Confirmation of Bosutinib Structure; Demonstration ... - ACS Publications

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Confirmation of Bosutinib Structure; Demonstration of Controls To Ensure Product Quality Paul Bowles,† Frank R. Busch,*,† Kyle R. Leeman,‡ Andrew S. Palm,‡ and Karen Sutherland† †

Chemical Research and Development and ‡Analytical Research and Development, Pfizer Worldwide R&D-Groton Laboratories, 558 Eastern Point Road, Groton, Connecticut 06340, United States S Supporting Information *

ABSTRACT: Nonbranded/unauthorized vendors had been manufacturing/selling what they described as bosutinib, while the material supplied was actually an isomer of bosutinib. This raised concerns within the worldwide research community around the established control strategies for bosutinib. This manuscript summarizes that the appropriate testing was in place to ensure product quality, along with additional experimentation that was performed to confirm that testing (methods) can differentiate the potential isomeric compounds. Testing includes the use of IR for identity confirmation of raw materials, material characterization by NMR, single crystal X-ray to confirm structure, and evaluation of several potential isomers by HPLC, melting point, and IR, thus demonstrating the control strategy needed to ensure the product controls.

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this work, a goal was to enable identification of analogues by melting point alone; however, this was found to be insufficient. Melting points are provided on 2a and related analogues to provide a straightforward method for screening synthetic precursors, although testing by at least two methods is recommended. Literature melting points of the starting anilines are provided for additional control of raw materials. Use of IR for identity testing is recommended herein. The bosutinib manufacturing process is illustrated in Scheme 1. The halogenated aniline headpiece is introduced via the cyanoacetamide 2a condensation reaction with aniline 1 and triethyl orthoformate (TEOF). The aniline can be used as an incoming process stream in a suitable solvent or as the disuccinate salt. The condensation reaction provides compound 3 as a mixture of isomers. Both isomers of 3 undergo ring closure in a POCl3 mediated reaction in sulfolane, and after aqueous quench, crude bosutinib is isolated as a hexahydrate. This crude material is recrystallized and converted to the monohydrate. This synthesis was used for the desired analogues with modifications in the halogenated aniline headpiece incorporated into the cyanoacetamide variants of 2a. One route to compound 2a is via dichlorination of 3-methoxyaniline, followed by amidation with ethyl cyanoacetate. Thus, the control of the regiochemistry is based upon the selection of 3methoxyaniline rather than 4-methoxyaniline followed by the ortho/para directing effects during the bis-chlorination.

n May 2012, C&E News reported on confusion around the structure of bosutinib1 and that potentially multiple isomers of bosutinib had been marketed by unauthorized vendors.2 In order to eliminate confusion, this paper provides characterization information on the correct structure and data to assure the research community that only the correct bosutinib structure was used for patients in Wyeth/Pfizer clinical studies and for commercial supplies. Control strategies are presented herein for the differentiation of potential isomers by the research community/sellers of bosutinib, while providing an approach for control strategies that may be used for other products. Specifically this paper describes the use of IR as a test to control raw material identity, as well as a summary of the controls in place to ensure the product quality. The synthesis of bosutinib developed by Wyeth (now Pfizer) is shown in Scheme 1. This synthesis provides a convergent route that incorporates the more advanced 2,4-dichloro-5-methoxyaniline cyanoacetamide as the source of the aniline and does not use the 2,4-dichloro-5-methoxyaniline in the process. Bosutinib (Bosulif) is a potent inhibitor of Abl and Src kinases that inhibit metastasis3−6 and has progressed through registration. It was approved in the United States 4 Sept. 2012, for the treatment of chronic myeloid leukemia (CML)7 and received marketing authorization in the EU in March 2013.8 Routes to bosutinib have been published by Wyeth3,4 including a summary of the routes.9 More recently several presentations have been given to show the improvements made to the process, including a talk at the 2010 Pacific Chemical Society conference,10 an OPR&D paper,11 a talk delivered at Princeton University,12 and most recently a talk delivered at the 2013 New Orleans ACS meeting.13 Herein is reported the structural confirmation of bosutinib based on a single crystal structure. Data for distinguishing the analogues of bosutinib by IR, NMR, HPLC retention times, and melting point data; along with controls is provided. Melting points of bosutinib and several of the isomers/des-chloro analogues are provided so samples can be quickly screened by other laboratories. At the initiation of © XXXX American Chemical Society

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CONFUSION ON THE STRUCTURE OF BOSUTINIB ISOMERS As bosutinib became more advanced as a candidate, additional researchers wanted to obtain a supply of the material for testing against other kinases. The May 21st 2012 issue of C&E News1 reported that an isomer of bosutinib had been supplied to several research organizations, including Stanford UniverReceived: July 31, 2015

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DOI: 10.1021/acs.oprd.5b00244 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Scheme 1. Synthesis of Bosutinib

sity.14,15 The Stanford group, following months of research, realized something was wrong and characterized the samples by NMR and X-ray,16 determining the isomeric structure 4d shown in Scheme 2. Their supplier, LC Laboratories, stated on

3. Analyze the desired cyanoacetamide aniline and bosutinib versus corresponding potential isomers using currently registered methods (HPLC, IR) along with NMR. 4. Determine if a simple technique (e.g., melting point, IR) could be used to differentiate the isomers, and report the results, thus providing potential controls within the research community. As pointed out in the C&E News article, Levinson and Boxer conducted 1H and 13C NMR experiments to determine the sample they had purchased was in fact the isomer. “It’s only when you use 13C NMR that the symmetrical nature of the aniline group in the isomer becomes clear.”16 We have repeated the NMR characterization of the “bosutinib isomer” and agree with the Stanford group. As was noted in points 3 and 4, simpler methods than 13C, multidimensional NMR, or X-ray are needed to simplify compound identification. Additional confusion, as pointed out by Levinson and Boxer, has occurred with the route published in Molecules by Fei Li and co-workers.18 It appears that they used 3,5-dichloro-4methoxyaniline in place of 2,4-dichloro-5-methoxyaniline; please see Table S4 under Supporting Information for the evaluation of the bosutinib isomer 13C NMR vs the Molecules published data.

