The Development of Practical Synthetic Routes to a CB2 Agonist

Jan 2, 2013 - An efficient and scalable process for the preparation of a purine-based CB2 agonist was developed. The production route to the requisite...
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The Development of Practical Synthetic Routes to a CB2 Agonist: Efficient Construction of a Densely Substituted Purine Core Amy C. DeBaillie, Chauncey D. Jones, Michael E. Laurila, Nicholas A. Magnus, and Michael A. Staszak* Chemical Product Research and Development Division, Eli Lilly and Company, Indianapolis, Indiana 46285, United States ABSTRACT: An efficient and scalable process for the preparation of a purine-based CB2 agonist was developed. The production route to the requisite purine core relies on N-acylation and sequential substitution of a 5-amino-4,6-dichloropyrimidine with two amine building blocks followed by a cyclocondensation reaction. The chemistry was successfully employed to rapidly prepare over 5 kg of the active pharmaceutical ingredient. To further improve efficiencies, postproduction development resulted in a rearranged synthesis which reduced the need for pressure reactors and introduced the most costly reagent in the final step.

1. INTRODUCTION The search for orally active compounds for treating pain has been a longstanding goal of the pharmaceutical industry.1 In particular, osteoarthritic pain presents a large and challenging field, with much effort expended to discover pharmacological agents which exhibit limited side effects during potentially longterm treatments. The cannabinoid receptors CB1 and CB2 have been studied extensively;2 in broad terms, the CB1 site is expressed mainly in the brain,3 whereas the CB2 receptor is expressed in the immune system and hematopoietic cells.4 A 2004 Pfizer patent described the use of purine compounds as CB1 receptor antagonists and their use as anti-obesity agents.5 The therapeutic potential of the CB2 receptor has been reviewed, and that receptor site is considered a prime target for treating pain.6 More recent Lilly patents7 describe the use of certain purine compounds as selective CB2 agonists, which could provide a means of treating pain while limiting side effects. Herein we describe the process development leading to a scalable synthesis of LY2828360 (1), a CB2 agonist which was being developed by Eli Lilly and Company as an oral treatment for osteoarthritis pain.8

Scheme 1. Initial route to CB2 agonist 1

philes) for screening and subsequently to synthesize gram amounts of select compounds for early-stage testing. Finally, the chemistry outlined was used to produce several hundred grams of the lead molecule 1 (as the phosphate salt 1a) to fund toxicological evaluations. The discovery route began with a SNAr reaction of the appropriate amine nucleophile and methylpyrimidine 2. This first step was typically high yielding (∼90%) but required extended reaction times (>5 days) and multiple charges of the nucleophile.11 The second step included imine formation, an oxidative cyclization, and a second SNAr reaction, all in one reaction vessel. The process required upwards of 7 days at elevated temperature to afford the desired purine. Nitrobenzene was used as the oxidant in the system, with anisole as the highboiling solvent (bp 154 °C). Although the crude weight yields were acceptable (∼75%), the product potency at the free base stage was unacceptably low. Salt formation was used both to

2. DISCUSSION AND RESULTS 2.1. Discovery Chemistry Synthesis. The route shown below (Scheme 1) was developed by discovery chemists at Lilly’s Erl Wood Laboratories.9 This route is illustrated using the synthesis of 1, although it was employed to synthesize a number of SAR10 candidates (using various amine nucleo© XXXX American Chemical Society

Received: October 7, 2012

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Scheme 2. Generic route to purines14

