mTOR Inhibitor

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A Practical, Protecting Group Free Synthesis of a PI3K/mTOR Inhibitor Qingping Tian, Ursula Hoffmann, Theresa Humphries, Zhigang Cheng, Pirmin Hidber, Herbert Yajima, Maud Guillemot-Plass, Jane Li, Ulrike Bromberger, Srinivasan Babu, David Askin, and Francis Gosselin Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op500366s • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 17, 2015

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Organic Process Research & Development

A Practical, Protecting Group Free Synthesis of a PI3K/mTOR Inhibitor Qingping Tian,*+ Ursula Hoffmann, # Theresa Humphries, + Zhigang Cheng, + Pirmin Hidber, # Herbert Yajima, + Maud Guillemot-Plass#, Jane Li,+ Ulrike Bromberger,# Srinivasan Babu+, David Askin+ and Francis Gosselin+ +

Small Molecule Process Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA

94080 #

Pharma Technical Development PTDCA, F. Hoffmann-La Roche AG, Grenzacherstrasse 124,

CH-4070 Basel, Switzerland *Corresponding author: [email protected] Table of contents graphic

ACS Paragon Plus Environment

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ABSTRACT We report a practical and protecting group free synthesis amenable to produce multikilogram amounts of PI3K/mTOR inhibitor GDC-0980. The route employed metalation / formylation and reductive amination followed by a metal catalyzed Suzuki-Miyaura cross-coupling.

The

metalation was performed via triarylmagnesiate intermediates allowing formylation under noncryogenic conditions. 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 selective mono-amidation of piperazine with (S)-lactate to produce the piperazine lactamide starting material. The boronic acid was produced from 2-amino-5bromopyrimidine in a one step and protecting group free approach. The final crystallization in 1propanol and water afforded API in 59% overall yield in 4 steps and > 99% purity by HPLC. Introduction The PI3K/Akt/mTOR pathway plays a central role in cell proliferation, survival, migration and metabolism. The lipid kinases of the PI3K family are critical for the phosphorylation of the 3'hydroxyl group of phosphatidylinositols, leading to the activation of the serine / threonine protein kinase Akt and further downstream oncogenes.1 The PI3K pathway is one of the most frequently activated pathways in tumors, with mutations in one of its components detected in a significant percentage of human cancers.2 The kinase mTOR is activated downstream of Akt and leads to increased protein synthesis and growth.3 Thus, considerable attention has been drawn to the development of compounds that inhibit both kinases.4 GDC-0980 is a novel small molecule PI3K/mTOR inhibitor pursued at Genentech as an anticancer agent (Figure 1).5 To support the safety assessment, pharmaceutical development, and clinical studies, we have developed a practical synthesis suitable for the preparation of the API on large scale.


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Figure 1. Structure of PI3K/mTOR inhibitor GDC-0980 First-Generation Route to the API.6

The first-generation route commenced with the

metalation/formylation of thienopyrimidine 1 which is prepared from commercially available methyl 3-amino-4-methylthiophene 2-carboxylate drawing inspiration from a similar sequence we developed for another project (Scheme 1).7 Treatment of thienopyrimidine 1 with n-BuLi at – 70 °C, followed by warming to –50 °C achieved complete deprotonation as confirmed 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 2 in 87% yield.


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Scheme 1. First-Generation Route to GDC-0980 O

O

S

N N

Me

N

1) n-BuLi, -70 o C

N

O

2) DMF, -70 oC 3) 0.25 M aq. HCl 87%

Cl

1

S

H

N N

Cl

Me

O S

2

+

B

Na(OAc) 3BH 88%

3 O

PdCl2 (PPh3 )2 /Na 2CO3 2-Propanol/H 2O

N N

Florisil/Thio-Silica

NHBoc 4

N

S

N N

N

Cl

Me

N

O

N

N

90%

Me 1) L-(+)-Lactic Acid HOBt H 2O, EDC HCl HO DIPEA, CH 2Cl2

N

HCl S

N 2) MeOH/THF slurry

EtOH

N Me 7

O

O

N

N N

S

N N

NHBoc

6

O

HN

N

Me

5

HCl

N

N H

Boc N

N N

HC(OCH3 )3 HOAc

+

O

Boc N

Boc N

NH 2

61%

N N

N

Me N GDC-0980

NH2

The reductive amination of aldehyde 2 with Boc-piperazine 3 was performed using trimethyl orthoformate as the dehydrating agent.

Next, the Suzuki-Miyaura coupling reaction of

intermediate 4 and the boronate 5 led to intermediate 6. A protecting group was needed for the boronate 5 to improve the solubility of the Suzuki-Miyaura coupling product 6 and facilitate the removal of the residual Pd. As such, a common Boc protection scheme was introduced for the boronate 5. After a brief screening of reaction conditions, PdCl2(PPh3)2 and Na2CO3 were identified as the suitable catalyst and base for the reaction and 1.2 equiv of the boronate and 0.01 equiv of the Pd catalyst were sufficient to drive the reaction to completion. 1,4-Dioxane, a ICH Class 2 solvent, was initially used as the solvent. After a brief survey of solvents, 2-propanol was identified as the suitable alternative solvent for this reaction. The reaction proceeded faster


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in 2-propanol/water and was complete in 2–3 h. The crude product typically contained 200– 1000 ppm residual Pd which 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 featured two chemical transformations; the deprotection of the two Boc groups and the amidation with L-(+)-lactic acid as shown in Scheme 1. Deprotection was readily achieved without any issues by using HCl in ethanol. However, the amidation reaction turned out to be problematic. Coupling agents, including 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT),

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC•HCl)

and

additive such as 1-hydroxy-1,2,3-benzotriazole hydrate (HOBt•H2O), have been explored;8 however, no desired product was observed when these coupling reagents were employed individually in the reaction. 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 on kilogram scale, it failed to reach completion after 24 h, with 10–20% of intermediate 7 remaining unconsumed, and required additional amounts of EDC•HCl and L-(+)-lactic acid to drive the reaction to completion. Another issue for the coupling step was the formation of impurities, with the major two being identified as the des-lactate 8 and di-lactate 9 (Figure 2), found at 1.8A% and 3.7A% levels by HPLC, respectively. The crude product was then re-slurried in a mixture of methanol (7.5 vol) and THF (2.5 vol) and the impurities were reduced to 0.53A% and 1.1A%, respectively, with the overall purity being improved to 97A%.