Scheme 2. Bosutinib and “Bosutinib Isomer”

their Web site that “at least two different chemical compounds have been described as ‘bosutinib’”14 and that 18 of the unauthorized suppliers were also supplying an isomer. The formation of bosutinib isomer(s) is related to the structure of the cyanoacetamide aniline (compound 2a in Scheme 1). The chlorination reaction to form the dichloroaniline precursor controls the position of the substituents, which carries through the synthesis to the product with the same substituent regiochemistry. In response to the C&E News article, our research team reviewed the data previously generated on clinical and commercial supplies within our organization. Our laboratories had never observed any positional isomers of the 2,4-dichloro5-methoxyaniline cyanoacetamide; although we thought our methods would likely resolve isomers based upon the deschloro 2b being resolved by over 4.8 min from 2a, the article challenged our research team to review the established controls: 1. Characterize “Bosutinib Isomer”, 4d, from LC Laboratories using NMR and our registered identity tests (HPLC, IR) and demonstrate that the tests provide appropriate differentiation and could distinguish the regioisomer (4d) from bosutinib (4a). 2. Prepare samples of several other likely dichloro-isomers17 of the cyanoacetamide aniline, as well as samples of the two monochloro intermediates, expected from partial chlorination, 2b and 2c. Then, convert each of the cyanoacetamide materials to analogues for further characterization and confirmation of the specificity of the analytical methods.

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CHARACTERIZATION OF ANILINE ISOMERS This paper describes the analytical controls and additional studies conducted to ensure appropriate and consistent quality. The desired isomer for the synthesis of bosutinib (2,4-dichloro5-methoxy cyanoacetamide or 2a), shown in Scheme 3, and des-chloro 2b are known in the literature, the other analogues were obtained from the known anilines. Following the preparation of the des-2-chloro and the other isomers shown, IR analysis was conducted. Characterization of the isomers by IR and HPLC analysis was conducted to ensure that the test method(s) were specific at identifying any of the monochloro analogues or dichloro isomers of 2a shown in Scheme 3. An example of the IR is shown in Figure 1; compounds 2a and 2d are compared. As predicted the “finger print” region shows differences. The mid-IR spectral differences between 2a and 2d can be attributed to the positions of the methyl ether and chlorines on the phenyl ring. The proximity of the chlorine next to the secondary amide causes bands to shift, particularly the Amide I band from 1686 to 1669 cm−1 and bands in the Cl stretching vibration region from 800 to 700 cm−1. The band position at B



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bosutinib manufacture. Figure 2 shows a chromatogram obtained running the method, with each analog added as a

Scheme 3. Monochloro Impurities and Dichloro Isomers of 2a

Figure 2. Chromatographic resolution of compound 2a from deschloro impurities and isomers with retention times.

minor component. Evaluation of the potential isomers of 2a showed that they are effectively differentiated by the IR identity test and HPLC purity methods. Each peak, as shown in Figure 2 is clearly resolved from the desired 2a by at least 1 min retention time. All of the analogues are well resolved from 2a, the retention times of the two key compounds 2a and 2d are 14.38 and 15.44 min respectively and show good peak resolution. It is recommended that if HPLC is used as the only characterization that authentic standards be obtained. We also wanted to investigate the two potential monochloro impurities; peaks for the monochloro compound 2c (at 11.22 min.) and isomer 2h (at 11.54 min) are close in retention time. As these compounds are not isomeric, HPLC with mass spec detection could be used, if needed, to confirm the identity of each compound. During development, the team’s concern was

869 cm−1 in the spectrum of 2a is indicative of the CH out-ofplane deformation of a 1,2,4,5-tetrasubstituted ring, whereas the band position at 855 cm−1 is indicative of the CH out-of-plane deformation of a 1,2,3,5-tetrasubstituted ring.19 As illustrated in Figure 1, the IR method shows sufficient differences between the isomers to differentiate the wrong isomer from the desired compound. The comparison of the IR spectra was repeated for each of the compounds depicted in Scheme 3, and overlaid as in Figure 1. In each case the IR spectroscopy was found to differentiate the structures, see Supporting Information. A robust chromatography method had been developed for the evaluation of purity of 2a. The method was appropriately validated and registered as part of the process controls for

Figure 1. Overlay of mid-IR scans of 2a (blue) vs 2d (red), scale expanded for mid-IR to show details of fingerprint region. C



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Figure 3. Chromatographic resolution of bosutinib, des-chloro compounds, and isomers.

Figure 4. Single crystal X-ray of bosutinib monohydrate.

Scheme 4. Preparation of Bosutinib Isomers

isomer were mistakenly present. The bosutinib method may not differentiate 4b−4f and 4c−4h on the basis of Rt; however, HPLC/MS could differentiate the two peaks. The single crystal X-ray provides confirmation of the structure of bosutinib; the 2,4-dichloro-5-methoxy structure of the aniline is clearly distinguished. X-ray parameters are provided in the Supporting Information. X-ray provides assurance that the substitution pattern is correct, see Figure 4. The properties of the compounds prepared, via the route shown in Scheme 4, are summarized in Table 1. The table includes if the compound 2 analog is known or if there was not a CAS number; along with melting point data on compounds 2a-h. The CAS numbers for known compounds are listed prior to the Experimental Section. Compounds 2a−h are herein characterized by mp and IR scans. The conversion of each of the compounds 2c−h to the corresponding 3c−h was conducted as shown in Scheme 4.20 The compound 3 column in Table 1 provides retention time (R t ), with the HPLC methodology provided in the experimental; of the two geometric isomers of compound 3,

