Manufacturing Development and Genotoxic ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Full Paper

Manufacturing Development and Genotoxic Impurity Control Strategy of the Hedgehog Pathway Inhibitor Vismodegib Remy Angelaud, Mark Reynolds, Cadapakam J. Venkatramani, Scott Savage, Huldreich Trafelet, Thomas Landmesser, Peter Demel, Michael Levis, Olivier Ruha, Baerbel Rueckert, and Heinz Jaeggi Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00208 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Organic Process Research & Development is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

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

Organic Process Research & Development

Manufacturing Development and Genotoxic Impurity Control Strategy of the Hedgehog Pathway Inhibitor Vismodegib Remy Angelaud,*,† Mark Reynolds, † Cadapakam Venkatramani, ‡ Scott Savage, † Huldreich Trafelet,§ Thomas Landmesser, § Peter Demel, § Michael Levis, § Olivier Ruha, § Baerbel Rueckert§ and Heinz Jaeggi§ †

Small Molecule Process Chemistry, ‡Small Molecule Analytical Chemistry, Genentech, A Member of the Roche Group, 1 DNA Way, South San Francisco, CA 94080, USA § Siegfried AG, Untere Brühlstrasse 4, CH-4800 Zofingen, Switzerland

ACS Paragon Plus Environment

1


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

Page 2 of 35

Table of Contents Graphic


2

Page 3 of 35

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


ABSTRACT The development work towards the robust and efficient manufacturing process to Vismodegib the active pharmaceutical ingredient (API) in Erivedge® is described. The optimization of the 4stage manufacturing process was designed to produce the API with the required critical quality attributes: 1) the selective catalytic hydrogenation reduction of the nitro compound 3 to the corresponding aniline 4 while minimizing the formation of potential genotoxic (mutagenic) impurities; 2) the control of the polymorphic phase and multipoint specification for particle size distribution. KEYWORDS. Vismodegib, nitro aromatic reduction, particle size distribution, genotoxic impurity, hedgehog.


3


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

Page 4 of 35

INTRODUCTION The Hedgehog1 signalling pathway transmits information to embryonic cells and is required for proper development. This signalling regulates embryonic development, ensuring that tissues reach their correct size and location, maintain tissue polarity and cellular content. In the skin, the Hedgehog pathway is critical for regulating hair follicle and sebaceous gland development. Hedgehog signalling normally remains inactive in most adult tissues. Inappropriate reactivation of the Hedgehog pathway has been linked to several human cancers, most often caused by exposure to UV radiation. In basal cell carcinoma (BCC), abnormal Hedgehog pathway signalling is the key molecular driver of the disease and more than 90% of BCCs have abnormal activation of Hedgehog pathway signalling. Vismodegib, the active pharmaceutical ingredient (API) in Erivedge® is the first inhibitor2 of the Hedgehog pathway to be approved for the treatment of metastatic or locally advanced BCC and represents an important treatment option for patients where surgery is not recommended. Vismodegib 1 is synthesized in a 4-step manufacturing process from two designated starting materials, 2-(2-chloro-5-nitrophenyl)pyridine 3 and 2-chloro-4-(methylsulfonyl)benzoic acid 2 (Scheme 1). The development and optimization towards the manufacturing process faced two major hurdles: (1) the control of the numerous potential residual genotoxic (mutagenic) impurities (GTIs) in the API introduced by the nitro starting material 3; (2) the control of the polymorph phase and the particle size distribution (PSD) of the API since it impacted the dissolution of the corresponding capsule drug product. Vismodegib is assigned as a Biopharmaceutics Classification System (BCS) Class 2 compound with low solubility and high permeability.


4

Page 5 of 35

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


Scheme 1. Vismodegib Synthesis

RESULTS AND DISCUSSION Starting Materials Manufacturing Starting material acid 2 is commercially available and was directly purchased in >99.5 A% HPLC purity with a reproducible impurity profile. Nitro starting material 3 is custom made and two manufacturing routes to this product were developed.3 In the first route,4 iodonitrobenzene 7 is produced from the corresponding nitroaniline 6 via a Sandmeyer reaction followed by crystallization from water. Metal halogen exchange on 2-bromopyridine with isopropyl Grignard followed by transmetalation with ZnCl2 furnishes the corresponding organozinc reagent which is used directly in the subsequent Negishi palladium catalyzed Negishi cross-coupling with aryl iodide 7 in THF to provide 3 after treatment with activated charcoal to scavenge residual palladium, in >99.5A% HPLC (Scheme 2).


5


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

Page 6 of 35

Scheme 2. Negishi Route to 3

In the second route5, 2-(2-chloro-5-nitrophenyl)pyridine 3 is synthesized through the sequence depicted in Scheme 3, in which 2-chloro-5-nitroacetophenone intermediate 9 is obtained by Knoevenagel condensation of the corresponding acyl chloride of 2-chloro-5-nitrobenzoic acid 8 with dimethyl malonate followed by decarboxylation and subsequent purification by crystallization in methanol/water. Condensation of 1,1,3,3-tetramethoxypropane and N,N’dimethyl urea provides pyrimidinium chloride 10 after crystallization from 2-propanol. Biphenyl 3 is finally obtained by condensation of 9 and oxo-dihydropyrimidinium chloride in presence of ammonium acetate in acetic acid in >99.5A% purity by HPLC after crystallization from acetone/water.


