Process Development and Multikilogram Syntheses of XL228 Utilizing

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Process Development and Multikilogram Syntheses of XL228 Utilizing a Regioselective Isoxazole Formation and a Selective SNAr Reaction to a Pyrimidine Core Nathan R. Guz,*,† Helena Leuser,§,⊥ and Erick Goldman‡,† †

Chemical Development, Exelixis, Inc., South San Francisco, California 94080, United States Chemical Development, CARBOGEN-AMCIS, CH-5001 Aarau, Switzerland

§

S Supporting Information *

substrate in the SNAr additions of amines, phenols, thiols, and other nucleophiles.6 Using established precedent,7 the SNAr addition between 3 and 2 was quickly optimized (8 vol of IPA, 1.5 equiv of TEA, 20 °C, 3 h then 14 vol of H2O at 20 °C) on small scale and demonstrated on a 100-g scale to yield 4 in 89% yield and 98.5% purity with 99% warranted a new approach to a kilogram-scale process. Second-Generation Approach to 1. To overcome the poor selectivity encountered with the SNAr addition of 5 to 4, a new method to attach the substituted isoxazole subunit to the pyrimidine ring was necessary. During the process development of the modified medicinal chemistry route (Scheme 1), [3 + 2] cycloaddition reactions were explored in-house as a method to synthesize 5. Scheme 2 describes one method that proved to be an efficient and cost-effective approach to 5.9

ABSTRACT: Route scouting, process development, and multikilogram syntheses of an IGF-1R/Src/Bcr-Abl inihibitor are reported. Key aspects of the developed route are a regioselective [3 + 2] isoxazole formation on a pyrimidine core and a selective SNAr addition of an aryl amine to a symmetrical dichloro substituted pyrimidine. The route contains six synthetic steps and was demonstrated twice on scale, delivering 4.6 and 11.2 kg (25% and 16% overall yield), for Phase I clinical studies.



INTRODUCTION Pyrimidine 1 is a potent, reversible, and ATP competitive smallmolecule inhibitor developed in 2004 at Exelixis, Inc. This molecule targets the IGF-1R, Src, and Bcr-Abl pathways. IGF1R and Src both play important roles in cell proliferation and survival in colon, breast, prostate, ovarian, lung, and hepatocellular cancers.1,2 The Bcr-Abl gene is responsible for most patients afflicted with the hematological malignancies CML (chronic myelogenous leukemia), ALL (acute lymphoblastic leukemia), and AML (acute myelogenous leukemia).3 XL228 also targets the T315I mutant form of Bcr-Abl, a mutant that shows resistance to conventional Bcr-Abl therapies.4 Discovery Synthesis. The medicinal chemistry approach to synthesize 1 (Scheme 1) utilized 2 as the core building block for three separate nucleophilic aromatic substitution (SNAr) reactions. This short synthesis was capable of producing 35 g of 1 for toxicological range-finding studies, but chromatography or multiple hot slurry procedures were necessary to purify the intermediates and 1 to high-performance liquid chromatography (HPLC) purities of >95%. These purification techniques would not be amenable for manufacturing the approximately 4 kg of 1 necessary to initiate IND enabling toxicology and Phase I clinical studies. However, the brevity of the medicinal chemistry synthesis was quite attractive, and initial route optimization for a multikilogram synthesis of 1 focused on this approach.5



RESULTS AND DISCUSSION Initial Route Optimization. In order for the route presented in Scheme 1 to be successful in providing kilogram quantities of 1, the SNAr reactions would need to be selective and the resulting products amenable to straightforward crystallizations. The pyrimidine 2 is well investigated as a © XXXX American Chemical Society

Received: May 29, 2013

A

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Scheme 1. Medicinal chemistry routea

a

Reagents and conditions: (a) 1-BuOH, TEA, 80 °C, 2 h, 92%, 11.8:1 4/4a; (b) 1-BuOH, TEA, 120 °C, 16 h, 39%, 3.1:1 6/6a; (c) 110 °C, 3 h, 69%.