2b, as an impurity in 2a, due to the potential of partial chlorination while preparing the desired dichloro-product; the method has excellent resolution for compounds 2a vs 2b and 2c. Following the procedures provided in the experimental, each of the compound 2 analogues were converted to compounds 3c−h which were then converted to compounds 4c−h. Samples of 3a and 4a were obtained from the manufacturing campaign. Compounds 4a−h were characterized by melting point, IR, NMR, and HPLC and then the results reviewed to confirm that the methods are specific. For compounds 4b−h, the resolution of each from 4a was confirmed using the registered HPLC method; an overlay chromatogram is shown in Figure 3. The chromatogram shows that each of the compounds 4b−h are suitably differentiated from the parent, 4a. The identity criterion of the method requires that the relative retention time be within 3% of the standard. In all cases, each isomer and the two des-chloro compounds was outside of that criteria and therefore would fail the identity test for bosutinib, if a bosutinib D



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Table 1. Properties of 2a−h, 3a−h, and 4a−h substitution on 2a−h no.

R1

R2

R3

R4

R5

2

mp of 2

IR of 2

a Bosutinib b

Cl

H

Cl

OCH3

H

known

199 °C

Figure 1

Cl

H

H

OCH3

H

known

157 °C

Supporting Information

c

H

H

Cl

OCH3

H

known

184 °C


d “Isomer” e

H

Cl

OCH3

Cl

H

none

188 °C

Figure 1

Cl

OCH3

Cl

H

H

none

157 °C


f

Cl

OCH3

H

H

Cl

none

218 °C


g

H

Cl

Cl

OCH3

H

none

226 °C


h

Cl

H

OCH3

Cl

H

none

193 °C


3 retention time (min)

mp of 4

a

125 °Cb

major 3.29 minor 3.29 major 3.03 minor 3.03 major 2.92 minor 3.11 major 3.19 minor 3.56 major 3.44 minor 3.82 major 2.68 minor 2.91 major 3.24 minor 3.56 major 3.32 minor 3.71

168 °C 88 °C 207 °C 108 °C 212 °C 210 °C 145 °C

a

Both of the E and Z isomer have the same retention time for intermediate 3a, both cyclize to the desired 4a. bMelting point of 4a was measured at 125 °C, bosutinib is a monohydrate, the dehydration occurs as measured by DSC, between 100 to 160 °C.

Table 2. Melting Point Comparison of Desired and Cyanoacetamide Isomers

the “bosutinib isomer” and other potential isomers has shown that these materials can be distinguished via typical/ straightforward analytical methodology and a review of the chemical literature. We want to assure the research and medical community that there is only one bosutinib produced by Pfizer. Literature Review. In addition to the analytical controls, the outcome of a synthesis is determined by conducting the appropriate literature review of the materials used in the process. The incorrect synthesis of the bosutinib isomer by vendors could have been avoided by characterization of the 2,4dichloro-5-methoxyaniline or characterization of its precursor 3-methoxyaniline. The web posting by PKC Pharmaceuticals correctly suggests that “...to enable others to replicate the chemical and pharmacological results obtained with a compound obtained from a synthesis, it is necessary to do “sufficient” additional unambiguous, relatively inexpensive, non-X-ray characterizations of material...”;21 results included here-in provide linkage to the authentic structure of bosutinib.22 The desired control of the process with a simple melting point determination would not have been possible in 2012 as compound 2d was not known in the literature. As Table 2 shows the melting points of 2a and 2d an 11 degree difference would appear to be enough to differentiate these isomers; although, the melting point we obtained differs from that seen by prior workers.3,23 Thus, the difference between samples of 2a and 2d should not be distinguished based upon melting point measurement alone; instead use of IR for identity is recommended. As there are

the ratio does not matter as both cyclize to the desired product. Mass spectral data was not obtained on 3a−h as it was not deemed necessary as 3a,d−h are isomeric and mass spectroscopy would not distinguish isomers. The determination of the melting point for 4a was 125 °C; this does not match the reported literature value.4 The standard used for the melting point determination was bosutinib monohydrate (4a) which is the commercial form, while the discovery synthesis yielded the slightly lower melting dihydrate form. The use of melting points offers the advantage of a simple method to assist in screening of the isomers; however, as noted previously,11 bosutinib is a promiscuous solvate former, thus the melting point needs to take potential solvates into consideration. The melting point difference between 4a and 4d when screened would indicate if observed, that additional testing is needed. Control of the synthesis by evaluation of compound 2a, at the start of the synthesis, is preferred.

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DISCUSSION C&E News quoted LC Laboratories (a division of PKC Pharmaceuticals, Inc.), stating that two “Bosutinibs” are known, resulting in confusion among researchers.1 The error appears to be due to poor quality controls in the manufacture of supplies, by unauthorized vendors, where 3,5-dichloro-4-methoxyaniline was apparently mistakenly used instead of the desired 2,4dichloro-5-methoxyaniline.16 This error progressed through the process unrecognized, giving the isomer of the intermediate (3d) followed by the isomer of bosutinib (4d). Investigation of E



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multiple routes to bosutinib, we cannot assume that the vendors producing the bosutinib isomer 4d were operating the route shown in Scheme 1; thus literature evaluation back to the dichloromethoxy aniline isomers is warranted. The cyanoacetamides are derived from the corresponding dichloromethoxy anilines, as shown in Table S3. The desired material 2,4-dichloro-5-methoxyaniline (literature mp: 50.5− 51.5 °C)24 vs the precursor to the bosutinib isomer25 3,5dichloro-4-methoxyaniline (literature mp: 80−80.5 °C) melts ∼30 °C higher. Other potential synthetic routes provide for the direct introduction of the 2,4-dichloro-5-methoxyaniline, so the melting point of this material can be a key control, thus permitting utilization of an inexpensive and simple test to ensure the desired regio-isomer is obtained in the product.26