6

Page 7 of 35

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


Scheme 3. Pyrimidinium Route to 3

Both starting materials 2 and 3 are well characterized and their respective manufacturing processes routinely produce them in high purity with no single impurities >0.2A% HPLC. However, toxicology assessment of nitro aromatic precursors 6, 7, 8, 9 and nitro starting material 3 were performed using Quantitative Structure-Activity Relationship ((Q)SAR)6 methodologies that predict the outcome of a bacterial mutagenicity assay. All five nitro compounds gave structural alerts for predicted mutagenicity and were then submitted to a biological assay to assess their mutagenic potential (Ames test)7. Although starting material 3 was Ames-negative, nitroaromatic intermediates 6, 7, 8 and 9 were found to be Ames-positive and are therefore considered genotoxic impurities (GTIs) in the process. In order to set appropriate specifications for these four GTIs, a more thorough process understanding was needed and their respective purging will be discussed later in this paper.


7


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

Page 8 of 35

Manufacturing Process Optimization The original Discovery route8 to Vismodegib involved the following 2-step process: reduction of 3 with iron filings in acetic acid to the corresponding aniline 5, followed by amide coupling with acid 2 mediated by HBTU/HOAt. Because of the large waste generation and its poor scalability this early route was not suitable for production and we decided to develop and optimize a more efficient route as follows (Scheme 1): 1) Stage 1: Catalytic hydrogenation of the nitro starting material 3; 2) Stage 2: Activation of acid 2 to the corresponding acyl chloride; 3) Stage 3: Standard Schotten-Baumann type acylation; 4) Stage 4: Control of the physical properties (polymorphic form, residual solvents and PSD). Stage 1: Hydrogenation of 3 to amine Amine 4 Selective catalytic hydrogenation of nitroaromatic compounds to the corresponding aromatic amines is a well-known process in the literature and numerous examples have been reported9. The initial process development on the Stage 1 hydrogenation involved an exploratory experimental design evaluating the effect of catalyst type (Pd(OH)2/C, Pd/C, and Pt/C), pressure and temperature of the reaction (Table 1). Table 1. Effect of catalyst, H2 pressure and temperature on reduction of 3

Entrya

Catalyst

Pressure Temperature 4 (A%)e 3 (A%) (psi) (°C)


8

Page 9 of 35

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


1

Pd(OH)2/Cb

110

80

92.2

ND

2

Pd(OH)2/C

50

80

93.5

ND

3

Pd(OH)2/C

110

25

26.9

65

4

Pd(OH)2/C

50

25

22

68.7

5

Pd/Cc

110

80

95.4

ND

6

Pd/C

50

80

96.2

ND

7

Pd/C

110

25

59.5

36.7

8

Pd/C

50

25

14.5

81.1

9

Pt/Cd

110

80

93.6

ND

10

Pt/C

50

80

95.6

ND

11

Pt/C

110

25

99.0

ND

12

Pt/C

50

25

96.7

ND

a

Reactions run on 2.3 g (9.8 mmol) of 3 in THF (25 mL). b20% Pd(OH)2/C (50% wet; 2.5 wt%).

c

5% Pd/C (60% wet; 10 wt%). d5% Pt/C (60% wet; 10 wt%). eArea % by HPLC10 after 20 h

reaction time. Analysis of the results in Table 1 showed that in THF, 5% Pt/C was the optimal catalyst (entry 912). Poor conversion to 4 was observed at 25 °C with Pd(OH)2 (entries 3 and 4) and with Pd/C (entries 7 and 8) and the reactions had to be heated to go to completion. This had a negative impact on the impurity profile of the reaction. In order to further understand the process, we identified the main impurities generated during the screen in Table 1 and a general trend was observed (Scheme 4). When the hydrogenation is performed at room temperature/low pressure, an incomplete reduction generates hydroxylamine 11 which then generates11 12 which in turn, is further reduced to aza and hydrazine by-products 13 and 14, respectively. On the other hand,


9


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

Page 10 of 35

when the hydrogenation is performed at high temperature/high pressure, over-reduction leads to des-chloro amine 15 and piperidine 16. Scheme 4. Under- and over-reduced impurities generated during hydrogenation of 3

With the impurity markers in hand, we found out that hydroxylamine 11 co-eluted with desired amine 4 with the HPLC method used at the time of this development and thus, conversions for entries 9-12 (Table 1, Pt/C) were deceptively high. Furthermore, 11 was found to be Amespositive12 and is considered a GTI. Therefore a new HPLC method for reaction monitoring was implemented and we designed another screen. A medium temperature (60 °C) and pressure (88 psi) were chosen in order to minimize over- and under-reduction. Methanol and ethanol were chosen as solvents based on solubility studies and to facilitate the work up. Acid additives (HCl and AcOH) were also tested and 1% Pt +2% V/C catalyst was introduced based on literature precedent13. The results with acetic acid are reported in Table 2.


10

Page 11 of 35

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


Table 2. Effect of catalyst, solvent and acetic acid on reduction of 3 Entrya

Catalyst

Solventb

4 (A%)c

11 (A%)

12 (A%)

13 (A%)

14 (A%)

15 (A%)

16 (A%)

1

1%Pt+2%V/C

MeOH

96.7

ND

0.51

0.32

1.51

0.38

ND

2

1%Pt+2%V/C

MeOH/AcOH

97.4

ND

0.34

0.24

1.26

0.16

ND

3

1%Pt+2%V/C

EtOH/AcOH

97.4

ND

0.31

0.34

1.08

0.19

ND

4

5%Pd/C

MeOH/AcOH

91.3

80

0.49

0.33

0.27

1.81

ND

5

5%Pt/C

MeOH/AcOH

94.0

25

0.30

0.19

3.03

0.41

0.19

20%Pd(OH)2/C MeOH/AcOH

83.9

25

0.37

0.26

1

8.64

ND

6 a

Reactions run on 1 g (4.3 mmol) of 3 in 10 mL of solvent at 60 °C and 88 psi for 6 hours.

b

ROH/AcOH 9:1. cArea % by HPLC after 3 h reaction time.