Scheme 2. In-house method to synthesize 5a

Reagents and conditions: (a) 50% NH2OH in H2O, 20 °C, 8 h, 90%; (b) N-chlorosuccinimide, DMF, 20 °C, 12 h; (c) CH2Cl2, TEA, 20 °C, 2 h; (d) 4 N HCl/IPA, 0 °C, 12 h, 74% from 8.

a

Scheme 3. Isoxazole ring formationa

Reagents and conditions: (a) Ac2O, DMA, 130 °C, 5 h, 67%; (b) propargyl bromide (80% in PhMe), K2CO3, DMA, 40 °C, 16 h, 79%; (c) 9, DMA, 40 °C, 10 h, 97% conversion by LC.

a

experienced with the aforementioned reaction may be translated to an alternative system. The pyrimidine 10 was selected as a template to build the appropriate partner for a reaction with 9 (Scheme 3).11 Starting material 10 was protected with an acyl group12 and alkylated with propargyl

The syntheses of 8 and 9 were straightforward, and the addition of Boc-propargylamine to 9 produced 5 as a single regioisomer (both 5 and reaction medium were analyzed).10 If an intermediate electronically similar to Boc-propargylamine was chosen and reacted with 9, the high regioselectivity B

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Scheme 4. SNAr addition of 3 and 7 to 13 under various conditionsa

Reagents and conditions: (a) 1.15 equiv of 3, DMA, TEA, or KHCO3, 20 or 46 °C, 12 h, >99.9:0.1 14/15; (b) 1.15 equiv of 3, DMA, TEA, or KHCO3, 80 °C, 12 h, 91.3:8.7 14/15; (c) 1.15 equiv of 7, DMA, TEA, or KHCO3, 20 °C, 12 h, 80.5:19.5 16/17; (d) 1.15 equiv of 7, DMA, TEA, or KHCO3, 80 °C, 12 h, 66.4:33.6 16/17. a

Scheme 5. Initial GMP route to manufacture 1.a

Reagents and conditions: (a) Ac2O, DMA, 130 °C, 3 h, 62%; (b) propargyl bromide (80% in PhMe), K2CO3, KHCO3, DMA, 30 °C, 24 h, 98% conversion by LC; (c) 9, DMA, 30 °C, 10 h, 87% conversion by LC; (d) 3, DMA, 30 °C, 12 h, 97% conversion by LC; (e) CH3CN, HCl/IPA, 15 °C, 4 h, 57% from 11. (f) iPrOAc, H2O, K2CO3, 20 °C, then 7, iPrOAc, reflux, 4 h, 99% conversion by LC; (g) K2CO3, MeOH, 20 °C, 6 h, >99% conversion by LC; (h) MtBE/iPrOAc, 25 to 10 °C, 58% yield from 14; (i) MtBE/iPrOAc, 25 to 10 °C, 92% yield, 98% purity. a

bromide (80% in PhMe)13 to produce the desired cycloaddition partner 12. Addition of 9 to 12 at 40 °C readily produced the desired isoxazole 13. Regioisomer 13a was not detected in either the reaction medium or isolated product. With a procedure in place to regioselectively form the desired substituted isoxazole, the additions of 3 and 7 to 13 were investigated (Scheme 4). An important advantage of proceeding through 13 as compared to 4 was the symmetrical structure of the 4,6-dichloropyrimidine core for addition reactions. Judicious choice of reactant order and reaction conditions could facilitate the selective addition of 3 or 7 to 13. Development continued to focus on SNAr reactions because

of the broad knowledge gained during the initial scouting work as well as the extensive literature precedent. Aromatic metalation14 and transition metal-mediated reactions15 were not investigated. Reactivity/selectivity principles played a key role in potentiating the SNAr addition of 3 and 7 to 13. The acetoamino group provided enough electron-withdrawing character to the pyrimidine ring (13) to promote the selective monoaddition of 3 to 13 under mild reaction conditions (Scheme 4, conditions (a)). Only at increased temperatures (Scheme 4, conditions (b)) did the overaddition product with 3 appear (91.3:8.7 14/15). C

