PROCESS FOR THE PREPARATION OF PURINE DERIVATIVES EXHIBITING CDK INHIBITORY ACTIVITY

Abstract

The present invention relates to a process for preparing a compound of formula [I], or a pharmaceutically acceptable salt thereof, said process comprising the steps of: (i) forming a reaction mixture comprising a compound of formula [II] and a compound of formula [III]; (ii) heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [I]; (iii) isolating said compound of formula [I] from the mixture and optionally recovering unreacted compound of formula [III]; and (iv) optionally converting said compound of formula [I] into salt form; wherein: R.sup.1 and R.sup.2 are each independently H, alkyl or haloalkyl; R.sup.3 and R.sup.4 are each independently H, alkyl, haloalkyl or aryl; R.sup.5 is alkyl, alkenyl, cycloalkyl or cycloalkyl-alkyl, each of which may be optionally substituted with one or more OH groups; R.sup.6 is selected from cyclopropylamino, cyclopropylmethylamino, cyclobutylamino, cyclobutylmethylamino and where one of X, Y and Z is N and the remainder are CR.sup.9; R.sup.7, R.sup.8 and each R.sup.9 are independently H, alkyl or haloalkyl, wherein at least one of R.sup.7, R.sup.8 and R.sup.9 is other than H. Further aspects of the invention relate to a process for preparing intermediates of formula [II], and other intermediates useful in the synthesis of compounds of formula [I].

##STR00001##

Claims

1. A process for preparing a compound of formula [I], or a pharmaceutically acceptable salt thereof, ##STR00033## wherein: R.sup.1 and R.sup.2 are each independently H, alkyl or haloalkyl; R.sup.3 and R.sup.4 are each independently H, alkyl, haloalkyl or aryl; R.sup.5 is alkyl, alkenyl, cycloalkyl or cycloalkyl-alkyl, each of which may be optionally substituted with one or more OH groups; R.sup.6 is selected from cyclopropylamino, cyclopropylmethylamino, cyclobutylamino, cyclobutylmethylamino and ##STR00034## where one of X, Y and Z is N and the remainder are CR.sup.9; R.sup.7, R.sup.8 and each R.sup.9 are independently H, alkyl or haloalkyl, wherein at least one of R.sup.7, R.sup.8 and R.sup.9 is other than H; said process comprising the steps of: ##STR00035## (i) forming a reaction mixture comprising a compound of formula [II], and a compound of formula [III]; (ii) heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [I]; (iii) isolating said compound of formula [I] from the mixture and optionally recovering unreacted compound of formula [III]; and (iv) optionally converting said compound of formula [I] into salt form.

2. A process according to claim 1 wherein the reaction takes place in the absence of a solvent.

3. A process according to any preceding claim wherein the reaction mixture in step (i) is heated to a temperature of from about 135° C. to about 175° C., more preferably from about 150° C. to about 175° C.

4. A process according to any preceding claim wherein the reaction mixture in step (i) is heated for a period of at least 24 hours.

5. A process according to any preceding claim wherein the reaction mixture in step (ii) comprises from about 4 to about 7 mole equivalents of compound [III] relative to compound [II], more preferably about 5 mole equivalents of compound [III] relative to compound [II].

6. A process according to any preceding claim wherein step (iii) comprises extracting the reaction mixture from step (ii) into aqueous HCl and an organic solvent, separating the organic phase and concentrating the filtrate.

7. A process according to any preceding claim which comprises preparing a compound of formula [II] by the steps of: ##STR00036## (i) treating a compound of formula [VI] with R.sup.6—NH.sub.2, or a salt thereof, to form a compound of formula [VII]; and (ii) treating said compound of formula [VII] with R.sup.5Br to form a compound of formula [II]; where R.sup.5 and R.sup.6 are as defined in claim 1.

8. A process according to any one of claims 1 to 6 which comprises preparing a compound of formula [II] by the steps of: ##STR00037## treating a compound of formula [VI] with R.sup.5Br to form a compound of formula [VIII]; and (ii) treating said compound of formula [VIII] with R.sup.6—NH.sub.2, or a salt thereof, to form a compound of formula [II]; where R.sup.5 and R.sup.6 are as defined in claim 1.

9. A process according to claim 7 or claim 8 wherein step (ii) of claim 7 or step (i) of claim 8 is carried out in the presence of DMSO and K.sub.2CO.sub.3.

10. A process according to claim 7 or claim 8 wherein step (i) of claim 7 or step (ii) of claim 8 is carried out in the presence of nBuOH and a base, preferably, DIPEA.

11. A process according to any preceding claim wherein step (iii) of claim 1 further comprises the step of crystallizing compound [I], preferably from a mixture of n-butyl acetate and heptane.

12. A process according to any one of claims 1 to 10 which comprises converting said compound of formula [I] into salt form.

13. A process according to any preceding claim which comprises converting said compound of formula [I] into the L-tartrate salt.

14. A process according to claim 13 wherein the L-tartrate salt is in crystalline form, preferably form E.

15. A process according to claim 14 which comprises refluxing said compound of formula [I] in ethanol and adding dropwise thereto a solution of L-tartaric acid in a mixture of water and ethanol.

16. A process according to claim 15 wherein the ratio of ethanol:water in the final mixture after addition of the L-tartaric acid solution is at least about 15:1, more preferably about 37.5:1.

17. A process according to claim 15 or claim 16 which comprises maintaining the temperature at 75 to 78° C. during the addition of the solution of L-tartaric acid.

18. A process according to any one of claims 1 to 17 which further comprises the step of preparing compound [III], where R.sub.2 is H, by: ##STR00038## (a) treating a compound [V] with (S)-2-Me-CBS-oxazoborolidine and borane-N,N-diethylaniline complex in a solvent comprising THF to form a compound [IV]; and (b) removing the protecting group PG from said compound [IV] to give compound [III], wherein PG is a protecting group, preferably Boc, R.sup.1 is alkyl or haloalkyl, and R.sup.3 is alkyl, haloalkyl or aryl.

