Discovery and Process Development of Class I PI3K and Class I

Chapter 8

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Discovery and Process Development of Class I PI3K and Class I PI3K/mTOR Inhibitors GDC-0941 and GDC-0980 Srinivasan Babu,1 Francis Gosselin,1 Theresa Humphries,1 Alan Olivero,2 Daniel Sutherlin,2 and Qingping Tian*,1 1Small

Molecule Process Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States 2Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States *E-mail: [email protected].

The PI3K signaling pathway has attracted significant attention in drug development due to its role in tumorigenesis. Two orally available clinical compounds which have been developed include GDC-0941 (pictilisib), a selective Class I PI3K inhibitor, and GDC-0980 (apitolisib), a selective Class I PI3K/mTOR inhibitor. Chemical process development has been conducted to support both compounds, which share a common thienopyrimidine core, from the pre-clinical development stage to the clinical study. An enabling process (the first-generation route) was employed to produce multikilogram of API for the GLP and initial GMP production while an improved process (the second-generation route) was developed as the projects were advanced to late stage development. The development of a practical and protecting-group-free synthesis for GDC-0980 and an efficient synthesis of a key intermediate for GDC-0941 through aminoalkylation is discussed in detail in this chapter.

Discovery Chemistry The phosphoinositide-3-kinase ( PI3K) pathway is involved in the transmission of growth and survival signals from the outside of the cell to the nucleus (1). As such, this signaling pathway has received a great © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


deal of interest as a potential target for cancer therapy where unregulated cell growth and survival plays a large role in the pathogenesis of the disease (2). Functionally, signal transduction occurs when PI3K proteins are recruited to the cell membrane whereupon PI3Ks phosphorylate the membrane-bound lipid phosphatidylinositol-(4,5)-bis-phosphate (PIP2) to phosphatidylinositol-(3,4,5)-tris-phosphate (PIP3) which serves to activate and recruit the oncogene Protein Kinase B (AKT). Subsequently, AKT activates a number of pathways that ultimately result in the transcription of genes that promote cell survival and growth (2). PI3Ks are lipid kinases, distinguishing them from serine and threonine kinases, and exist in four different isoforms, PI3Kα, β, δ, and γ (Class I PI3Ks). While these isoforms have been implicated in cancer to varying degrees, PI3Kα is considered to be influential in tumorigenesis based on the identification of PI3Kα activating mutations and through observation that Phosphatase and Tensen Homolog (PTEN), a negative regulator of PI3K signaling, is deleted in many tumor samples (2). Based on this strong genetic evidence for PI3Ks’ significant role in cancer, we sought to develop inhibitors of this pathway. In our early efforts to identify potent inhibitors of PI3K signaling, we developed two clinical molecules: GDC-0941/pictilisib and GDC-0980/apitolisib (Figure 1) in collaboration with Piramed Pharmaceuticals (later acquired by Roche) (3, 4). While both compounds were potent inhibitors of all four Class I PI3K isoforms, they differed in their selectivity for mTOR, an additional kinase that plays an important role in cell signaling and cancer. We hypothesized that these biological differences could lead to a unique efficacy, and safety profiles that would require further exploration in the clinic to truly understand the therapeutic potential for each compound.

Figure 1. Structures for Class I PI3K inhibitor GDC-0941 and Class I PI3K/mTOR inhibitor GDC-0980. Early discovery efforts initiated by Piramed began with thienopyrimidine 1 (Table 1), a compound that was chosen for its excellent potency for PI3K and good overall properties (3). Analysis of a molecular model and literature data for other morpholine-containing compounds led to speculation that the morpholine oxygen bound to the hinge region of the kinase and would position the phenol in the affinity pocket to make a hydrogen bond contact to the protein via the phenol OH. Both of these interactions were observed to be critical for PI3K potency. 238 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Unfortunately regardless of the R1 substitution, every compound with a phenol at R3 had unacceptable oral bioavailability. A thorough investigation of phenol replacements that might retain the same back-pocket donor interaction led to discovery of indazole 2. Although indazole 2 lost four-fold potency compared to phenol 1, we were gratified to see the oral bioavailability in rats improve from 2% to 37%. Once the imidazole was discovered, R1 substitutions were explored to optimize solubility, potency, and PK properties. Ultimately the sulphonylated piperazine was incorporated to yield GDC-0941 (3). This change improved potency significantly while also improving microsomal stability relative to 2.

Table 1. Summary of Major Structural Changes in the Transformation of Lead Compound 1 to Identify GDC-0941 and GDC-0980

GDC-0941 was shown to be a broad class I PI3K inhibitor (IC50s for PI3Kα, 3 nm; PI3Kβ, 33 nM; PI3Kδ, 3 nM; PI3Kγ, 75 nM). Consistent with results from many lipid kinase inhibitors, this compound was found to be very selective relative to a large number of serine/threonine kinases in the kinome. The potent biochemical activity for both PI3Kα and PI3Kβ isoforms contributed to cellular potency of this compound in cell lines with activating mutations of 239 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

PI3Kα (MCF7.1) and with deleted PTEN (PC3-NCI) (5, 6). This trend was consistent across a larger panel of cell lines. Specifically, GDC-0941 inhibited the downstream phosphorylation of AKT with an IC50 of 28 nM and 37 nM in PC3-NCI and MCF7.1 cells respectively. Based on potent inhibition of PC3 and MCF7 cell proliferation and significant efficacy in their corresponding xenograft models, GDC-0941 was nominated for clinical development. Additional preclinical PK and safety data also supported this nomination (7). The subsequent discovery of GDC-0980 (6) began with the identification of 4 (Table 1), where the indazole of GDC-0941 is replaced with an aminopyrimidine that occupies the affinity pocket (8). This change was significant for two reasons:


•

•

First the aminopyrimidine lowered the cLogP of otherwise identical indazoles by nearly two log units which contributed to an increase in stability in rat microsomes, and a decrease in plasma protein binding ultimately combining to lower the human dose projection. Secondly, this change brought about an approximate 20-fold increase in mTOR potency, a property that was anticipated to increase efficacy and broaden the scope of activity across cell lines.

