Process Development and Crystallization in Oiling-Out System of a Novel Topical Antiandrogen

Article abstract of DOI:10.1021/acs.oprd.6b00392

An efficient route to (S)-N-(2-bromo-6-methoxypyridin-4-yl)-2-hydroxy-2,4-dimethylpentanamide 1, a new topical antiandrogen, is described. The target compound has been manufactured on kilogram scale with an overall yield of 25% (HPLC purity 98.8% and >99% ee) from citrazinic acid. The key amide coupling between aminopyridine 4 and α-hydroxy-acid 6 was performed using a temporary protecting group to facilitate the acyl chloride formation. Aminopyridine 4 was manufactured from commercially available citrazinic acid via dibromide formation using phosphorus(V) oxybromide followed by mono S_NAr reaction with sodium methoxide and a final Hofmann rearrangement. Enantiopure α-hydroxy-acid 6 was obtained using an enantioselective cyanosilylation followed by salt resolution with (S)-α-methyl benzylamine. The absolute configuration of compound 1 was determined with anomalous scattering and the final crystallization of API was performed after seeding a liquid-liquid mixture below the monotectic temperature and afforded a crystalline powder presenting a "desert rose" shape clusters.

Full text of DOI:10.1021/acs.oprd.6b00392

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Full Paper
Process Development and Crystallization in Oiling-
Out System of a Novel Topical Antiandrogen
Sebastien Daver, Nicolas Rodeville, Francois Pineau, Jean-Marie Arlabosse, Christine Moureou, Franck
Muller, Romain Pierre, Karinne Bouquet, Laurence Dumais, Jean-Guy Boiteau, and Isabelle Cardinaud
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00392 • Publication Date (Web): 21 Dec 2016
Downloaded from http://pubs.acs.org on December 29, 2016
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Process Development and Crystallization in Oilingꢀ
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Sébastien Daver, Nicolas Rodeville, Francois Pineau, JeanꢀMarie Arlabosse, Christine
Moureou, Franck Muller, Romain Pierre, Karinne Bouquet, Laurence Dumais, JeanꢀGuy
Boiteau*, and Isabelle Cardinaud.
Galderma R&D Les Templiers, 2400 Route des colles BP 87, 06902 SophiaꢀAntipolis CEDEX,
France
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TABLE OF CONTENTS :
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ABSTRACT : An efficient route to (S)ꢀNꢀ(2ꢀbromoꢀ6ꢀmethoxypyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀ
dimethylpentanamide 1, a new topical antiandrogen, is described. The target compound has been
manufactured on kilogram scale with an overall yield of 25 % (HPLC purity 98.8% and >99%
ee) from citrazinic acid. The key amide coupling between aminopyridine 4 and αꢀhydroxyꢀacid
6 was performed using a temporary protecting group to facilitate the acyl chloride formation.
Aminopyridine 4 was manufactured from commercially available citrazinic acid via dibromide
formation using phosphorus(V) oxybromide followed by mono SNAr reaction with sodium
methoxide and a final Hofmann rearrangement. Enantiopure αꢀhydroxyꢀacid 6 was obtained
using an enantioselective cyanosilylation followed by salt resolution with (S)ꢀαꢀmethyl
benzylamine. The absolute configuration of compound 1 was determined with anomalous
scattering and the final crystallization of API was performed after seeding a liquidꢀliquid mixture
below the monotectic temperature and afforded a crystalline powder presenting a “desert rose”
shape clusters.
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KEY WORDS : Antiandrogen, oilingꢀout, monotectic, anomalous scattering, Hofmann
rearrangement, enantioselective cyanosilylation, amide coupling.
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INTRODUCTION : Hormones and androgens play an important role in skin disorders such as
oily skin or acne.1 Multiple hormonal modulators are available for the treatment of acne such as
spironolactone, flutamide, cyproterone acetate and finasteride by oral route.2 However, due to
systemic side effects such as fetus feminization, they can only be used in females and
contraception has to be set up. Oral antiandrogens have also been applied topically3 to reduce
systemic side effects and are promising agents to treat acne in females, nevertheless long term
safety is an issue with these hepatic stable drugs. The design of a topical antiandrogen with a fast
clearance and a fast metabolism is of great interest, because it could have a local antiꢀseboreic
activity in skin without any systemic sideꢀeffects. Compound 1 is a potent androgen receptor
antagonist in the nanomolar range that has been designed and developed for the treatment of both
excess sebum and acne treatment.4 Its structure shares some analogy with known nonsteroidal
antiandrogens (Flutamide, RUꢀ58841 or RꢀBicalutamide) but displays an original pyridine core
(Figure 1). Based on its interesting properties, compound 1 was selected as the clinical candidate
and multiꢀkilogram batches of API were required to support preclinical and clinical studies.
Figure 1 : Non Steroidal Antiandrogens and Compound 1 Structure
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Background :
The discovery route to compound 1 is depicted in Scheme 1. Access to this triꢀsubstituted
pyridine is not trivial and the initial chemical pathway relied on oxidation of commercially
available 2,6ꢀdibromopyridine with hydrogen peroxide, followed by nitration at the 4 position to
yield Nꢀoxide 2. Concomitant iron reduction of nitro group and Nꢀoxide afforded the dibromo
aminopyridine 3. SNAr of one bromine atom with a methoxy group was carried out with sodium
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hydroxide in methanol under microwave irradiation at 120 °C and 7 bars of pressure to give
aminopyridine 4. Acylation of compound 4 with pyruvic acid and DCC followed by addition of
isoꢀbutyl magnesium chloride afforded the racemic compound racꢀ1 which was engaged in a
preparative chiral HPLC. Compound 1 was obtained with 99% ee, but as an oil, with an overall
yield lower than 1% from 2,6ꢀdibromopyridine and the absolute configuration of the stereogenic
center was not determined at this stage of development.
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Scheme 1 : Discovery Route to Compound 1.
a) H2O2, TFA, 80%. b) H2SO4, HNO3, 73%. c) Fe, NH4Cl, EtOH, 51%. d) MeOH, NaOH,
MW 120 °C, 7 bars 84%. e) DCC, pyruvic acid, NMP, 29%. f) iꢀBuMgCl, THF, 34% g) Chiral
HPLC separation, 45%.
This chemical pathway based on a functionalization of key intermediate 5, was appropriate to
have fast access to libraries of compounds and build quickly structureꢀactivity relationships;
However considerable improvements needed to be performed in order to manufacture kilogram
batches of drug substance to support preclinical and clinical trials. The major drawback of this
approach was the final construction of racemic compound 1 that was subjected to chiral HPLC
separation along with the use of energetic nitropyridine 2 as well as high pressure SNAr reaction.