h with isopropylamine and 1.4 equiv of isopropylethyl amine (DIPEA or, Huenig’s base) at 100 °C in sealed pressure vessels. Yields for this step were typically >90%.16 Once supplies of 4-amino-THP allowed, the knowledge gained from the use of isopropylamine was applied to the preparation of 3 with only minor modifications. Laboratoryscale trials showed this first SNAr reaction required approximately 36−40 h at 100 °C (again in sealed vessels) with 1.6 equiv of the amine to reach completion. The reaction could be performed in as few as 5 volumes17 (vol) of IPA with 90−94% isolated yields after an ethyl acetate extractive workup. However, the workup was cumbersome due to the need for multiple large-volume extractions. Thus, a simple precipitation method was developed using water, which afforded the product in 80−84% recovery on 50-g scale. The lower recovery was viewed as a fair trade-off for the simplicity achieved for largescale synthesis. For the amide formation step (III to IV in Scheme 2), the Pfizer patent procedure employed a slight excess of an acid chloride at 0−5 °C with dimethylacetamide (DMAc) as solvent. Adding water to the ending reaction mixture had afforded their amides in good yields (80−82%) and high purity. Although we initially tested a number of logical replacements for DMAc,18 that solvent was found to be superior in terms of both yield and product purity. For our derivatives (see middle step of Scheme 4), we found the reactions worked best in 7 vol of DMAc with 1.2 equiv of 2-chlorobenzoyl chloride 4. Reversing the quench order resulted in a more easily filterable, crystalline product with an increase of purity. Yields of 5 were typically 80−85%. Significantly, to avoid overacylation, no amine base was used to scavenge the HCl byproduct. The Pfizer patent procedures (Et3N, IPA, 80 °C) afforded only very low conversions to the desired material from our compound (5) reacting with 1-methylpiperazine. Switching to N-methylpyrrolidinone (NMP) as solvent, using DIPEA as base, and heating the reaction mixture to 120 °C in a sealed vessel, we were able to isolate the desired (penultimate) intermediate 6 in 54% yield (Scheme 3). Thus, with this

render a crystalline product and as an attempted aid in purification. Overall, the assay-corrected yields for this one-pot reaction were 99% HPLC purity. Although our original goal was to reliably produce 1 which could then be converted to a pharmaceutically acceptable salt (i.e., 1a or 1b), the crystalline free base had never been available for testing. After performance of comparative microscopy, XRPD, DSC/TGA, and hygroscopicity studies on the free base and the phosphate salt, the data indicated that the free base exhibited preferable physicochemical characteristics. Additionally, a polymorph screen for the crystalline free base indicated that the polymorph obtained via our new process appeared to be the most stable form.19 To date, no other crystal forms, hydrates nor solvates have been identified for this material. On the basis of these data, the free base 1 became our target for scale-up. The finalized synthetic route to 1 is shown in Scheme 4. This chemistry was laboratory tested using 50 g of the starting pyrimidine 2. The two steps requiring high temperature/ pressure (the conversions of 2 to 3 and 5 to 1) were performed

portion of the synthesis demonstrated, we were ready to test the final cyclization to the purine. Scheme 3. Initial two-step sequence to 1b

The Pfizer patent conditions for the cyclization of V to VI employed 3 equiv of conc H2SO4 in refluxing IPA. For our THP derivative, we found it necessary to use 9 equiv of H2SO4 to achieve conversion. Yields of 1b (the sulfate of 1) were, at best, 68% when the reaction was performed in ethanol at 100 °C in a sealed vessel (Scheme 3). Again, this chemistry was demonstrated to be feasible with our derivative (6), and we were poised to further develop the synthetic route. Scheme 4. Finalized laboratory route to 1

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Scheme 5. Proposed formation of impurity 7

isolation. Ultimately, LC/MS data confirmed that the impurity was the dimethylamino derivative 7 (Scheme 5). Considering these results, especially in the context of our development work on this step, where DMAc was found to be an excellent solvent for this reaction, we proposed the following cascade of events. First, DMAc is likely not just a solvent for this reaction but is also a participant in a Vilsmeier− Haack-22type activation. Outlined in Scheme 5, the acid chloride could react with DMAc to form an iminium ion, which then reacts with the poorly basic and poorly nucleophilic amine in our system. During the water quench/crystallization portion of the process, the byproducts (including any iminium chloride from excess acid chloride reagent) are ultimately hydrolyzed to dimethylamine hydrochloride and acetic acid.23 The higher concentrations, more occlusive crystallization, and less efficient water washes of the large-scale run likely all contributed to significant amounts of dimethylamine hydrochloride remaining in/on the amide product. Still, this impurity might not have been problematic (due to its low boiling point) in downstream chemistry if not for the fact that the second SNAr and cyclization steps are performed in sealed vessels. In that process, the DIPEA employed would neutralize the dimethylamine hydrochloride to its free base form which then acts as just another nucleophile for the SNAr reaction, hence, the formation of the dimethylamino analogue (7) of the API when the production lot of amide 5 was laboratory tested. To purify the crude bulk amide, the material was refluxed in IPA and then cooled, filtered, and rinsed with IPA. The material was recovered in an overall 81% yield and >99% HPLC purity for this step. Significantly, the 1H NMR of the material showed