Despite these advantages, 4 had high clearance in dogs. This liability was addressed by the addition of a methyl group to the thienopyrimidine scaffold to yield 5, that served to reduce in vivo clearances in multiple species and across multiple scaffolds (9). In an effort to improve the intrinsic solubility of 5, the sulfonamide moiety was replaced with a (S)-lactic amide to yield GDC-0980 (6), which increased the solubility of GDC-0980 by nearly 10-fold relative to 5. Like GDC-0941, GDC-0980 was found to be a broad class I PI3K inhibitor (5, 27, 7, and 14 nM IC50s for PI3Kα, β, δ, γ, respectively), very selective in a large kinase panel, and potent in proliferation assays for cell lines with both PI3K activating mutations and PTEN deletions (MCF7-neo/HER-2, 240 nM and PC3-NCI, 120 nM IC50s). GDC-0980 was active in a larger number of cell lines when compared to GDC-0941 (10, 11) and was attributed to the addition of mTOR inhibition to the scaffold. Based primarily on the improved properties of GDC-0980 relative to GDC-0941 (better overall PK properties and a higher free-drug fraction), GDC-0980 was found to be active at doses as low as 5 mg/kg in xenograft efficacy studies. With this differentiated profile relative to GDC-0941, GDC-0980 was also selected for clinical development (12, 13). The medicinal chemistry route to both GDC compounds and their analogs began with thiophene amino esters 7 and 8, for GDC-0941 and GDC-0980 respectively (Scheme 1). High temperature condensation with molten urea gave the thienopyrimidones 9 and 10. Bis-chlorination was effected by treatment with POCl3 to generate the corresponding dichloro intermediates 11 and 12, followed by a selective displacement of the most electrophilic chlorine by morpholine at room temperature, yielding monochloro-morpholines 13 and 14. Selective deprotonation of the CH adjacent to sulfur followed by addition of DMF gave the late stage aldehydes 15 and 16 which could be used to rapidly generate analogs that had varying functionality in two important areas of the binding pocket, the solvent exposed region through functionalization of the aldehyde, and the affinity 240

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


pocket through aromatic substitution and cross coupling reactions with the aryl chloride (indicated by R1 and R3 in Table 1). For GDC-0941, a reductive amination was performed with aldehyde 15 and methylsulphonylpiperazine to yield 17. Next, a Suzuki-Miyaura coupling reaction with indazole boronic ester 19 furnished the final molecule. A similar reductive amination sequence with Boc-piperazine on 16, followed by Suzuki-Miyaura coupling with commercially available aminopyrimidine boronic ester 20 provided 18. The synthesis of GDC-0980 was completed through Boc removal under acidic conditions followed by an amide coupling with (S)-lactic acid. This consisted of the Med Chem route to these PI3K inhibitors.

Scheme 1. Medchem Synthesis of GDC-0941 and GDC-0980 241 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Process Chemistry Development Process chemistry development has been pursued as both compounds (GDC-0941 and GDC-0980) were advanced to the clinic. We employed a similar synthetic strategy for both compounds due to the structural similarity of GDC-0941 and GDC-0980 (14, 15). As such, the process development for GDC-0980 will be discussed in this chapter and some interesting route scouting work on GDC-0941 will be added.


The Enabling Synthesis As the GDC-0980 project was advanced to the development stage, it was important to produce multikilogram quantities of API to support the GLP tox study and the clinical study. Due to the tight timeline, we elected to modify the medicinal chemistry route (Scheme 1) to enable the synthesis on large scale. Several issues were identified when we were attempting to scale up the medicinal chemistry synthesis. • • •

Step 1 was run neat at 200 °C. Long reaction time (3 days) was required for step 2. Microwave radiation (MW) was used in the Pd-catalyzed SuzukiMiyaura coupling reaction which was not practical on large scale.

Those issues required resolution prior to the scale-up of the synthesis. Synthesis of intermediate 10 relied on condensation of commercially available methyl 3-amino-4-methylthiophene 2-carboxylate 8 with urea at 200 ºC (Scheme 1). Although the reaction afforded a good yield of the desired product, there were concerns about the safety of this reaction on large scale. We noted that urea could solidify in the condenser and block the outlet of the NH3 gas formed in the reaction. To avoid this potential hazard, we sought milder conditions for the condensation reaction. When urea was replaced with potassium cyanate in aqueous AcOH, the reaction proceeded smoothly at room temperature to afford 10 in 84% yield (Scheme 2) (16). Pyrimidinone 10 was subsequently chlorinated with POCl3 to afford the dichloropyrimidine 12. The yield was significantly improved in the presence of N,N-dimethylaniline (0.70 equiv) and the reaction time was reduced from 3 d to 24 h. Subsequent site-selective SNAr reaction (17, 18) with morpholine in MeOH proceeded under mild conditions and gave thienopyrimidine 14 in 96% yield and 98% purity by HPLC. This process was then scaled up to >100 g scale in our lab and subsequently at contract manufacturing organizations (CMO) to >10 kg scale. The synthesis continued with the metalation/formylation of thienopyrimidine 14. Thus, 14 was treated with n-BuLi at –70 °C and the mixture was warmed up to –50 °C to achieve complete deprotonation, as ascertained by 1H NMR spectroscopic analysis of aliquots quenched into D2O. Subsequent formylation of the resulting organolithium compound with DMF at –70 °C, followed by quenching into cold aqueous HCl, afforded aldehyde 16 in 87% yield (Scheme 3). 242