The new synthetic route envisaged was based on an amide coupling of aminopyridine 4 with
enantiopure αꢀhydroxyꢀacid 6 (Scheme 2). In order to realize this route, a scalable process to
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make the aminopyridine 4 using a Hofmann rearrangement, as well as a simple manufacture of
αꢀhydroxyꢀacid 6 via enantioselective cyanosilylation of MIBK were considered.
Scheme 2 : Retrosynthesis of Compound 1.
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Characterization of API and absolute configuration determination:
In addition to the synthesis of compound 1, effort was focused on the crystallization of this
new API. This small molecule (Mw = 331 g/mol) was fairly soluble (>40 mg/mL) in 42 common
solvents screened except in water and in alkanes (2 mg/mL), however crystals of racemic form
(m.p. 106 °C, ꢁHR = 31264 J.molꢀ1) were obtained after crystallization from nꢀ
heptane/diisopropyl ether 95/5 mixture. Crystallization of enantiopure 1 obtained from chiral
HPLC separation was more troublesome than anticipated but was finally achieved from pure nꢀ
heptane. This first solid had a melting point of 52 °C, but DSC analysis showed a second fusion
around 80 °C.5 After recrystallization experiment from nꢀHeptane, this first solid phase turned to
be a metastable solid form and a new crystalline phase was obtained with a melting point of 81
°C (ꢁHE = 20137 J.molꢀ1). This 25 °C difference between enantiopure 1 and rac-1 indicated that
we might have an unfavorable racemic compound behavior system as depicted in Figure 2. An
extensive polymorph screen was undertaken in order to identify other crystalline forms of
enantiopure 1 being accessible under processꢀrelevant conditions and only one solid phase was
obtained. The construction of binary diagram using DSC measurement and calculated liquidus
curves from simplified equations of SchröderꢀVan Laar and PrigogineꢀDefay (see experimental
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support) was performed. The experimental data obtained were in accordance with the calculated
ones, and confirmed our hypothesis of an eutectic with a value of about 95 mole percent.6 In
addition, superimposition of Xꢀray powder diffraction (XRPD) patterns solids with different
molar fractions confirmed the racemic compound behavior system with two distinct patterns for
the enantiopure 1 and the racemic solids (Figure 3).
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Figure 2 : Binary Phase Diagram of Compound 1.
Figure 3 : XRPD Patterns of Solids with Different Molar Fractions.
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Consequently, the control of the enantiopurity of αꢀhydroxyꢀacid 6 was mandatory because
crystallization of enantioꢀenriched 1 below 95% ee would not be an option to provide
enantiopure API. The other key was the determination of the absolute configuration of the
quaternary center in compound 1. The usual strategies to determine absolute configuration were
investigated but as the pKa of this substituted pyridine was predicted to be ꢀ0.4,7 salt formation
using chiral acids was unsuccessful, even attempted salt formation with camphor sulfonic acid
gave poor results. In addition, attempts to derivatize the alcohol with chiral auxiliaries such as
Mosher's ester failed as well, probably due to the steric hindrance and low reactivity of tertiary
alcohol. The presence of a heavy bromine atom on the API structure was clearly an opportunity
to perform an anomalous scattering experiment and access directly the absolute configuration
without derivatization. Slow crystallization from nꢀheptane generated a single crystal that was
subjected to anomalous scattering and we were able to establish the (S) absolute configuration of
quaternary center (Figure 4).
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Figure 4 : Absolute Configuration of Compound 1
Synthesis of Aminopyridine 3 and Hydroxy-acid 6.
The synthesis of aminopyridine 4 was also revisited, and particularly the SNAr of the methoxy
group that was the key step. The substitution reaction with 2,6ꢀdibromoꢀaminopyridine 3, in the
original route with sodium methoxide worked well in methanol in a sealed tube at 120 °C.
However all experiments to realize the substitution reaction at lower temperature or in another
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solvent afforded a complex mixture composed of selfꢀreaction sideꢀproducts of aminopyridine 4.
Attempts to perform the substitution, on the related electron deficient 2,6ꢀdibromo 4ꢀ
nitropyridine failed because the bromides substituents were this time too reactive, leading to
mixtures of monoꢀ and diꢀsubstituted products. Selective monoꢀsubstitution was rather difficult
to achieve and the 2,6ꢀdimethoxyꢀ4ꢀnitropyridine was always present in appreciable quantity
even when sodium methoxide was used as the limiting reagent. Therefore, we completely
changed the synthesis route to aminopyridine 4 and decided to introduce the amino group using a
Hofmann rearrangement of the corresponding amide 9 (Scheme 3).
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Scheme 3 : Manufacture of Aminopyridine 4
a) POBr3, sulfolane, 125 °C then methanol b) MeONa, MeTHF, 44% over 2 steps. c) Aqueous
NH3 6.4 M, 25 °C, 87%. d) NaOCl 13.9% w/v, then NaOH 80 °C, 86%.
Synthesis of compound 7 was described in literature from commercially available citrazinic
acid, but only on small scale, in neat phosphorus(V) oxybromide at 140ꢀ180 °C and with a
chromatographic purification.8 We investigated this reaction and during laboratory experiments,
we noticed formation of sticky black syrup probably composed of polyphosphoric residues that
were difficult to separate from reaction mixture. We found that the purity profile of this reaction
was greatly improved when sulfolane (8 Vol) was used as a solvent with only 1.5 equivalents of
phosphorus(V) oxybromide. To control the exothermic event, the brominating agent was added
at 90 °C to the sulfolane solution of citrazinic acid and then heated to 125 °C for 2 hours. The
mixture was quenched with methanol at 45 °C, diluted with MeTHF, treated with charcoal and
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filtered through Clarcel®. Due to the difficult isolation of compound 7 the MeTHF solution was
directly treated with sodium methoxide at 60 °C to afford compound 8. During this step, the
water content of the MeTHF solution was carefully controlled below 0.03% in order to avoid
saponification of the methyl ester. The crude mixture was recrystallized from isoꢀpropanol to
afford compound 8 in 44% overall yield from citrazinic acid and in 99% purity on a kilogram
scale. Methyl ester 8 was suspended in an aqueous solution of ammonia 6.4 M at 25 °C for 48
hours and afforded the corresponding amide 9 in 87% yield. The process safety for the Hofmann
rearrangement was a concern that we wanted to investigate before starting kilogram
manufacture.9 During laboratory experiments on a 10 g scale, after the complete addition of
bleach and heating of the chloramine crude mixture to 55 °C, we observed an exotherm rising the
temperature up to 65 °C. To control this exothermic phenomenon the reaction was performed in
two separated reactors. The formation of chloramine was realized at 20 °C with bleach and was
slowly transferred into a second reactor containing a hot (80 °C) sodium hydroxide solution to
effect the rearrangement. Under this process, the rearrangement was instantaneous without heat
accumulation and the control of reaction was monitored by the addition rate of the chloramine
solution. During the decarboxylation step with concentrated hydrochloric acid we also noticed
foam formation due to CO2 evolution and concomitant precipitation. Addition of one volume of
ethanol to the reaction mixture reduced significantly the formation of foam, probably by
reducing the surface tension of the aqueous solution. Compound 4 crystallized nicely after a
basic workꢀup in 86% yield. Impurities 3 and 10 derived from unreacted material or double SNAr
with sodium methoxide were observed at 1.1%, and 0.1% respectively. Impurity 11 was obtained
from an aromatic Finkelstein reaction10 of residual chlorides during Hofmann rearrangement and
was observed at around 0.3% (Scheme 4).