19. A process according to claim 18 wherein step (b) comprises treating said compound [IV] with gaseous HCl in methanol, concentrating in vacuo, dissolving in ethyl acetate and then sparging with NH.sub.3.

20. A process according to any preceding claim for preparing a compound of formula [1], or a pharmaceutically acceptable salt thereof: ##STR00039## said process comprising the steps of: ##STR00040## (i) forming a reaction mixture comprising a compound of formula [2], and a compound of formula [3]; (ii) heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [1]; (iii) isolating said compound of formula [1] from the mixture and optionally recovering unreacted compound of formula [3]; and (iv) optionally converting said compound of formula [1] into salt form.

21. A process according to claim 20 which further comprises the step of preparing a compound of formula [2] by: ##STR00041## (i) treating a compound of formula [6] with a compound of formula [9], or a salt thereof, to form a compound of formula [7]; and (ii) treating said compound of formula [7] with isopropyl bromide to form a compound of formula [2].

22. A process according to claim 20 which further comprises the step of preparing a compound of formula [2] by: ##STR00042## (i) treating a compound of formula [6] with isopropyl bromide to form a compound of formula [8]; and (ii) treating said compound of formula [8] with a compound of formula [9], or a salt thereof, to form a compound of formula [2].

23. A process according to claim 21 or claim 22, wherein step (ii) of claim 21 or step (i) of claim 22 is carried out in DMSO in the presence of K.sub.2CO.sub.3.

24. A process according to claim 21 or claim 22, wherein step (i) of claim 21 or step (ii) of claim 22 is carried out in .sup.nBuOH in the presence of a base, preferably, DIPEA.

25. A process according to any one of claims 20 to 22 wherein compound [3] has a diastereomeric excess of at least 85%, more preferably, at least 90%, even more preferably, at least 95%.

26. A process according to any one of claims 20 to 25 which further comprises the step of preparing a compound of formula [3] by: ##STR00043## (a) treating a compound of formula [5] with (S)-2-Me-CBS-oxazoborolidine and borane-N,N-diethylaniline complex in a solvent comprising THF to form a compound of formula [4]; and (b) removing the protecting group PG from said compound of formula [4] to give a compound of formula [3], where PG is a protecting group, preferably Boc.

27. A process according to any one of claims 20 to 26 which comprises refluxing the product isolated in step (iii) of claim 20 in ethanol and adding dropwise thereto a solution of L-tartaric acid in a mixture of water and ethanol.

28. A process according to claim 27 which further comprises the step of polish filtering the mixture, warming the filtrate to a temperature of about 60 to about 65° C. and seeding with crystalline [1]-L-tartrate form E.

29. A process according to claim 28 which comprises stirring the seeded filtrate at a temperature of about 60 to about 65° C. for at least 1 hour.

30. A process according to claim 29 which further comprises the step of cooling the mixture to a temperature of about 15 to about 20° C. and stirring at that temperature for at least 1 hour to induce crystallisation of compound [1]-L-tartrate.

31. A process according to claim 30 wherein the cooling rate is about 5 to about 10° C./hour.

32. A process according to any one of claim 30 or 31 wherein the compound [1]-L-tartrate is filtered, washed with ethanol and dried in vacuo.

33. A process for preparing a compound of formula [I], or a pharmaceutically acceptable salt thereof, ##STR00044## wherein R.sup.1 and R.sup.2 are each independently H, alkyl or haloalkyl; R.sup.3 and R.sup.4 are each independently H, alkyl, haloalkyl or aryl; R.sup.5 is alkyl, alkenyl, cycloalkyl or cycloalkyl-alkyl, each of which may be optionally substituted with one or more OH groups; R.sup.6 is selected from cyclopropylamino, cyclopropylmethylamino, cyclobutylamino, cyclobutylmethylamino and ##STR00045## where one of X, Y and Z is N and the remainder are CR.sup.9; R.sup.7, R.sup.8 and each R.sup.9 are independently H, alkyl or haloalkyl, wherein at least one of R.sup.7, R.sup.8 and R.sup.9 is other than H; said process comprising the steps of: ##STR00046## (a) treating a compound of formula [VI] with R.sup.6—NH.sub.2 or a salt thereof to form a compound of formula [VII]; (b) treating said compound of formula [VII] with R.sup.5Br to form a compound of formula [II]; (c) forming a reaction mixture comprising a compound of formula [II], and a compound of formula [III] and heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [I]; (d) isolating said compound of formula [I] from the mixture and optionally recovering unreacted compound of formula [III]; and (e) optionally converting said compound of formula [I] into salt form.

34. A process of preparing a compound of formula [1], or a pharmaceutically acceptable salt thereof, said process comprising the steps of: ##STR00047## (a) treating a compound of formula [6] with a compound of formula [9], or a salt thereof, to form a compound of formula [7]; (b) treating said compound of formula [7] with isopropyl bromide to form a compound of formula [2]; (c) forming a reaction mixture comprising a compound of formula [2], and compound of formula [3] and heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [1]; (d) isolating said compound of formula [1] from the mixture and optionally recovering unreacted compound of formula [3]; and (e) optionally converting said compound of formula [1] into salt form.

35. A process for preparing a compound of formula [I], or a pharmaceutically acceptable salt thereof, ##STR00048## wherein: R.sup.1 and R.sup.2 are each independently H, alkyl or haloalkyl; R.sup.3 and R.sup.4 are each independently H, alkyl, haloalkyl or aryl; R.sup.5 is alkyl, alkenyl, cycloalkyl or cycloalkyl-alkyl, each of which may be optionally substituted with one or more OH groups; R.sup.6 is selected from cyclopropylamino, cyclopropylmethylamino, cyclobutylamino, cyclobutylmethylamino and ##STR00049## where one of X, Y and Z is N and the remainder are CR.sup.9; R.sup.7, R.sup.8 and each R.sup.9 are independently H, alkyl or haloalkyl, wherein at least one of R.sup.7, R.sup.8 and R.sup.9 is other than H; said process comprising the steps of: ##STR00050## (i) forming a reaction mixture comprising a compound of formula [II], and a compound of formula [III]; (ii) heating said reaction mixture to a temperature of at least about 130° C. to form a compound of formula [I]; (iii) optionally isolating said compound of formula [I] from the mixture and optionally recovering unreacted compound of formula [III]; and (iv) optionally converting said compound of formula [I] into salt form.