in Hastelloy C Parr reactors. The overall yield to crystalline 1 from 2 was 63%, with 99% purity by HPLC. 2.3. Production Results. A production plan to prepare 5 kg of 1 was devised in conjunction with our outsourcing partner.20 The following paragraphs summarize the successes and additional learning points achieved while performing the first scale-up of this chemistry. For the first step, SNAr, several minor process modifications were initiated, which led to increased yields over those of laboratory experiments. The reaction was performed using 1.4 equiv of the 4-amino-THP and 2 equiv of DIPEA in only 3.3 vol of IPA at 100 °C. The desired product 3 was isolated by simple concentration followed by precipitation with water. Although the reaction did take slightly longer on scale, complete conversion of 2 was reached in 2.5 days, and the yields improved. Two combined 20-L autoclave batches afforded 5.9 kg of 3 in 96% yield and >99% purity by HPLC. To prepare for the large-scale amide formation, the reaction was demonstrated in the laboratory to work well in 3.5 vol of DMAc (versus the original 7 vol). This reduced volume would allow all the required amide to be formed in one production run, saving processing time. Unfortunately, this volume change for production purposes led to a slower filtration (on scale) and formation of a new impurity in isolated 5, which exhibited a sharp singlet (2.7 ppm) in the 1H NMR (CDCl3) spectrum of the amide product.21 When a small portion of the amide 5 was forward processed through the final steps, a new impurity was also observed in 1. This new impurity was rather similar in behavior to 1 and was difficult to reject during workup and D

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Scheme 6. Rearranged synthesis to amide 5

(0.5 equiv) prior to telescoping this step. HPLC analysis indicated an 89% solution yield of the amide, and the small amount of residual water did not have a deleterious effect on the desired substitution reaction. As in the two-step process, the conversion was complete in less than 4 h at 80 °C in NMP. The yield of 5 for the two-step process was 82% from the pyrimidine 2, and product purity was 99% (HPLC). This material behaved identically to that from the earlier route when forward processed (using the pressure protocol) to 1. Towards the goal of conserving 4-amino-THP through its later use in the synthesis, the isolated amide 8 was reacted with 1-methylpiperazine in NMP (Scheme 7). This reaction did not

no dimethylamine hydrochloride signal, and a laboratory test using purified 5 afforded 1 free of dimethylamino impurity 7. The final SNAr and cyclization steps were completed in an autoclave in two successful production runs, each requiring approximately 48 h at 160 °C to reach completion. The yield for these last steps was 92%, and a total of 5.16 kg of 1 was produced. The purity (HPLC) of the final material was >97%, which was within our specifications. Thus, the overall production-scale yield for this first campaign was 72% from the starting pyrimidine 2. 2.4. Post Campaign Development Efforts. The first campaign to synthesize 1 was deemed successful on the basis of the goals of the project. Anticipating a larger second campaign, we critically reviewed the new route for additional development opportunities, and several were identified. First, using DMAc as solvent has both health and environmental issues. Furthermore, as we discovered during production, the solvent also has the potential to lead to impurity formation. Second, although the new processes were shorter than the original reactions, two steps still required >48 h at elevated temperatures to reach completion. Third, the most expensive reagent (4-amino-THP) was used early in the synthesis, and we wondered if rearranging the order of the steps would be possible, thus potentially leading to lower cost of goods for the final product. Finally, with high temperature and pressure reactions prominent in the synthesis, we wanted to investigate how to make the entire route more amenable to standard plant reactors. While developing the amide formation step, a number of alternatives to DMAc had been screened, including CH3CN, MTBE, DMSO, CH2Cl2, THF, and N-methylpyrrolidinone (NMP). Of these solvents, NMP was the only one to allow product formation of reasonable quality and yield. We wondered if NMP would work better when the amidation was performed at an earlier stage.24 Significantly, we showed that the methylpyrimidine 2 (Scheme 6) could react with a slight excess of the acid chloride 4 in NMP at 70 °C. The reaction was complete in 5 h, and 8 could be isolated as a solid in 91% yield. Subsequently, we demonstrated that compound 8 could be converted to intermediate 5 by reaction with 4-amino-THP in NMP with DIPEA. This SNAr reaction was significantly more facile with 8 than when forming our original intermediate 3 (Scheme 4). The conversion was affected at 80 °C in ∼3 h without the need for pressure equipment. After precipitation using water, intermediate 5 was recovered in 90% yield from 8. To further streamline the synthesis, we showed that amide 8 could be carried directly into the first SNAr reaction (using 4amino-THP). The slight excess of acid chloride present at the end of the amide formation needed to be quenched using water

Scheme 7. Rearranged synthesis from amide 8 to 1

require elevated temperature nor pressure, as intermediate 9 was formed in 2.5 h at room temperature and was isolated in 92% yield. Subsequently, compound 9 was treated with 4amino-THP (1.75 equiv) in IPA (5 vol) using 3 equiv of DIPEA. After 26 h at 160 °C in a pressure vessel, the desired product 1 was obtained in 87% yield, providing proof of concept for the reordered synthesis from pyrimidine 2 (Scheme 7).