Residual HOBt (0.89wt%) and N,N-

diisopropylethylamine (DIPEA) (0.1wt%) were also observed in the crude product, but these could be readily purged by an acid/base extraction procedure; however, the overall yield of this step was only 61% due to loss of the product during the above purification procedures.


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O HN

N N

S

Me

O

HO

N

O N N

N N

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S

N N

N

N

O

Me

Me N Des-lactate 8

N

NH2

N H

Me OH

Di-lactate 9

Figure 2. Process Impurities in Final Step of First-Generation Route While the first-generation synthesis was successful for the production of the GLP tox batch and initial GMP batches, it suffered from several shortcomings that were not ideal for large-scale implementation. The synthesis was not convergent and also required protection/deprotection steps. In addition, the yield of the final step was low. Thus, a more convergent and efficient synthesis was needed to produce large amount of API for the advanced clinical studies.9 Second-Generation Route.

Since the reductive amination and Suzuki-Miyaura coupling

reactions performed well in the first-generation synthesis, we elected to keep both for future syntheses but strive to overcome the existing problems. As such, we envisioned that the API could be assembled in a highly convergent manner via Suzuki-Miyaura coupling of unprotected boronic acid 10 and

2-chloro-thienopyrimidine 11 (Scheme 2). After the metalation and

formylation of thienopyrimidine 1, piperazine lactamide 12 would be appended via the reductive amination.

By a simple reordering of steps, this would avoid the problematic late-stage

lactamide formation encountered in the first-generation synthesis.


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Scheme 2. Retrosynthetic Analysis of the Second-Generation Route

Improved Formylation Process. The cryogenic conditions employed in the first generation route were not ideal for large scale synthesis. We recently reported that deprotonation of a thienopyrimidine heterocycle could be achieved under non-cryogenic conditions through use of a lithium trialkylmagnesiate.7,10

To our delight, we were able to also use this method for

thienopyrimidine 1. It is noteworthy that the presence of the adjacent methyl group significantly improved the stability of the lithium triarylmagnesiate 13 and thus led to the excellent yield of aldehyde 2 (Scheme 3).11 Scheme 3. Non-cryogenic Conditions for Metalation and Formylation

Isolation of the aldehyde product proved challenging because of slow filtration of the crude mixture. We found that removal of THF was beneficial to the filtration. The filtration rate was also significantly improved through an ripening process. After aging for 1–2 h at 50 °C, the filtration was about 10× faster as the specific cake resistance was reduced from 1.3×1011 m/kg to


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4.2×109 m/kg. This is the result of a crystal 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 12 (Scheme 2). Initially, the HCl salt of 12 was tested in the reaction,12 but a significant amount of the starting material 2 did not react, presumably due to the fact that the HCl salt was deliquescent under normal lab conditions and introduced water into the reaction. After screening a variety of acids, we found that the corresponding oxalic acid salt 14, was wellbehaved, much less hygroscopic and thus easier to handle.13 Therefore, piperazine lactamide oxalate 14 was chosen as the starting material for the reductive amination (Scheme 4). Scheme 4. Reductive Amination of the Second-Generation Synthesis Method A: 1) NaOAc, HOAc MS 3Å, ACN O 2) Na(OAc) 3BH OH 75%

O N O

S

H

N N

NH HO

Me N

+ HO Cl

O

HO

N

O

O

Me 2

Me

14

Method B: 1) NaOAc, HOAc MeOH, HC(OCH 3 )3 2) 2-Picoline BH 3 82%

O

O

N

N N

S

+ HO

N N

Me 11

Cl

S

N N

Cl

Me 15

Initially, Na(OAc)3BH was employed as the reducing reagent (Scheme 4, Method A). With acetonitrile as solvent, we observed the best conversion and the least amount of the alcohol impurity 15. After a brief survey of bases, which is required to form the free base of oxalate 14 in situ, we identified sodium acetate as optimal.14 We also found that addition of acetic acid (0.5 equiv) was critical in order to suppress the formation of alcohol impurity 15. This impurity 15 was further controlled by using 1.5 equiv of 14 and adding the Na(OAc)3BH in multiple portions.