Description

[0226] The present invention is further described with reference to the following figures, wherein:

[0227] FIG. 1 shows the reaction scheme for preparing compound [1] as disclosed in WO 2008/122767.

[0228] FIG. 2 shows the reaction scheme for preparing compound [1]-L-tartrate as disclosed in WO 2011/089401.

[0229] FIG. 3 shows the reaction scheme for preparing compound [1]-L-tartrate as disclosed in WO 2018/138500.

[0230] The present invention is further described with reference to the following non-limiting Examples.

EXAMPLES

Abbreviations

[0231] THF tetrahydrofuran
EtOAc ethyl acetate
PMA phosphomolybdic acid
MeOH methanol
DCM dichloromethane
TBME (MTBE) tertiary butyl methyl ether
DCM dichloromethane

DIEA N,N-diisopropylethylamine

[0232] DMSO dimethyl sulfoxide
.sup.1H NMR: .sup.1H NMR spectra were collected using a JEOL ECX 400 MHz spectrometer equipped with an auto-sampler. The samples were dissolved in D.sub.6-DMSO for analysis and the spectrum was acquired at ambient temperature using a standard proton experiment acquiring 16 scans using Delta NMR Processing and Control Software version 4.3. The data were then processed using ACD labs 1D NMR processor version 12.0.
DSC: DSC data were collected on a PerkinElmer Pyris 6000 DSC equipped with a 45 position sample holder. The instrument was verified for energy and temperature calibration using certified indium. A predefined amount of the sample, 0.5-3.0 mg, was placed in a pin holed aluminium pan and heated at 20° C..Math.min.sup.−1 from 30 to 350° C., or varied as experimentation dictated. A purge of dry nitrogen at 20 ml.Math.min.sup.−1 was maintained over the sample. The instrument control, data acquisition and analysis were performed with Pyris Software v11.1.1 Revision H.
XRPD: X-Ray Powder Diffraction patterns were collected on a PANalytical diffractometer using Cu Kα radiation (45 kV, 40 mA), 6-6 goniometer, focusing mirror, divergence slit (½″), soller slits at both incident and divergent beam (4 mm) and a PIXcel detector. The software used for data collection was X′Pert Data Collector, version 2.2f and the data was presented using X′Pert Data Viewer, version 1.2d. XRPD patterns were acquired under ambient conditions via a transmission foil sample stage (polyimide-Kapton, 12.7 μm thickness film) under ambient conditions using a PANalytical X′Pert PRO. The data collection range was 2.994-35° 2θ with a continuous scan speed of 0.202004° s.sup.−1.

HPLC: Method A

Sample Solution Preparation:

[0233] Accurately weigh 50 mg of sample into a 100 ml volumetric flask. Add 50 ml of Methanol to the flask, dissolve via sonication if necessary, dilute to volume with Purified Water and mix the resulting solution thoroughly. [0234] Column: 150×4.6 mm Luna C18 (2), 5 μm particle size, (ex-Phenomenex; #OOF-4252-EO) [0235] Mobile Phase: A—0.01 M Ammonium Acetate Buffer (pH 8.0) [0236] B—Acetonitrile [0237] Flow Rate: 1.0 ml.Math.min.sup.−1 [0238] Injection Volume: 5 μl [0239] Detection: UV@ 254 nm [0240] Column Temp: 30° C. [0241] Post Run: 5 minutes

HPLC: Method B

Sample Solution Preparation:

[0242] Accurately weigh 50 mg of sample into a 100 ml volumetric flask. Add 50 ml of acetonitrile to the flask, dissolve via sonication if necessary, dilute to volume with Purified Water and mix the resulting solution thoroughly. [0243] Column: 150×4.6 mm XBridge Phenyl, 3.5 μm particle size, (ex-Waters; #186003335) [0244] Mobile Phase: A—Purified Water:Trifluoroacetic acid (100:0.1) [0245] B—Acetonitrile:Trifluoroacetic acid (100:0.1) [0246] Flow Rate: 1.0 ml.Math.min.sup.−1 [0247] Injection Volume: 5 μl [0248] Detection: UV@ 268 nm [0249] Column Temp: 30° C. [0250] Post Run: 5 minutes

Chiral HPLC

[0251] Column: 250×4.6 mm Chiralpak AD-H, 5 μm particle size, (ex-Daicel Chemical Industries, Ltd; #DAIC 19325) [0252] Mobile Phase: Ethanol:Hexane (50:50) [0253] Flow Rate: 1.0 ml.Math.min.sup.−1 [0254] Injection Volume: 20 μl [0255] Detection: UV@ 268 nm [0256] Column Temp: 40° C. [0257] Run Time: 20 minutes

HRGC:

Sample Solution Preparation

[0258] Accurately weigh 50 mg of sample into a 10 ml volumetric flask. Dissolve in 5 ml of dichloromethane, using sonication if required, dilute to volume with dichloromethane and mix the resulting solution thoroughly. [0259] Column: DB-1 30m×0.32 mm; 1.0 μm film thickness (ex-J&W Scientific #123-1033) [0260] Oven Program: 40° C. (Hold 5 mins) then 10° C..Math.min.sup.−1 to 300° C. (Hold 10 mins) [0261] Injector Temperature: 200° C., split [0262] Column Temperature: 250° C., F.I.D. [0263] Head Pressure: 12 psi, constant pressure [0264] Carrier Gas: Nitrogen [0265] Split Ratio: 50:1 [0266] Injection Volume: 2 μl [0267] Liner: SGE Focusliner with glass wool insert

Enantiomeric Excess by HRGC:

Standard Solution Preparation

[0268] Accurately weigh 10 mg of each enantiomer [(2R,3S); (2S,3R); (2R,3R); (2S,3S)] into a suitable container. Dissolve in about 1 ml of HPLC grade dichloromethane, sonicating if necessary. Add 500 μl of trifluoroacetic anhydride and 500 μl of trifluoroacetic acid and allow to derivatise for 15-30 minutes at room temperature. Inject this solution

Sample Solution Preparation

[0269] Accurately weigh, in duplicate, 10 mg of sample into a suitable container. Dissolve in about 1 ml of HPLC grade dichloromethane, sonicating if necessary. Add 500 μl of trifluoroacetic anhydride and 500 μl of trifluoroacetic acid and allow to derivatise for 15-30 minutes at room temperature. Inject this solution. [0270] Column: Gamma-TA Cyciodextrin 30 m×0.32 mm; [0271] 0.125 μm film (ex-Astec; Cat no. 73033) [0272] Oven Program: 80° C. (Hold 10 mins) then 2° C..Math.min.sup.−1 to 90° C. [0273] (Hold 20 mins) then 10° C..Math.min.sup.−1 to 80° C. [0274] Injector Temperature: 200° C., split [0275] Column Temperature: 250° C., F.I.D. [0276] Head Pressure: 20 psi, constant pressure [0277] Carrier Gas: Nitrogen [0278] Spilt Ratio: 50:1 [0279] Injection Volume: 1 μl [0280] Liner: SGE Focusliner with glass wool insert

[00001]$\%\mathrm{enantiomeric}\mathrm{excess}=\frac{\mathrm{Peak}\mathrm{area}\mathrm{of}\left[\left(2R,3S\right)-\left(2S,3R\right)\right]}{\mathrm{Peak}\mathrm{area}\mathrm{of}\left[\left(2R,3S\right)+\left(2S,3R\right)\right]}\times 100\%$$\%\mathrm{diastereomeric}\mathrm{excess}=\frac{\mathrm{Peak}\mathrm{area}\mathrm{of}\left[\left(2R,3S\right)-\left(2S,3S\right)\right]}{\mathrm{Peak}\mathrm{area}\mathrm{of}\left[\left(2R,3S\right)+\left(2S,3S\right)\right]}\times 100\%$

Example A

(2R,3S)-3-aminopentan-2-ol synthesis

Preparation of Compound [4]

[0281] ##STR00026##

[0282] (S)-2-Methyl-CBS-oxaborolidine (1M solution in toluene, 59.6 mL, 0.06 mol) was diluted with THF (171 mL) in a dry, nitrogen purged vessel. Borane N,N-diethylaniline complex (102 mL, 0.57 mol) was added dropwise at room temperature and the solution was allowed to stir for 15 minutes. Compound [5] (115.0 g, 0.57 mol) was dissolved in THF (345 mL) and added dropwise over 4.5 h. After the addition was complete the reaction was allowed to stir overnight at room temperature under a nitrogen atmosphere. Thin layer chromatography (20% EtOAc in heptane, visualised by PMA) indicated the complete consumption of starting material. The reaction was carefully quenched via dropwise addition of methanol (174 mL) over 1 h. The temperature was maintained at <20° C. throughout the quench. The solution was concentrated in vacuo before additional methanol (174 mL) was added. The solution was concentrated under reduced pressure to afford a white waxy solid. The crude product was recrystallised from heptane (202 mL). The recrystallised product was filtered and rinsed with heptane (2×156 mL) to yield a white solid. This was dried in a vacuum oven at 40° C. overnight to give compound [4] as a white solid (99.2 g, 85%). Analysis was by HRGC and chiral HRGC as described above.

Example B

Deprotection of Compound [4]

[0283] ##STR00027##

[0284] MeOH (645 mL) was gassed with HCl for 1 hour at <20° C. in a 20 L flask under N.sub.2. The solution was 3.85M by titration. The flask was cooled to <15° C. Compound [4] (101.1 g, 0.50 mol) was charged portion-wise at <15° C. The solution was stirred overnight. The reaction was complete by TLC (5% MeOH/DCM, visualised with PMA). The solution was concentrated in vacuo at 35-40° C. The oil was azeotroped with EtOAc (4×75 mL) and triturated to give a white solid. The solid was taken up in EtOAc (588 mL). The reaction mixture was cooled to 0-5° C. and sparged with NH.sub.3 (g) for 1 hour at 0-5° C. under N.sub.2. At the end of the addition the pH was 8. The mixture was filtered and the filter cake was washed with EtOAc (147 mL). The filtrate was concentrated in vacuo at 35-40° C. to give the desired product [3] as a light yellow oil (50.7 g). .sup.1H NMR confirmed the identity of the product and indicated ˜4% residual EtOAc to be present giving an active yield of 48.7 g, 95%.

Example C

Synthesis of Compound [7]

[0285] ##STR00028##

[0286] Compound [6] (90 g, 0.48 mol) and compound [9] (100 g, 0.48 mol) are charged to a vessel along with n-butanol (1.98 L) and DIEA (295 mL, 1.68 mol). The reaction mixture was heated to 110° C. under N.sub.2 for 22 hours. The reaction was monitored by .sup.1H NMR to confirm that the starting material had been consumed. The reaction was cooled to room temperature and stirred at 15-25° C. for 30 min. The reaction mixture was filtered and washed 3× with TBME (3×125 mL). The product was dried in vacuo at 40° C. for 68 hours to yield compound [7] (124 g, 89%). .sup.1H NMR confirmed the identity of the product and HPLC (Method A) indicated a purity of 99.2%.

Example D

Synthesis of Compound [2]

[0287] ##STR00029##

[0288] Compound [7] (120 g, 0.416 mol), potassium carbonate (115 g, 0.832 mol) and DMSO (1.2 L) are charged to a vessel. 2-bromopropane (117 ml, 1.248 mol) is added portion wise over 2 minutes. The reaction mixture is heated to 60° C. and stirred for 50 minutes. The reaction was monitored by HPLC (Method A) to confirm that the starting material had been consumed. The reaction was cooled and water (1.2 L) was added and mixture was extracted 3× with ethyl acetate (3×0.9 L). The combined organic phase was washed 6× with water (6×1.1 L) and dried with magnesium sulphate. The magnesium sulphate was filtered off and the organic phase was evaporated to dryness on a rotary evaporator. The crude product was purified through a plug of silica using 15% methanol in ethyl acetate as eluent. The fractions containing the product were evaporated to dryness on a rotary evaporator to yield compound [2] (130 g, 93%). .sup.1H NMR confirmed the identity of the product and HPLC (Method A) indicated a purity of 99.2%.

