 4 in accordance with scheme 1.

In this example, pentafluorophenyl diphenylphosphinate was used as activation agent.

Procedure starting from crude Epothilone B-acetic hydrazide adduct;

200 mg (1.0 eq) crude acetic hydrazide adduct was dissolved in 10 ml degassed DMF under nitrogen gas and cooled to 0° C. Then 3 eq DIPEA was added and subsequently 1 ,2 eq pentafluorophenyl diphenylphosphinate. The reaction mixture was left to reach room temperature and stirred for 12 h. Then the mixture was diluted with ethyl acetate and washed with water followed by brine solution.

HPLC - MS [Column: Xbridge C-18 (50x3.0mm, 3,5um), Mobil phase: A; 5.0mM NH ₄ OAc B; AcN (Gradient), Time/B%: 0.01/10 0.5/10 4/90 8/90, Flow: 0.8ml/Min, MS: ESI; positive mode] : Ret. time: 2.65 (59%), m/z: 564 (M+l , 100%). Example 5 One pot reaction forming Ixabepilone from compound 2 according to scheme 1.

X = N HCOCH3

In this example, Ixabepilone was formed in a one pot reaction by activation of the compound 2 (formula II) by activation using diphenyl phosphoryl azide as activation agent, resulting in the formation of compound 4 of scheme 1 , followed by spontaneous formation of Ixabepilone:

100 mg (1.0 eq) crude Epothilone B - acetic hydrazide adduct was dissolved in 80 ml dry and degassed DMF and cooled to 0° C under nitrogen gas. Then 7.7 eq sodium bicarbonate and subsequently 4.0 eq diphenyl phosphoryl azide was added and stirring at 0° C maintained for 24 h. The reaction mixture was then diluted with 80 ml of cold phosphate buffer solution and extracted with ethyl acetate. Subsequent washing of the organic layer with cold 10% aqueous LiCl and drying with Na ₂ S0 ₄ followed by concentration in vacuo then furnished the crude product. HPLC - MS [Column: Xbridge C-18 (50x3.0mm, 3,5um), Mobil phase: A; 5.0mM NH ₄ OAc B; AcN (Gradient), Time/B%: 0.01/10 0.5/10 4/90 8/90, Flow: 0.8ml/Min, MS: ESI; positive mode] : Ret. time: 4.55 (43%), m/z: 507 (M+l , 7%).