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Scheme 6. Alternative [3 + 2] cycloaddition conditions to form 13a

a

Reagents and conditions: (a) cat. TEA, iPrOAc, reflux, 6 h, 92% yield.

with acceptable purity, but there were three aspects of the synthesis that required optimization for future scale-up campaigns. The synthesis of 9 was facile on scale for use in the [3 + 2] cycloaddition reaction, but intermediate 9 was found to have limited stability above 30 °C, which is approximately the lowest temperature required for the cycloaddition reaction to proceed. For the toxicology campaign, 92.6% conversion of 12 to 13 was achieved, and during the initial GMP campaign, only 86.8% conversion was realized, both using 2.1 equiv of 9.17 Intermediate 14·HCl was effective as a short-term isolation point, but the long-term stability of this salt was not as robust as initially anticipated. After one month of storage at ambient temperature, a sample of 14·HCl from the initial GMP campaign partially deliquesced to a mixture of deacylated 14 and numerous small, unidentified impurities (starting purity = 83.8%,18 purity at one month time point = 72.9%). A similar phenomenon was seen with development and toxicology batches (LC purity ≥ 91.0%) after ∼4 months. The lower conversion of 12 to 13 and subsequent lower purity of 14·HCl realized from the initial GMP campaign may be responsible for the decreased stability of 14·HCl when compared to that of 14· HCl samples from previous campaigns. Anticipating that similar purities of 14·HCl may be obtained in future GMP campaigns and hold times of 14·HCl may increase during multibatch processing, an alternative counterion for 14 or a different control point in the second GMP synthesis would need implementation. Finally, 1 was isolated as a mixture of an anhydrous and a hydrated crystal form. The physiochemical properties of the anhydrous form were far superior to the hydrate and other known forms. The anhydrous form was not hygroscopic and had the highest melting point (152 °C) of all forms isolated (115 °C for the hydrate, other forms had melting points below 125 °C). The delivery of 1 as a single, anhydrous crystal form was desirable to ensure a robust stability profile upon storage and a consistent dissolution performance during drug product formulation. Second-Generation Process Development. The concerns of the first GMP batch were addressed through development work prior to the initiation of the second GMP campaign. The formation of the isoxazole subunit on 13 was successfully switched to an analogous 1,3-dipolar cycloaddition procedure using 1,4-phenylene diisocyanate, 2-methyl-1-nitropropane (∼$1000/kilo, 1−3 week lead time), and catalytic TEA in iPrOAc under reflux conditions (Scheme 6).19 For this key reaction, 12, the diisocyanate, base, and solvent were charged together, and the mixture was heated at reflux temperature. The 2-methyl-1-nitropropane was dosed to the reaction mixture over ∼30 min, and heating was continued until the desired reaction end point was reached. The 1,4-