3. CONCLUSIONS In summary, we have presented an efficient, safe, and scalable synthesis of 1. The synthesis was successfully demonstrated on ∼5 kg scale, producing 1 of 97% purity and in overall yield of 72% from pyrimidine 2. Additionally, the route has been developed postproduction to afford further yield and processing gains. These gains were realized on laboratory scale but due to loss of project funding were not tested on pilot-plant scale. E

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at reflux for 48 h. After cooling to 25 °C and filtration, the product cake was washed with IPA (12.5 L) and dried under vacuum at 80 °C. Compound 5 was isolated as a white solid (7520 g, 81.6%, 99.6% purity by HPLC). 1H NMR (DMSO-d6, 400 MHz): δ 1.50 (m, 2H), 1.78 (dd, 2H), 2.36 (s, 3H), 3.38 (t, 2H), 3.83 (dd, 2H), 4.14 (m, 1H), 6.45 (d, 1H), 7.50 (m, 3H), 7.75 (d, 1H), 9.74 (bs, 1H). LC/MS data showed an M + 1 of 382. 4.1.3. 8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin1-yl)-9-(tetrahydro-2H-pyran-4-yl)-9H-purine, LY2828360 (1). A 20-L autoclave was charged with 5 (2021 g, 5.3 mol), 1-methylpiperazine (897 g, 8.95 mol, 1.75 equiv), DIPEA (758 g, 5.9 mol, 1.1 equiv), and IPA (10 L). The reaction mixture was heated to 160 °C. After 58 h, HPLC analysis of a reaction aliquot indicated >99% conversion of starting material 5. The reaction mixture was cooled to 20 °C and transferred to a 200L vessel, and the autoclave was rinsed out with IPA (10 L). Concurrently, a second run (2033 g of the amide starting material) was performed in a 20-L autoclave in like fashion. The combined runs were diluted with IPA (62 L) and heated to 80 °C to obtain a clear solution. After 1 h the mixture was cooled to 70 °C and filtered through an in-line filter.25 The tank and transfer line were rinsed with warm (∼45 °C) IPA (5 L), and the clear solution was concentrated by distillation (∼76 L of distillate was collected). The mixture was cooled to 20 °C over 4 h. Water (70 L) was added to the suspension over 1 h, and stirring was continued for 2 h. The contents of the tank were filtered, and the cake was washed with water (2 × 20 L). The cake was dried overnight on the funnel under a nitrogen flow and then further dried under vacuum at 80 °C. 1 was obtained as a crystalline white solid (4.13 kg, 92.2%, 97.4% purity by HPLC). Mp (DSC) (10 °C/min) onset 185.6 °C; peak 187.8 °C. 1H NMR (DMSO-d6, 400 MHz, VT = 75 °C): δ 1.70 (bd, 2H), 2.21 (s, 3H), 2.41 (bs, 4H), 2.47 (bs, 3H), 2.7 (m, 2H), 3.24 (t, 2H), 3.90 (d, 2H), 4.02 (m, 1H), 4.17 (bs, 4H), 7.51 (m, 1H), 7.63 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 26.6, 30.8, 40.6, 46.2, 54.2, 55.1, 66.8, 118.0, 128.0, 130.0, 130.3, 132.5, 133.1, 134.0, 145.8, 152.5, 153.3, 160.5. 4.2. Laboratory-Scale Procedures for the Re-ordered Synthesis (Post API-1). 4.2.1. 2-Chloro-N-(4,6-dichloro-2methylpyrimidin-5-yl)benzamide (8) (cf. Scheme 6). Under a nitrogen atmosphere, 2 (10.0 g, 56.2 mmol) was dissolved in NMP (50 mL). 2-Chlorobenzoyl chloride 4 (10.8 g, 61.8 mmol, 1.1 equiv) was added and the mixture heated to 70−75 °C. After 5 h, the mixture was cooled to ambient temperature and water (100 mL) was added over 10 min. The resulting slurry was stirred for 1 h, filtered, and the solids were washed with water (2 × 100 mL). After vacuum drying at 50 °C, compound 8 was recovered as white solid (16.1 g, 90.7%). 1H NMR (DMSO-d6, 400 MHz): δ 2.63 (s, 3H), 7.45−7.61 (m, 4H), 10.80 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): δ 25.3, 125.9, 127.7, 129.4, 130.4, 130.6, 132.2, 135.6, 160.1, 165.6, 167.1. HRMS (ESI+): calcd for C12H9Cl3N3O (M+): 315.9806, found 315.9803. 4.2.2. 2-Chloro-N-(4-chloro-2-methyl-6-((tetrahydro-2Hpyran-4-yl)amino)pyrimidin-5-yl)benzamide 5 (using a stepwise process; cf. Scheme 6). 8 (13.5 g, 42.6 mmol, prepared as described in the previous procedure) was combined with 4amino-THP (6.0 g, 59.7 mmol, 1.4 equiv), DIPEA (14.9 mL, 85.4 mmol, 2.0 equiv), and NMP (72 mL). The mixture was heated to 80 °C, stirred for 3.25 h, and cooled to ambient temperature. Water (140 mL) was added over 15 min, and the resulting slurry was stirred for 1 h. The mixture was filtered, and