Scheme 2. Synthesis of the Thienopyrimidine 14

Scheme 3. First-Generation Route to GDC-0980 The reductive amination of aldehyde 16 with Boc-piperazine was performed in the presence of trimethyl orthoformate as the dehydrating agent. The resulting aryl-chloride 21 proceeded in a Suzuki-Miyaura coupling reaction with the boronate 22 to produce coupled-compound 23. A protecting group was needed for the primary amino group of boronate 22 to improve the solubility of the product 23 and avoid interference during the removal of the residual Pd. Since a Boc protecting group was already placed in aryl-chloride 21, it was convenient to use the same protecting group for boronate 22. The main task on the Suzuki-Miyaura coupling reaction was to replace the microwave conditions used in the medicinal chemistry route. We conducted a brief screening of reaction conditions and identified PdCl2(PPh3)2 and Na2CO3 as the suitable catalyst and base for the reaction. It was proved that 1.20 equiv of the boronate 22 and 0.01 equiv of the Pd catalyst were sufficient to drive the reaction to completion. 1,4-Dioxane was initially used as the solvent, however, 243



1,4-dioxane is not a preferred solvent (19). Therefore, it was desirable to explore other solvents in the reaction. After a survey of several solvents, we identified 2-propanol as a suitable solvent for this reaction. The reaction proceeded faster in 2-propanol/water and was complete in 2–3 h. The use of 2-propanol/water also simplified the workup procedure as the crude product was isolated by simply filtering the slurry after further dilution with more water. When 1,4-dioxane was used as the solvent, a solvent swap from 1,4-dioxane to acetonitrile was needed before the filtration. The crude product typically contained 200–1000 ppm residual Pd and was then treated with Florisil®(2.0 wt) and Thio-Silica® (0.40 wt) in dichloromethane for 16 h to reduce residual Pd to < 20 ppm. The final step of the synthesis incorporated two chemical transformations; the deprotection of the two Boc groups and the amidation with (S)-lactic acid (Scheme 3). Deprotection was readily achieved without any issues by using HCl in ethanol. However, the amidation reaction turned out to be problematic. We explored several coupling agents including 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and an additive such as 1-hydroxy-1,2,3-benzotriazole hydrate (HOBt•H2O) (20). However, no desired product resulted when these coupling reagents were employed. It appeared that the combination of EDC•HCl and HOBt•H2O would work as the reaction proceeded well on lab scale. However, when we performed the reaction at kilogram scale, the reaction did not reach completion after 24 h as 10–20% of intermediate 24 was not consumed. Additional amounts of EDC•HCl and (S)-lactic acid were added to drive the reaction to completion. Another issue was the isolation and purification of the product. HOBt (0.89 wt%) and N,N-diisopropylethylamine (DIPEA) (0.1 wt%) appeared in the crude product, so an acid/base extraction procedure was performed to purge out those two residual reagents. Two major process impurities in the tox lot were identified as the des-lactate 25 at 1.80 % and di-lactate 26 at 3.70 %, (Figure 2). When the crude product was re-slurried in a mixture of methanol (7.5 vol) and THF (2.5 vol), the impurities were reduced to 0.53 % and 1.10 %, respectively, with the overall purity being improved to 97%. However, the overall yield of this step was only 61% due to loss of the product during the above purification process. We anticipated this loss could still be lessened once additional process research was conducted.

Figure 2. Process impurities in final step of first-generation route. 244 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


API Salt Selection, Polymorph Screening, and Particle Size The salt screening of GDC-0980 (measured pKa: 3.1 and 4.7) was conducted and several salts were identified, but all of them including the HCl and phosphate salt were hygroscopic. On the other hand, the free base was not hygroscopic and showed good physical properties. The dog PK data did not show any difference between the free base, the HCl salt, and the phosphate salt. Therefore, the free base was selected for bulk drug preparation. As for the polymorph screening, a total of 15 crystalline forms of GDC0980 have been identified. The initial observed form (anhydrous form A) was the predominant crystalline form identified in the screening and also the most stable one. Thus, anhydrous crystalline form A was selected for development. Investigations have also been made on the particle size and the morphology of GDC-0980. Both factors had significant impact on the flow property of the API, which was a critical parameter for making the powder in capsule (PIC) and the initial Phase I formulation. In the tox lot preparation, the crude product was triturated with 10 volumes of 3:1 methanol:THF. The resulting API had a poor flow property, presumably due to fine particle size (Table 2, entry 1). When the total solvent volume was increased to 25 and the ratio of MeOH/THF was changed from 3:1 to 1:1, the resulting particle size was significantly larger (Table 2, entry 2). Also, SEM confirmed the transformation from small particles to large ones. In Table 2, entry 3, the reference standard lot was prepared through a heating/ cooling crystallization, i.e., the crude GDC-0980 was dissolved in 100 volumes of 1:1 MeOH:THF and the resulting solution was gradually cooled to ambient temperature, followed by filtration to isolate the product with a purity of > 99.0% by HPLC. The reference standard lot showed a good flow property in the formulation process; however, the yield of the crystallization process was low (58%). To improve the yield, a significant amount of solvent (65 vol) was removed and the product was obtained in 86% yield while retaining good flow property (Table 2, entry 4) and acceptable purity. Therefore, this procedure (first-generation crystallization process) was selected for the first GMP production. The SEM results suggested agglomeration of GDC-0980 crystals. Therefore, the API was milled through a Fitzmill to break the agglomerates. While the first-generation synthesis was successfully employed in the preparation of the GLP tox batch and first GMP batch, it suffered from several shortcomings that were not ideal for large-scale implementation:

• • • •

The synthesis was not convergent and also required protection /deprotection steps. One of the starting materials, the boronate 22, was very expensive ($25000/kg). The yield of the final step was low. It was difficult to remove impurities 25 and 26 from the API generated in the final step. 245



Table 2. Crystallization Process and the API Properties (PSD, SEM, and Flow)

Thus, a more convergent and efficient synthesis was needed to produce the large amount of API required for the advanced clinical studies. Second-Generation Route. Since the reductive amination and SuzukiMiyaura coupling reactions performed well in the first-generation synthesis, we elected to utilize both reactions for the second-generation synthesis. We envisioned that GDC-0980 could be assembled in a highly convergent manner via Suzuki-Miyaura coupling of unprotected boronic acid 27 and 2-chloro-thienopyrimidine 28 (Scheme 4). The thienopyrimidine core 28 would then be assembled through metalation and formylation of 14 followed by reductive amination of the resulting aldehyde 16 with piperazine 29 bearing the lactamide moiety. The key feature of the synthesis is to install the lactamide in the new starting material 29, thus avoiding the problematic late stage lactamide formation in the first-generation synthesis.

246 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 4. Retrosynthetic Analysis of the Second-Generation Route

Improved Formylation Process Although metalation of the thiophene ring could be performed with n-BuLi under cryogenic conditions, the instability of the resulting organolithium species precluded its use on large scale. Lithium trialkylmagnesiates have been used successfully in halogen-magnesium exchange (21–23), and for deprotonation of a variety of heterocycles including furans and thiophenes (24–26). Lithium triarylmagnesiates are generally more thermally stable than the corresponding organolithium species, and thus reactions can be performed under non-cryogenic conditions. To our delight, we found that use of n-Bu2i-PrMgLi allowed for deprotonation and formylation under non-cryogenic conditions (–10 °C) and provided aldehyde 16 in 96% yield (Scheme 5, R = CH3). Both the resulting lithium triarylmagnesiate 31 and the components of the reaction mixture (after addition of DMF) were very stable at reaction temperature between −10 °C and –5 °C for an extended time (>15 h). In an optimized procedure, i-PrMgCl and n-BuLi were added sequentially to a solution of 14 in THF at –10 °C. This operationally simple process proved easy to perform on 50 kg scale and obviated the need for a separate vessel to prepare n-Bu3MgLi as reported previously (26). It is noteworthy that the presence of the adjacent methyl group significantly improved the stability of the lithium triarylmagnesiate 31 and thus led to the excellent yield of aldehyde 16. Without the adjacent methyl group (Scheme 5, R = H), the resulting lithium triarylmagnesiate 30 and the components of the reaction mixture (after addition of DMF) were stable at −5 °C for > 6 h, and the desired product 15 was produced in 87% yield. 247



Scheme 5. Non-Cryogenic Conditions for Metalation and Formylation The aldehyde product 16 was isolated by filtration; however, the filtration of the crude mixture was slow. We found that removal of THF was beneficial to the filtration. The filtration was also significantly improved through an ripening process. After ripening for 1–2 h at 50 °C, the filtration was about 10× faster. This is the result of a crystalline form change confirmed by DSC and XPRD. We also noticed that the ripening process afforded larger crystals as indicated by the microscopy data. Under the optimal conditions, the desired product was reproducibly produced in 94‒98% yield. Reductive Amination As previously mentioned, the lactamide moiety was incorporated into the new starting material 29 (Scheme 4); however, piperazine 29 was an oil, so salt formation was required to obtain the material in a preferable solid state. Initially, the HCl salt of 29 was tested in the reaction, but a significant amount of the starting material 16 was not consumed, presumably due to the fact that the HCl salt was deliquescent under normal lab conditions and water was thus brought into the reaction. After screening a variety of acids, we found that the corresponding oxalic acid salt 32 (Scheme 6), was less hygroscopic and thus easier to handle, and it performed well in the reaction (27). Therefore, piperazine lactamide oxalate 32 was chosen as the starting material for the reductive amination. Initially, Na(OAc)3BH was employed as the reducing reagent (Scheme 6). Several solvents including dichloromethane, acetonitrile, methanol, and tetrahydrofuran were examined in the reaction. We observed the best conversion and the least amount of the alcohol impurity 33 when acetonitrile was used as the solvent. A brief survey of bases, which was needed to free base the oxalate 32 in situ, identified sodium acetate as optimal (28). Addition of acetic acid (0.50 equiv) was critical in order to suppress the formation of alcohol impurity 33. This impurity 33 was further controlled by using 1.50 equiv of 32 and adding the Na(OAc)3BH in multiple portions. A dehydrating reagent was needed for the iminium ion formation in the reaction. When HC(OCH3)3 was employed, the outcome of the reaction was not reproducible. In some runs, we observed significant amounts of the unconsumed starting material 16 and alcohol impurity 33. We eventually resolved this problem by the use of molecular sieves, with the reaction proceeding consistently well when powdered 3Å molecular sieves were employed. Use of 100 wt% of sieves was sufficient to suppress the formation of alcohol impurity 33. 248 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 6. Reductive Amination of the Second-Generation Synthesis