In contrast, when 7 and 13 were reacted, overaddition of 7 occurred under both mild (Scheme 4, conditions (c), 80.5:19.5 16/17) and more forcing conditions (Scheme 4, conditions (d), 66.4:33.6 16/17). Deacylated 13 was inactive to the SNAr addition of 3, and only under the most forcing conditions (K2CO3, DMA, 120 °C) was 7 added to deacylated 13 in low conversion (11.1%). This collection of SNAr reaction data demonstrated that the proper order of addition of the nucleophiles (3 to 13, followed by 7) and selection of an appropriately activated pyrimidine ring (11) can yield two consecutive selective SNAr reactions. With the major goal of demonstrating selective reactions achieved, the end-game development was completed by isolating 14 as an HCl salt. This control point allowed for the removal of the unreacted reagents from the alkylation, cycloaddition, and SNAr reactions. The final SNAr addition of 7 to the pyrimidine core and an acyl deprotection yielded 1 as expected. With a suitable process route in place, scale-up activities commenced. First GMP Campaign. The process route and conditions described in Scheme 5 were successfully employed in a smallscale demonstration campaign that delivered 25 g of 1. A campaign to deliver 100 g of 1 for IND enabling toxicology studies using this route (Scheme 5) was also accomplished. With the confidence gained from these two campaigns, the route was scaled up and demonstrated in the first manufacture of 1 under GMP. The yields and description presented below are from that initial GMP campaign (Scheme 5). The acylation of 10 proceeded smoothly, providing 11 in modest yield and high purity. The alkylation of 11, [3 + 2] cycloaddition of 9 and 12, and SNAr addition of 3 to 13 were telescoped by employing DMA as the solvent for all three reactions. The bases for the three telescoped reactions, K2CO3 (freshly milled prior to charge) and KHCO3, were charged prior to the initial alkylation reaction to minimize the number of solid charging manipulations. The alkylation occurred without incident although reaction times were longer on scale (24 vs 11 h) when compared to the toxicology campaign. Intermediate 9 was synthesized in a separate reactor using the conditions outlined in Scheme 2 and was successfully added over 2 h to 12 to provide 13 as a single regioisomer. The SNAr addition of 3 to 13 was successful under mild conditions, and the resulting product, 14, was isolated as an HCl salt. Intermediate 14·HCl was converted to the free base, 7 was added to 14, the acetoamino group was removed, and 1 was crystallized to 96.5% purity from a 30-vol 2:1 MtBE/ iPrOAc solution. A subsequent 17-vol 1.4:1 MtBE/iPrOAc crystallization provided 4.59 kg of 1 in 92% yield and 98% purity (18.4% yield from 10).16 First GMP Campaign Assessment. The first GMP synthesis was capable of delivering the desired amount of 1 D

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Scheme 7. Second GMP campaign to manufacture 1a

Reagents and conditions: (a) Ac2O, DMA, 130 °C, 10 h, 71%; (b) propargyl bromide (80% in PhMe), K2CO3, KHCO3, DMA, 30 °C, 15 h, 65% yield; (c) 1,4-phenylene diisocyanate, TEA, 2-methyl-1-nitropropane, iPrOAc, 80 °C, 6 h, 99% conversion by LC; (d) 3, KHCO3, DMA, 38 °C, 13 h, 98% conversion by LC; (e) 7, iPrOAc, 90 °C, 255 min, >99% conversion by LC; (f) K2CO3, 20 °C, 12 h, >99% conversion by LC; (g) iPrOAc, 60 to 20 °C, 24 h then −10 °C, 12 h, 40% yield from 12, 98% purity; (h) iPrOAc/EtOH, 53 to 2 °C, 86% yield, 16.4% from 10, 99% purity. a

phenylene diisocyanate-derived polymers were easily filtered away from the reaction solution upon cooling and water addition. This alternative cycloaddition reaction did not require a separate vessel to form the cycloaddition partner (9), and triethylamine and unreacted 2-methyl-1-nitropropane were innocuous in the downstream chemistry. Importantly, 13a was not detected in the cycloaddition reaction during development. A safety assessment of this reaction using reaction calorimetry revealed the process to be safe under the conditions presented in the experimental details.20 Investigations into a new volume-efficient crystallization system that would provide 1 in high purity and as a single, nonsolvated crystal form were also conducted. A crystallization solvent screen with crude 1 revealed that numerous solvent systems (iPrOAc and iPrOAc with MeOH, EtOH, or IPA) were capable of improving the purity of crude 1 from as low as 80% to >97.0% purity. These systems also yielded the desired anhydrous crystal form of 1 and an impurity profile that was similar or better to the material obtained in the toxicological and first GMP campaign. Crystallization and recrystallization of 1 in 10 vol of iPrOAc was thus chosen as the final isolation method for the second GMP campaign. Armed with the excellent impurity purging capability of the 10 vol iPrOAc crystallization and recrystallization, small-scale (25-g) development batches proved that the control point of 14·HCl was not necessary, and the process could be telescoped from isolated 12 to crude 1 successfully. After recrystallization of the crude material, 1 was isolated in improved purity compared to the first GMP campaign (>99.0% vs 97.2%) and with no new impurities above 0.1% when compared to the material isolated from the toxicological and first GMP campaign. Second GMP Campaign. The second GMP campaign was run on a 26-kg scale based on 10 and is described in Scheme 7. The acyl protection of 10 proceeded as previously described, but the yield was improved by employing 1:1 IPA/H2O as the antisolvent instead of water to precipitate 11.