4. EXPERIMENTAL SECTION HPLC analyses were performed using a reverse-phase technique as follows. Instrument: Agilent 1200 series. Column: YMC Pack Pro C18 100 mm × 4.6 mm, 3 μm. Flow: 1.0 mL/ min. Solvent A = 0.05% TFA in H2O; Solvent B = 0.05% in CH3CN. Gradient: 90% A to 40% A over 10 min; change to 5% A over 1 min and hold at 5% A for 2 min. Change back to 90% A over 2.5 min and hold for 2.5 min. Column temperature: 40 °C. Wavelength: 230 nm. LC/MS analyses were performed as follows. Instrument: Agilent 1200 MSD. Column: SeQuant ZIC-HILIC 150 mm × 4.6 mm, 5 um. Mobile phase consisting of solvent A: 2 mM aqueous NH4Cl; Solvent B: 2 mM NH4Cl in 95/5 CH3CN/H2O; Gradient: 90% B for 5 min, then change to 50% B over 5 min and hold for 6 min. Change to 90% B over 2 min and hold for 2 min. Flow rate: 1.0 mL/min; Column temperature 40 °C. Wavelength: 230 nm; Ionization mode: ESI Positive. 4.1. Production Scale Procedures (cf. Scheme 4). 4.1.1. 6-Chloro-2-methyl-N4-(tetrahydro-2H-pyran-4-yl)pyrimidine-4,5-diamine (3). A 20-L autoclave was charged with 2 (2243 g, 12.6 mol), 4-amino-THP (1782 g, 17.6 mol, 1.4 equiv), DIPEA (3263 g, 25.2 mol, 2 equiv), and IPA (3.7 L). The reaction mixture was heated to 100 °C. After 48 h, HPLC analysis of a reaction aliquot indicated 100% conversion of 2. The reaction mixture was cooled to 20 °C and transferred to a 100-L vessel; the autoclave was rinsed out with IPA (5 L). Concurrently, a second run (2240 g of starting material 2) was performed in a 20-L autoclave using the same procedure. The combined runs were concentrated in vacuo to remove ∼20 L of the solvent. The internal temperature was adjusted to 75 °C, and H2O (33 L) was added over 45 min. A suspension formed, and concentration in vacuo was continued to collect another 10 L of solvent. The resulting suspension was filtered and the cake washed with H2O (23 L) and dried on the filter using vacuum and a nitrogen flow for 22 h. The cake was further dried under vacuum at 80 °C to a constant weight. Compound 3 was recovered as an off-white solid (5870 g, 95.9%, 99.3% purity by HPLC). 1H NMR (DMSO-d6, 400 MHz): δ 1.42 (m, 2H), 1.83 (dd, 2H), 2.19 (s, 3H), 3.96 (t, 2H), 3.85 (dd, 2H), 4.08 (m, 1H), 4.78 (bs, 2H), 6.45 (bs, 1H). LC/MS data showed an M + 1 of 243. 4.1.2. 2-Chloro-N-(4-chloro-2-methyl-6-((tetrahydro-2Hpyran-4-yl)amino)pyrimidin-5-yl)benzamide (5). To DMAc (17.6 L) was added 3 (5854 g, 24.1 mol), and the mixture was stirred at 25 °C to attain a solution (∼10 min) and then cooled to 0 °C. 2-Chlorobenzoyl chloride 4 (5180 g, 29.6 mol, 1.23 equiv) was added over 30 min, and the transfer line was rinsed with DMAc (2.9 L). After 1 h at 0−5 °C, the mixture was warmed to 20 °C over 5 h, stirred for 8 h, and checked by HPLC, which indicated >99% conversion of starting material. The reaction mixture was transferred to a separate addition tank, and the transfer line was rinsed with DMAc (1.3 L). The original reaction vessel was charged with water (60 L), which was cooled to 15 °C. The reaction mixture was added to the water over 1.5 h. The resulting suspension was warmed to 25 °C and stirred for 20 h. The suspension was filtered, washed with water (28 L), and dried on the funnel under a nitrogen purge for 48 h. The overweight product cake was divided into three portions and further dried under vacuum at 80 °C; the resulting solids were then manually crushed into smaller-sized particles. The combined materials were suspended in IPA (78 L) and heated F