Besides the alcohol impurity 33, other impurities were also observed in the reaction mixture (Figure 3). Considerable efforts were made to control these and the downstream impurities. The alcohol impurity 33 would be purged out in the work-up and its derivative in the downstream process would be purged out in the following step. As for other impurities, it was also critical to control them in this step since the impurities derived from these impurities in the following steps would be difficult to remove.

Figure 3. Impurities of the reductive amination. 249 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


It was anticipated that all three impurities 34-36 would be generated from the corresponding impurities in the starting material 32, such as piperazine 37 and acetyl piperazine 38 (Figure 4). Therefore, we set the specification for the starting material 32 with a limit of ≤ 0.20% for each of these two impurities.

Figure 4. Impurities in starting material 32.

Another source for the impurities was the hydrolysis of the product 28 to afford the piperazine impurity 34, which would be further converted to the acetyl impurity 35 under the reaction conditions in the presence of acetic acid. Therefore, the reaction conditions and the workup process were extensively studied. We found out that the amounts of the piperazine impurity 34 and the acetyl piperazine impurity 35 were significantly increased when the reaction mixture was aged at 55–60 °C. It was particularly striking that the amount of the piperazine impurity 34 was increased to 3.38% after the mixture was aged at 60 °C for 23 h. Therefore, the reaction temperature was lowered from 55–60 °C to 35–45 °C so as to suppress 34. We also discovered that the amount of the piperazine impurity 34 was increased 2x from 1.73% to 3.36% at pH 0.4 after 24 h. As such, the acidic aqueous workup should be operated at a higher pH 1.2 while maintaining the temperature at 0–10 °C. The optimal procedure using Na(OAc)3BH has been successfully scaled up to 50 kg; however, it was not desirable to use molecular sieves and add Na(OAc)3BH in multiple portions as solid via a special solid dosing unit or as a slurry. To address these shortcomings, we explored other reducing agents such as sodium borohydride, pyridine•BH3 and 2-picoline•BH3 (29, 30). A significant amount of the alcohol impurity 33 (7–10%) formed when sodium borohydride was used. On the other hand, the reaction with pyridine•BH3 or 2-picoline•BH3 proceeded smoothly and fewer impurities were observed, although the level of alcohol impurity 33 was slightly higher than that when Na(OAc)3BH was used. This prompted us to further investigate the reductive amination reaction using 2-picoline•BH3 (31). We examined the reaction with 2-picoline•BH3 at different temperatures, and in the presence or absence of the dehydrating reagent HC(OCH3)3 (Scheme 7 and Table 3): 250


•

• •

•


•

We were able to achieve the best conversion and the lowest % of the alcohol impurity 33 when the reaction was run in the presence of 10 equiv of HC(OCH3)3 at 50 °C (entry 3). We demonstrated that the addition of 2-picoline•BH3 in 3 portions was effective. We explored other solvents such as ACN, THF, and EtOH in the reaction, but higher amounts of the alcohol impurity 33 were observed (entries 4-6). We determined that 1.20 equiv of 2-picoline•BH3 and 10 equiv of HC(OCH3)3 would be needed to drive the reaction to completion (entries 3, 7, 8 and 10). We were able to charge 2-picoline•BH3 as solutions in either methanol or THF and both reactions performed well (entries 9-10).

Since 2-picoline•BH3 in THF is commercially available, it was selected for large scale production.

Scheme 7. Reductive Amination with 2-Picoline•BH3

2-Picoline•BH3 has several advantages over Na(OAc)3BH: • •

•

The reaction proceeded well in the presence of HC(OCH3)3, so no molecular sieves were needed. 2-Picoline•BH3 could be added as a solution in THF in a continuous mode. As a result, the time required for the addition of the reducing reagent was significantly reduced. The reaction was cleaner and fewer impurities were generated, presumably due to the improved stability of 28 in the presence of 2-picoline.


Table 3. Reductive Amination of 16 and 32 with 2-Picoline•BH3a)


Entry

Temp (°C)

2-Picoline•BH3 (equiv)

Solvent

% of the reaction mixture by HPLC 28

33

16

1

rt

1.50 (solid)

MeOH

84.0

9.6

6.4

2

rt

1.50 (solid)

MeOH

93.0

7.0

0.0

3

50

1.50 (solid)

MeOH

95.1

4.9

0.0

4

50

1.50 (solid)

ACN

84.3

13.6

2.1

5

50

1.50 (solid)

THF

18.6

72.7

8.7

6

50

1.50 (solid)

EtOH

63.0

36.8

0.2

7

50

1.25 (solid)

MeOH

94.5

5.0

0.5

8

50

1.00 (solid)

MeOH

93.8

5.6

0.6

9

50

1.25 (13wt% solution in MeOH)

MeOH

94.9

4.4

0.6

10

50

1.20 (30wt% solution in THF)