Alkylation of 11 produced a crude LC reaction profile that showed an almost 8-fold increase (10.6%) of an unidentified impurity as compared to the initial GMP campaign (1.4%). The reaction conditions between the two campaigns were unchanged. LC/MS analysis of the enhanced impurity returned a m/z+ of 433, a mass that matched the dimerized product 18 (Figure 1). This impurity was possibly the result of the hydrolysis of 12 and subsequent SNAr addition of another molecule of 12.

Figure 1. Impurity formed during the alkylation of 11.

Examination of historic in-process control chromatograms revealed that the impurity 18 was present in all alkylation reactions. The toxicology campaign and the initial GMP campaign contained 1.1% and 1.4% of 18, respectively. All route scouting and development reactions had 18 at levels of 0.2% to 1.5% in the final alkylation reaction solutions and in examples where crude 18 was precipitated from a 10−15 °C water addition to the bulk DMA solution. LC/MS analyses of 1 manufactured for the toxicology and first GMP campaign revealed that 18 was not detected. To challenge the newly implemented iPrOAc crystallization and recrystallization system, a small portion (500 mL) of bulk reaction solution containing 12 was removed, diluted with water, and extracted with MEK, and the desired product was precipitated from a room temperature 1.3:1.0 water/IPA solution. Isolated intermediate 12, containing 12.0% 18, was carried through the process (Scheme 7), and the new iPrOAc crystallization and recrystallization were utilized. Crude 1 was readily purified >99.0% with impurity 18 not detected, and E

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and practical cycloaddition procedure utilizing 12, 1,4-phenylenediisocyanate, catalytic TEA, and 2-methyl-1-nitropropane to synthesize 13 was successfully introduced at the second GMP campaign. Both GMP campaigns used reactivity/selectivity principles to add 3 by SNAr to a symmetrical dichloropyrimidine (13) without overaddition. A volume-efficient crystallization/recrystallization procedure was also developed to yield the most stable crystal form of 1. The final process was capable of producing 11.24 kg (16% yield) of 1 with a 99.35% LC purity.

impurity 19 (Figure 2) below 0.1%. The GMP campaign was thus continued.