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the solids were washed with water (3 × 140 mL). After vacuum drying at 50 °C, compound 5 was recovered as a white solid (14.6 g, 89.9%). 1H NMR (DMSO-d6, 400 MHz): δ 1.47−1.56 (m, 2H), 1.80 (d, 2H), 2.36 (s, 3H), 3.38 (t, 2H), 3.83 (d, 2H), 4.09−4.19 (m, 1H), 6.45 (d, 1H), 7.43−7.56 (m, 3H), 7.74 (dd, 1H), 9.74 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 25.8, 32.7, 47.2, 66.3, 110.1, 127.5, 130.0, 130.3, 130.8, 131.9, 136.0, 155.8, 159.5, 165.7, 165.8. HRMS (ESI+): calcd for C17H19Cl2N4O2 (M+): 381.0880, found 381.0888. 4.2.3. 2-Chloro-N-(4-chloro-2-methyl-6-((tetrahydro-2Hpyran-4-yl)amino)pyrimidin-5-yl)benzamide (5) (using a telescoped process; cf. Scheme 6). Under a nitrogen atmosphere, 2 (9.9 g, 55.5 mmol) was dissolved in NMP (45 mL). 4 (10.3 g, 57.2 mmol, 1.03 equiv) was added and the mixture heated to 80 °C. After 4.5 h, water (0.5 mL, 27.7 mmol, 0.5 equiv) was added, and stirring was continued at 80 °C for 0.5 h. DIPEA (29.0 mL, 166.3 mmol, 3.0 equiv) was added followed by 4-amino-THP (7.9 g, 78.6 mmol, 1.4 equiv) and NMP (10 mL rinse). After 3.25 h at 80 °C, the mixture was cooled to ambient temperature, and water (110 mL) was added over 15 min. The resulting slurry was stirred for 0.5 h and filtered, and the solids were washed with water (3 × 100 mL). After vacuum drying at 50 °C, compound 5 was recovered as an off-white solid (17.4 g, 82.3%). 1H NMR (DMSO-d6, 400 MHz): δ 1.47−1.56 (m, 2H), 1.80 (d, 2H), 2.36 (s, 3H), 3.38 (t, 2H), 3.83 (d, 2H), 4.09−4.19 (m, 1H), 6.45 (d, 1H), 7.43− 7.56 (m, 3H), 7.74 (dd, 1H), 9.74 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 25.8, 32.7, 47.2, 66.3, 110.1, 127.5, 130.0, 130.3, 130.8, 131.9, 136.0, 155.8, 159.5, 165.7, 165.8. HRMS (ESI+): calcd for C17H19Cl2N4O2 (M+): 381.0880, found 381.0888. 4.2.4. 2-Chloro-N-(4-chloro-2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-5-yl)benzamide (9) (Scheme 7). 8 (15.0 g, 47.4 mmol) was dissolved in NMP (75.0 mL) under a nitrogen atmosphere. 1-Methylpiperazine (5.5 mL, 49.8 mmol, 1.05 equiv) was added over 5 min, and this material was rinsed into the flask with NMP (1.5 mL). After 10 min, DIPEA (9.9 mL, 56.8 mmol, 1.2 equiv) was added followed by an NMP rinse (1.5 mL), and the resulting mixture was stirred at ambient temperature for 2.5 h. Water (150 mL) was added over 10 min followed by ethyl acetate (75 mL), and the resulting slurry was stirred for 1 h. After filtration, water wash (3 × 150 mL), and ethyl acetate wash (30 mL), the isolated solids were vacuumdried at 50 °C. Compound 9 was recovered as a white solid (16.5 g, 91.5%). 1H NMR (DMSO-d6, 400 MHz): δ 2.15 (s, 3H), 2.35 (dd, 4H), 2.39 (s, 3H), 3.72 (bm, 4H), 7.44−7.59 (m, 4H), 10.15 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 25.6, 46.0, 46.7, 55.0, 111.2, 127.7, 129.3, 130.5, 131.9, 136.1, 160.0, 161.2, 164.8, 165.8. HRMS (ESI + ): calcd for C17H20Cl2N5O (M+): 380.1039, found 380.1045. 4.2.5. 8-(2-Chlorophenyl)-2-methyl-6-(4-methylpiperazin1-yl)-9-(tetrahydro-2H-pyran-4-yl)-9H-purine, LY2828360 (Scheme 7). A Hastelloy C Parr reaction vessel was charged with 9 (3.5 g, 9.2 mmol), 4-amino-THP (1.7 g, 16.6 mmol, 1.8 equiv), DIPEA (1.8 mL, 10.2 mmol, 1.1 equiv), and IPA (17.5 mL). The vessel was purged with nitrogen and sealed, and the internal temperature was raised to 160 °C. After 26 h, the contents were cooled to 45−50 °C, transferred to a separate reaction flask, and treated with cold water (60 mL). The resulting slurry was stirred for 1.75 h and filtered. The solids were washed with cold water (17.5 mL) and dried under vacuum at 50 °C. LY2828360 was obtained as a white solid (3.4 g, 86.6%). 1H NMR (DMSO-d6, 400 MHz, VT = 75 °C): δ