The results of these screening experiments show that the 1% platinum/2% vanadium on carbon catalyst was the superior catalyst (entries 1-3) giving > 97% conversion to 4 and very low levels of over- and under-reduced impurities. MeOH/AcOH (9:1) was the best solvent system based on the level of over-reduced products 15 and 16 (entry 2). A final screen was then performed in order to define optimum ranges for temperature, concentration, molar equivalents of AcOH while narrowing catalyst loading ranges and monitoring reaction completion and formation of des-chloro impurity 15 and hydroxylamine 11 (Table 3).


11


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

Page 12 of 35

Table 3. Optimum ranges screen for reduction of 3

Temp (°C)

MeOH AcOH Catalyst Amount Amount (wt%) (g/g) (equiv)

Entry 1 25 3 4 1.50 2 25 7 4 1.50 3 25 3 8 1.50 4 25 7 8 1.50 5 25 3 4 6 6 25 7 4 6 7 25 3 8 6 8 25 7 8 6 9 60 3 4 1.50 10 60 7 4 1.50 11 60 3 8 1.50 12 60 7 8 1.50 13 60 3 4 6 14 60 7 4 6 15 60 3 8 6 16 60 7 8 6 17 40 5 6 3.70 18 40 4 6 3.70 19 40 6 6 3.70 20 40 5 5 3.70 21 40 5 7 3.70 22 40 5 6 1.60 23 40 5 6 5.90 24 55 5 6 3.70 25 55 3 4 1.50 a Reactions run on 1 g (4.3 mmol) of 3 at 88 psi for 3 hours.

4

15

11

15.59 93.17 49.69 95.48 8.59 43.64 21.88 95.90 7.95 95.97 67.41 97.40 6.60 96.50 88.63 97.05 97.05 73.11 97.20 98.82 96.94 96.77 87.11 95.01 15.59

(A%) 0.05 0.35 0.13 0.35 0.03 0.08 0.07 0.27 ND 0.32 0.07 0.17 ND 0.22 0.08 0.21 0.17 0.12 0.16 0.16 0.15 0.18 ND ND 0.35

0.03 0.12 0.08 ND 0.02 0.01 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.03

From these experiments, it is clear that when using the 1% platinum/2% vanadium on carbon catalyst in MeOH/AcOH:


12

Page 13 of 35

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


Formation of 11 (GTI) is only seen in the low temperature experiments (entries 1-3) and even then is not formed to any significant extent (entry 2)

Amounts of catalyst < 5% gave low levels of conversion within the investigated reaction time of 3 hours (entries 1, 5, 9, 13 and 25)

Increased reaction temperatures gave higher levels of the des-chloro impurity 15 (entries 10, 14, 16 and 25)

The impact of acetic acid concentration is small within the investigated range

The level of 15 is highest when the conversion to product 4 is also highest (entries 4, 8, 16, 17, 20 and 21)

Entry 24 shows the best compromise between conversion and impurity profile with 95% conversion and not detectable amounts of both under-reduced 11 and over-reduced 15

Based on these results, the manufacturing parameters for the hydrogenation were defined as followed: MeOH (9 wt)14, AcOH (1 wt), 1% Pt + 2% V/C (0.05 wt), temperature 50-55 °C (target: 51 °C), pressure 60-88 psi (4-6 bar) for approximatively three hours15. The higher percentage of methanol relative to the screened conditions ensured good dissolution of the starting material. With MeOH/AcOH chosen as the solvent system for the hydrogenation reaction, experiments were performed to optimize the isolation of 4 directly from the reaction after removal of the catalyst by filtration. Isolation of the freebase was accomplished by distillation of a portion of the MeOH/AcOH and addition of an aqueous base. Sodium carbonate was used initially, but as this produced significant off-gassing, sodium hydroxide and aqueous ammonia were investigated. While both allowed isolation of the freebase directly from the reaction mixture, aqueous ammonia proved better at rejecting residual platinum and vanadium to 10. The resulting slurry is aged for one hour, centrifuged and the solid washed with water until the pH of the washing liquor is 99.9 A% purity by HPLC. Table 4 summarizes the operating ranges and criticality assessment for the Stage 1 hydrogenation. Table 4. Stage 1 operating ranges comparison and criticality assessment

Parameter Target 1% Pt+2% V/C (~50% wet) 0.05 wt Methanol 9.0 wt Acetic Acid 1.0 wt Water 4 wt 25% Aqueous NH4OH 4 wt Reaction temperature 51 °C Reaction pressure 5 bar a The catalyst type is a critical material attribute

Normal Operating Range ± 5% ± 5% ± 5% ± 5% ± 5% 50–55 oC ~5 bar

Key for Critical Yield or Process Purity Parameter Yesa Yesa No No No No No No No No Yes Yes No No


14

Page 15 of 35

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


Stage 2: Activation of 2 to acid chloride 5 Standard conditions to form the acid chloride (SOCl2, catalytic DMF in CH2Cl2) were chosen to initiate process development of this step and screening DOEs to better understand the reagent stoichiometry and to identify regions of optimum reaction space were performed initially. Table 5. Effect of SOCl2 stoichiometry and reaction concentration on the conversion to 5

a

Entry

SOCl2 CH2Cl2 (molar equiv) (vol)