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Scheme 4 : Hofmann Rearrangement Intermediates.
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a) NaOCl 13.9% w/v, 25 °C. b) NaOH, 80 °C. c) HCl 36%, EtOH. d) NaOH, 86% from 9
Access to enantiopure αꢀhydroxyꢀacid 6 was a key step for the manufacture of our API.
Several routes have been described in the literature using BaeyerꢀVilliger oxidation of a
ketoester,11 or enantioselective 1,2ꢀaddition of isoꢀbutyl magnesium bromide on a methyl vinyl
ketone followed by ozonolysis,12 but none of them was suitable for multiꢀkilogram production.
Enantioselective cyanosilylation is a useful method to introduce quaternary centers on prochiral
ketones,13 nevertheless the chiral catalysts described are generally substrate dependent and
enantiomeric excesses are in the 80% range. We applied the Feng et al. conditions on methyl isoꢀ
butyl ketone, with the catalyst system composed of an aluminum salen ligand and an Nꢀoxide coꢀ
catalyst.14 Results are summarized in Table 1.
Table 1 : Effect of Catalyst, Solvent and Temperature on Enantioselectivity
Entrya
catalyst
L1
solvent
THF
temperature Yield [%]
ee [%]b
8 (S)
1
2
3
25 °C
25 °C
25 °C
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90
91
THF
68 (S)
56 (S)
L2
THF
L3
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THF
0 °C
<1%
91
ND
L2
L2
DCM
25 °C
68 (S)
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[a] All reactions were carried out with 0.4% of chiral Alꢀcomplex and 0.2% of N,Nꢀ
dimethylaniline oxide over 18 h. [b] determined by chiral GC analysis on Astec Chiraldex BꢀDM
column.
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When catalyst L1 was used, only a moderate 8% ee was obtained with MIBK in favor of the
(S) enantiomer, whereas switching to catalyst L2, increased enantioselectivity to 68% ee. Bulky
catalyst L3 was also investigated but in this case the ee obtained was not improved compared to
the previous result. Another option to increase the selectivity was to perform the reaction at a
lower temperature, but interestingly this had a dramatic impact on the reaction rate as at 0 °C,
only a very low conversion (<1%) was achieved after 18 h. During the workꢀup, solvents were
removed under vacuum; the volatility of the OꢀTMS cyanohydrin 13 prompted us to evaluate a
low boiling point solvent such as dichloromethane to replace THF. In dichloromethane, the
reaction worked smoothly in 18 h and afforded the (S)ꢀOꢀTMS cyanohydrin 13 in 91% yield
with 68% ee. To avoid release of cyanhydric acid (HCN), TMSCN was always used as the
limiting reagent compared to MIBK. As αꢀhydroxyꢀacid 6 was also fairly water soluble, the
amount of water used in the hydrolysis step was decreased by using 36% HCl solution. The
reaction mixture was heated under reflux for 1 hour, then the crude acid crystallized upon
cooling at around 50 °C and afforded αꢀhydroxyꢀacid 6 in 77% yield and 68% ee. (Scheme 5).
Since only a moderate enantiomeric excess was obtained from the cyanosilylation reaction, a
further enantioselective crystallization step was necessary for the preparation of enantiopure 6.
The crude acid was crystallized with (S)ꢀαꢀmethyl benzylamine in ethyl acetate to give the
ammonium salt 6.mba that was filtered off and treated with 36% HCl to yield enantiopure acid 6
with an overall yield of 24% from MIBK. Direct crystallization of racemic αꢀhydroxyꢀacid 6
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with (S)ꢀαꢀmethyl benzylamine was also possible, however to obtain an enantiopure compound
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in one crystallization, a yield of 10% was the maximum we could achieve.
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Scheme 5 : Developed Route to Enantiopure Hydroxy-Acid 6.
a) N,NꢀDimethyl aniline oxide, TMSCN, DCM, 91%. b) HCl 36%, reflux, 77%. c) (S)ꢀαꢀ
MBA, AcOEt 53% d) HCl 36%, 64%.
Development of final coupling step:
With the two key intermediates 4 and 6 in hand, we investigated the amide coupling in order to
obtain the final API. Despite coupling of αꢀhydroxyꢀacids structurally related to our compound
being described in literature,15 in our case this amide bond proved to be very difficult to make.
The steric hindrance of acid 6 and the weak nucleophilic behavior of aminopyridine 4 were
suspected to be responsible for this lack of reactivity. Activation of acid 6 with common reagents
such as thionyl chloride, oxalyl chloride, EDC/HOBT, T3P® or CDI were unsuccessful and no
evidence of the expected coupled product was observed. An alternative coupling of αꢀhydroxyꢀ
acids with weakly nucleophilic amines has been described using thionyl chloride as an activator
of the amino group. In this case, a Nꢀsulfinyl amine intermediate is generated and reacts with the
hydroxyl group to generate a cyclic intermediate that finally gives the amide bond with release of
SO2 (Scheme 6).16 Activation of the aminopyridine 4 with thionyl chloride and imidazole in
dichloromethane at ꢀ10 °C produced the Nꢀsulfinyl amine 14, that was used in the coupling
reaction directly after a solvent switch from dichloromethane to acetonitrile. At room
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temperature, no reaction occurred but when the mixture was heated under reflux, compound 1
was obtained with 50% conversion. This was a great improvement, but the conversion seemed to
be blocked at 50% whatever the reaction conditions.