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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, significant amounts of the unconsumed starting material 2 and alcohol impurity 15 were observed. 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 suppressed the formation of alcohol impurity 15. Although the reductive amination process using Na(OAc)3BH has been successfully scaled up to 50 kg, it was not desirable to use sieves and add Na(OAc)3BH in multiple portions. Na(OAc)3BH needs to be added as solid via a special solid dosing unit or as a slurry. To address these shortcomings, other reducing agents such as sodium borohydride, pyridine•BH3 and 2picoline•BH3 were explored in the reaction.15 A significant amount of the alcohol impurity 15 (7– 10%) occurred when sodium borohydride was used. On the other hand, the reaction with pyridine•BH3 or 2-picoline•BH3 proceeded smoothly and fewer impurities formed, although the level of alcohol impurity 15 was slightly higher than that when Na(OAc)3BH was used.16 We examined the reactions with 2-picoline•BH3 at different temperatures, and in the presence or absence of the dehydrating reagent HC(OCH3)3 (Scheme 4, Method B and Table 1). We were able to achieve the best conversion and the lowest A% of the alcohol impurity 15 when the reaction was run in the presence of HC(OCH3)3 at 50 oC (entry 3). It was also shown that addition of 2-picoline•BH3 in 3 portions was effective. Other solvents such as ACN, THF, and EtOH were examined leading to higher amounts of the alcohol impurity 15 (entries 4 ̶ 6). We determined that 1.2 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). Moreover, we were able to charge 2picoline•BH3 as solutions in methanol and THF eliminating the former problem of solids


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addition (entries 9 ̶ 10). Since 2-picoline•BH3 in THF is commercially available, it was selected for large scale production. Table 1. Optimization of the Reaction with 2-Picoline•BH3a)

Entry

Temp (°C)

2-Picoline•BH3 (equiv)

Solvent

1

rt

1.50 (solid)

2

rt

3

A% of the reaction mixture by HPLC 11

15

2

MeOH

84.0

9.6

6.4

1.50 (solid)

MeOH

93.0

7.0

0.0

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

a)

The reaction of entry 1 was run without HC(OCH3)3. All other reactions were operated in the presence of 10 equiv of HC(OCH3)3.

The reductive amination reaction using 2-picoline•BH3 produced cleaner intermediate compared to the preceding process and has been successfully scaled up to 8.75 kg with a reproducible yield of 79‒86%. Synthesis of Oxalate 14. The initial synthesis of oxalate 14, illustrated in Scheme 5, employed 5 steps with an overall yield of 40% (Scheme 5). Several steps were utilized for the protection and


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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. Scheme 5. The Initial Synthesis of Oxalate 14

(1) 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 oC); however, these reactions did not reach completion. (S)-Methyl lactate was also investigated, as we reasoned the lower boiling point of the methanol by-product could aid the reaction conversion, but this 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 21 in ~ 13A% (Figure 3).

Figure 3. Ester By-product 21 Next, 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.17 Thus, we explored several alkoxide bases and


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selected NaOMe (25wt% in MeOH) since erosion of enantiomeric ratio (er) was observed when other bases were employed (Table 2 and Scheme 6). Table 2. Effect of Alkoxide Bases on Enantiomeric Ratio of the Amidation Reaction Entry

Base (0.15 equiv)

(S)-Ethyl lactate (equiv)

HPLC assay yield (%)

Enantiomeric ratio (er)

1

t-BuONa (solid)

3.00

90

85.0:15.0

2

t-PentONa 1.4 M solution in THF

1.03

61

96.7:3.3

3

NaOEt 21wt% solution in EtOH

1.03

37

98.9:1.1

4

NaOMe 25wt% solution in MeOH

1.03

34

99.3:0.7

Scheme 6. Benzylpiperazine Route to Oxalate 14

Optimization of the reaction conditions were focused on the amount of base and (S)-ethyl lactate. We identified the optimal conditions to be NaOMe (25wt% in MeOH) (0.75 equiv) and (S)-ethyl lactate (3 equiv). 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. We also


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investigated the effect of temperature on the reaction and found that at elevated temperature (40 °C and 70 °C), the reaction did progress at a faster rate, achieving 99% purity. The piperazine route to oxalate 14 is significantly more efficient with a 55% reduction in process mass intensity (PMI).21a Not only was the volume of waste dramatically reduced, but more importantly, we were able to obviate the use of the hazardous and toxic reagents used in the enabling synthesis.

We were also able to achieve 53% reduction in total solvent used in the

process. More significantly, the undesirable solvent, dichloromethane, heavily used in 3 of the 5 steps of the enabling synthesis, was replaced with preferred solvents and a small amount of THF (4.3%).21b 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, and thus producing oxalate 14 in a single chemical step and a protecting group free synthesis.21c Overall, the piperazine route is more concise, cleaner and safer.


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Suzuki-Miyaura Cross-Coupling. We next turned our attention to the Suzuki-Miyaura crosscoupling reaction (Scheme 8). As previously illustrated in Scheme 2, a boronic acid 1022 was used to replace the expensive boronate 5 employed in the first-generation route. But unlike the first-generation synthesis, no protecting group was employed for boronic acid 10. In addition, boronic acid 10 was prepared directly from 2-amino-5-bromopyrimidine in a one-step and protecting group free synthesis.23 Scheme 8. Suzuki-Miyaura Cross-Coupling Reaction of the Second-Generation Synthesis

Me

O

HO

N

Me

O

HO

N

O

O B(OH) 2 N N

S

+

N N

Cl

N

N NH 2

Me 11

PdCl2(PPh 3) 2 K3PO4 , n-PrOH

N N

S

N

85 o C N 81%

N

Me N

10

NH 2

crude GDC-0980

Several solvents including 2-propanol, 1-propanol and acetonitrile were evaluated for the reaction. 1-Propanol was chosen as the reaction solvent due to the better solubility of the product GDC-0980 and its higher boiling point which allowed us to run the reaction at a higher temperature. A variety of bases had been investigated in the reaction when boronate ester 5 was used. We found that in the presence of Na2CO3 or Cs2CO3, a significant amount of amide hydrolysis byproduct des-lactate 8 (7–17%, Figure 2) was generated.