10 (A%)b

1

1.9

6.0

99

2

1.5

11.5

99

3

1.5

6.0

99

4

1.7

11.5

99

5

1.7

6.0

99

6

1.2

11.5

99

7

1.2

6.0

99

a

Reactions run at a scale of 1 g of 2 for 6 hours at 40 °C. bConversion to 5 is monitored by HPLC through derivatization to the corresponding methyl ester 10.17

First we studied the impact of SOCl2 and DMF stoichiometry on the conversion to 5 in DCM at 40 °C and it was clear that the ratio of SOCl2 / DMF has no impact18 on the conversion (>99%) and that the reaction performs well with as little as 0.05 weight equivalent of DMF and 1.9 molar


15


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

Page 16 of 35

equivalent of SOCl2. Next, we studied the impact of the molar equivalents of SOCl2 and the reaction concentration on the conversion to 5 while holding temperature (40 °C) and equivalents of DMF (0.05 wt) constant (Table 5). The results demonstrated that the reaction could be successfully run with 1.2 molar equivalent of SOCl2, 0.05 weight of DMF in 6 volumes of CH2Cl2. While this first-generation process performed well at 20 kg scale, we opted to use of toluene instead of CH2Cl2 for safety (vide infra), environmental reasons and for isolation purposes since 5 crystallizes out from this solvent.19 In toluene, the acid chloride formation is run as a suspension at a calculated concentration of 18.7 wt%. Despite the low solubility of starting material 2 in the reaction medium it completely dissolved as it was consumed during the reaction to the acid chloride intermediate 5. The low end of the reaction temperature range in toluene is limited to 65 °C for safety reasons, ensuring that the reagents are consumed faster than they are charged, avoiding a potential adiabatic exothermic runaway reaction if a build-up were to occur. Thus, the reaction is routinely run at 70 °C. The feed rate of the thionyl chloride has been set at >45 minutes for safety reasons, as stated above. Additionally, a large volume of HCl and SO2 by-product gases have to be removed from the production vessel (191 L/kg of 2). This gas flow has to be low enough to ensure that there is no build up of pressure in the vessel, and this is ensured using the addition rate described above. After the reaction is complete, the solution is partially concentrated at atmospheric pressure (~111 °C). This serves mainly to remove excess thionyl chloride and HCl and SO2 reaction gases. The distillation is controlled by the amount of distillate (~0.5 wt) per kilogram of starting material. To test whether the acid chloride intermediate 5 remains stable in the reaction solution, the reaction mixture was held at reflux at two different concentrations (prior and after distillation for 24 hours). During these experiments, the only observed impurity was a small amount of the


16

Page 17 of 35

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


N,N-dimethylamide impurity 17 (Table 5). This impurity has never been detected in normal processing and can be purged during the isolation of acid chloride 5 so it is not of concern. The mixture is then cooled20 to 10 °C and aged for 2 hours, to enable full crystallization of 5, centrifuged, and the solid washed with toluene (1.2 vol). Due to its reactive nature, the isolation of 5 requires significant care and it had been dried originally at 40 °C under reduced pressure. However, because of its reactive and corrosive nature, metals were quickly corroded in the drying equipment. Furthermore, in order to reduce the potential for hydrolysis during isolation, drying and handling, it was decided not to incorporate a drying step and to store acid chloride 5 as a toluene-wet cake cold21 (2–8 °C) in sealed polyethylene bags purged with an inert gas (nitrogen or argon) and used as-is in the amide coupling Stage 3. On scale, this step is routinely run on 23 kg of 2 and produce acid chloride 5 in 92-95 % yield (corrected for residue on drying) with >99.8 A% purity by HPLC. Table 6 summarizes the operating ranges and criticality assessment for the Stage 2 acid chloride formation. Table 6. Stage 2 operating ranges and criticality assessment

Key for Yield or Purity No

Critical Process Parameter No

Parameter Thionyl Chloride

Target 0.6 wt

Normal Operating Range ± 5%

DMF

0.05 wt

± 5%

No

No

Toluene

4.22 wt

± 5%

No

No

Reaction Temperature

70 °C

± 5 oC

No

No


17


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

Page 18 of 35

The only impurity detected at this Stage is the starting acid 2 in 15 wt% at 20 °C) and because we could isolate crude Vismodegib directly from THF / water mixture through crystallization. Our main area of development focused on the type and stoichiometry of the inorganic base and thus we designed the experiments in Table 7 with the following assumptions: use of acid chloride 5 in 1.1 molar equivalent excess to ensure that all amine 4 is consumed and the slight excess of 5 will be hydrolyzed to the corresponding acid and purged with the basic aqueous waste stream as a salt. The solvent ratio (THF/water) was designed so amine 4 is fully soluble at the beginning of the reaction. Before addition of 5, the THF/water ratio is 60:40 and solubility of 4 is ~7.5 wt% and well in range of the solubility22 of ~5.3 wt% in 50:50 THF/water at 20 °C. Table 7. Impact of type and stoichiometry of the base on the Schotten-Baumann acylation

Entrya

Base

Molar 1 (A%) 4 (A%) Yieldb (%) equivalent


18

Page 19 of 35

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


1

Na2CO3

0.60

94.4

5.4

83

2

Na2CO3

0.66

94.5

5.4

96

3

Na2CO3

0.72

98.4

1.4

87

4

K2CO3

0.66

99.8

ND

101

5

K2CO3

0.72

99.8

ND

99

a

Reactions run on ~72 g (0.35 mol) of 4, in 14 volumes of THF/water (60:40), cooling to 5 °C, adding 1.1 molar equivalent of 5 in 11 volumes of THF, then warming to 20 °C for 1-2 hours. b Crude yield not corrected for purity. The results from Table 7 screen clearly shows that potassium carbonate is a more robust base for the amidation, giving higher yields and purities (entries 4 and 5). Also, purging of excess acid 2 seems to be more efficient with potassium carbonate presumably because of the higher solubility of the potassium salt of 2 vs the corresponding sodium salt in water (entries 4-5 vs 1-3). In order to ensure good purging of the potassium salt of 2, a pH check to verify that the aqueous layer is on the basic side (pH > 7) was implemented. Thus, 0.66 molar equivalent of K2CO3 was chosen based on the resulting yield and purity (entry 4). For the isolation, the aqueous layer is drained after reaction completion (0.05 A%. The acylated derivatives of amines byproduct 11, 14, 15 and 16 from Stage 2 do not present any structural alert from in silico assessment and are therefore not considered GTIs. The acid chloride 5 is considered a GTI and its purging and fate will be discussed in the genotoxic assessment section.


