Early Process Development of a Squaramide-Based CXCR2 Receptor

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Organic Process Research & Development

Early process development of a squaramide-based CXCR2 receptor antagonist Benjamin Martin†*, Xinzhong Lai§, Urs Baettig‡, Eva Neumann†, Thomas Kuhnle†, David Porter‡, Richard Robinson‡, Julia Hatto‡, Anne-Marie D’Souza‡, Oliver Steward‡, Simon Watson‡, and Neil J. Press‡ †

Novartis Pharma AG, Fabrikstrasse 14, 4002 Basel, Switzerland

§

Suzhou Novartis Pharma Technology Company Limited, Changshu, Jiangsu 215537, China

‡

Novartis Pharmaceuticals UK Limited, Wimblehurst Road, Horsham Sussex RH12 5AB, United

Kingdom

*Corresponding Author E-mail: [email protected]

ACS Paragon Plus Environment

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Table of Contents Graphic


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ABSTRACT

The synthesis of a CXCR2 antagonist is presented, highlighting the process changes made from research synthesis to clinical supply. The target compound is the choline salt of a nonsymmetrical squaramide, and the modifications to the synthetic route which have effect on chemical purity are discussed with reference to the isolated by-products. Although drug substance quality was shown to increase following optimisation of the linear sequence, an alternative one-pot, convergent sequence was introduced, which makes dual use of choline hydroxide as both base and salt-forming agent. The overall benefits of the strategic change is discussed in terms of overall yield and economy.

KEYWORDS: squaramide; choline; nucleophilic vinylic substitution; CXCR2; COPD; one-pot


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1.

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Introduction

Chronic obstructive pulmonary disorder (COPD) is now the fourth leading cause of death in the world1 and further increases in its prevalence and mortality is predicted in the coming decades.2 Disease progression is traditionally estimated by lung-function tests which are closely related to mortality rate, with nearly 35% of severe COPD patients dying within 12 years. Despite this, the disease remains poorly treated because few agents have been developed specifically for the treatment of the disease. Available treatments for COPD are mainly palliative and include shortand long acting β-adrenergic bronchodilators, inhaled anticholinergics (muscarinic antagonists) and inhaled corticosteroids to treat the symptoms and exacerbations of the disease. The chemokine IL-8, originally called neutrophil activating protein, primarily activates CXCR1 and CXCR2, both of which are expressed on neutrophils.3 Novartis is developing a CXCR2 selective antagonist 1 (Figure 1) which has the potential to become a first in class anti-inflammatory treatment for COPD.4 Figure 1. A selective CXCR2 antagonist in development at Novartis

Synthetic routes to squaramides typically start with squarate diesters which are the electrophilic partner in sequential nucleophilic vinylic substitutions by amines.5 The retrosynthesis of the squaramide target 1 therefore led us back to the squarate di-ethyl ester 2 and two amines 3 and 4 (Figure 2). While 3-pentylamine 4 is commercially available, aniline 3 required synthesis from


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simpler building blocks. The anilino and phenolic functional groups of 3 originate in the protected form of a benzoxazole 5 which is prepared from a one-pot sequence which has been described previously.6,7 As such this publication will focus on the forward synthesis of compound 1 from the functionalized benzoxazole 5. Figure 2. Retrosythesis of target compound 1

2.

Results and Discussion

2.1 Pre-clinical Supply An initial route was developed within the research department and based on this a first scale up was inititated to supply toxicological studies, which was then later repeated on kilogram scale. The route consisted of four chemical steps (Figure 3) with overall yield of 56 %. Deprotection of the tert-butyl benzoxazole 5 was achieved with an excess of sulfuric acid with 1,4-dioxane/water as heated solvent mixture. This step is common to all the explored routes to compound 1, but


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attempts were later made to move away from use of dioxane as it is has undesirable toxicological properties.8 Despite confirmation on laboratory scale that alcohols would be good solvent substitutes, a process was not put into practice due to the possible formation of genotoxic sulfonates deriving from sulfuric acid.9 Figure 3. Stepwise route to compound 1 originating from research

For the first amidation addition of the aniline 3 to the squarate diester 2 requires mild base for reasonable reaction times. Triethylamine was chosen and an ethanol/ethylacetate solvent mixture employed at room temperature which gave a suspension of the intermediate 7 as the triethylammonium phenolate salt. For the initial 90 g campaign the free-acid phenol was then released by addition of HCl to adjust the pH to 6. The yield of the isolated yellow solid was only