1.70 (bd, 2H), 2.21 (s, 3H), 2.41 (bs, 4H), 2.47 (bs, 3H), 2.7 (m, 2H), 3.24 (t, 2H), 3.90 (d, 2H), 4.02 (m, 1H), 4.17 (bs, 4H), 7.51 (m, 1H), 7.63 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 26.6, 30.8, 40.6, 46.2, 54.2, 55.1, 66.8, 118.0, 128.0, 130.0, 130.3, 132.5, 133.1, 134.0, 145.8, 152.5, 153.3, 160.5.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Special thanks to the process chemistry group at CarboGen AG, Aarau, Switzerland, and in particular to Dr. Marc Lanz and Dr. Jurgen Beyer for their development efforts and scale demonstrations of the chemistry shown in Scheme 3.



REFERENCES

(1) Dronsfield, A.; Brown, T.; Ellis, P. Pain Relief: From Coal Tar to Paracetamol. Education in Chemistry (Royal Society of Chemistry) 2005, 42 (4), 102−105. (2) For example, see (a) Mackie, K. Cannabanoid Receptors: Where They Are and What They Do. J. Neuroendrocrinol. 2008, 20 (Supplement 1), 10−14. and (b) Graham, E. S.; Ashton, J. C. Cannabanoid Receptors: A Brief History and ‘What’s Hot’. Front. Biosci. 2009, 14, 944−957. (3) Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Nature 1990, 346, 561−564. (4) Pacher, P.; Mechoulam, R. Prog. Lipid Res. 2011, 50, 193−211. (5) Griffith, D. A. (Pfizer Corp.) Preparation of Purines as Cannabinoid Receptor Ligands (CB1 Receptor Antagonists). WO/ 2004/037823, 2004; Chem. Abstr. 2004, 140, 391293. (6) Guindon, J.; Hohmann, A. G. Cannabinoid CB2 Receptors: A Therapeutic Target for the Treatment of Inflammatory and Neuropathic Pain. Br. J. Pharmacol. 2008, 153, 319−334. (7) (a) Astles, P. C.; Guidetti, R.; Tidwell, M. W.; Hollinshead, S. P. (Eli Lilly and Co.). Preparation of Piperazinyl Purine Compounds for the Treatment of Pain. U.S. Patent Appl. 0160288, 2010; Chem. Abstr. 2010, 153, 115955. (b) Hollinshead, S. P. (Eli Lilly and Co.). Preparation of Purine Compounds for Use as CB2 Agonists. WO/ 2011/123482, 2011; Chem. Abstr. 2011, 155, 483848. (8) Ibid. 7a. (9) The discovery chemistry route was developed by Dr. Jeffrey Richardson at Eli Lilly’s Erl Wood Laboratories in Surrey, England. We are indebted to Dr. Richardson for the groundwork he laid for the chemistry of these CB2 agonists, the insight he afforded us from a synthetic standpoint, and the samples of intermediates and reagents he supplied. (10) SAR is shorthand for structure−activity relationship (the relationship between the chemical structure of a molecule and its biological activity). (11) In the case of the 4-aminotetrahydropyran reagent, the reagent itself was unstable to extended periods of heating, and thus extra equivalents of the material were necessary for this process. (12) Domestically, there were two companies which had supplies of this material. Combi-Blocks Corporation had two 1 g samples of the HCl salt available at $134/g. Austin Chemicals had prepared the HCl salt of the 4-amino-THP for the discovery chemistry efforts, but had only 5 g of the material remaining. (13) For a general treatise on purines and pyrimidines, see Cumulative Index of Heterocyclic Systems; Brown, D. J., Evans, R. F., Cowden, W. B., Fenn, M. D., Eds.; Chemistry of Heterocyclic Compounds: The Pyrimidines, 2008; Vol. 65, p 52 (DOI: 10.1002/ 9780470187395). For more specific syntheses, see the following: (a) Young, R. C.; Jones, M.; Milliner, K. J.; Rana, K. K.; Ward, J. G. J. G