20

Page 21 of 35

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


Stage 4: Control of Vismodegib Physical Properties (polymorphic phase and PSD) This Stage was designed to deliver the API with the required critical quality attributes: 1) the desired polymorph form of the API (polymorph B) with acceptable levels of residual solvents and 2) the required particle size distribution (PSD). Polymorph B is the thermodynamically most stable polymorph observed and is the only form used in clinical trials. However, a less thermodynamically stable form (Form A) has been observed during development but has never been produced on scale. Figure 1 shows a differential scanning calorimetry (DSC) trace containing a mixture of polymorph Form A (melting point, 177 °C) and Form B (melting point, 187 °C). The formation of Form B is controlled by seeding the crystallization step with the appropriate polymorphic form and by drying to ensure low solvate and thus low residual solvent levels. If a mixture of Form A and B is used for seeding, the crystallization also furnishes a mixture of both forms. Figure 1. DSC of form A and B mixture

Vismodegib has poor solubility24 in most organic solvents and eventually methyl isobutyl ketone (MIBK) was chosen because it offered high solubility of 1 at reflux (11.8 wt%, 117 °C) and


21


down to 10,000 ppm of MIBK, it was assumed that the MIBK solvate was a channel solvate. In order to test this hypothesis, we ran new crystallization experiments with water as additive. We were pleased to


23


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

Page 24 of 35

find that by using MIBK with 0.5 wt% of water in the crystallization we could isolate the API with very low level of MIBK. As illustrated in Figure 1, the MSZW of Vismodegib in MIBK plus 0.5 wt% of water shows solubility curves almost identical to pure MIBK making the 95 °C seeding temperature still a robust set point. While the crystallization with water significantly reduced residual MIBK, drying at 90 °C under vacuum was required to reach the low acceptable levels. On production, this process always reduced MIBK to 99.95 A% purity by HPLC and the desired polymorph form B. Finally, development was undertaken to develop milling parameters to provide API with a particle size range as required for the Drug Product (formulated capsules) manufacturing. Per ICH Q6A, the API particle size distribution is considered a critical quality attribute since it impacts the dissolution of the corresponding formulated capsules. The correlation of the PSD of the API and the dissolution profile of the capsules is illustrated in Figure 3, in which bigger particles significantly slow down the time needed for the capsules to reach 85% dissolution.


24

Page 25 of 35

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


Figure 3. Correlation between PSD of the API and capsule dissolution profiles

Thus, a multipoint specification for the API particle size distribution was established to ensure Drug Product quality as follows: d(v,0.1) = 1 µm to 15 µm, d(v,0.5) = 20 µm to 70 µm and d(v,0.9) = 100 µm to 200 µm. Vismodegib is milled using a hammer mill and the milling parameters were systematically investigated with the goal to provide a robust process meeting the above multipoint specification of the PSD. Experiments were performed to assess the impact of the hammer speed on the PSD on different lots of unmilled Vismodegib and the results are illustrated in Figure 4.


25


Figure 4. Correlation of Hammer Speed on Particle Size

Particle Size (µm)

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

Page 26 of 35

Hammer Speed (rpm)

The data shows a good correlation between the hammer speed and the final mean PSD27, with a faster hammer speed producing a smaller mean PSD. For production, a hammer speed in the range of 3750 - 4250 rpm produces a very reproducible PSD meeting the multipoint PSD specification and ensures Drug Product quality and manufacturability.

Genotoxic Impurity Control Strategy ICH M728 limits the exposure of genotoxic impurities (GTIs) in patients to a maximum of 1.5 micrograms (µg)/day (Threshold of Toxicological Concern or TTC) corresponding to a theoretical 10-5 excess lifetime risk of cancer. Based on the TTC and on a Vismodegib dose of 150 mg/day, a maximum daily exposure can be calculated: 1.5 µg/150 mg = 10 ppm. Therefore, any GTI must have a 10 ppm maximum limit (acceptance criterion) in the API specifications. To


26

Page 27 of 35

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


ensure that the 10 ppm limit in Vismodegib will be met, a better understanding of the fate and purging of the GTI’s in the process for each identified GTI was needed. First, we addressed the fate of the nitroaromatic GTIs 6 and 7 originated from the Negishi route (Scheme 2) and nitroaromatics 8 and 9 originated from the pyridinium route to 3 (Scheme 3). We designed spiking experiments with all 4 GTIs in the Stage 1 (hydrogenation) to assess the purging capability of the process. Thus in different experiments, 1 wt% (10,000 ppm) of each 6, 7, 8 and 9 were spiked in the starting material 3 and submitted to the Stage 1 process. A GC-MS method was developed and validated for the quantitation of 6, 7 and 9, while a LC-MS was developed for 8. Both methods with selective ion monitoring29 have a limit of quantitation 4 ppm, giving a purging factor for each GTI of >2,500. Based on this data, impurities 6, 7, 8 and 9 were specified at 1 ppm, giving a purging factor of >10,000. This purging factor justifies the specification for 11 of 80% overall yield and with >99.95% purity on 20 kg scale, with the desired polymorphic phase and the specified particle size distribution. A control strategy in which genotoxic impurities are specified at the different stages of the manufacturing process was implemented and ensures that the 10 ppm limit for GTIs in the API will be met.