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Scheme 6 : Coupling Reaction with N-Sulfinyl Amine
a) Imidazole, SOCl2, DCM, ꢀ10 °C. b) 6, ACN reflux, 18 h, 50% from 4.
In order to improve yield and purity profile of this coupling reaction we came back to
activation of the acid 6. We demonstrated that the lack of reactivity was mainly due to the αꢀ
hydroxy function as the coupling reaction of aminopyridine 4 with 2,4ꢀdimethylpentanoic acid
activated with oxalyl chloride worked nicely. Early studies to temporary protect the free alcohol
on αꢀhydroxyꢀacid 6, using AcCl gave poor results but when we used TMS as a protecting group,
we were able to generate the acyl chloride and then to add it to the aminopyridine 4 to provide
the target compound in good yield17 (Scheme 7). The protocol used was to protect both
carboxylic acid and alcohol with two equivalents of TMSCl in pyridine at 10 °C. The water
content of αꢀhydroxyꢀacid 6 was carefully monitored (< 1%) in order to achieve the desired
protection. The acyl chloride was synthesized in situ with oxalyl chloride and a catalytic amount
of DMF while maintaining the temperature below 5 °C. The crucial amide bond was realized by
addition of a solution of aminopyridine 4 in dichloromethane at 25 °C and gave the targeted
compound in 30 min. Final deprotection of the silyl group was achieved by adding a solution of
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acetic acid in ethanol. The crude mixture was quenched with hydrochloric acid and extracted
with dichloromethane, submitted to polish filtration and stored in solution.
Scheme 7 : Manufacture of Final API
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HO
H
TMSO
Cl
HO
O
Br
N
b,c,d
a
HO
N
O
O
OMe
6
1
a) TMSCl, DMAP, pyridine, DCM, 10 °C then (COCl)2, DMF b) 4, pyridine, DCM. c) EtOH,
AcOH. d) Cyclohexane, 5% compound 1 seeds, 74% from 6
Crystallization in oiling-out system:
The main challenge of this synthesis resulted in the crystallization process of the final API, as
this small molecule was highly soluble in every solvent tested except in alkanes or in water.
Attempts to recrystallize compound 1 (m.p. = 81 °C) in water gave a liquidꢀliquid phase
separation (LLPS) when the temperature reached 60 °C and this emulsion remained stable at 25
°C. At this temperature, seeds of crystalline material 1 were added and the oily phase slowly
turned into a gummy solid. In nꢀheptane the behavior was slightly different as a clear solution
was obtained when temperature reached 65 °C and oilingꢀout was observed upon cooling at
around 60 °C (Figure 5). This oilingꢀout system was stirred at 25 °C for several days to perform
a slurry ripening and submitted to heat cycles from 20 °C to 40 °C but no crystallization
occurred.18 This solubility gap (e.g. liquidꢀliquid phase separation) was indicative of a
monotectic system, and in our case the observation of stable emulsions at room temperature
indicated that the nucleation step was the limiting step.
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Figure 5 : Oiling-out Phenomenon in n-Heptane
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A) Suspension of pure 1 in nꢀHeptane (10 V/P) at 25 °C. B) Homogenous solution at 65 °C. C)
Oilingꢀout at 60 °C.
Oilingꢀout often occurs for small organic molecules with a low melting point and
crystallization of such systems is generally unwanted as crystals obtained are usually impure and
purging of impurities remains a challenge.19 In such systems, a liquidꢀliquid phase separation
occurs upon cooling, leading to two liquid phases: a solute lean phase (LI) and a solute rich phase
(LII) (Figure 6a). The knowledge of the monotectic temperature is essential and seeding the
liquidꢀliquid mixture to induce nucleation can only be performed below the monotectic
temperature where crystals of compound 1 can thermodynamically exist.20
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Figure 6 : Schematic Representation of a Monotectic System and Crystals of Compound 1
Obtained after Seeding
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6a
6b
6c
6d
6a) Simplified representation of a monotectic system 6b) Microscopic observation of
compound 1, magnification 10X observed between crossed polarizing filters and a ¼ wavelength
plate 6c) Scanning Electron Microscopy, magnification 100X 6d) Scanning Electron
Microscopy, magnification 600X
To approximate the monotectic temperature, we observed the LLPS temperature upon cooling
homogenous solutions, and seeding with crystalline material was performed 5 °C below LLPS
temperature. Several alkanes were tested and the LLPS temperature was recorded: nꢀhexane, (61
°C) nꢀheptane (60 °C), methylcyclohexane (36 °C), cyclohexane (33 °C).21 Despite the fact that
crystallization occurred in nꢀheptane and nꢀhexane, the solids obtained were greasy and mainly
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stuck on the reactor glassware. The behavior observed in cyclohexane or methylcyclohexane was
much better and afforded an even crystallization.
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For our process, cyclohexane was chosen based on price and ICH solvent residual limits
allowed in the API (cyclohexane 3800 ppm; methylcyclohexane : 1180 ppm). The scale up of the
laboratory procedure was performed incrementally from a 0.1 L to 10 L reactor to ensure that
the LLPS and the crystals shapes were not dependent of stirrer type and rotational speed.22 This
first study prompted us to perform the reaction in a 100 L reactor at kilogram scale, the crude
dichloromethane solution of compound 1 was azeotroped with cyclohexane until temperature of
condensates reached 75 °C, cyclohexane was added to the reactor to reach a final volume of
about 10 V/P and cooled until oilingꢀout occurred at around 33 °C. At 28 °C seeds of compound
1 were added (5%) and the mixture was stirred gently for 16 hours at 28 °C. Microscopic
observation of the powder displayed “desert rose” shape clusters of platelet crystals
demonstrating that nucleation occurred in the soluteꢀrich droplets of the emulsion (Figure 6b, 6c,
6d).23 Compound 1 was obtained as a crystalline powder with 74% yield, an HPLC purity of
98.8% with less than 50 ppm of cyclohexane. The main impurities identified in compound 1
were the acylated compounds 14, 15 and 16 arising from aminopyridines 3, 10 and 11. (Scheme
8) During the crystallization process, the impurity 14 was efficiently purged, whereas levels of
compounds 15 and 16 remained in the same range in compound 1 indicating that these two
impurities have to be controlled in aminopyridine 4. The purge of compound 14 was unexpected
as crystallizations of systems exhibiting an oilingꢀout are usually not recommended for
purification purpose. The conversion of coupling step was followed by TLC, thus the precise
amount in crude mixture of impurity 14 was not determined. The coupling reaction conditions
were applied to aminopyridine 3 with αꢀhydroxyꢀacid 6 and afforded compound 14 in a similar
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yield (66% vs 74%) indicating that nucleophilicity of aminopyridines 3 and 4 were comparable
and that compound 14 might be present in the crude mixture around 1%. The lack of
incorporation of compound 14 in the compound 1 crystal lattice might be explained by the steric
bulk of two bromines atoms on the structure or by a different solubility in cyclohexane.