To our delight, with

K3PO4 no des-lactate 8 was detected in the reaction with the boronate ester 5 or when we later made the switch to the corresponding boronic acid 10. Different amounts of boronic acid were examined in the reaction. We found that 1.18 equiv of boronic acid 10 were sufficient for the reaction.


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Expensive scavengers were employed for the residual Pd removal in the first-generation route, which would not be practical on large scale. To resolve this issue, we reduced the Pd catalyst loading to 0.15 mol% permitted by the faster reaction at higher temperature with 1-propanol as the solvent, in combination with an activated carbon cartridge filtration of crude API solution in 1-propanol and water, to reduce residual Pd to 20–40 ppm. Three major impurities, des-lactate impurity 8 (Figure 2) in ~ 0.60A%, homo-coupling impurity 25 (Figure 4) in ~ 0.65A% and alcohol impurity 26 (Figure 4) in ~ 0.25A%, stemming from alcohol impurity 15 in the precursor, were generated in the reaction.

The removal of these

impurities proved challenging in particular for 25. We found that all three impurities could be depleted to < 0.20A% through a crystallization process in 25:75 w/w mixture of 1-propanol and water. The product was produced on 16 kg scale reproducibly in 79‒83% yield.

Figure 4. Homo-coupling Impurity 25 and Alcohol Impurity 26 API Crystallization. We developed the API crystallization to further deplete residual Pd and process impurities and achieve the desired crystalline form and particle size distribution. We found that the API has relatively low solubility 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 5). The solubility and super-saturation curves in this solvent composition are displayed in Figure 6.


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140 5wt% API

Clear Point Temperature (°C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7wt% API

120 11wt% API

100

80

60 0

10

20

30

40

50

60

70

80

Water in 1-Propanol (wt%)

Figure 5. Solubility Curves of Various Concentrations of GDC-0980 as a Function of Water/1-Propanol Composition


19


17 clear point cloud point

15

13 boiling point at normal pressure

Solution Concentration wt%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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11

9

7

5 45

50

55

60

65

70

75

80

85

Figure 6. Clear and Cloud Curves of GDC-0980 in Water/1-Propanol (40:60 w/w) The boiling point of the water/1-propanol mixture (40:60 w/w) was 87–88 °C. Under normal pressure the maximum achievable solubility is ~14 wt% as shown in Figure 6. For practical purposes and to ensure that the API would not spontaneously precipitate out during the polish filtration, we set the concentration for dissolution at 11.7wt%, resulting in a final concentration of 10wt% after the polish filtration and rinses. The solution was then cooled slowly to ‒10 oC while spontaneous nucleation started at a temperature between 60 °C and 65 °C. The suspension was filtered and dried to afford API of > 99.0% by HPLC, all identified impurities within the specifications and all unidentified impurities < 0.15A% (Scheme 9). The crystallization process has been scaled up reproducibly in multiple batches (13.9 kg to 15.7 kg) in 89 ̶ 96% yields. Scheme 9. API Crystallization


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Me HO

O

O

N

1) n-PrOH/Water 88 oC

N N

S

N

2) Cool to -10 oC

N

N

92%

Me N

NH2

Me

O

HO

N

O N N

S

N N

N

Me N

crude GDC-0980

GDC-0980

NH2

Conclusion We have developed a practical and protecting group free synthesis for GDC-0980 (Scheme 10). Noncryogenic conditions were employed in the formylation of 1 via a triarylmagnesiate intermediate.

The synthesis of the key intermediate 11 was achieved through a reductive

amination in the presence of 2-picoline•BH3. A concise one-step synthesis was developed for the direct mono-lactamidation of piperazine with (S)-lactate to afford the key starting material, oxalate 14.

The Boc protected boronate 5 was replaced with boronic acid 10 which was

prepared in one step directly from 2-amino-5-bromopyrimidine obviating the use of a protecting group. We developed the crystallization process to produce the API in 59% overall yield in four steps and >99A% HPLC purity on 10 kg scale.


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Scheme 10. Manufacturing Scale Synthesis of GDC-0980

Experimental General. All reactions were performed under a nitrogen (or Argon) atmosphere. Melting points were measured by differential scanning calorimetry (DSC). HPLC method for purity and assay analysis are listed below. HPLC method for compounds 1 and 2: column, Water XBridge Phenyl (150 mm × 4.6 mm, 3.5 µm); temperature: 10 oC; mobile phase A, water; mobile phase B, CH3CN; gradient (17 min) 60:40 A/B to 40:60 A/B over 10 min, then change to 20:80 A/B over 2 min, then change to 60:40 A/B in 0.1min, hold for 5 min; flow rate = 1.2 mL/min; detection, 210 nm; injection volume, 10 µL; tR of 1 = 6.12 min, tR of 2 = 6.95 min.