Experimental Section General. Unless otherwise noted, all reactions were run under a nitrogen atmosphere, and solvents and reagents were without further purification. 1H NMR (400 MHz) and 13C NMR (100 MHz) were recorded on a Bruker Avance 3 spectrometer. Chemical shifts are reported in ppm (δ


28

Page 29 of 35

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


units) downfield of internal tetramethylsilane [(CH3)4Si] or residual CHCl3; coupling constants are reported in hertz (Hz). Multiplicities are as follows: s = singlet, d = doublet, t =triplet, q = quartet, dd = doublet of doublets, m = multiplet. Melting points were measured by differential scanning calorimetry (DSC, Büchi B-540). Compounds 2 (CAS#53250-83-2), 6 (CAS#6283-256), 7 (CAS#74534-15-9), 8 (CAS#2516-96-3), 9 (CAS#23082-50-0), 10 (CAS#623927-88-8) and 15 (CAS#15889-32-4) are commercially available.

Stage

1,

Synthesis

of

4-chloro-3-(pyridin-2-yl)aniline

4.

A

mixture

of

2-(2-chloro-5-nitrophenyl)pyridine 3 (18.0 kg, 76.7 mol, 1 wt) and a 50 wt% wet catalyst with approximately 1% platinum/2% vanadium on carbon (0.90 kg, 0.05 wt) in methanol (162 kg, 9 wt) and acetic acid (18 kg, 1 wt) is hydrogenated at a temperature of 50-55 °C under a pressure of ~5.0 bar (~75 psi) for 3 hours (until hydrogen uptake is complete, as determined by a flow of ≤ 50 L/hour). After cooling to 20 °C, the catalyst is removed by filtration. The remaining solution is concentrated (~50%) under vacuum at a jacket temperature of a maximum of 60 °C and then cooled to approximately 13 °C. A 1:1 mixture of water (72 kg) and 25 wt% aqueous ammonia (73 kg) is added at 15 °C over 1.5 hours (until the pH is ≥ 10).

The resulting

suspension is aged for at least 1 hr at 8-10 °C and centrifuged. The solid is washed with water (397 kg) until the pH of the washing liquor is ≤ 7.5 and 15.9 kg of 4 is obtained (97.9% residue on drying, 99.2% corrected yield, >99.9A% by HPLC). The wet 4 intermediate, is stored at ≤ – 15 °C in polyethylene bags placed in a fiberboard drum and used as is in Stage 3. 1H NMR (400 MHz, CDCl3) δH 8.72-8.68 (ddd, J = 4.9, 1.8, 1.0 Hz, 1H), 7.74 (td, J = 7.6, 1.8 Hz, 1H), 7.66 (dt, J = 7.9, 1.1 Hz, 1H), 7.27 (ddd, J = 7.5, 4.9, 1.3 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 6.93 (d, J


29


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

= 2.9 Hz, 1H), 6.65 (dd, J = 8.5, 2.9 Hz, 1H), 3.73 (s, 2H).

13

Page 30 of 35

C NMR (CDCl3, 100 MHz): δC

157.00, 149.42, 145.42, 139.57, 135.73, 130.71, 124.88, 122.31, 121.00, 117.75, 116.35. Stage

2,

Synthesis

of

2-chloro-4-(methylsulfonyl)benzoyl

chloride

5.

2-Chloro-4-(methylsulfonyl)benzoic acid 2 (23.0 kg, 98.0 mol,1 wt) is suspended in toluene (97 kg, 4.22 wt) and a catalytic amount of N,N-dimethylformamide (DMF) (1.15 kg, 15.7 mol, 0.05 wt) is charged. Thionyl chloride (13.9 kg, 116.8 mol, 0.60 wt; 1.19 molar equiv.) is charged over ≥ 45 minutes at 70 °C, and the mixture is held at 70 °C until dissolution is complete (by visual monitoring) and the evolution of gas has ceased, signaling the end of the reaction. Excess thionyl chloride and a portion of the toluene (0.5 wt) are removed by distillation at atmospheric pressure (73-111 °C). The mixture is cooled to 10 °C, allowing 5 to precipitate, and aged for ≥ 2 hours. The precipitate is isolated by filtration and washed with toluene (28 kg) to give 27.4 kg of 5 (83.6% residue on drying, 92.4% corrected yield, >99.8A% HPLC). The solvent-wet acid chloride cake 5 is stored cold (2–8 °C) in sealed polyethylene bags purged with an inert gas (nitrogen or argon) and placed in a polyethylene drum and used as is in Stage 3. Acid choride 5 is characterized as the corresponding methyl ester 10: 1H NMR (400 MHz, CDCl3) δH 8.04 (d, J = 1.7 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.88 (dd, J = 8.1, 1.8 Hz, 1H), 3.98 (s, 3H), 3.09 (s, 3H). 13

C NMR (CDCl3, 100 MHz): δC 164.81, 144.01, 135.08, 134.70, 132.13, 129.80, 125.38, 53.01,

44.25.