[0289] In an alternative embodiment, compound [2] is prepared by treating compound [6] with 2-bromopropane to form compound [8] using similar conditions and reagents to those described above. Compound [8] is then reacted with compound [9] in the presence of n-butanol and DIEA using similar conditions to those described above to form compound [2].

Example E

Synthesis of Compound [1] (Crude)

[0290] ##STR00030##

[0291] Compound [2] (25 g, 0.0768 mol) and compound [3] (39.6 g, 0.384 mol) were charged to a vessel and heated to 170° C. (heating block temperature) under nitrogen for 48 hours. The reaction was monitored by HPLC (Method B) for the disappearance of Compound [2].

Recovery of Compound [3] by Distillation

[0292] The reaction was cooled to 60° C. and PEG400 (80 mL) and 8M sodium hydroxide solution (9.4 mL) were added. The reaction mixture was fractionally distilled in vacuo at 30 to 50 mBar and at temperatures from 80 to 170° C. A fraction was collected at a head temperature of 88° C. which contained Compound [3] (25 g, 71% recovery). The fraction was analysed by .sup.1H NMR and indicated a purity of >95%. The fraction was also analysed for what content by Karl Fischer titration which indicated that the fractioned contained 12% water.

[0293] “Butyl acetate (330 mL) and brine (650 mL) were added to the distillation pot residue and stirred. The organic phase was separated and the aqueous phase was re-extracted with “Butyl acetate (170 mL). The combined organic layers were washed 4× with water (4×250 mL) and dried over magnesium sulphate, filtered and the solvent was removed in vacuo to give the crude Compound [1] (29 g, 86% yield). The product was analysed by .sup.1H NMR which indicated that it contained 11% “Butyl acetate) and also by HPLC (Method B) which indicated a purity of 95.5%.

Example F

Crystallisation of Crude Compound [1]

Example F.1

[0294] Crude Compound [1] (28 g) was dissolved in “Butyl acetate (130 ml) and heated to 70° C. and then cooled to 66° C. and seed crystals of compound [1] were added and stirred for 1h. The seed crystals of compound [1] are designated as Form A and are as described in WO 2011/089401 (Cyclacel Limited; see in particular, Example 1—reproduced below), the contents of which are hereby incorporated by reference. The mixture was cooled to 25° C. over 14 hours whereupon a slurry formed. The slurry was stirred for 6 hours and then heptane (60 ml) was added over 1 hour 20 min and then stirred for 1 hour. The heptane functions as an antisolvent to increase the yield. The slurry was then cooled to 0° C. over 1 hour and stirred for 1 hour. The product was filtered in vacuo and washed 2× with 2:1 “Butyl acetate:heptane (2×20 ml) and then dried in vacuo at 45° C. for 18 hours to give Compound [1] (20.8 g, 69% overall yield (83% (crystallisation yield). The product was analysed by HPLC (Method B) which indicated a purity of 98.3% and also by XRPD which indicated that the product was crystals of Form A (in accordance with Example 1 of WO 2011/089401). XRPD peaks for crystalline free base (Form A) of Compound [1] are shown in Table 1.

Example F.2

Preparation of Seed Crystals of Compound [1] (Form A) as Described in Example 1 of WO 2011/089401

[0295] Compound [1] was crystallised from MTBE by the following method. MTBE (2 vol) was added to compound [1] and heated to reflux. The mixture was held at reflux for 30-60 minutes before the temperature was reduced to 50° C. (held for 2 hours). The suspension was allowed to cool slowly to room temperature before being filtered and rinsed with MTBE (3×1 vol). The solids were dried in vacuum oven at 40° C. for 8 hours to afford the desired crystalline free base (mass recovery 84.5%, LC purity 97.4%).

Example F.3

Alternative Crystallisation Conditions for Crude Compound [1] Isopropyl Acetate, 5 Volumes, 85° C., 6 g Scale

[0296] Crude Compound [1] (6.09 g) was suspended in isopropyl acetate (30 ml, 5 volumes) in a suitable glass vessel under N.sub.2 fitted with reflux condenser and stirrer bead agitation at 420 RPM. The beige suspension was heated to 85° C. (target 80-85° C.) upon which full dissolution to a dark brown solution was achieved after ca. 15 minutes at temperature.

[0297] The hot mixture was clarified through a heated large nylon syringe filter (0.44 μm) into a Mya4 100 ml process vessel with overhead U-shape agitation at 200 RPM pre-heated to 95° C. (target internal temperature=80-85° C.). The dark brown solution was cooled to 74.5° C. over 15 minutes and seeded with crystals of compound [1] (Form A) (ca. 6 mg 0.1% wt). Cooling was continued to 70.1° C. and seeding repeated, which was observed to maintain in solution after 15 minutes at temperature. A T=0 solubility measurement was taken (189.83 mg/ml) before the mixture was then cooled to 0° C. at a rate of 5° C./hour (14 hours, 840 minutes). Noticeable amounts of off-white precipitate were observed ca. 61-62° C. Following cooling, the mixture was held at 0° C. for ca. 3 hours. The solids were isolated via filtration, with no evidence of fouling of the vessel or agitator. The vessel and filter cake were washed with the isolated filtrates and air dried for 15 minutes before collection of the off-white solids and dark brown liquors. The solids were collected and dried in vacuo at 45° C. for ca. 4 hours to yield the title compound. 4.88 g of crystalline Compound [1] was successfully isolated in 80.1% yield. The maximum theoretical recovery based upon measured solubility pre-isolation at 0° C. was 94.2%.