MeOH

96.0

3.5

0.5

All the reactions were run with compound 16 (10.0 g, 1.00 equiv) and compound 32 (12.5 g, 1.50 equiv) in 190 mL of solvent for 20 h. The reaction of entry 1 was run without HC(OCH3)3. All other reactions were performed in the presence of 10 equiv of HC(OCH3)3. a)

The reductive amination reaction using 2-picoline•BH3 has been successfully scaled up to 8.75 kg with a reproducible yield of 79‒86% and >99.0% purity by HPLC. Synthesis of Oxalate 32. The enabling synthesis of oxalate 32, illustrated in Scheme 8, employed five steps with an overall yield of 40%. Several steps were utilized for the protection and deprotection of both the hydroxyl group and piperazine. As the project was advancing to late stage process development, we developed a more concise and efficient synthesis. N-Benzylpiperazine Route We initially envisioned a one-pot amidation-deprotection process starting from N-benzylpiperazine with (S)-ethyl lactate as solvent at elevated temperatures (70–100 °C) to produce benzylpiperazine lactate 43 (Scheme 9). However, these reactions proved difficult to reach completion with up to 15% by HPLC of N-benzylpiperazine remaining unconsumed. (S)-Methyl lactate was also investigated, as we reasoned the lower boiling point of the methanol by-product and its distillation could aid the reaction conversion. The reaction also failed to progress to completion and therefore offered no advantages over the cheaper (S)-ethyl lactate. A further issue with these reactions was the formation of the ester impurity 44 in ~ 13% where the hydroxyl group of the desired product was esterified in the presence of the excess (S)-ethyl lactate. 252



Scheme 8. The Enabling Synthesis of Oxalate 32

Scheme 9. Amidation of (S)-ethyl Lactate and N-Benzylpiperazine Next, in an attempt to eliminate the formation of the ester impurity 44, we decided to concentrate our efforts on reaction conditions that could be carried out at ambient temperature. The amidation between amines and (S)-ethyl lactate in the presence of an alkoxide base has previously been reported (32–34). Thus, we explored this method in the synthesis of oxalate 32 (Scheme 10).

Scheme 10. Benzylpiperazine route to oxalate 32 253 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Several alkoxide bases were examined in the amidation reaction (Table 4). We selected NaOMe (25wt% in MeOH) (entry 1) as the base since significant erosion of enantiomeric ratios (er) resulted when other bases were employed (entries 2-4).

Table 4. Effect of Alkoxide Bases on Enantiomeric Ratio of the Amidation Reactiona)


Entry

Base (0.15 equiv.)

(S)-Ethyl lactate (equiv)

HPLC assay yield (%)

Enantiomeric ratio (er)

1

NaOMe 25wt% solution in MeOH

1.03

34

99.3:0.7

2

t-BuONa (solid)

3.00

90

85.0:15.0

3

t-PentONa 1.4 M solution in THF

1.03

61

96.7:3.3

4

NaOEt 21wt% solution in EtOH

1.03

37

98.9:1.1

The reaction was run with N-benzylpiperazine (10.0 g, 1.00 equiv), (S)-ethyl lactate (20.1 g, 3.00 equiv) and base (0.15 equiv) at ambient temperature for 23 h.

a)

Table 5. Optimization of N-Benzylpiperazine Reactiona) Entry

NaOMe (25wt% in MeOH) (equiv)

(S)-Ethyl lactate (equiv)

Assay yield of 43

Remaining N-benzylpiperazine (wt%)

Enantiomeric ratio

1

0.25

3.00

73

9.0

99.0:1.0

2

0.50

3.00

90

2.0

99.1:0.9

3

0.75

3.00

91

0.9

99.2:0.8

4

0.75

2.00

64

1.4

99.1:0.9

5

0.75

2.50

70

1.5

99.2:0.8

6

0.75

3.50

82

0.9

99.2:0.8

7

0.75

4.00

84

1.0

99.2:0.8

The reaction was run with N-benzylpiperazine (10.0 g, 1.00 equiv), (S)-ethyl lactate (2.50–4.00 equiv) and base (0.25–0.75 equiv) at ambient temperature for 20 h.

a)