Stage 3, Crude Vismodegib 1. 4-Chloro-3-(pyridin-2-yl)aniline 4, (13.9 kg dry basis, 67.8 mol, 1 molar equiv) is dissolved in THF (100 kg), an aqueous solution of K2CO3 is added (6.2 kg, 0.66 equiv., in 70 kg of water + 11.5 kg for rinse), and the biphasic mixture is cooled to 2 °C. A


30

Page 31 of 35

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


solution of 2-chloro-4-(methylsulfonyl) benzoyl chloride 5 (18.9 kg, 75.6 mol, 1.1 molar equiv.) in THF (132 kg + 14 kg for rinse) is charged over ≥ 35 minutes, and the mixture is warmed to 20 °C. Upon reaction completion as determined by in-process control (IPC) testing (≤ 1.0A% of 4 by HPLC), the aqueous layer is confirmed to have a pH of ≥ 7 and is then removed. Water (80 kg) is then added and the resulting mixture is distilled at atmospheric pressure (~67 °C) while water (~80 kg) is added maintaining constant volume. Once all water has been added, the distillation is continuing until a final volume of ~ 250 L is reached and the batch is then seeded with Vismodegib (0.38 kg, 0.013 molar equiv.), aged at reflux for a further 1–2 hours, followed by additional distillation until the temperature reaches 82 °C (~180 L final volume). The suspension is cooled to approximately 15 °C and held for at least 1 hour. The crude vismodegib is isolated by centrifugation, washed with water (~119 kg) to give 29.3 kg of crude 1 (93.4% residue on drying, 96% corrected yield, >99.9A% HPLC) and stored wet at a temperature of ≤ – 15 °C in polyethylene bags placed in a fiberboard drum and used as is in Stage 4. Stage 4, Pure Vismodegib 1. Crude vismodegib (12.5 kg dry basis, 29.7 mol, 1 wt) is dissolved in methyl isobutyl ketone (MIBK) (162.5 kg, 13 wt), and approximately 40% of the solvent is azeotropically distilled at atmospheric pressure (115 °C) to remove residual water from the Stage 3 intermediate. The solution is cooled to 105 °C and the amount of MIBK removed by the distillation is replaced with fresh MIBK (~65 kg). The solution is then checked for complete dissolution and filtered through a 1 µm filter as a final particulate-removing polish filtration and rinse with MIBK (18.8 kg, 1.5 wt). This solution is concentrated by distillation to approximately 9.5 volumes of MIBK and cooled to 95 °C. Approximately 0.5% water relative to total volume is added, and the solution is visually checked for undissolved solids and seeded with the Form B polymorph of vismodegib (85 g, 0.67 wt%). The suspension is aged for 2 hours at 95 °C, and


31


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

Page 32 of 35

then cooled to 5 °C at a gradient of approximately 18 °C/hour. The batch is aged for 3–24 hours at 5 °C before isolation by centrifugation. The wet cake is washed with MIBK (~2.7 vol), dried under vacuum, first at approximately 65 °C for about 5 hours and then at approximately 90 °C for ≥ 6 hours, until the residue on drying and water levels meet IPC specifications to give 10.7 kg of pure Vismodegib 1 (85.5% yield, >99.95A% HPLC) as a white solid. The dried, unmilled, vismodegib is stored at ≤ 30 °C until it is milled. The particle size of the dried vismodegib (23.1 kg) is reduced by milling using a hammer mill with a screen size of approximately 1650 µm (0.065 in), a feed rate of 35 rpm and a hammer speed of 4000 rpm to give 22.5 kg (97.4% yield) of milled 1. MP: 187 °C. HRMS: 421.0170 (M+1). 1H NMR (CD3CN, 600 MHz): δΗ 9.09 (s, 1H), 8.67 (d, J= 4.84 Hz, 1H), 8.03 (d, J= 1.68 Hz, 1H), 7.93 (dd, J= 7.92, 1.62 Hz, 1H), 7.88 (d, J= 2.65 Hz, 1H), 7.85 (td, J= 7.73, 7.67, 1.86 Hz, 1H), 7.78 (d, J= 7.92 Hz, 1H), 7.73 (dd, J= 8.70, 2.70 Hz, 1H), 7.69 (dd, J= 7.92, 1.03 Hz, 1H), 7.53 (d, J= 8.70 Hz, 1H), 7.37 (td, J= 7.59, 4.89, 1.09 Hz, 1H), 3.11 (s, 3H).

13

C NMR (CD3CN, 150 MHz): δC 165.06, 157.17, 150.61,

144.35, 141.75, 140.76, 138.34, 137.27, 132.79, 131.59, 130.93, 129.73, 127.93, 127.13, 125.80, 123.97, 123.68, 122.08.

ASSOCIATED CONTENT Supporting Information. Experimental and HPLC method for nitroaryl 3, Effect of SOCl2 / DMF ratio on the conversion to 5, Solubility of Vismodegib in organic solvents, Vismodegib spectroscopy data (NMR, IR, HRMS, single crystal X-ray, XRPD), MIBK crystallization of Vismodegib cooling curves, PSD diagram, Vismodegib milling studies comparing screen size,


32

Page 33 of 35

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


hammer speed, and feed rate, HPLC method descriptions for Stage 1, 2 and 3/4, GC-MS method for quantitation of 6, 7 and 9, LC-MS method for quantitation of 8, LC-MS method for quantitation of 11, 12, 13, 14 and 16 in Vismodegib,

Solubility of amine 4 in THF and

THF/Water Mixtures, spectroscopic data for 3, 4, 10, 11, 12, 13, 14, 15 and 16. AUTHOR INFORMATION Corresponding Author *[email protected]