Example F.4

Ethyl Acetate, 5 Volumes, 75° C., 6 g Scale

[0298] Crude Compound [1] (6.00 g) was suspended ethyl acetate (30 ml, 5 volumes) in a suitable glass vessel under N.sub.2 fitted with reflux condenser and stirrer bead agitation at 420 RPM. The beige suspension was heated to 75° C. (target 70-75° C.) upon which full dissolution to a dark brown solution was achieved during heating at ca. 68° C. The hot mixture was clarified through a heated large nylon syringe filter (0.44 μm) into a Mya4 100 ml process vessel with overhead U-shape agitation at 200 RPM pre-heated to 85° C. (target internal temperature=70-75° C.). The dark brown solution was cooled to 65.2° C. over 15 minutes (target 62.5-67.5° C.) and seeded with crystals of compound [1] (Form A) (ca. 6 mg 0.1% wt). Cooling was continued to 59.8° C. (target 57.5-62.5° C.) and seeding repeated, which was observed to maintain in solution after ca. 20 minutes at temperature. A T=0 solubility measurement was taken (184.35 mg/ml) before the mixture was then cooled to 0° C. at a rate of 5° C./hour (12 hours, 720 minutes). Noticeable amounts of an off-white precipitate were observed at ca. 52-53° C. Following cooling, the mixture was held at 0° C. for ca. 5 hours before solubility was measured indicating 90.06% development (18.84 mg/ml). The solids were isolated via filtration. The vessel and filter cake were washed with the isolated filtrates and air dried for 12 minutes before collection of the off-white solids and dark brown liquors. The solids were collected and dried in vacuo at 45° C. for ca. 4 hours to yield the title compound. 4.59 g of crystalline Compound [1] was successfully isolated in 76.4% yield. The maximum theoretical recovery based upon measured solubility pre-isolation at 0° C. was 90.6%.

Example F.5

[0299] n-Butyl Acetate, 5 Volumes, 85° C., 6 g Scale

[0300] Crude Compound [1] (5.96 g) was suspended n-Butyl acetate (30 ml, 5 volumes) in a suitable glass vessel under N.sub.2 fitted with reflux condenser and stirrer bead agitation at 420 RPM. The beige suspension was heated to 85° C. (target 80-85° C.) upon which full dissolution to a dark brown solution was achieved during heating at ca. 82° C. The hot mixture was clarified through a heated large nylon syringe filter (0.44 μm) into a Mya4 100 ml process vessel with overhead U-shape agitation at 200 RPM pre-heated to 95° C. (target internal temperature=80-85° C.). The dark brown solution was cooled to 74.6° C. over 15 minutes (target 72.5-77.5° C.) and seeded with crystals of compound [1] (Form A) (ca. 6 mg 0.1% wt). Cooling was continued to 69.5° C. (target 67.5-72.5° C.) and seeding repeated, which was observed to maintain in solution after 45 minutes at temperature. A T=0 solubility measurement was taken (175.84 mg/ml) before the mixture was then cooled to 0° C. at a rate of 5° C./hour (14 hours, 840 minutes). Development of a small amount of large particulates were observed at ca. 60-62° C. Following cooling, the mixture was held at 0° C. for ca. 3 hours before solubility was measured indicating 91.7% development (16.61 mg/ml). The solids were isolated via filtration. The vessel and filter cake were washed with the isolated filtrates and air dried for 10 minutes before collection of the grey solids and dark brown liquors. The solids were collected and dried in vacuo at 45° C. for ca. 4 hours to yield the title compound. 4.64 g of crystalline Compound [1] was successfully isolated in 77.9% yield. The maximum theoretical recovery based upon measured solubility pre-isolation at 0° C. was 91.7%.

Example F.6

[0301] Crystallisation of Crude Compound [1] in n-Butyl Acetate (5 Volumes, 85° C.) Using Heptane Anti-Solvent Addition

[0302] The crystallisation procedure outlined in Example F.5 was followed with the following modifications: [0303] Heptane (2.5 volume) anti-solvent addition performed at 25° C. at a rate of 1 volume/hour following controlled cool at 5° C./hour; [0304] Heptane (2.5 volume) anti-solvent addition performed at 70° C. following seeding at a rate of 1 volume/hour, followed by controlled 5° C./hour cool to 25° C.

[0305] As the final composition of the crystallisation contains a mixed solvent system, the impact of a standard 2×2 volume vessel and filter cake rinse using the final solvent composition of n-Butyl acetate/Heptane [2:1] was assessed.

n-Butyl Acetate, 5 Volumes, 85° C., 6 g Scale Crystallisation with Heptane (2.5 Volume) Anti-Solvent Addition Performed at 25° C. at a Rate of 1 Volume/Hour Following Controlled Cool at 5° C./Hour

[0306] 4.73 g of crystalline Compound [1] was successfully isolated in 78.5% yield. The maximum theoretical recovery based upon measured solubility pre-isolation at 25° C. was 92.4%.

n-Butyl Acetate, 5 Volumes, 85° C., 6 g Scale Crystallisation with Heptane (2.5 Volume) Anti-Solvent Addition Performed at 70° C. at a Rate of 1 Volume/Hour Followed by Controlled Cool at 5° C./Hour to 25° C.

[0307] 4.73 g of crystalline Compound [1] was successfully isolated in 76.0% yield. The maximum theoretical recovery based upon measured solubility pre-isolation at 25° C. was 92.4%.