Scheme 8 : Identified Impurities
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Impurities in
aminopyridine 4
3 : 1.1%
Related Impurities in
compound 1
14 : 0.1%
10 : 0.1%
15 : 0.1%
11 : 0.3%
16 : 0.3%
Conclusion :
In conclusion, we have developed a scalable process for the manufacture of compound 1 with
an overall yield of 25% (HPLC purity 98.8% and > 99% ee) from citrazinic acid widely available
commercially. A stable polymorphic form has been obtained and the absolute configuration has
been established with anomalous scattering experiment thanks to the bromine atom on the
structure. A new access to aminopyridine 4 has been developed through a dibromide formation
of citrazinic acid followed by SNAr reaction and Hofmann rearrangement. A simple synthesis of
enantiopure αꢀhydroxyꢀacid 6 has been devised using an enantioselective cyanosilylation
followed by (S)ꢀαꢀmethyl benzylamine salt resolution. The key amide coupling was achieved
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with a TMS protection followed by acyl chloride formation and final crystallization was
successfully achieved by seeding an oilingꢀout system in cyclohexane below the monotectic
temperature. Our final API has been successfully manufactured several times at multiꢀkilogram
scale to support preclinical and clinical studies.
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General Information.
All solvents and reagents were purchased from the suppliers and used without further
1
13
purification. H NMR and C NMR were recorded on a Bruker Avance 400 MHz, 100 MHz.
Chemical shifts are expressed in parts per million (ppm) downfield from residual solvent peaks,
and coupling constants are reported in hertz (Hz). Splitting patterns are indicated as follows: br,
broad; s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet. Xꢀray
powder diffraction (XRPD) analyses were performed by Holodiag using Panalytical X’Pert Pro
MPD Xꢀray powder diffractometer. Differential scanning calorimetry (DSC) were performed
using Perkin Elmer Diamond thermogravimetric analyzer, using 50 µL aluminum caps. Hold for
2.0 min at 20.0 °C, heat from 20.0 °C to 110.0 °C at 10.0 °C/min then hold for 2.0 min at 110.0
°C. Enantiomer ratios were determined by chiral GC analysis with Astec Chiraldex BꢀDM
column, or by chiral HPLC analysis on Daicel Chiralpak ID or Chiralpack IC columns in
comparison with racemates. The HRMS measurement was performed on a LCT (Time Of Flight)
mass spectrometer from Waters in the positiveꢀion mode. Microscopic observation were
performed with Zeiss AXIO microscope with 10X magnification and observed between crossed
polarizing filters and ¼ wavelength plate. SEM observations were realized with a JEOL JCM
5000 Neoscope with an accelerating voltage of 10 kV.
Binary Phase Diagram. The binary phase diagram was constructed from DSC data’s based on
simplified SchröderꢀVan Laar equation for the portion of the liquidus curve between the eutectic
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and the pure enantiomer and from PrigogineꢀDefay equation for the liquidus curve between the
two eutectics. with x = mole fraction, R = gas constant (J.molꢀ1.Kꢀ1), TE, TR = melting
temperature (°K) of enantiomer and racemic compounds, and ꢁHE, ꢁHR = enthalpy of fusion of
enantiomer and racemic compounds (J.molꢀ1) see also Supporting Information. The theoretical
eutectic was obtained from the intersection of the two liquidus curves.
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ꢀꢁ ꢂ = ∆ꢃꢄ ꢆꢈꢇ − ꢈꢇꢉ : SchröderꢀVan Laar
ꢅ
ꢄ
ꢌ∆ꢃ
ꢅ
ꢊ
ꢋ
ꢀꢁ 4ꢂ 1 − ꢂ =
ꢍ ꢆꢈꢇ − ꢈꢇꢉ : PrigogineꢀDefay
ꢍ
Methyl 2-bromo-6-methoxyisonicotinate (8)
In a 20 L double jacketed reactor, citrazinic acid (1.0 kg, 6.4 mol, 1 eq.) and sulfolane (6.3 kg,
5 vol.) were charged. The reaction mixture was heated to 90 °C then a solution of phosphorus(V)
oxybromide (2.77 kg, 9.7 mol, 1.5 eq.) in sulfolane (3.7 kg, 3 vol.) was added over 39 min. The
reaction mixture was then heated to 125 °C and stirred to this temperature for 2 h. Consumption
of citrazinic acid was monitored by HPLC. The reaction mixture was chilled to 40 °C, then
methanol (2.6 L, 10 eq.) was introduced over 30 min and stirred at 45 °C for 30 min, methanol
was distilled off under vacuum (V = 0.64 L). In a 20 L double jacketed reactor, water (8.0 kg, 8
vol.) was charged, then the previous reaction mixture was charged at 20 °C to allow
precipitation. The reaction mixture was shilled to 0 °C then stirred at this temperature for 1 h.
The solid was filtered off and washed with water (4 x 3 L.). The solid was reꢀcharged into the
reactor then MeTHF (4.30 kg, 5 vol.) was added with 0.68 kg of charcoal. The reaction mixture
was heated at 50 °C and stirred at this temperature for 30 min. The reaction mixture was filtered
on Clarcel®, and washed with dichloromethane (3 x 1 L) and with MeTHF (4 x 1.7 kg). The
organic phase was washed with brine (1.0 L, 1 vol.) then azeotroped with MeTHF (8 L) until
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water content reach 0.03%, to yield a methyl 2,6ꢀdibromoisonicotinate solution (4.84 Kg). The
previous solution was heated to 60 °C then a sodium methoxide solution 25%w/w in MeTHF
(0.73 kg, 6.5 mol, 1 eq.) was added over 1 hour. The reaction mixture was shilled to 30 °C, then
washed with saturated NaHCO3 (8.0 L), and with another saturated NaHCO3 (2.0 L), and finally
with brine (3.0 L). The organic phase was concentrated and the residue (m = 0.90 kg) was
recrystallized from isopropanol (5.0 L). The solid was filtered off, washed with isopropanol (1.2
L) and dried under vacuum at 45 °C. 691 g of methyl 2ꢀbromoꢀ6ꢀmethoxyisonicotinate were
obtained. Overall yield = 44%.