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69 % with 95.0 area% purity10 due to the formation of the bis-amide by-product 9 (Figure 4) resulting from double addition of the aniline 3 (for a comparative overview of the levels of this by-product over various campaigns see Table 1). Figure 4. Major by-products obtained during synthesis by the original and alternative routes

Further reduction of 9 was achieved by directly isolating the triethylammonium phenolate salt from the reaction mixture. Eliminating the salt exchange operation led to an increase in isolated yield (81%) and purity (99%). Furthermore, the salt is a competent reactant in the next step without the need to add additional base; simply charging 3-pentylamine 4 to a mixture containing this salt and heating to reflux provided 8 in near-quantitative yield and very high purity (>99%) following acidification and crystallization. Curiously when ethanol was used as reaction solvent then an ethanol solvate of the isolated product was obtained. However since the salt of the drug substance 1 was itself not prone to solvate formation this did not lead us to be concerned at this stage. A cause for concern did come during the kilogram campaign where a new by-product containing a structural isomer of the 3pentylamine unit was observed alongside the squaramide product 8. Very poor purge efficiency in later steps led us to rapidly develop a recrystallisation from acetonitrile/ethanol (reduction to 1.1 area%), but at the cost of yield on this step (74 %). Tighter control at the supplier of the 3pentylamine 4 was sufficient to control quality downstream in later campaigns.


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The drug substance 1 is a choline salt of the phenolate. Choline is commercially available as a solution of choline hydroxide in either methanol or water. In this early campaign the methanolic solution was used in mild excess, but a true crystallisation was not achieved since there was incomplete dissolution of substance 1 in ethanol prior to addition of n-heptane as anti-solvent. Isolated purity was nonetheless high at 99.03 area% with 93 % recovered yield. For the second pre-clinical campaign an additional crystallisation of 1 from methanol/isopropanol was initiated, which substantially purged the structural isomer present as major impurity (98.5 area%, Table 2).

2.2 Clinical supply

Integral to any scale-up is a thermal safety assessment of the raw materials and processes to be run. The squaramide motif is suggestive of a strained ring system, and indeed compound 2 undergoes decomposition from 170 oC releasing considerable energy sufficient to lead to an adiabatic temperature rise of approximately 474 oC in pure form. However calorimetry pointed towards the aromatic moeity of the compounds 5, 3, 7, 8 and 1 as the source of more significant energy release, with each pure compound providing greater than -1000 J/g at temperatures starting from 100 oC in the case of compound 5 (results and profiles are included in the experimental section and supporting information). As such maximal jacket tempertures were implemented for each chemical step, and for the pilot set-up to form compound 8 a headtank of solvent was connected to provide an additional boiling-barrier in case of thermal runaway. Comprehensive hazard analyses were put in place for all steps and no incidents were observed on scale.


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The goal of the first clinical campaign was to produce a 10 kg batch of compound 1 for clinical studies. The deprotection of 5 was optimised by a refinement of the work-up, paying particular attention to the pKa difference of the pivalic acid by-product and the product. Following complete hydrolysis and partial solvent switch from dioxane to water, the reaction mixture was basified with sodium hydroxide to pH 5.5, sufficient to form the soluble sodium salt of pivalic acid (pKa of 5.05).11 At this pH the desired compound 3 can be isolated by filtration. Raising the target pH by 0.5 during the basification, the yield of this step in the first clinical campaign was raised to 94 %, from 91 %. Control of the bis-amide impurity 9 in the following amination step remained a key concern. In past campaigns the various modes of addition of the aniline 3 onto the squarate diester 2 had been explored in order to identify the key parameters which might lead to a more limited overaddition to form the bis-amide (see Table 1). Table 1. Control of the level of bis-amide impurity 9 in compound 7.

Campaign Research

1st Pre-clinical

2nd Pre-Clinical

Clinical

solid

solution in hot EtOAc

the TEA salt in THF

solution in THF

Squarate 2 in reactor:

with TEA in EtOH

with TEA in EtOH

in EtOAc

with TEA in EtOH

Compound 7 isolated as:

free-acid

free-acid

TEA salt

TEA salt

Bis-amide byproduct 9 in 7

12 area%

1.4 area%

0.5 area%

Early Process Development of a Squaramide-Based CXCR2 Receptor

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