CAS Numbers, 2a: [846023-24-3]; 2b: [1307530-78-4]; 2c: [879644-03-8]; 4a (anhydrous) [380843-75-4] and 4a (monohydrate) [918639-08-4; 4d [1391063-17-4]. Compounds have been made available for research purposes: 4a (PF-05208763, catalog no. PZ0192), 4b (PF-05883083, catalog no. PZ0286), 4c (PF-06658959, catalog no. PZ0292), 4d (PF-06651481 catalog no. PZ0289), 4e (PF-06663827, catalog no. PZ0291), 4f (PF-06663181, catalog no. PZ0290), 4g (PF-06663829, catalog no. PZ0288), 4h (PF-06665105, catalog no. PZ0287) from Sigma-Aldrich.27

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EXPERIMENTAL SECTION General. NMR were run on 500 or 600 or 700 MHz instruments, in DMSO-d6 or CDCl3 as indicated for each sample. IR analysis of mono- and dichloro isomers; comparison to compound 4a: The method is consistent with USP for sample preparation using a diamond ATR accessory. The infrared spectrum was obtained using a Thermo Scientific Nexus 470 Fourier-transform infrared spectrometer (FT-IR) and a Specac Golden Gate d-ATR accessory at 4 cm−1 resolution and a spectral range of 4000 cm−1 to 525 cm−1. X-ray data was obtained using a Bruker single crystal diffractometer. Parameters are provided under Supporting Information. The structure was solved using SHELXTL, Version 5.1, Bruker AXS, 1997. Melting point determinations were obtained using a Thomas−Hoover “Unimelt” capillary melting point apparatus. Supplies. Compound 4b was prepared previously to screen for des-chloro method specificity and was available from the internal Pfizer sample bank. A sample of “Bosutinib Isomer” (4d) was obtained from LC Laboratories, Woburn, MA, for NMR comparison. Samples of compounds 2b−h were obtained from WuXi AppTec (Shanghai) Co., Ltd. We thank Steve Guinness for providing samples of 3a and 4a. HPLC Analysis. (a) The analysis of isomers 3b−h was performed using a gradient UPLC with a Waters BEH C18; 1.7 μm, 2.1 × 50 mm column at 45 °C. UPLC conditions utilized mobile phase A−10 mM ammonium bicarbonate in water and mobile phase B: acetonitrile. Gradient elution was employed using an initial composition of 95% A and 5% B with a 0.5 min hold. The gradient profile included a ramp to 0% A and 100% B over 4.5 min with an additional 0.5 min hold. The flow rate was 0.6 mL/min, with an injection volume of 1 μL, and a detection wavelength of 266 nm. (b) HPLC conditions of 2a−h per Figure 2: The separation of the test mixture was on an YMC Pro C18; 3.0 μm, 4.6 × 150 mm column at 40 °C. HPLC conditions utilized mobile phase A−0.1% phosphoric acid in water/acetonitrile (95:5) and mobile phase B−0.1% phosphoric acid in water/acetonitrile (5:95). Gradient elution was employed using an initial composition of 80% A and 20% B with a linear gradient to 5% A and 95% B over 47 min. The flow rate was 1.0 mL/min, with an injection volume of 10 μL, and a detection wavelength of 210 nm. (c) HPLC of 4a−h separation of the test mixture was on a Waters Sunfire C18; 3.5 μm, 4.6 × 150 mm column at 55 °C. HPLC conditions utilized mobile phase A, 30 mM potassium phosphate in water (pH 2.0)/acetonitrile (95:5) and mobile phase B, 10 mM potassium phosphate in water (pH 2.0)/ acetonitrile (15/85). Gradient elution was employed using an initial composition of 100% A and 0% B with a 4 min hold. The

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CONCLUSION Evaluation of the analogues 2b−h of 2a demonstrated that each isomer or des-chloro compound is effectively differentiated by the IR identity test and HPLC purity method used by Pfizer. The methods are described, and the IR overlays are shown in Figure 1 and in the Supporting Information, providing methodology for the identification and differentiation of potential isomers/des-chloro analogues for the bosutinib synthesis. Based on these established methods, compounds 2b−h are clearly distinguishable from 2a and would be rejected before use in the bosutinib commercial manufacturing process. The use of IR spectra is a powerful and straightforward tool to confirm identity. In addition, the bosutinib identity tests (IR and HPLC) demonstrate acceptable selectivity between the desired and potential isomer(s). Based upon the established acceptance criteria for identity, compounds 2b−h would be deemed unacceptable and would fail release testing. Additional testing of 4a provides confirmation that the synthesis provides the authentic material and is clearly distinguished from 4b−h. Thus, a control strategy continues to be in place to ensure that the synthesis of bosutinib only utilizes the correct starting material, 2a. There are two “bosutinibs available in the research chemical market place” (italicize research chemical added for emphasis for clarification from pharmacy supplies). One bosutinib produced under the strict process controls following Good Manufacturing Procedures (cGMP) by Wyeth (now Pfizer) and an isomeric compound which was marketed by several vendors. Authentic samples of bosutinib are available from Sigma-Aldrich sourced from Pfizer for external nonclinical use.27 The identification and control of the starting materials clearly demonstrates the advantages of the use of identity testing of samples vs a standard (a routine Pfizer practice), thus providing assurance that the correct raw material is utilized in the synthesis. This work produced standards of four additional potential bosutinib isomers and the two monochloro-bosutinib analogues that would result from incomplete chlorination of the mmethoxyaniline, in addition to the “bosutinib isomer” which started the confusion. Each of the precursor intermediate isomers or monochloro compounds shown in Schemes 3 and 4 were converted to the corresponding bosutinib analogues. In addition to characterization of each sample, the melting points are provided, so that a simple and inexpensive test may be used for initial screening. However, the experience with confusion on the bosutinib isomer shows the importance of characterization of samples by at least two orthogonal methods. F