Example G

Synthesis of Compound [1]-L-Tartrate Salt

[0308] ##STR00031##

[0309] Crystalline compound [1] free base (29.9 g, 75.22 mmol) was dissolved in ethanol (420 mL) and the resulting solution heated at reflux. A solution of L-tartaric acid (11.29 g, 75.22 mmol) in water (12 mL)/ethanol (30 mL) was added dropwise maintaining the batch temperature at 75-78° C. The solution was polish filtered (cooled to 57° C. during filtration with no evidence of crystallisation). The filtered solution was warmed to 60-65° C. and seeded with compound [1]-L-tartrate salt form 11 (Form E) (0.003 g) prepared in accordance with Example 5 of WO 2011/089401 (reproduced below; Cyclacel Limited). The mixture was stirred at 60-65° C. for 1 hour during which time crystallisation initiated. The suspension was then cooled to 15-20° C. at 10° C./h. After stirring at 15-20° C. for 1 hour, the solid was filtered, washed with ethanol (3×60 mL) and pulled dry. Further drying in a vacuum oven yielded [1]-L-tartrate salt as a white solid (36.0 g, 87% from free base). .sup.1H NMR confirmed the identity of the product and HPLC (Method B) indicated a purity of 98.80%. The product was also analysed by chiral HPLC. DSC analysis (peak 182.73° C., onset 179.61° C.) and XRPD confirmed form E in accordance with WO 2011/089401. XRPD peaks for L-tartrate (Form E) of Compound [1] are shown in Table 2.

Preparation of Seed Crystals of L-Tartrate Salt (Form E) of Compound [1] According to Examples 4 and 5 of WO 2011/089401

Example 4: Preparation of L-Tartrate Salt (Form D) of Compound [1]

[0310] Compound [1] (500 mg, 1.26 mmol, 1 equiv.), L-tartaric acid (193 mg, 1.28 mmol, 1.02 equiv) and ethyl acetate (5 ml, 10 vol) were charged to a flask and stirred under ambient conditions for 2 hours, precipitation occurred inside 1 hour. The white precipitate was isolated by vacuum filtration, washed with EtOAc (3×0.5 ml, 2×1 ml) and dried in a vacuum oven at 40° C. for 16 hours to yield the L-tartrate salt as a white solid (565 mg, 82% yield; Form D).

Example 5.1

[0311] A suspension of Form D L-tartrate salt of compound [1] as prepared in Example 4 of WO 2011/089401 above (1.0 g) in ethanol (12 ml) was heated at reflux. Acetonitrile (3 ml) was added portion wise over 30 minutes. After this addition, a solution was not obtained. Further portions of ethanol (4.5 ml) and acetonitrile (1 ml) were added until a solution was obtained. The solution was polish filtered (hot) then cooled to room temperature at a rate of 10° C./hour (crystallisation initiated at ˜65° C.). After stirring at room temperature overnight, the resulting solid was filtered, washed with cold ethanol (5 ml) and pulled dry. Further drying in a vacuum oven at 50° C. yielded the desired product as a white crystalline solid (0.725 g, 73%). .sup.1H NMR analysis confirmed a 1:1 salt and XRPD confirmed Form E.

Example 5.2

[0312] A suspension of Form D L-tartrate salt of compound [1] as prepared in Example 4 of WO 2011/089401 above (10.2 g) in ethanol (120 ml) was heated to 65° C. Acetonitrile (20 ml) was added and the suspension heated at reflux for 10 minutes after which time a solution was obtained. The solution was cooled to room temperature over 2-3 hours with crystallisation initiating at −50° C. The resulting suspension was stirred at room temperature overnight. The resulting solid was filtered, washed with ethanol (10 ml) and pulled dry. Further drying in a vacuum oven at 50° C. yielded the desired product as a white crystalline solid (8.76 g, 88%). .sup.1H NMR analysis confirmed a 1:1 salt and XRPD confirmed Form E.

Example 5.3—Slurry Conversion

[0313] Form E of the L-Tartrate salt of compound [1] was also prepared by slurry conversion from four different solvents (ethyl acetate, IPA, IMS or acetonitrile). A 1:1 mixture of Form by weight of D:Form E L-Tartrate salt (200 mg total) was heated at 45° C. over 48 hours in 2 ml of solvent prior to filtration and analysis. Form E was produced in each slurry (purity 98%).

Example 5.4—Seeding

[0314] A suspension of Form D L-tartrate salt compound [1] as prepared in Example 4 of WO 2011/089401 above (10.2 g) in ethanol (120 ml) was heated to 65° C. Acetonitrile (20 ml) was added and the suspension heated at reflux for 10 minutes. The mixture was polish filtered through HPLC filter frits. No precipitation was observed in process. The material was then cooled from reflux and seeded at 70° C. with Form E L-tartrate salt (as prepared above), cooling at a rate of 10° C. every 1.5 hours. The first seed dissolved completely and seeding was repeated at 60° C. The seed remained and the solution changed to show a very faint opaque phase. Crystallisation began at approximately 50° C. An isolated yield of 80% was obtained.

Example 5.5—Formation from Free Base of Compound [1]

[0315] Compound [1] free base Form A (0.2 g) was dissolved in ethanol (9 vol, 1.8 mL) and heated at reflux. A solution of tartaric acid (1 eq, 0.076 g) in water (1.7 vol, 0.34 mL)/ethanol (1 vol, 0.2 mL) was added dropwise maintaining the temperature at reflux. The resulting solution was then polish filtered before cooling to 70° C. A seed of Form E was added giving a cloudy solution. The batch was stirred at 70° C. for 1 hour before cooling to room temperature. After stirring at room temperature for 2 hours, the solid was filtered, washed with ethanol (2×0.5 mL) and pulled dry. Further drying in a vacuum oven at 50° C. yielded Compound [1]-L-tartrate salt Form E as a white solid (0.2 g, 72%). .sup.1H NMR confirmed a 1:1 salt and HPLC indicated a purity of 97.97%. XRPD and DSC confirmed Form E.

Example H

[0316] Preparation of Compound [1] with Direct Isolation as the L-Tartrate Salt

##STR00032##

[0317] Compound [2] (25 g, 0.0768 mol) and compound [3] (39.6 g, 0.384 mol, 5 mol eq) were charged to a vessel and heated to 170° C. (heating block temperature) under nitrogen for 48 hours. The reaction was monitored by HPLC (Method B) for the disappearance of Compound [2].