EXPERIMENTAL SECTION General. An HP 1110 liquid chromatograph equipped with a photodiode array detector was used for in-process analyses and final analytical release. The analysis method used a 32-min method ramping from 85:15 to 35:65 (A:B; solvent A = 10 mM borate buffer, pH = 8.65 in 95:5 DI water/ACN; solvent B = ACN) over 25 min, and 35:65 to 5:95 (A:B) from 25 to 32 min with a Gemini C-18, 150 mm × 4.6 mm, 5 μm column at 1.0 mL/min, column temperature of 25 °C, and detection at 235 nm. Approximate retention times (min): 1 (17.4), 3 (3.3), 10 (10.8), 11 (7.5), 12 (20.3), 13 (24.5), 14 (21.4). Reported yields are uncorrected. N-(4,6-Dichloropyrimidin-2-yl)acetamide (11). A mixture of 10 (26.56 kg, 161.9 mol) and DMA (90 L) was heated to 130 °C and acetic anhydride (49.6 kg, 486 mol) charged over 35 min. The reaction mixture was stirred at 130 °C for 10 h and cooled to 20 °C, and a 20 °C 1:1 IPA/H2O solution (264 L) was charged to the reaction mixture over 20 min. The resulting suspension was stirred at 20 °C for 60 min and at 4 °C for 135 min; the solids were collected, washed with a cold 1:1 IPA/H2O solution, and dried under reduced pressure at 45 °C for 5 days to yield 23.65 kg (70.9% yield, 97.8% purity) of 11. 1 H NMR (400 MHz, DMSO-d6): δ 11.12 (s, 1H), 7.58 (s, 1H), 2.15 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 168.8, 161.3, 157.1, 115.4, 24.8. HRMS (ESI): [M + Na]+ m/z Calcd for C6H5Cl2N3ONa 227.9707; Found 227.9703. Mp 190−197 °C (dec.) N-(4,6-Dichloropyrimidin-2-yl)-N-(prop-2-ynyl)acetamide (12). A mixture of 11 (23.63 kg, 114.7 mol), milled potassium carbonate (19.86 kg, 143.4 mol), potassium bicarbonate (22.96 kg, 229.4 mol), and DMA (72 L) was heated to 30 °C. Propargyl bromide (80% in PhMe, stabilized with MgO, 20.45 kg, 137.6 mol) was charged to the mixture over 35 min and stirred at 30 °C for 15 h. CAUTION: Neat propargyl bromide is shock sensitive and must be handled as a stock solution (see reference 13). The reaction mixture was cooled to 20 °C and diluted with water (483 L) and 2butanone (482 L), and the layers were separated, and the organic phase was concentrated to 45 kg. The concentrated organic solution was diluted with IPA (15.5 L) and water (20 L), and seeds of 12 were charged. The resulting suspension was stirred at 20 °C for 50 min and 6 °C for 75 min, yielding 12 in suspension. The solids were collected on a filter and washed with two portions of cold 1:1 H2O/IPA (48 L), and 12 was held in the filter at 25 °C under a stream of nitrogen until the next step (18.1 kg, 64.7% isolated yield). An analytical sample of 12 was prepared by a 2:1 H2O/IPA recrystallization to yield 12 as pale-yellow flakes. 1H NMR (400 MHz, CDCl3): δ 7.12 (s, 1H), 4.87 (d, J = 2.4 Hz, 2H), 2.61 (s, 3H), 2.13 (t, J = 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 171.7, 162.0, 159.1, 115.9, 79.2, 70.8, 35.1, 27.0. HRMS (ESI): [M + H]+ m/z

Figure 2. Impurity derived from 18.

Intermediate 12 was isolated as described above (18 = 12.0%) and submitted to the cycloaddition reaction yielding 13. Regioisomer 13a was not detected. The two SNAr reactions, the deprotection, and the initial crystallization to supply crude 1 also occurred smoothly, but the crude isolated 1 contained a red tint. The red tint of the batch was attributed to the starting material 3. While the same vendor was used to supply 3 for the toxicology and the first and second GMP campaigns, the lot used in the second GMP campaign was a red oil, while 3 used in all previous development work was a yellow oil. Small-scale test crystallizations with crude 1 showed that the originally planned iPrOAc recrystallization was not capable of removing the red tint. Changing the final purification to a 40:1 iPrOAc/EtOH recrystallization successfully removed the red tint and yielded 11.2 kg (16.4% from 10) of 1 in 99.35% purity (18 = ND, 19 = 0.09%) as an off-white solid. DSC and XRPD analyses confirmed that the desired single, anhydrous crystal form was isolated. Control of Alkylation Impurity 18.21 A brief survey of process changes to the formation of 12 (drying the milled K2CO3, 10 °C reaction temperature, 10 reaction vol.) showed that formation of 18 could be reduced to less than 1.9% by LC analysis in numerous examples, but never eliminated. Alternative bases (KH 2 PO 4 , K 3 PO 4 , NaHCO 3 , DBU, DABCO, and 2,6-lutidine) were also examined, but impurity 18 was formed in all cases (≥0.9% by LC), except for 2,6lutidine, where only starting material was detected by LC analysis. Rather than placing stringent water content controls on the input materials, a robust recrystallization of crude 12 that was capable of eliminating 18 at levels encountered during the second GMP campaign was investigated. A 2:1 H2O/IPA recrystallization (14 vol H2O, 7 vol IPA, 50 °C, seed at 1:1 H2O/IPA), was found to be capable of purging up to 13.1% of 18 to