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13
NMR H (400 MHz, CDCl3): 7.54 (s, 1H); 7.22 (s, 1H); 3.91 (s, 3H); 3.89 (s, 3H). NMR C
(100 MHz, CDCl3) : 164.0, 163.6, 142.4, 138.7, 119.1, 109.5, 54.7, 53.1. ESIꢀHRMS(m/z) calcd
for C8H9BrNO3 [M + H]+ 245.9760 found 245.9760
+
2-Bromo-6-methoxyisonicotinamide (9)
In a 10 L double jacketed reactor, methyl 2ꢀbromoꢀ6ꢀmethoxyisonicotinate 8 (450 g; 1.83 mol;
1.0 eq.) were charged followed with an ammonia solution 6.4 M (4 L). The suspension was
stirred at 20 °C for 48 h, then the solid was filtered off, washed with water (7 x 900 mL) until pH
of filtrate reached pH=8. The solid was dried under vacuum at 40 °C. 370 g de 2ꢀbromoꢀ6ꢀ
methoxyisonicotinamide were obtained. Yield = 87%
NMR 1H (400 MHz, DMSOꢀd6): 8.23 (sl, 1H); 7.80 (sl, 1H); 7.57 (s, 1H); 7.23 (s, 1H); 3.88 (s,
13
3H). NMR C (100 MHz, DMSOꢀd6) : 164.5, 163.9, 147.2, 138.4, 118.5, 108.1, 54.5. ESIꢀ
+
HRMS(m/z) calcd for C7H8BrN2O2 [M + H]+ 230.9764 found 230.9764
2-Bromo-6-methoxypyridin-4-amine (4)
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In a 1L double jacketed reactor 2ꢀbromoꢀ6ꢀmethoxyisonicotinamide 9 (100 g; 0.44 mol; 1.00
eq.) and water (400 mL) were charged, and then the mixture was shilled to 10 °C. A sodium
hydroxide solution was prepared from NaOH (21 g, 0.51mol) in 50 mL water, then this solution
was added to the previous solution maintaining temperature below 10 °C. A bleach solution (279
mL; 139.50 g/L; 0.52 mol; 1.2 eq.) was added while keeping temperature below 10 °C, and then
the reaction mixture was stirred at 20 °C for 2 h. In a 4 L double jacketed reactor NaOH (20.96
g; 0.51 mol; 1.16 eq.) were dissolved in 400 mL water, and heated to 80 °C. Onto this solution,
the previous chloramine solution was added over 20 minutes (T = 77 °C) the mixture was stirred
2 hours at 80 °C then shilled to 25 °C. Water (700 mL) and ethanol (100 mL) were added and
the reaction mixture was shilled to 10 °C. Hydrochloric acid 36% (140 mL; 12 M; 1.69 mol; 3.88
eq.) was added while keeping temperature below 6 °C then the reaction mixture was heated to 20
°C. Another Hydrochloric acid solution made from concentrated HCl 36% (36 mL; 12 M; 0.44
mol; 1.00 eq.) in 500 mL water was added and the suspension was filtered on dicalite® and
finally recharged into the reactor. Sodium hydroxide (99.90 g; 2.42 mol; 5.55 eq.) was added
portionwise while keeping temperature below 30 °C. The reaction mixture was stirred 1 hour at
25 °C and filtered off; the cake was washed with water (3 x 1L) and dried at 45 °C. 76 g of 2ꢀ
bromoꢀ6ꢀmethoxypyridinꢀ4ꢀamine were obtained. Yield = 86%.
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1
RMN H (400 MHz, DMSOꢀd6): 6.38 (s, 1H); 6.26 (sl, 2H); 5.82 (s, 1H); 3.71 (s, 3H). RMN
13C (100 MHz, DMSOꢀd6): 164.2, 158.6, 138.4, 106.9, 90.7, 53.3. ESIꢀHRMS(m/z) calcd for
C6H8BrN2O+ [M + H]+ 202.9815 found 202.9816
(S)-1-Phenylethan-1-aminium (S)-2-hydroxy-2,4-dimethylpentanoate (6.mba)
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In a 15 L double jacketed reactor trimethylsilyl cyanide (3 162 mL; 24.86 mol; 0.95 eq.) and
N,Nꢀdimethylaniline oxide (8.97 g; 65.42 mmol; 0.004 eq.)14 were charged and this solution was
stirred 1 hour at 23 °C. In a 10 L double jacketed reactor 2,2'ꢀ((1E,1'E)ꢀ(((1R,2R)ꢀcyclohexaneꢀ
1,2ꢀdiyl)bis(azanylylidene))bis(methanylylidene))bisꢀ4ꢀbromophenolate ethylaluminium (55.92
g; 104.67 mmol; 0.006 eq.)14 in MIBK (3 276 mL; 26.17 mol; 1.00 eq.) and DCM (7.86 L) was
stirred 1 hour at 23 °C. This solution was added to the trimethylsilyl cyanide solution at 23 °C
and the resulting reaction mixture was stirred 1 hour at this temperature. The mixture was
concentrated until minimum volume then a mixture of nꢀheptane/ethyle acetate 80/20 with 1% of
diisopropylethylamine (3 L) was added. This solution was filtered through a pad of silica and
washed with a mixure nꢀheptane/ethyle acetate 80/20 with 1% of diisopropylethylamine (1 L).
The filtrate was charged in the reactor and concentrated under reduced pressure to yield 2,4ꢀ
dimethylꢀ2ꢀ((trimethylsilyl)oxy)pentanenitrile as a pale yellow oil (4 772 g) Yield = 91%, 68%
ee (Chiral GC, Astec Chiraldex BꢀDM, 0.25 mm x 30 m, column temperature = 42 °C
(isothermal), inject temperature = 105 °C, IonSource temperature = 150 °C, inlet pressure =
112.7 kPa) : tr (minor) = 38.5 min, tr (major) = 39.2 min.