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The crude E/Z isomer products were not purified further as both converted to desired product in the next step. General Procedure for the Preparation of Bosutinib Isomers. The preparation of 4h is provided as an example. Workup varied depending upon whether the material crystallized or needed alternate isolation. The following methods utilized for the isolation are specified for each compound. Preparation of 4-(2,5-dichloro-4-methoxyphenylamino)6-methoxy-7-(3-(4-methylpiperazin-1yl)propoxy) quinoline3-carbonitrile. Compound 3h (20 g, 1.0 equiv, 36.5 mmol) was charged to a vessel (under an inert atmosphere throughout) containing premelted sulfolane (100 mL, 5 vol.), and the contents were heated to 85 °C. Phosphorus oxychloride (7.3 g, 4.4 mL, 1.3 equiv, 48 mmol) was added dropwise at a rate allowing the exotherm to increase the reaction contents to 105 °C. At the end of the addition, the temperature was adjusted to 105 °C and stirred overnight. Reaction completion was checked using UPLC using method a. After the reaction was judged complete, the contents were cooled to 75 °C. Using 5.5% potassium hydroxide (325 mL), the pH was adjusted to 10 and then contents stirred at 75 °C for 30 min. Methyl isobutyl ketone (240 mL, 15 vol.) was charged to the flask stirring hot for 15−30 min, then separated layers hot. Note: The meniscus is difficult to see without a flashlight. The upper organic product layer was transferred to a vessel and water (240 mL, 15 vol) was added. Vessel contents were brought to 75 °C and stirred for 15 min before separating the layers. Bosutinib isomer isolation/purification of this example, and listed bosutinib isomers/des-chloro analogues, was carried out in one of five methods: 1. If solids were present upon cool down, they were granulated several hours, filtered slurry, washed cake with IPA, and dried product in vacuum oven overnight at 55 °C. 2. If slurry looked thin, or if a solution was present, after cooling to room temperature, the solution was concentrated to a viscous oil, dissolved in 5−15 volumes ethyl acetate, silica gel plug filtration loaded plug with ethyl acetate, poured product solution through and rinsing with ethyl acetate, increasing polarity to 9:1 ethyl acetate−methanol, then eluting product using 1:1 ethyl acetate−methanol, concentrate product fractions to dryness. 3. If last hot organic layer was emulsified, it was filtered hot through filter aid, concentrated the filtrate to a viscous oil and dissolved the residue in ethyl acetate (5 volumes), heated to 55 °C for solution, cooled to room temperature, granulated several hours, filtered the slurry, washed cake with small amount of cold ethyl acetate, and dried in a vacuum oven at 55 °C. 4. If a solution, it was concentrated to solids or oil, dissolved residue in ethyl acetate (10 vol.), heated to 70 °C, then slow cooled to room temperature. It was granulated several hours, filtered, and washed cake with cold ethyl acetate. 5. If ethyl acetate crystallization conditions did not generate solids, it was concentrated to an oil, added 10 mL of ethyl acetate, added 10 volumes dropwise of water for precipitation, heated to 40 °C stirred for 15 min, slow cooled to 23 °C, granulated several hours, filtered, and washed cake with water, dried in vacuum oven at 55 °C.

gradient profile included a ramp to 94% A and 6% B over 0.5 min, 87% A and 13% B over 25.5 min, 44% A and 56% B over 20 min, 0% A and 100% B over 2.5 min with a final 5 min hold. The flow rate was 1.5 mL/min, with an injection volume of 15 μL, and a detection wavelength of 266 nm. Note: We have used the term HPLC throughout this paper, as the shorter abbreviation for liquid chromatography (LC) could be confused with LC laboratories. Preparation Procedure 3c−h. The following procedure can be used to make all of the bosutinib isomer/des-chloro precursors: 3c−h. Here, the preparation of 3c is used as an example. Workup varied for 3d−h and is specified for each compound below. Procedure for the Preparation of 3c. N-(2-Chloro-5methoxyphenyl)-2-cyano-3-(4-methoxy-3-(3-(4-methylpiperazin-1-yl)propoxy)phenylamino)acrylamide (E and Z Isomers, E Is Major Product). Compound 1 (20 g, 1.0 equiv, 39 mmol), IPA (132 mL, 6.5 vol.), and tributylamine (33.8 g, 44 mL, 4.7 equiv, 182 mmol) were charged to a reaction vessel (which had a nitrogen inert atmosphere throughout). The contents were stirred for 30 min at 20−25 °C. Compound 2c (10.6 g, 1.05 equiv, 40.7 mmol) and triethyl orthoformate (26.3 g, 29.6 mL, 4.6 equiv, 178 mmol) were added to the flask. The contents were heated to 83 °C and stirred for a minimum of 18 h. Reaction monitoring using UPLC method a showed completion. The contents of the flask were cooled to 23 °C. The product isolation of 3c−h, each pair of isomer intermediates, was carried out in one of two ways: 1. If solids were present upon cool down, they were granulated several hours, filtered the slurry, washed the cake with IPA, and dried the product in vacuum oven overnight at 55 °C. 2. If slurry looked thin or a solution was present after cooling to room temperature, then it was concentrated to a viscous oil, dissolved in 10 volumes of ethyl acetate, performed silica gel plug filtration, loaded the plug with ethyl acetate, poured the product solution through and rinsed with ethyl acetate, increased the polarity to 9:1 ethyl acetate−methanol, then eluted the product using 1:1 ethyl acetate−methanol, and concentrated the product fractions to dryness. The following are the specific product isolation methods used and yield for each intermediate. Preparation of Compound 3c. Slurry at room temperature; isolated using method 1. 80% weight yield, 95% combined E/Z isomer purity. Preparation of Compound 3d. Solution at room temperature; isolated using method 2. 81% weight yield, 95% combined E/Z isomer purity. Preparation of Compound 3e. Solution at room temperature; isolated using method 2. 80% weight yield, 91% combined E/Z isomer purity. Preparation of Compound 3f. Solution at room temperature; isolated using method 2. Quantitative weight yield, 92% combined E/Z isomer purity. Preparation of Compound 3g. Slurry at room temperature; isolated using method 1. 78% weight yield, 95% combined E/Z isomer purity. Preparation of Compound 3h. Sticky slurry at room temperature; isolated using method 2. Quantitative weight yield, 77% combined E/Z isomer purity. G