Further optimization of the reaction conditions was focused on the amount of base and (S)-ethyl lactate (Table 5). 0.75 Equivalents of the base was required to drive the reaction to completion and control the residual N-benzyl piperazine at < 1.0% (entries 1−3). On the other hand, excess of (S)-ethyl lactate (3.00 equiv) was needed to fully consume the N-benzyl piperazine (entries 3-7). Thus, the reaction conditions selected were N-benzylpiperazine (limiting reagent), (S)-ethyl lactate (3.00 equiv) and sodium methoxide (25 wt% in methanol) (0.75 equiv) at ambient temperature (entry 3). Monitoring the reaction progress hourly by HPLC showed that >7 h of reaction time was required. The reaction was complete at ambient temperature in ~ 20 h, affording less than 1% of residual N-benzylpiperazine and an enantiomeric ratio of 99.2:0.8. The effect of temperature on the reaction was also investigated. At both 40 °C and 70 °C, the reaction did progress at a faster rate, achieving 70% when 3.0 equiv of water was added as the result of the conversion of the bis-lactamide 45 to the desired product 29 presumably by hydrolysis (35). Our next task was to isolate the product from the reaction mixture. We thus needed to purge out the residual sodium salt, present as sodium ethoxide and /or sodium methoxide, residual piperazine and (S)-ethyl lactate, as well as the by-products, the bis-lactamide 45 and lactic acid, the latter presumably being generated from the hydrolysis of (S)-ethyl lactate and the bis-lactamide 45. Since piperazine 29 is soluble in water, an aqueous work up was not an option to remove the residual salts. Our initial attempt was to use an Amberlite IRC-748 resin treatment to remove sodium salts. However, we observed significant loss of product on the resin (~ 20%), even when using the minimum amount of resin (0.80 equiv) that was required. We next investigated formation of a sodium salt as a way to remove residual sodium. We chose oxalic acid since the final product 32 is an oxalate. After exploring different amounts of oxalic acid, we discovered that 0.25 equiv of oxalic acid was able to generate the di-sodium oxalate salt which is insoluble in ethanol and could therefore be readily removed by filtration (36). To the resulting filtrate, additional oxalic acid was then added to adjust the pH to 7–7.5. The residual piperazine was precipitated out as the oxalate salt and subsequently removed through a filtration while the product 29 remained in the mother liquor. Direct treatment of the filtrate with excess oxalic acid (1.12 equiv) afforded the desired oxalate 32 in 59% isolated yield and > 99% purity. The piperazine route to oxalate 32 is significantly more efficient than the enabling synthesis with a 55% reduction in process mass intensity (PMI) (37). We were also able to achieve a 53% reduction in total solvent volume used in the process. More significantly, the undesirable solvent, dichloromethane, heavily used in three of the five steps of the enabling synthesis, was replaced with preferred solvents and a small amount of a usable solvent, THF (4.3%) being used (38). 257


Another improvement for the piperazine route is a 41% increase in atom economy as the result of the elimination of the non-value adding steps (installation and removal of the protecting group), and thus producing oxalate 32 in a single chemical step and a protecting group-free synthesis (39). Overall, the piperazine route is more concise, cleaner and safer, and ready for scale-up.


Aminoalkylation Approach The reductive amination reaction performed well; however, there were concerns about the alcohol impurity 33 which was carried in the downstream chemistry resulting in formation of additional impurities that were difficult to remove. We therefore explored an alternative route involving aminoalkylation to avoid the formation of the alcohol impurity 33. As shown in Scheme 13, we envisioned that the aminoalkylation could be performed by direct addition of lithium triarylmagnesiate 31 to iminium salt 46 to produce intermediate 18. The same strategy could potentially be employed for the synthesis of 17, an intermediate in the synthesis of GDC-0941 (Scheme 13).

Scheme 13. Synthesis of 17 and 18 by Aminoalkylation

Our investigation on the aminoalkylation commenced with the preparation of the iminium salt 47. As shown in Scheme 14, the iminium salt 47 was generated from the aminal 49 or aminol ether 50 (40, 41). The resulting iminium salt was then subjected to reaction with lithium triarylmagnesiate 30 to afford the desired product 17. However, a significant amount of the starting material 48 was observed in the crude product, possibly due to the impurities present in the iminium salt (42). 258 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 14. Synthesis of Intermediate 17 via the Iminium Salt In another approach the iminium salt was also generated in situ by treating the aminol ether 50 with a Lewis acid (Scheme 15), followed by addition of the lithium triarylmagnesiate 30 which was generated using non-cryogenic conditions. We identified ZnCl2 as the preferred Lewis acid with the desired product being obtained in ~ 80% yield.

Scheme 15. Synthesis of 17 from Iminium Salt Generated in Situ from Aminol Ether To further improve the aminoalkylation process, our efforts were then turned towards the benzotriazole substrates that have been widely used in the aminoalkylation reactions (43–45). Treatment of 48 with benzotriazole, paraformadehyde and MeOH in the presence of KHCO3 afforded benzotriazolyl-piperazine 51 in 90% yield after isolation by simple filtration 259 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


(Scheme 16). Unlike the aminol ether 50, compound 51 is not hygroscopic and can be isolated as a bench-stable solid. Treatment of compound 51 with ZnCl2 followed by addition to a solution of lithium triarylmagnesiate 30 afforded the desired product 17 in 93% yield.

Scheme 16. Aminoalkylation via Benzotriazolyl-Piperazine 51

This route achieved a slightly higher yield than the reductive amination route (Scheme 17) and did not generate the corresponding alcohol impurity 52. The synthesis has been demonstrated on kilogram scale. Although a large excess of ZnCl2 (4.00 equiv) was needed, the aminoalkylation route offered a complementary process to the reductive amination route which has been scaled up to > 35 kg.

Scheme 17. Synthesis of 17 by the Reductive Amination Route 260 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

As for the synthesis of GDC-0980, we also explored the aminoalkylation strategy for the synthesis of intermediate 28 using the optimal conditions for the synthesis of intermediate 17 shown in Scheme 16; however, the strategy did not work for intermediate 18 as the reaction suffered with low conversion, presumably due to presence of the hydroxyl group of piperazine lactate 32. Therefore, the reductive amination process was selected for the synthesis of intermediate 28 (Scheme 7).


Suzuki-Miyaura Cross-Coupling With intermediate 28 in hand, our attention was then turned to the final carboncarbon bond-forming step of the synthesis of GDC-0980, the Suzuki-Miyaura cross-coupling reaction (Scheme 18). We used the boronic acid 27 to replace the expensive boronate 22 employed in the first-generation route. Unlike the firstgeneration synthesis, no protecting group was employed for boronic acid 27 which was prepared directly from 2-amino-5-bromopyrimidine in a one-step synthesis (46).