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In a 15 L double jacketed reactor, 2,4ꢀdimethylꢀ2ꢀ((trimethylsilyl)oxy)pentanenitrile (2 406 g;
12.07 mol; 1.00 eq.) and concentrated HCl 36%. (6 L; 72 mol; 2.50 V) were charged, then the
reaction mixture was stirred under reflux for 5 hours. Water (6 L) was added onto the reaction
mixture to allow the temperature to reach 50 °C. Crystallization occurred and the mixture was
allowed to cool to 23 °C. The solid was filtered off, washed with water and dried under vacuum
at 45 °C. (S)ꢀ2ꢀHydroxyꢀ2,4ꢀdimethylpentanoic acid was obtained as a white crystalline powder.
(1 368 g), Yield = 77%, 68% ee. HPLC (Daicel Chiralpak ID 250x4.6 mm, 5µm,
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Water/MeOH/HCOOH 600:400:2, 0.7 mL.minꢀ1, 25 °C, UV 215 nm) : tr (major) = 7.6 min, tr
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(minor) = 9.4 min.
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In a 15 L double jacketed reactor (S)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylꢀpentanoic acid (1 150 g; 7.87
mol; 1.00 eq.) and ethyle actate (11.5 L) were charged then (S)ꢀ1ꢀphenylethylamine (912 mL;
7.08 mol; 0.90 eq.) was added while maintaining temperature below 30 °C. Crystallization was
started with a seed of enantiopure (S)ꢀ1ꢀphenylethanꢀ1ꢀaminium (S)ꢀ2ꢀhydroxyꢀ2,4ꢀ
dimethylpentanoate (10 g) and the reaction mixture was stirred 3 hours at 20 °C, The solid was
filtered off, washed with ethyl acetate (1 L) and dried under vacuum at 50 °C. (S)ꢀ1ꢀPhenylethanꢀ
1ꢀaminium (S)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanoate (1 122 g) was obtained as a white powder.
Yield = 53%, 99.5% ee.
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NMR H (400 MHz, DMSOꢀd6): 7.48 (m, 2H); 7.35 (m, 3H); 4.29 (q, J = 6.7 Hz, 1H); 1.71
(non, J = 6.7 Hz, 1H); 1.55 (dd, J = 13.4 & 6.6 Hz, 1H); 1.45 (d, J = 7 Hz, 3H); 1.33 (dd, J =
13
13.6 & 5.6 Hz, 1H); 1.12 (s, 3H); 0.87 (d, J = 6.6Hz, 3H); 0.81 (d, J = 6.6 Hz, 3H). NMR C
(100 MHz, DMSOꢀd6) : 180.1, 141.4, 128.5, 127.9, 126.7, 73.6, 49.9, 48.7, 28.3, 24.7, 24.3,
23.7, 21.8.
(S)-2-Hydroxy-2,4-dimethyl-pentanoic acid (6)
In a 15 L double jacketed reactor (S)ꢀ1ꢀphenylethanꢀ1ꢀaminium (S)ꢀ2ꢀhydroxyꢀ2,4ꢀ
dimethylpentanoate (3 081 g; 11.52 mol; 1.00 eq.) and water (3 L) were charged. Concentrated
hydrochloric acid 36%. (7.7 L) was added while maintaining temperature below 40 °C. The
reaction mixture was chilled to 10 °C, the solid was filtered off, washed with shilled water, and
dried under vacuum at 45 °C. (S)ꢀ2ꢀHydroxyꢀ2,4ꢀdimethylꢀpentanoic acid (1 078 g) was obtained
as a white powder Yield = 64%, 99.5% ee.
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NMR 1H (400 MHz, DMSOꢀd6): 12.36 (sl, 1H); 4.74 (sl, 1H); 1.74 (hept, J = 6.1 Hz, 1H); 1.74
(dd, J = 13.7 & 6.7 Hz, 1H); 1.46 (dd, J = 13.6 & 5.6 Hz, 1H); 1.25 (s, 3H); 0.88 (d, J = 6.6Hz,
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3H); 0.83 (d, J = 6.6Hz, 3H). NMR C (100 MHz, DMSOꢀd6): 178.2, 73.3, 48.5, 27.5, 24.4,
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ꢀ
24.0, 23.4. ESIꢀHRMS(m/z) calcd for C7H13O3 [M ꢀ H]ꢀ 145.0870 found 145.0856
(S)-N-(2-Bromo-6-methoxypyridin-4-yl)-2-hydroxy-2,4-dimethylpentanamide (1)
In a 100 L glass lined reactor (S)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylꢀpentanoic acid 6 (2019 g; 13.8 mol;
1.00 eq.), 4ꢀdimethylaminopyridine (84.2 g; 0.69 mol; 0.05 eq.), dichloromethane (16 L) were
charged. The mixture was shilled to 0 °C, and then pyridine (2.4 L) and dichloromethane (4 L)
were added while maintaining temperature below 5 °C. Chlorotrimethylsilane (3.3 Kg; 30 mol;
2.20 eq.) was added while maintaining temperature below 5 °C and the reaction mixture was
allowed to warm to 20 °C and then stirred at this temperature for 4 hours. The mixture was
shilled to 0 °C and N,Nꢀdimethylformamide (35 g; 0.17 mol; 0.03 eq.) was added followed by
oxalyl chloride (1583 g; 12.5 mol; 0.90 eq.) keeping temperature below 5 °C. The reaction
mixture was stirred 1 hour at 5 °C and another N,Nꢀdimethylformamide (18 g; 0.09 mol; 0.02
eq.) was added. The mixture was allowed to warm to 20 °C and stirred 1 hour to this
temperature. A solution of 2ꢀBromoꢀ4ꢀAminoꢀ6ꢀMethoxyꢀPyridine 4 (2328 g; 11.4 mol; 0.83
eq.) in dichloromethane (18 L) and pyridine (1.2 L) was prepared and added onto the previous
reaction mixture while keeping temperature below 25 °C. The mixture was stirred 30 min at this
temperature and complete conversion was monitored TLC. A solution of acetic acid (1659 g; 2
eq.) in ethanol (13 L) was prepared and added onto the reaction mixture while keeping
temperature below 25 °C and the reaction mixture was stirred 18 h. A solution of hydrochloric
acid (1.6 L; 12.00 M) in water (18 L) was prepared and added to the reaction mixture, the
aqueous phase was discarded and the organic phase was washed with water (20 L) then with a
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solution of NaOH (773 g) in water (20 L) and finally with water (20 L). The organic phase was
filtered through a filter packed with charcoal (600 g) and the filter was washed with
dichloromethane (6 L). The resulting solution was charged into the reactor and concentrated, 20
L were removed and cyclohexane (28 L) was added and concentration was pursued until
condensate temperature reached 75 °C. Extra cyclohexane (7 L) was added to reach a final
volume of about 30 L and the reaction mixture was cooled to 28 °C. A seed of (S)ꢀNꢀ(2ꢀbromoꢀ
6ꢀmethoxypyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanamide (99 g; 0.29 mol; 0.05 eq.) was
added and the reaction mixture was shilled to 20 °C and stirred 16 hours at this temperature. The
solid was filtered off, washed with cyclohexane (7 L) and dried under vacuum at 45 °C to yield
(S)ꢀNꢀ(2ꢀbromoꢀ6ꢀmethoxypyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanamide (2.8 Kg). Yield =
74%, HPLC purity 98.8%, 99.5% ee HPLC (Daicel Chiralpak IC 250x4.6 mm, 5µm, nꢀ
Heptane/IPA/TFA 930:70:1, 1 mL.minꢀ1, 25 °C, UV 210 nm) : tr (minor) = 5.1 min, tr (major) =
5.9 min.