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C NMR (DMSO-d6, 150 MHz, 25 °C). δ 156.1, 153.1, 150.6, 149.6, 148.3, 146.4, 140.7, 132.2, 117.3, 115.1, 114.6, 114.3, 109.2, 105.5, 101.8, 89.9, 66.9, 56.7, 56.1, 54.7 × 2, 54.3, 52.7, 45.7 × 2, 26.0. HRMS. (ESI+) Calcd for C26H30O3N5Cl2 (M + H)+: 530.17202, Found: 530.17212. Preparation of 4h. Due to an emulsified product organic layer, 4h was isolated using method 3; 45% weight yield, 99% purity by HPLC. 1 H NMR (DMSO-d6, 600 MHz, 25 °C). δ 9.50 (bs, 1H), 8.37 (s, 1H), 7.81 (s, 1H), 7.68 (s, 1H), 7.42 (s, 1H), 7.29 (s, 1H), 4.18 (t, J = 6.5 Hz, 2H), 3.942 (s, 3H), 3.937 (s, 3H), 2.50− 2.18 (b, 8H), 2.44 (t, J = 7.1 Hz, 2H), 2.15 (s, 3H), 1.95 (quint, J = 6.8 Hz, 2H). 13 C NMR (DMSO-d6, 150 MHz, 25 °C). δ 154.4, 152.7, 150.9, 150.1, 149.3, 145.6, 132.1, 130.9, 129.3, 119.9, 117.0, 113.3, 112.0, 109.3, 101.7, 85.1, 66.8, 56.9, 56.2, 54.8 × 2, 54.3, 52.7, 45.7 × 2, 26.0. HRMS. (ESI+) Calcd for C26H30O3N5Cl2 (M + H)+: 530.17202, Found: 530.17116. The sample of 4h contains impurity signals at δH 2.99, 2.07 and at δC 50.5, 22.1, consistent with residual sulfolane. Characterization of 4b, 3-Quinolinecarbonitrile, 4-[(2Chloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl1-piperazinyl)propoxy]. 1H NMR (DMSO-d6, 600 MHz, 25 °C). δ 9.53 (s, 1H), 8.39 (s, 1H), 7.81 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.06 (s, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.78 (s, 3H), 2.50−2.18 (b, 8H), 2.45 (t, J = 7.0 Hz, 2H), 2.15 (s, 3H), 1.95 (quint, J = 6.8, Hz, 2H). 13 C NMR (DMSO-d6, 150 MHz, 25 °C). δ 158.6, 152.7, 150.8, 149.5, 149.3, 145.7, 137.0, 130.2, 122.8, 116.8, 114.7, 114.5, 112.4, 109.3, 101.8, 86.4, 66.8, 56.2, 55.7, 54.8 × 2, 54.3, 52.7, 45.7 × 2, 26.0. HRMS. (ESI+) Calcd for C26H31O3N5Cl1 (M + H)+: 496.21099, Found: 496.20985. Characterization of 4a, 3-Quinolinecarbonitrile, 4-[(2,4Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4methyl-1-piperazinyl) propoxy], Hydrate (1:1). 1H NMR (DMSO-d6, 500 MHz, 25 °C). δ 9.63 (bs, 1H), 8.40 (s, 1H), 7.82 (s, 1H), 7.73 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 2.45 (t, J = 7.1 Hz, 2H), 2.49−2.19 (b, 8H), 2.15 (s, 3H), 1.95 (quint, J = 6.8 Hz, 2H). 13 C NMR (DMSO-d6, 125 MHz, 25 °C). δ 154.0, 152.7, 150.6, 149.4, 149.3, 145.3, 136.6, 129.8, 122.8, 120.1, 116.9, 113.2, 112.6, 109.0, 101.9, 86.2, 66.8, 56.7, 56.2, 54.7 × 2, 54.3, 52.7 × 2, 45.7, 26.0. HRMS. (ESI + ) Calcd for C 26 H 30 O 3 N 5 Cl 2 (M+H) + : 530.17202, Found: 530.17122. 13