Scheme 18. Suzuki-Miyaura Cross-Coupling Reaction of the Second-Generation Synthesis

After a brief screening of solvents (2-propanol, 2-propanol/water, acetonitrile and 1-propanol), we identified 1-propanol as the reaction solvent due to the better solubility of the product GDC-0980 and the solvent’s higher boiling point which allowed us to run the reaction at a higher temperature. We also evaluated a variety of bases in the reaction when the corresponding boronate ester 22 was used. We found that a significant amount of amide hydrolysis byproduct des-lactate 25 (7–17%, Figure 2) was generated when either Na2CO3 or Cs2CO3 was used. To our delight, the use of K3PO4 as the base has significantly reduced the formation of the des-lactate 25 in the reaction with the boronate ester 22 or when we later made the switch to the corresponding boronic acid 27. Further optimization showed that only 1.18 equiv of boronic acid 27 were sufficient to achieve complete conversion of 28 to GDC-0980. 261 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Expensive scavengers were employed in the first-generation route for the removal of the residual Pd, which would not be practical on large scale. To resolve this issue, we first reduced the Pd catalyst loading from 0.25 mol% to 0.15 mol% as the result of a faster reaction at higher temperature with 1-propanol as the solvent. Further, we eliminated the use of scavengers by filtering the crude GDC-0980 solution in 1-propanol and water through an activated carbon cartridge, and the resulting solution was concentrated followed by filtration to afford the crude product which contained 20-40 ppm of the residual Pd. The crude product contained three major impurities: • • •

des-lactate impurity 25 (Figure 2) in ~ 0.60%. homo-coupling impurity 53 (Figure 5) in ~ 0.65%. alcohol impurity 54 (Figure 5) in ~ 0.25%, stemming from alcohol impurity 33 in the precursor.

The levels of those impurities were reduced to < 0.20% after the crude product went through a recrystallization process from 25:75 w/w mixture of 1-propanol and water, and the product was produced on ~ 10 kg scale reproducibly in 79‒83% yield.

Figure 5. Homo-coupling Impurity 53 and Alcohol Impurity 54.

API Recrystallization The recrystallization process of the first-generation synthesis employed large volumes of methanol and THF (total 100 vol) in order to dissolve the API, and a distillation was subsequently required to remove the excess solvent to improve the yield. To develop a more efficient recrystallization process, we started with the investigation of the solubility of GDC-0980. We found that GDC-0980 has relatively low solubility (< 1 mg/mL) in organic solvents; however, the API did show good solubility in a mixture of water and 1-propanol, with maximum solubility in 40:60 w/w (Figure 6). The solubility and super-saturation curves in this solvent composition are displayed in Figure 7 262 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 6. Solubility of various concentrations of GDC-0980 in water/1-propanol.

Figure 7. Clear and cloud curves of GDC-0980 in water/1-propanol (40:60 w/w). 263 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The boiling point of the water/1-propanol mixture (40:60 w/w) was 87–88 °C. As shown in Figure 7, the maximum achievable solubility is ~14 wt% under atmospheric pressure. For practical purposes and to avoid the possible precipitation of API during the polish filtration, we set the concentration for dissolution at a slightly lower level (11.7wt%), affording a final concentration of 10 wt% after the polish filtration and rinses. The resulting solution was then cooled slowly to ‒10 °C while spontaneous nucleation started at a temperature between 60 °C and 65 °C. The solid product was collected by filtration and dried to afford the API. The implementation of this new API recrystallization process has consistently produced the desired crystalline form and particle size distribution as well as it further depleted the levels of residual Pd and process impurities. The purity of the API was >99.0% by HPLC and all identified impurities were within the specifications and all unidentified impurities < 0.15% (Scheme 19). The residual Pd was < 10 ppm. The recrystallization process has been scaled up reproducibly in multiple batches (13.9 kg to 15.7 kg) in 89 ̶996% yields.

Scheme 19. API Crystallization

Summary Two clinical compounds, GDC-0941(pictilisib) and GDC-0980 (apitolisib), have been discovered. While both compounds inhibit the Class I PI3Ks, GDC-0980 also inhibits the kinase mTOR. Chemical process development has been conducted to support both compounds in the clinical study. For GDC-0980, the first-generation route was employed to produce API for the GLP tox and the initial clinical study while the second-generation route (Scheme 20) was developed as the project advanced to the late stage of the clinical study. The second-generation route was convergent, efficient and robust. The metalation was performed under non-cryogenic conditions via triarylmagnesiate intermediates. 2-Picoline•BH3 was employed to replace Na(OAc)3BH in the reductive amination and to eliminate the use of molecular sieves. A concise one-step synthesis was developed for the piperazine lactamide starting material through the selective mono-amidation of piperazine with (S)-ethyl lactate. The second-generation route has been demonstrated on >10 kg scale affording GDC-0980 in 59% overall yield in four steps and >99.0% purity by HPLC. 264



Scheme 20. The Second-Generation Route to GDC-0980

Employing a similar synthetic strategy, we have developed a practical synthesis for GDC-0941 (Scheme 21). Non-cryogenic conditions were employed in the formylation of 13 via a triarylmagnesiate intermediate. The synthesis of the key intermediate 17 was also achieved through an aminoalkylation approach, a complementary process to the reductive amination. The THP deprotection and salt formation were combined in one operation in the final step to afford GDC-0941 in >99.0% purity by HPLC and 54% overall yield over four steps.



Scheme 21. A Practical Synthesis of GDC-0941

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Discovery and Process Development of Class I PI3K and Class I

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