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HPLC method for compounds 14: column, Sielc Primesep 100 C18 (4.6 mm × 150 mm, 5 um); temperature: 40 oC; mobile phase A, 0.5% H3PO4 in water; mobile phase B, CH3CN; gradient (18 min) 50:50 A/B for 8 min, then change to 10:90 A/B over 4 min, hold for 3 min, then change to 95/5 A/B in 0.1 min, hold for 3 min, then change to 50/50 A/B in 0.1 min; flow rate = 1.2 mL/min; detection, 210 nm; injection volume, 5 µL; tR of 14 = 7.0 min. HPLC method for compound 11: column, Water XBridge C18 (150 mm × 4.6 mm, 3.5 µm); temperature: 40 oC; mobile phase A, water, 0.1% (v/v) 25% aqueous NH4OH; mobile phase B, CH3CN, 0.1% (v/v) 25% aqueous NH4OH; gradient (43 min) 95:5 A/B to 80:20 A/B over 5 min, then change to 71:29 A/B over 20 min, then change to 16:84 A/B over 8 min, then hold for 5 min, then change to 95/5 A/B in 0.1 min, hold for 5 min; flow rate = 1.4 mL/min; detection, 270 nm; injection volume, 10 µL, tR of 11 = 25.0 min, tR of 2 = 27.5 min. HPLC method for GDC-0980: column, Water XBridge C18 (150 mm × 4.6 mm, 3.5 µm); temperature: 40 oC; mobile phase A, 1000 ml of water, 560 µL of 25% aqueous NH4OH, pH 9.2 (adjusted with HOAc); mobile phase B, CH3CN; gradient (43 min) 95:5 A/B to 80:20 A/B over 5 min, then change to 71:29 A/B over 20 min, then change to 16:84 A/B over 8 min, then hold for 5 min, then change to 95/5 A/B in 0.1 min, hold for 5 min; flow rate = 1.2 mL/min; detection, 270 nm; injection volume, 15 µL, tR of GDC-0980 = 16.9 min. 2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde (2). A mixture of 4-(2-chloro-7-methylthieno[3,2-d]pyrimidin-4-yl)morpholine (1) (9.45 kg, 35.0 mol) and anhydrous THF (94.5 L) was cooled to below –8 °C. A 20wt% solution of i-PrMgCl in THF (8.64 kg, 16.8 mol) was added, followed by addition of a 25wt% solution of n-BuLi in heptane (9.84 kg, 38.4 mol) while maintaining the temperature below –8 °C. The mixture was stirred at below –8 °C for 2 h. Anhydrous DMF (5.11kg, 69.9 mol) was added while keeping the temperature below –8 °C. The reaction mixture was stirred for 1 h, transferred to a mixture of


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acetic acid (23.6 kg), 37% aqueous hydrochloric acid (9.10 kg), isopropanol (94.4 L) and water (91.7 L). The resulting slurry was heated to 50−55 °C and stirred for 1 h. The suspension was concentrated under reduced pressure to remove THF. The suspension was then cooled to rt, filtered and rinsed with water (2 × 20 L). The cake was dried under reduced pressure at 40−60 °C to afford 2 as a yellow solid (10.0 kg, 96% yield): mp 198.0 oC; 1H NMR (300 MHz, CDCl3) 10.37 (s, 1H), 4.16–3.97 (m, 4H), 3.93–3.70 (m, 4H), 2.75 (s, 3H); 13C NMR (75 MHz, CDCl3) 183.83, 162.20, 158.96, 157.50, 142.26, 140.95, 116.37, 66.59, 46.55, 11.50; HRMS calcd for C12H12CN3O2S [M+H] 298.0412, found 298.0408. (S)-2-hydroxy-1-(piperazin-1-yl)propan-1-one (14) (Benzylpiperazine Route, Scheme 6). A flask charged with benzyl piperazine (100 g, 0.57 mol 1.00 equiv) was cooled to 10 °C. (S)-ethyl lactate (22) (201 g, 1,67 mol, 3.00 equiv) was added at a rate to maintain the temperature below 20 °C. Sodium methoxide (25 wt% in MeOH) (97.3 mL, 0.43 mmol, 0.75 equiv) was added while maintaining the temperature below 20 °C. The reaction mixture was allowed to warm to ambient temperature and was aged for 16 h. The mixture was diluted with ethanol (500 mL) and treated with Amberlite® IRC-748 resin (Na+ form 630 g, 1.8meq/g, 2 equiv, pre-conditioned to H+ form using 5% aq HCl). The suspension was stirred at ambient temperature for 2 h. The resin was removed by filtration through a pad of Celite® (70 g) and the pad was washed with EtOH (2 × 677 mL). The filtrate and washes were combined and concentrated under reduced pressure to 1000 mL. To the solution was added activated charcoal (70 g), the suspension was stirred at ambient temperature for 18 h, then filtered through a pad of Celite® (70 g) and the pad was washed with EtOH (2 × 677 mL). The filtrate and washes were combined and concentrated under vacuum to afford a residue containing (S)-1-(4-Benzylpiperazin-1-yl)-2-hydroxypropan-1-one (23).


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The residue was diluted with EtOH (450 mL) and the solution was degassed three times through a nitrogen and vacuum cycle. Palladium (10% on activated carbon, 56.14% water wetted) (41.4 g, 0.017 mol, 0.03 equiv) was added and the mixture was degassed five times through a nitrogen and vacuum cycle. The mixture was heated to 55 °C and cyclohexene (233 g, 2.84 mol, 5.00 equiv) was slowly added. The reaction mixture was heated to reflux and stirred for 2.5 h. After cooling to ambient temperature, the mixture was filtered through a pad of Celite® (70 g) and washed with EtOH (2 × 675 mL). The filtrate and wash were combined and concentrated under reduced pressure to afford a residue containing (12) as an oil which was dissolved in a 50:50 (v/v) mixture of EtOH/THF (352 mL each) and cooled to 10 °C. A solution of oxalic acid dihydrate (143 g, 1.23 mol, 2.00 equiv) in a 50:50 (v/v) mixture of EtOH/THF (282 mL each) was slowly added. The suspension was allowed to warm to ambient temperature and aged for 13 h. The suspension was cooled to 10°C and the solid was collected by filtration. The solid was washed with cold EtOH (2 x 550 mL) and dried under vacuum at 50 °C for 24 h to afford oxalate salt 14 as a white solid (103 g, 73%): mp 115.2

o

C; 1H NMR (300 MHz, D2O) 4.73–4.51 (m,

1H), 3.93–3.59 (m, 4H), 3.26 (dd, J = 8.8, 4.0 Hz, 4H), 1.27 (d, J = 6.7 Hz, 3H). 13C NMR (75 MHz, D2O) 174.48, 165.55, 64.36, 43.02, 42.94, 41.96, 38.91, 19.17.