The following are the specific isolation methods used and characterization results for each bosutinib analog made: Preparation of 4c. Solution present; isolated using method 4; 30% weight yield, 97.6% purity. 1 H NMR (DMSO-d6, 600 MHz, 25 °C). δ 9.55 (bs, 1H), 8.49 (s, 1H), 7.73 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.03 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.4, 2.4 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.92 (s, 3H), 3.85 (s, 3H), 2.44 (t, J = 7.1 Hz, 2H), 2.48−2.17 (b, 8H), 2.14 (s, 3H), 1.95 (quint, J = 6.8 Hz, 2H). 13 C NMR (DMSO-d6, 150 MHz, 25 °C). δ 154.7, 152.8, 150.7, 149.3, 148.9, 146.1, 140.4, 129.8, 117.3, 116.5, 115.8, 113.8, 109.2, 107.9, 101.8, 88.5, 66.8, 56.0 × 2, 54.7 × 2, 54.2, 52.6 × 2, 45.7, 25.9. HRMS. (ESI + ) Calcd for C 26 H 31 O 3 N 5 Cl 1 (M+H) + : 496.21099, Found: 496.21094. Preparation of 4d, CAS [1391063-17-4]. Solids present; isolated using method 1; 26% weight yield, 95% purity. 1 H NMR (DMSO-d6, 700 MHz, 25 °C). δ 9.54 (bs, 1H), 8.54 (s, 1H), 7.67 (s, 1H), 7.36 (s, 2H), 7.34 (s, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.92 (s, 3H), 3.82 (s, 3H), 2.55−2.17 (b, 8H), 2.44 (t, J = 7.1 Hz, 2H), 2.15 (s, 3H), 1.95 (quint, J = 6.8 Hz, 2H). 13 C NMR (DMSO-d6, 175 MHz, 25 °C). δ 153.1, 150.6, 149.5, 148.6, 148.2, 146.3, 137.9, 128.3 × 2, 123.1 × 2, 117.3, 114.0, 109.2, 101.7, 89.1, 66.9, 60.8, 56.1, 54.8 × 2, 54.3, 52.7 × 2, 45.8, 26.0. HRMS. (ESI+) Calcd for C26H30O3N5Cl2 (M + H)+: 530.17202, Found: 530.17126. Preparation of 4e. Crystallization conditions did not yield solids; they were isolated using method 5; 67% weight yield, 96% purity. 1 H NMR (CDCl3, 600 MHz, 25 °C). δ 8.71 (s, 1H), 7.43 (s, 1H), 7.18 (d, J = 8.9 Hz, 1H), 6.88 (s, 1H), 6.76 (s, 1H), 6.58 (d, J = 8.9 Hz, 1H), 4.26 (t, J = 6.6 Hz, 2H), 3.98 (s, 3H), 3.76 (s, 3H), 2.57 (t, J = 7.3 Hz, 2H), 2.66−2.34 (b, 8H), 2.30 (s, 3H), 2.11 (quint, J = 6.8 Hz, 2H). 13 C NMR (CDCl3, 150 MHz, 25 °C). δ 154.1, 153.3, 150.5, 149.6, 147.8, 147.5, 138.1, 128.0, 123.7, 121.1, 116.3 × 2, 115.2, 109.9, 101.2, 95.1, 67.7, 60.9, 56.1, 55.1 × 2, 54.8, 53.1 × 2, 46.0, 26.3. HRMS. (ESI + ) Calcd for C 26 H 30 O 3 N 5 Cl 2 (M+H) + : 530.17202, Found: 530.17139. Preparation of 4f. Solids were present; they were isolated using method 1; 34% weight yield, 100% purity. 1 H NMR (CDCl3, 600 MHz, 25 °C). δ 8.56 (s, 1H), 7.40 (s, 1H), 7.38 (d, J = 9.1 Hz, 1H), 7.03 (s, 1H), 6.61 (d, J = 9.1 Hz, 1H), 6.88 (s, 1H), 4.24 (t, J = 6.7 Hz, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 2.56 (t, J = 7.1 Hz, 2H), 2.69−2.33 (b, 8H), 2.30 (s, 3H), 2.10 (quint, J = 6.8 Hz, 2H). 13 C NMR (CDCl3, 150 MHz, 25 °C). δ 154.9, 153.6, 150.5, 150.2, 148.7, 146.8, 134.9, 128.0, 124.4, 121.9, 116.3, 113.4, 111.0, 110.0, 99.8, 90.0, 67.6, 56.7, 56.0, 55.1 × 2, 54.8, 53.1 × 2, 46.0, 26.3. HRMS. (ESI+) Calcd for C26H30O3N5Cl2 (M + H)+: 530.17202, Found: 530.17248. Preparation of 4g. Following layer separation, a solution was present, isolated using method 4; 52% weight yield, 98% purity. 1 H NMR (DMSO-d6, 600 MHz, 25 °C). δ 9.61 (bs, 1H), 8.57 (s, 1H), 7.69 (s, 1H), 7.36 (s, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 2.1 Hz, 1H), 4.20 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 2.50−2.18 (b, 8H), 2.44 (t, J = 7.1 Hz, 2H), 2.15 (s, 3H), 1.95 (quint, J = 6.8 Hz, 2H).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.oprd.5b00244. Preparation of starting materials, IR spectra, NMR spectra, X-ray parameters, reference melting points, and 13 C NMR comparison of an authentic sample with erroneous literature data (PDF) H



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Article

isomer to other researchers; accessed March 28, 2015. Their web site posting was helpful to understand the scope of the issue. (15) “Bosutinib: Don’t Believe the Label!” In the Pipeline by Derek Lowe, see: http://pipline.corante.com/archives/2012/05/14/ bosutinib, accessed May 16, 2012, site no longer active. (16) Levinson, N. M.; Boxer, S. G. PLoS One 2012, 7, e29828. (17) The potential compounds prepared for the evaluation started with the two monochloro compounds, the known 2b and 2c, which are expected from incomplete chlorination. The other isomers were selected based upon the expected ortho/para directing for chlorination of methoxyaniline or its precursor. (18) Yin, X. J.; Xu, G. H.; Sun, X.; Peng, Y.; Ji, X.; Jiang, K.; Li, F. Molecules 2010, 15, 4261−4266. (19) Socrates, G. Infrared and Raman Characteristic Group Frequencies, 3rd ed.; John Wiley & Sons: Chichester, UK, 2001. (20) The goal of this chemistry was to prepare authentic samples for testing and identification; the chemistry is not optimized for the analogues, and several of the yields were lower than expected when the compound(s) did not crystallize at the same point in the process relative to the synthesis of bosutinib (4a). Although this resulted in reduced yields, it illustrates that the properties of the compounds, although structurally similar do vary in solubility and/or crystallinity. (21) PKC Pharmaceuticals web site, Bosutinib Report #1, February 20, 2012; p 14; accessed March 28, 2015. (22) Due to the controversy when the Boxer and Levinson work was published showing the PKC Pharma error (see ref 16), our NMR laboratory tested the retained samples of the lots which had been prepared for the clinical studies on bosutinib and confirmed that all of the 1H NMRs matched, thus serving as a double check on the quality controls in place for the release testing which had previously been conducted. (23) Although melting points offer a rapid and simple screening technique for compound identification, one should be aware of variation due to heating rate and the method use, see for example: Shriner, R. L.; Fuson, R.C.; Curtin, D. Y. The Systematic Identification of Organic Compounds, 5th ed.; John Wiley and Sons: New York, 1964; Chapter 4 and p 306. (24) Three references are found upon a search: (a) Jacobs, W. A.; Heidelberger, M.; Rolf, I. P. J. Am. Chem. Soc. 1919, 41, 458−474. (b) Theodoridis, G.; Hotzman, F. W.; Scherer, L. W.; Smith, B. A.; Tymonko, J. M.; Wyle, M. J. Pestic. Sci. 1990, 30, 259−274. (c) Weibing, S., et al.; Preparation Method of Arylamine, CN 102617358A (but no melting point appeared to be given, reference in Chinese). (25) de Traz, C. Helv. Chim. Acta 1947, 30, 232−6. (26) It is possible that the bosutinib isomer issue arose as early as the chlorination step. The melting points and proton NMR data for the mmethoxyaniline (mp: −1 to 1 °C) are different than p-methoxyaniline (mp: 57−60 °C), thus providing additional potential control points for the precursor process. (27) Pfizer has an agreement to make research samples available through Sigma-Aldrich so that research groups can gain access to selected reference materials described in this article.