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Med. Chem. 1990, 33, 2073−2080. (b) Dang, Q.; Brown, B. S.; Erion, M. D. Tetrahedron Lett. 2000, 41, 6559−6562. (c) Harada, H.; Asano, O.; Kawata, T.; Inoue, T.; Horizoe, T.; Yasuda, N.; Nagata, K.; Murakimi, M.; Nagaoka, J.; Kobayashi, S.; Tanaka, I.; Abe, S. Bioorg. Med. Chem. 2001, 9, 2709−2726. (d) Tandel, S.; Bliznets, I.; Ebinger, K.; Ma, Y.; Bhumralkar, D.; Thiruvazhi, M. Tetrahedron Lett. 2004, 45, 2321−2322. (14) Ragan, J. A. (Pfizer Corp.). Intramolecular Cyclocondensation Process for Preparing Purine Compounds. WO/2006/043175; Chem. Abstr. 2006, 144: 412537. (15) API = active pharmaceutical ingredient. (16) Using isopropylamine as the surrogate nucleophile, the isolated yields of the corresponding product 6-chloro-N4-isopropyl-2-methylpyrimidine-4,5-diamine were typically 90−92% after concentration in vacuo. (17) A “volume” (vol) as used here and in all later occurrences, refers to milliliters of the solvent per gram of the limiting reagent (in this case, compound 4). (18) For a useful guide to solvents, see the American Chemical Society Green Chemistry Institute’s (ACS-GCI) Pharmaceutical Roundtable Solvent Selection Guide, version 2.0; 2011 (available online at www.acs.org/gcipharmaroundtable). For specific information on DMAc, see Kennedy, G. L.; Sherman, H. Drug Chem. Toxicol. 1986, 9 (2), 147−l70. (19) We are grateful to Dr. Susan Reutzel-Edens of Lilly’s preformulations Solid State Group for performing the polymorph screening. (20) The outsourcing partner chosen for these efforts was CARBOGEN AMCIS AG in Switzerland. (21) The HPLC assay (at 230 nm) of isolated 5 showed no signal other than that of the desired material. The impurity was evident in the 1 H NMR data as only the sharp singlet mentioned at 2.7 ppm. We were immediately suspicious that the impurity was dimethylamine hydrochloride. When a sample of 5 was spiked with a bonafide sample of dimethylamine hydrochloride, the 1H NMR data supported that suspicion. When 5 was forward processed in the chemistry of the final step, the identification of 7 confirmed that dimethylamine hydrochloride was indeed the impurity in the material and there is no other possibility for it to arise other than from the DMAc used as solvent. (22) For a general review of the Vilsmeier−Haack reaction, see Meth-Cohn, O.; Stanforth, S. Comp. Org. Synth. 1991, 2, 777−794. (23) This series of events effectively results in the acid-catalyzed hydrolysis of the solvent DMAc, which is the only plausible source of dimethylamine hydrochloride in the reaction sequence. (24) In theory, NMP is susceptible to the same type of acid hydrolysis mentioned earlier. However, this solvent is regarded as quite resistant to such hydrolysis, and our analyses of products from reactions using NMP showed no signs of hydrolysis-related impurities. (25) For GMP processing, once the final, in-line filtration is completed, all subsequent rinses with solvents and/or deionized water were performed with filtered materials.

H

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