HRMS calcd for

C7H14N2O2 [M+H] 159.1128, found 159.1122. (S)-2-hydroxy-1-(piperazin-1-yl)propan-1-one (14) (Piperazine Route, Scheme 7). A flask charged with piperazine (100 g, 1.16 mol) and (S)-ethyl lactate (22) (178 g, 48 mmol, 1.30 equiv) was cooled to 10 °C. Sodium methoxide (25 wt% in MeOH) (128 g, 0.59 mol, 0.50 equiv) was added while maintaining the temperature below 20 °C. The reaction mixture was allowed to warm to ambient temperature and aged for 19 h. Water (62.3 g, 3.46 mol, 3.0 equiv) was added and the mixture was aged for 16 h. The mixture was diluted with EtOH (400 mL) and concentrated under reduced pressure to half of the volume. The residue was flushed with EtOH


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(3 × 300 mL) to remove the water. The resulting solution (300 mL) was treated with a solution of oxalic acid dihydrate (37 g, 0.29 mol, 0.25 equiv) in EtOH (180 mL) and the resulting suspension was aged for 1 h. The suspension was cooled to < 10 °C and a solution of oxalic acid dihydrate (37 g, 0.29 mol, 0.25 equiv) in EtOH (180 mL) was added to adjust pH to 7.0–7.5. The suspension was cooled to 0–5 °C, aged for 1 h, filtered through a pad of Celite® (404 g) and washed with cold EtOH (2 × 350 mL). The filtrate and washes were combined and filtered through a 0.45 µm membrane filter, concentrated to 1300 mL. The solution was cooled to 10 °C and a solution of oxalic acid dihydrate (161 g, 1.28 mol, 1.12 equiv) in EtOH (750 mL) was slowly added. The suspension was allowed to warm to ambient temperature and stirred for 12 h. The suspension was cooled to 5 °C, aged for 1 h and the solid was collected by filtration, washed with cold EtOH (2 × 180 mL) and dried under reduced pressure at 50 °C for 24 h to afford oxalate 14 as a white solid (161 g, 59%). (S)-1-(4-((2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1yl)-2-hydroxypropan-1-one (11) 2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine-6carbaldehyde (2) (8.75 kg, 29.4 mol) was charged to a reactor, followed by MeOH (165 L), (S)-2hydroxy-1-(piperazin-1-yl)propan-1-one oxalate (14) (10.9 kg, 43.9 mol), sodium acetate (7.21 kg, 87.9 mol), glacial acetic acid (1.76 kg, 29.3 mol) and trimethylorthoformate (31.2 kg, 294 mol). The slurry was heated to 55−60 °C and stirred for 4 h.

A 30% solution of 2-

picoline•borane in THF (12.6 kg, 35.3 mol) was added and the slurry was stirred for 1 h. The reaction mixture was partially concentrated under reduced pressure. Toluene (134 L) was added and the reaction mixture was concentrated under reduced pressure. Toluene (66.7 L) was added and the reaction mixture was again partially concentrated under reduced pressure. To the residue was added toluene (113 L) and the mixture was cooled to 20−30 °C. Water (253 L) was added and the pH was adjusted to 7.5–8.5 with 10% aqueous sodium carbonate solution. The organic


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phase was separated, cooled to 0−5 °C and extracted with a mixture of water (105.7 L) and 96% sulfuric acid (3.55 kg). The aqueous phase was separated and toluene (69.6 L) was added. The pH was adjusted to 7.5−8.5 with 10% aqueous sodium carbonate solution (60.4 L) at 0−5 °C. The mixture was warmed to 20 °C and the organic phase was separated. The organic phase was diluted with toluene (58.6 L) and concentrated under reduced pressure to its original volume (approximately 80 L). The solution was warmed to 53−57 °C, and n-heptane (13.5 L) was added. The solution was seeded with (S)-1-(4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one (11) (30 g) and the suspension was stirred at 53−57 °C for 30 min. n-Heptane (48.2 L) was slowly added and the resulting slurry was cooled to 0−5 °C, filtered and washed with a mixture of toluene (6.9 L) and n-heptane (14.7 L) and subsequently with n-heptane (29.4 L). The cake was dried at 30−45 °C under reduced pressure to afford 11 (10.6 kg, 82% yield): mp 145.8 oC; 1H NMR (300 MHz, DMSO-d6) 4.84 (d, J = 6.9 Hz, 1H), 4.45–4.37 (m, 1H), 3.90–3.84 (m, 4H), 3.79–3.63 (m, 4H), 3.58–3.41 (m, 4H), 3.30 (s, 2H), 2.59–2.35 (m, 4H), 2.24 (s, 3H), 1.17 (d, J = 6.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) 172.06, 162.19, 157.90, 155.55, 145.57, 128.67, 111.59, 65.79, 63.98, 54.15, 52.90, 52.46, 45.86, 44.42, 41.37, 20.60, 10.83; HRMS calcd for C19H26ClN5O3S [M+H] 440.1518, found 440.1505. (S)-1-(4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one (GDC-0980). (S)-1-(4-((2-chloro-7-methyl4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxy propan-1-one (11) (16.5 kg, 37.5 mol) was charged to a reactor, followed by n-propanol (143 L), 2-aminopyrimidin5-ylboronic acid (10) (6.22 kg, 44.8 mol) and potassium phosphate (15.9 kg, 74.9 mol). The resulting mixture was degassed by vacuum/argon purge three times. Bis(triphenylphosphine)palladium (II) chloride (395 g, 56.3 mmol) was added and the slurry was again degassed by vacuum/argon purge three times. The mixture was heated within 2 h to 85 °C and stirred for 30