AUTHOR INFORMATION

Corresponding Author

*E-mail: frank.r.busch@pfizer.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Karen S. Bronk, Shengquan Duan, Steven Guinness, Gregory Withbroe, and Rajappa Vaidyanathan for their assistance and advice. The authors also thank Sergei Timofeevski for Oncology Biochemistry screening; Fangming Kong, Brian Marquez, and Bao Nguyen for NMR characterization; and Brian Samas for X-ray analysis. We thank the discovery group lead by Diane H. Boschelli for laying the groundwork for this project. We thank John A. Ragan for review of this manuscript.

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REFERENCES

(1) Halford, B. Chem. & Eng. News, v90 2012, 90 (21), 34−35. (2) Unauthorized vendor describes the vendors which supply small quantities of a patented compound for research rather than clinical use, without the patent holder’s permission. (3) Sutherland, K.; Feigelson, G. B.; Boschelli, D. H.; Blum, D. M.; Strong, H. L. Process for Preparation of 4-Amino-3-quinolionecarbonitriles. US 2005043537 A1; later US 7,297,795 B2. (4) Boschelli, D. H.; Ye, F.; Wang, Y. D.; Dutia, M.; Johnson, S. L.; Wu, B.; Miller, K.; Powell, D. W.; Yaczko, D.; Young, M.; Tischler, M.; Arndt, K.; Discafani, C.; Etienne, C.; Gibbons, J.; Grod, J.; Lucas, J.; Weber, J. M.; Boschelli, F. J. Med. Chem. 2001, 44, 3965. (5) (a) Boschelli, D. H.; Wang, Y. D.; Johnson, S.; Wu, B.; Ye, F.; Sosa, A. C. B.; Golas, J. M.; Boschelli, F. J. Med. Chem. 2004, 47, 1599. (b) Boschelli, D. H.; Wu, B.; Ye, F.; Wang, Y.; Golas, J. M.; Lucas, J.; Boschelli, F. J. Med. Chem. 2006, 49, 7868. (6) Additional studies showed activity of SKI-606 in Abl (tyrosine kinase), see: Puttini, M.; Coluccia, A. M. L.; Boschelli, F.; Cleris, L.; Marchesi, E.; Donella-Deana, A.; Ahmed, S.; Redaelli, S.; Piazza, R.; Magistroni, V.; Andreoni, F.; Scapozza, L.; Formelli, F.; GambacortiPasserini, C. Cancer Res. 2006, 66, 11314−11322. (7) FDA News Release Sept. 4, 2012, “FDA approves new orphan drug for chronic myelogenous leukemia”, see: http://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/ucm318160.htm (accessed March 27, 2015). (8) See Pfizer press release on the EU Commission adoption of the Decision granting a conditional marketing authorization for ″Bosulif Bosutinib″, an orphan medicinal product for human use in CML. March 27, 2013. (9) (a) Synthetic approach where the aniline “headpiece” installed early, permitting preparation of 7-amino analogues. Treatment of compound 2, in this reference, with propyloxy-N-methylpiperazine gives bosutinib in low yield: Boschelli, D. H.; Wu, B.; Ye, F.; Durutlic, H.; Golas, J. M.; Lucas, J.; Boschelli, F. Bioorg. Med. Chem. 2008, 16, 405−412. (b) Boschelli, D. H.; Boschelli, F. Drugs Future 2007, 32 (6), 481−490. (10) Vaidyanathan, R. “The Bosutinib Story”. Presented at the Pacific Chemical Society conference, Hawaii, Dec 29, 2010. (11) Withbroe, G. J.; Seadeek, C.; Girard, K. P.; Guinness, S. M.; Vanderplas, B. C.; Vaidyanathan, R. Org. Process Res. Dev. 2013, 17, 500−504. (12) Duan, S. Route Selection and Process Development of Bosutinib, A Src Kinase Inhibitor; presented at Princeton University, Princeton, NJ, June 13, 2012. (13) Sutherland, K., et al. ACS Meeting April 8, 2013, New Orleans, paper no. 344: Bosulif®: A summary of synthesis, isomers, control strategy, and assurance of product quality. (14) http://www.pkcpharma.com/TwoOrMoreBosutinibs.html. PKC Pharmaceuticals Inc. noted that they did not produce the isomer but were one of eighteen vendors who sold the incorrect bosutinib I


Confirmation of Bosutinib Structure; Demonstration ... - ACS Publications

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