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NMR 1H (400 MHz, DMSOꢀd6): 10.0 (sl, 1H); 7.73 (s, 1H); 7.33 (s, 1H); 5.70 (sl, 1H); 3.80 (s,
3H); 1.79ꢀ1.67 (m, 2H); 1.49 (dd, J = 13.6 & 5.2 Hz, 1H); 1.32 (s, 3H); 0.89 (d, J = 6.4 Hz, 3H);
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0.78 (d, J = 6.4 Hz, 3H). NMR C (100 MHz, DMSOꢀd6): 176.7, 164.0, 149.5, 138.1, 111.1,
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74.9, 53.9, 48.6, 27.5, 24.3, 23.6, 23.2. ESIꢀHRMS(m/z) calcd for C13H20BrN2O3 [M + H]+
331.0652 found 331.0654
(S)-N-(2,6-Dibromopyridin-4-yl)-2-hydroxy-2,4-dimethylpentanamide (14)
Using compound 1 procedure, but with 2,6ꢀDibromoꢀpyridinꢀ4ꢀylamine (5.00 g; 19.85 mmol;
1.00 eq.), 5 g of (S)ꢀNꢀ(2,6ꢀdibromopyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanamide were
obtained. Yield = 66%
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NMR 1H (400 MHz, DMSOꢀd6): 10.40 (sl, 1H); 8.17 (s, 2H); 5.77 (sl, 1H); 3.80 (s, 3H); 1.80ꢀ
1.67 (m, 2H); 1.50 (dd, J = 13.6 & 5.0 Hz, 1H); 1.33 (s, 3H); 0.90 (d, J = 6.3 Hz, 3H); 0.78 (d, J
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= 6.3 Hz, 3H). NMR C (100 MHz, DMSOꢀd6): 177.2, 149.3, 140.2, 116.4, 75.0, 48.7, 27.5,
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24.3, 23.7, 23.3. ESIꢀHRMS(m/z) calcd for C12H17Br2N2O2 [M + H]+ 378.9651 found 378.9653
(S)-N-(2,6-Dimethoxypyridin-4-yl)-2-hydroxy-2,4-dimethylpentanamide (15)
Using compound 1 procedure, but with 2,6ꢀDimethoxyꢀpyridinꢀ4ꢀylamine (2.5 g; 20 mmol;
1.00 eq.), 2.1 g of (S)ꢀNꢀ(2,6ꢀdibromopyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanamide were
obtained. Yield = 47%.
1
NMR H (400 MHz, DMSOꢀd6): 9.79 (sl, 1H); 6.87 (s, 2H); 5.62 (sl, 1H); 3.80 (s, 3H); 1.80ꢀ
1.67 (m, 2H); 1.48 (dd, J = 14.0 & 5.0 Hz, 1H); 1.32 (s, 3H); 0.90 (d, J = 6.6 Hz, 3H); 0.78 (d, J
= 6.6 Hz, 3H). NMR 13C (100 MHz, DMSOꢀd6): 176.3, 163.1, 149.7, 91.3, 74.8, 53.1, 48.7, 27.5,
24.4, 23.6, 23.2. ESIꢀHRMS(m/z) calcd for C14H23N2O4 [M + H]+ 283.1652 found 283.1650
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(S)-N-(2-Chloro-6-methoxypyridin-4-yl)-2-hydroxy-2,4-dimethylpentanamide (16)
Using compound 1 procedure, but with 2ꢀchloroꢀ6ꢀmethoxypyridinꢀ4ꢀamine (5.00 g; 31 mmol;
1.00 eq.), 5 g of (S)ꢀNꢀ(2,6ꢀdibromopyridinꢀ4ꢀyl)ꢀ2ꢀhydroxyꢀ2,4ꢀdimethylpentanamide were
obtained. Yield = 55%
NMR 1H (400 MHz, DMSOꢀd6): 10.1 (sl, 1H); 7.58 (s, 1H); 7.31 (s, 1H); 5.69 (sl, 1H); 3.82 (s,
3H); 1.81ꢀ1.67 (m, 2H); 1.49 (dd, J = 13.8 & 5.2 Hz, 1H); 1.33 (s, 3H); 0.90 (d, J = 6.6 Hz, 3H);
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0.78 (d, J = 6.6 Hz, 3H). NMR C (100 MHz, DMSOꢀd6): 176.8, 164.2, 149.8, 147.7, 107.3,
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97.6, 74.9, 53.9, 48.7, 27.5, 24.4, 23.6, 23.2. ESIꢀHRMS(m/z) calcd for C13H20ClN2O3 [M + H]+
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287.1157 found 287.1157
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ASSOCIATED CONTENT
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SUPPORTING INFORMATION
1H and 13C NMR spectra of the compound 1; procedures for synthesis, isolation and
characterization data. This information is available free of charge via the Internet at
http://pubs.acs.org/
AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed: Tel +33 4 92 95 29 48.
JeanꢀGuy.Boiteau@galderma.com
ACKNOWLEDGMENT
We thank Eric Gayon from M2I for bromo methoxypyridine 8 manufacture, Babak Sayah from
Provence Technologies for early studies and Pilar Franco from Chiral Technologies for early
chiral separation. We thank also Professor Gérard Coquerel from Rouen University for
anomalous scattering experiment and absolute configuration determination, Pierric Marchand
from Holodiag for crystallization support. We also thank AnneꢀLaurence Ghilini, Céline Mathieu
and Michel Connan for analytical support.
REFERENCES
[1] Gao, W.; Bohl, C. E.; Dalton, J. T. Chem. Rev. 2005, 105, 3352ꢀ3370.
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