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min. The reaction mixture was cooled to rt, water (152 L) was added and the pH was adjusted to 6.0–8.0 with 37wt% aqueous hydrochloric acid solution (6.1 kg). The biphasic mixture was heated to 80 °C and stirred for 1 h. The organic phase was separated and filtered over a preheated pressure filter loaded with an activated carbon pad. The filter unit was washed with a warm (80 °C) mixture of n-propanol (35.0 L) and water (18.5 L). The filtrate was concentrated under reduced pressure while keeping the volume constant by addition of water (98 L). The resulting slurry was cooled to 26–36 °C, filtered and rinsed with a mixture of n-propanol (20.0 L) and water (75.5 L). The cake was dried under reduced pressure at 45 °C to afford the crude product as a yellowish white solid (15.1 kg, 81%). API Crystallization. The crude product (15.7 kg) was charged to a reactor, followed by npropanol (108 L) and water (56.0 L). The suspension was heated to 85 °C and stirred to afford a clear solution. The solution was filtered over a preheated polishing filter unit and rinsed with a hot mixture of n-propanol (23.1 L) and water (11.0 L). The filtrate was cooled to −10 °C, aged for 1 h and filtered. The filter cake was washed with cold n-propanol (110 L) and dried under reduced pressure at 60−70 °C to afford GDC-0980 as an off-white solid (14.5 kg, 92%): mp 232.8 oC; 1H NMR (300 MHz, DMSO-d6) 9.15 (s, 2H), 7.07 (s, 2H), 4.86 (d, J = 7.0 Hz, 1H), 4.49–4.32 (m, 1H), 4.00–3.89 (m, 4H), 3.84 (s, 2H), 3.81–3.71 (m, 4H), 3.64 ̶ 3.48 (m, 4H), 2.52 ̶ 2.49 (m, 4H), 2.34 (s, 3H), 1.17 (d, J = 6.5 Hz, 3H);

13

C NMR (75 MHz, DMSO-d6) 172.07,

164.04, 161.48, 157.76, 157.30, 156.36, 143.30, 129.59, 120.19, 110.91, 65.99, 64.03, 54.39, 52.89, 52.46, 45.80, 44.44, 40.33, 20.61, 10.95; HRMS calcd for C23H30N8O3S [M+H] 499.2234, found 499.2220. Supporting Information. Copies of 1H and experimental section.

13

C spectra for all the compounds listed in the

This material is available free of charge via the Internet at

http://pubs.acs.org


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Acknowledgment. We thank Mr. Hong Lin and Ms. Stefanie Gee for analytical support, Dr. Alan Deese for help with NMR analysis, and Dr. Christine Gu for HRMS analysis. We also thank Dr. Reinhard Reents and Dr. Mark Reynolds for their valuable suggestions and support. References (1) (a) Cantley, L. C. Science 2002, 296, 1655-1657. (b) Engelman, J. A. Nature Rev. Cancer 2009, 9, 550−562. (2) Luo, J.; Manning, B. D.; Cantley L. C. Cancer Cell, 2003, 4, 257−262. (3) Laplante, M.; Sabatini, D.M.J. Cell. Sci. 2010, 122, 3589–3594. (4) (a) Maira, S. M.; Stauffer, F.; Brueggen, J.; Furet, P.; Schnell, C.; Fritsch, C.; Brachmann, S.; Chene, P.; De Pover, A.; Schoemaker, K.; Fabbro, D.; Gabriel, D.; Simonen, M.; Murphy, L.; Finan, P.; Sellers, W.; Garcia-Echeverria, C. Mol. Cancer Ther. 2008, 7, 185−1863. (b) Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.; Darcy., M. G.; Donatelli, C. A.; Luengo, J. I.; Newlander, K. A.; Parish, C. A.; Ridgers, L. H.; Sarpong, M. A.; Schmidt, S. J.; Van Aller, G. S.; Carson, J. D.; Diamond, M. A.; Elkins, P. A.; Gardiner, C. M.; Garver, E.; Gilbert, S. A.; Gontarek, R. R.; Jackson, J. R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.; Sung, C.-M.; Sutton, D.; Tummino, P. J.; Wegrzyn, R. J.; Auger, K. R.; Dhanak, D. ACS Med. Chem. Lett. 2010, 1, 39–43. (c) Dehnhardt, C. M.; Venkatesan, A. M.; Santos, E. D.; Chen, Z.; Santos, O.; Ayral-Kaloustain, S.; Brooijmans, N.; Mallon, R.; Hollander, I.; Feldberg, L.; Lucas, J.; Chaudhary, I.; Yu, K.; Gibbons, J.; Abraham, R.; Mansour, T. S. J. Med. Chem. 2010, 53, 798– 810. (5) Sutherlin, D.P.; Bao, L.; Berry, M., Castanedo, G.; Chuckowree, I.; Dotson, J.; Folkes, A. J.; Friedman, L.; Goldsmith, R.; Gunzner, J.; Heffron, T.; Lesnick, J.; Lewis, C.; Mathieu, S.;