[0318] The reaction mixture was cooled to 60° C. The remaining compound [3] content was determined by .sup.1H NMR and 1 mol eq of HCl (as 4M HCl) relative to the amount of remaining compound [3] was charged. Ethyl acetate (10 vol) was charged and stirred to extract Compound [1] into the organic phase. The aqueous phase was separated and re-extracted with a further 10 vol of ethyl acetate. The organic phases were combined and washed with water (10 vol).

[0319] The organic phase was concentrated via distillation to approximately 5 volumes. Ethanol (10 vol) was charged and the distillation continued to remove the ethyl acetate. Further portions of ethanol were charged and the distillation continued until the ethyl acetate had been removed.

[0320] Sufficient ethanol was charged such that concentration of Compound [1] was 14 volumes. The mixture was heated to 75-78° C. L-tartaric acid (1 mol eq) was dissolved in purified water:ethanol (1:2.5 ratio, 1.4 vol relative to Compound [1]. The L-tartaric solution was added dropwise to the ethanol solution of Compound [1] at 75-78° C. The mixture was cooled to 60-65° C. and seeded with Tartrate Salt (Form E) of Compound [1]. The mixture was stirred at 60-65° C. for 1 hour until the crystallisation initiated. The suspension was cooled to 15-25° C. at 10° C./h. The suspension was stirred for 1 h at 15-25° C. and then filtered in vacuo, washed with ethanol (3×2 vol) and dried in vacuo at 50° C. Expected yield is 65-70% (29.4 g @ 70% yield).

[0321] Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

TABLE-US-00001 TABLE 1 XRPD peaks for crystalline free base (Form A) of Compound [1] Pos. Height FWHM d-spacing Rel. Int. Tip width [°2Th.] [cts] [°2Th.] [Å] [%] [°2Th.]  7.5313 21162.64 0.0768 11.73852 100.00 0.0921  9.6026 846.92 0.0768  9.21066 4.00 0.0921 10.2275 400.15 0.1023  8.64925 1.89 0.1228 11.2954 1863.94 0.1023  7.83384 8.81 0.1228 11.6652 300.53 0.1023  7.58631 1.42 0.1228 12.2672 3812.02 0.1023  7.21534 18.01 0.1228 12.6242 497.83 0.1023  7.01205 2.35 0.1228 13.1780 953.85 0.1023  6.71859 4.51 0.1228 14.0653 4092.77 0.1023  6.29672 19.34 0.1228 14.8535 1458.15 0.0768  5.96431 6.89 0.0921 15.1515 343.64 0.0768  5.84765 1.62 0.0921 15.5775 2894.50 0.1279  5.68868 13.68 0.1535 16.9914 2108.17 0.1023  5.21838 9.96 0.1228 17.6862 1501.97 0.1279  5.01490 7.10 0.1535 18.3040 644.51 0.0591  4.84701 3.05 0.0709 18.3954 1212.49 0.0768  4.82314 5.73 0.0921 18.6301 1666.18 0.1023  4.76289 7.87 0.1228 18.9784 1639.81 0.1279  4.67626 7.75 0.1535 19.3292 475.31 0.1023  4.59219 2.25 0.1228 20.2061 1067.39 0.1023  4.39483 5.04 0.1228

TABLE-US-00002 TABLE 2 XRPD peaks for L-tartrate salt (Form E) of Compound [1] Pos. Height FWHM d-spacing Rel. Tip width [°2Th.] [cts] [°2Th.] [Å] Int. [%] [°2Th.]  6.6675 15483.76 0.0768 13.25733 100.00 0.0921  8.2340 241.15 0.1023 10.73824 1.56 0.1228  9.7722 479.22 0.1023 9.05118 3.09 0.1228 11.9598 926.89 0.1023  7.40005 5.99 0.1228 12.3792 494.24 0.0768  7.15029 3.19 0.0921 13.0632 4104.92 0.0768  6.77739 26.51 0.0921 13.3777 2386.00 0.1023  6.61876 15.41 0.1228 13.9359 413.30 0.0768  6.35490 2.67 0.0921 14.9035 1349.55 0.1023  5.94439 8.72 0.1228 15.4032 975.17 0.0768  5.75266 6.30 0.0921 15.9507 949.23 0.1023  5.55642 6.13 0.1228 16.2665 488.77 0.1023  5.44926 3.16 0.1228 16.5423 792.08 0.1023  5.35902 5.12 0.1228 17.3614 2687.54 0.1023  5.10799 17.36 0.1228 17.5690 1410.91 0.1023  5.04809 9.11 0.1228 17.8630 201.26 0.1023  4.96566 1.30 0.1228 19.6395 1756.56 0.0768  4.52032 11.34 0.0921 19.8636 777.97 0.0768  4.46982 5.02 0.0921 20.1195 549.42 0.1023  4.41355 3.55 0.1228 20.7288 1423.91 0.1279  4.28518 9.20 0.1535 21.1373 389.18 0.1279  4.20327 2.51 0.1535 21.5804 674.89 0.1535  4.11797 4.36 0.1842 22.5683 459.02 0.1535  3.93989 2.96 0.1842 22.9541 780.05 0.1279  3.87454 5.04 0.1535 23.2869 904.34 0.1023  3.81992 5.84 0.1228 23.5693 1652.40 0.1535  3.77478 10.67 0.1842 24.0730 899.56 0.1535  3.69692 5.81 0.1842 24.6316 316.32 0.1791  3.61434 2.04 0.2149 25.2971 1357.36 0.1535  3.52074 8.77 0.1842 26.3772 346.67 0.1023  3.37898 2.24 0.1228 27.0905 141.69 0.1023  3.29160 0.92 0.1228 27.6723 474.86 0.1023  3.22371 3.07 0.1228 27.9727 708.87 0.1535  3.18977 4.58 0.1842 28.9051 262.52 0.1535  3.08896 1.70 0.1842 29.2843 136.18 0.1535  3.04982 0.88 0.1842 30.0801 73.71 0.1535  2.97092 0.48 0.1842 30.4059 137.17 0.1279  2.93982 0.89 0.1535 31.9006 27.79 0.1535  2.80541 0.18 0.1842 34.4898 70.18 0.2047  2.60050 0.45 0.2456