References 1

(a) Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Disc. 2006, 5, 1026-1033. (b) Stone, D. M.; Hynes, M.; Armanini, M.; Swanson, T. A.; Gu, Q.; Johnson, R. L.; Scott, M. P.; Pennica, D.; Goddard, A.; Phillips, H.; Noll, M.; Hooper, J. E.; de Sauvage, F.; Rosenthal, A. Nature 1996, 384, 129-134. (c) Hahn, H.; Wicking, C.; Zaphiropoulos, P. G.; Gailani, M. R.; Shanley, S.; Chidambaram, A.; Vorechovsky, I.; Holmberg, E.; Unden, A. B.; Gillies, S.; Negus, K.; Smyth, I.; Pressman, C.; Leffell, D. J.; Gerrard, B.; Goldstein, A. M.; Dean, M.; Toftgard, R.; ChenevixTrench, G.; Wainwright, B.; Bale, A. E. Cell 1996, 85, 841-851. (d) Johnson, R. L.; Rothman, A. L.; Xie, J.; Goodrich, L. V.; Bare, J. W.; Bonifas, J. M.; Quinn, A. G.; Myers, R. M.; Cox, D. R.; Epstein Jr, E. H.; Scott, M. P. Science 1996, 272, 1668-1671. 2

Erivedge® was the first Hedgehog signaling pathway targeting agent to gain U.S. Food and Drug Administration (FDA) approval in January 2012. 3

See Supporting Information for experimental details.

4

Gunzner, J. L.; Sutherlin, D. P.; Stanley, M. S.; Bao, L.; Castanedo, G.; Lalonde, R. L.; Wang, S.; Reynolds, M. E.; Savage, S. J.; Malesky, K.; Dina, M. S. WO2009126863. 5

See patents: (a) Sonoda, T.; Shintou, T.; Onoue, K. Nagayama, T. WO2008093392(A1) and (b) Kuwabara, H.; Zhang, C. S.; Saitoh, H.; Sonoda, T. EP1559711(A1). 6

Sutter, A.; Amberg, A.; Boyer, S.; Brigo, A.; Contrera, J. F.; Custer, L. L.; Dobo, K. L.; Gervais, V.; Glowienke, S.; van Gompel, J.; Greene, N.; Muster, W.; Nicolette, J.; Reddy, M. V.; Thybaud, V.; Vock, E.; White, A. T.; Müller, L. Regul. Toxicol. Pharmacol. 2013, 67, 39-52. 7

Ames, B. N.; Durston, W. E.; Yamasaki, E.; Lee, F. D. Proc. Natl. Acad. Sci. U. S.A. 1973, 70, 2281-2285.


33


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

Page 34 of 35

8

(a) Robarge, K. D.; Brunton, S. A.; Castanedo, G. M.; Cui, Y.; Michael S. Dina, M. S.; Goldsmith, R.; Gould, S. E.; Guichert, O.; Gunzner, J. L.; Halladay, J.; Jia, W.; Khojasteh, C.; Koehler, M. F. T.; Kotkow, K.; La, H.; LaLonde, R. L.; Lau, K.; Lee, L.; Marshall, D.; Marsters Jr., J. C.; Murray, L. J.; Qian, C.; Rubin, L. L.; Salphati, L.; Stanley, M. S.; Stibbard, J. H. A.; Sutherlin, D. P.; Ubhayaker, S.; Wang, S.; Wong, S.; Xie, M. Bioorganic & Medicinal Chemistry Letters 2009, 19, 5576-5581. (b) Gould, S. E.; Low, J. A.; Marsters Jr., J. A.; Robarge, K.; Rubin, L. L.; de Sauvage, F. J.; Sutherlin, D. P.; Wong, H.; Yauch, R. L. Expert Opinion on Drug Discovery 2014, 9, 969-984. 9

See for examples: (a) H. U. Blaser, U. Siegrist, H. Steiner, M. Studer, in Fine Chemicals through Heterogeneous Catalysis, ed. R. A. Sheldon, H. van Bekkum, Wiley-VCH, Weinheim, 2001, 389–406; (b) J. R. Kosak Catalysis in Organic Synthesis, ed. W. H. Jones, Academic Press, New York, 1980, 107–117. (c) Corma, A.; Serna, P. Science 2006, 313, 332. (d) Hoogenraad, M.; van der Linden, J. B.; Smith, A. A.; Hughes, B.; Derrick, A. M.; Harris, L. J.; Higginson, P. D.; Pettman, A. J. Org. Process Res. Dev. 2004, 8, 469-476. 10

At a later time this HPLC method was shown to not resolve hydroxylamine 11 from the product. 11

(a) Takenaka, Y.; Kiyosu, T.; Choi, J-C.; Sakakura, T.; Yasuda, H. Green Chemistry 2009, 11, 1385-1390. (b) Becker, A. R.; Sternson, L. A. J. Org. Chem., 1980, 45, 1708-1710. (c) Corma, A.; Concepción, P.; Serna, P. Angew. Chem. Int. Ed. 2007, 46, 7266-7269. (d) Siegrist, U.; Baumeister, P.; Blaser, H.-U. Chem. Ind. 1998, 75, 207-219.

12

Although by-products 15 and 16 also gave in silico structural alerts, they were found Amesnegative.

13

(a) Baumeister, P.; Blaser, H-U.; Studer, M. Catal. Lett. 1997, 49, 219-222. (b) Crump, B. R.; Goss, C.; Lovelace, T.; Lewis, R.; Peterson, J. Org. Process Res. Dev., 2013, 17, 1277-1286.

14

Weight (wt): grams of reagent per 1 gram of substrate.

15

End of reaction is monitored by hydrogen uptake as determined by a flow of

Manufacturing Development and Genotoxic ... - ACS Publications

Recommend Documents