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Murray, J.; Nonomiya, J.; Pang, J.; Pegg, N.; Prior, W.W.; Rouge, L.; Salphati, L.; Sampath, J.; Tian, Q.; Tsui, V.; Wan, N.; Wang, S.; Wei, B.; Weismann, C.; Wu, P.; Zhu, B.; Olivero, A. J. Med. Chem. 2011, 54, 7579‒7587. (6) The first generation route was based on modification of the route employed by discovery chemistry.5 (7) Tian, Q.; Cheng, Z.; Yajima, H.M.; Savage, S.J.; Keena, Green.; Humphries, T.; Reynolds, M.E.; Babu, S.; Gosselin, F.; Askin, D.; Kurimoto, I.; Hirata, N.; Iwasaki, M.; Shimasaki, Y.; Miki, T. Org. Process Res. Dev. 2013, 17, 97–107. (8) Valeur, E.; Bradley, M. Chem. Soc. Rev. 2009, 38, 606–631. (9) One of the starting materials, the boronate 5, was highly expensive ($25000/kg). (10) (a) Bayh, O.; Awad, H.; Mongin, F.; Hoarau, C.; Bischoff, L.; Trécourt, F.; Quéguiner, G.; Marsais, F.; Blanco, F.; Abarca, B.; Ballesteros, R. J. Org. Chem. 2005, 70, 5190−5196. (b) Mongin, F.; Bucher, A.; Baureau, J.; Bayh, O.; Awad, H.; Trécourt, F. Tetrahedron Lett. 2005, 46, 7989−7992. (c) Bayh, O.; Awad, H.; Mongin, F.; Hoarau, C.; Trécourt, F.; Quéguiner, G.; Marsais, F.; Blanco, F.; Abarca, B.; Ballesteros, R. Tetrahedron 2005, 61, 4779−4786. (11) Both the lithium triarylmagnesiate 13 and the reaction mixture after addition of DMF were very stable at reaction temperatures between –10 °C and –5 °C for extended time (> 15 h). (12) The free base 12 is a liquid, so salt formation was employed to produce the material in the preferred state. (13) The water content remained stable (1–3%) when the oxalic salt 14 was exposed to the atmosphere for a short period of time (2–3 h).


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(14) When other bases (potassium acetate, potassium carbonate and N,N-diisopropylethylamine) were employed, a greater amount of alcohol impurity 15 (27–48A% by HPLC) was generated. (15) (a) Sato, S.; Sakamoto, T.; Miyazawa, T.; Kikugawa, Y. Tetrahedron, 2004, 60, 7899–7906. (b) Wu, P-L.; Chen, H.; Line, M. J. Org. Chem. 1997, 62, 1532–1533. (16) Pyridine•BH3 was not further investigated due to its short, 6-month shelf life.

2-

Picoline•BH3 is more stable and was deemed suitable for large-scale production.15a (17) a) Ohshima, T.; Hayashi, Y.; Agura, K.; Fujii, Y.; Yoshiyamab, A.; Mashima, K. Chem. Commun., 2012, 48, 5434–5436. b) Pesti, J.; Chen, C-K.; Spangler, L.; Delmonte, A.J.; Benoit, S.; Berglund, D.; Bien, J.; Brodfuehrer, P.; Chan, Y.; Corbett, E.; Costello, C.; DeMena, P.; Discordia, R.P.; Doubleday, W.; Gao, Z.; Gingras,S.; Grosso, J.; Haas, O.; Kacsur, D.; Lai, C.; Leung, S.; Miller, M.; Muslehiddinoglu, J.; Nguyen, N.; Qiu, J.; Olzog, M.; Reiff,E.; Thoraval, D.; Totleben, M.; Vanyo, D.; Vemishetti, P.; Wasylak, J.; Wei, C. Org. Process Res. Dev. 2009, 13, 716–728. c) Tasaka, A.; Tamura, N.; Matsushita, Y.; Teranishi, K.; Hayashi, R.; Okonogi, K.; Itoh, K. Chem. Pharm. Bull. 1993, 41, 1035–1042. (18) The residual piperazine in 14 would react in the remaining synthesis to the API resulting in the des-lactate impurity 8 and di-lactate impurity 9 (Figure 2). To control the levels of these two impurities in the API within the limits, we set a specification of no more than 0.2% of piperazine in 14. (19) A key development was the observation of a significant change in the NMR spectra of the crude mixture in D2O after 18 h at ambient temperature. With careful examination of the NMR spectra, we discovered the conversion of the bis-lactamide 24 to the desired product 12


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presumably by hydrolysis. We therefore envisioned that we could improve the yield of 12 through the hydrolysis of the bis-lactamide 24. (20) Patnaik, P. Handbook of Inorganic Chemicals, McGraw-Hill: New York, 2003, p873. (21) (a) Anastas, P.; Warner, J. Green Chemistry: Theory and Practice, Oxford Univ. Press, 1998. (b) Federel, H. Green Chem., 2013, 15, 3105–3115. (c) Jimenez-Gonzalez, C.; Poechlauer, P.; Broxterman, Q. B.; Yang, B. S.; am Ende, D.; Baird, J.; Bertsch, C.; Hannah, R. E.; Dell’Orco, P.; Noorman, H.; Yee, S.; Reintjens, R.; Wells, A.; Massonneau, V.; Manley, J. Org. Process Res. Dev. 2011, 15, 900–911. (c) Trost, B.M. Angew. Chem. Int. Ed. Engl. 1995, 34(3), 259–281. (22) Boronic acid 10 was initially produced from 2-amino-5-bromopyrimidine in 38% overall yield through a sequence of Boc protection and metalation/borylation followed by the Boc deprotection. A concise and protecting group free synthesis was developed to produce boronic acid 10 directly from 2-amino-5-bromopyrimidine in one step in 40% yield. (23) Young, I. S.; Baran, P.S. Nature Chem. 2009, 1, 193-205.


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mTOR Inhibitor

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