Early Process Development of a Squaramide-Based CXCR2 Receptor Antagonist

Article abstract of DOI:10.1021/acs.oprd.5b00072

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 byproducts. Although drug substance quality was shown to increase following optimization 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.

Full text of DOI:10.1021/acs.oprd.5b00072

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Early process development of a squaramideꢀbased
CXCR2 receptor antagonist
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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
Eꢀmail: benjamin.martin@novartis.com
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Table of Contents Graphic
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ABSTRACT
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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 nonꢀ
symmetrical squaramide, and the modifications to the synthetic route which have effect on
chemical purity are discussed with reference to the isolated byꢀproducts. Although drug
substance quality was shown to increase following optimisation of the linear sequence, an
alternative oneꢀpot, convergent sequence was introduced, which makes dual use of choline
hydroxide as both base and saltꢀforming agent. The overall benefits of the strategic change is
discussed in terms of overall yield and economy.
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KEYWORDS: squaramide; choline; nucleophilic vinylic substitution; CXCR2; COPD; oneꢀpot
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1.
Introduction
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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 lungꢀfunction 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 shortꢀ
and long acting βꢀadrenergic bronchodilators, inhaled anticholinergics (muscarinic antagonists)
and inhaled corticosteroids to treat the symptoms and exacerbations of the disease. The
chemokine ILꢀ8, 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 antiꢀinflammatory
treatment for COPD.4
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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 diꢀethyl ester 2 and two amines 3 and 4
(Figure 2). While 3ꢀpentylamine 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 oneꢀpot 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.
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Figure 2. Retrosythesis of target compound 1
2.
Results and Discussion
2.1 Preꢀclinical 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 tertꢀbutyl benzoxazole 5 was achieved with an excess of sulfuric acid with 1,4ꢀdioxane/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
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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 freeꢀacid 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 bisꢀamide byꢀproduct 9 (Figure 4)
resulting from double addition of the aniline 3 (for a comparative overview of the levels of this
byꢀproduct over various campaigns see Table 1).
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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 3ꢀpentylamine 4 to a mixture containing
this salt and heating to reflux provided 8 in nearꢀquantitative 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 byꢀproduct containing a structural isomer of the 3ꢀ
pentylamine 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 3ꢀ
pentylamine 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 nꢀheptane as antiꢀsolvent.
Isolated purity was nonetheless high at 99.03 area% with 93 % recovered yield. For the second
preꢀclinical 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).
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2.2 Clinical supply
Integral to any scaleꢀup 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 setꢀup to form compound 8 a headtank of
solvent was connected to provide an additional boilingꢀbarrier 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 workꢀup, paying particular
attention to the pKa difference of the pivalic acid byꢀproduct 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 %.
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Control of the bisꢀamide 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 overꢀ
addition to form the bisꢀamide (see Table 1).
Table 1. Control of the level of bis-amide impurity 9 in compound 7.
Campaign
Operation &
Result
Research
solid
1st Preꢀclinical
2nd PreꢀClinical
Clinical
Amine 3 dosed
as:
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
TEA salt
0.5 area%
with TEA in
EtOH
Compound 7
isolated as:
freeꢀacid
freeꢀacid
TEA salt
Bisꢀamide byꢀ
12 area%
1.4 area%
<0.1 area%
product 9 in 7
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For the first clinical campaign it was rationalized that the ideal 1:1 stoichiometry between
starting materials should be more easily achieved by ensuring that a homogeneous solution of the
aniline was slowly added to a basic solution of the squarate diester in order that massꢀtransfer
limitations and accumulation would be minor in the local zone of addition. In complement, the
salt of product 7 was poorly soluble in the chosen ethanol/THF mixture and so was rapidly
precipitated from the solution thereby also limiting overꢀreaction. Applying these principles as
well as a direct crystallisation from solution, the product could be obtained in 99.9 area% purity
as well as in higher yield.
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For the second amine addition, the triethylammonium phenolate salt of compound 7 was again
directly taken, but due to the limited solubility in ethanol (the solvent used in the 2nd preꢀclinical
campaign), a switch was made to methanol which thereby increased the productivity of the
reaction and rendered it additionꢀcontrolled. Conveniently the product squaramide 8 could be
directly precipitated from solution as a methanol solvate by addition of aqueous HCl without the
need for antiꢀsolvent. Through these measures the yield could be raised to 98 % with high purity.
Final saltꢀformation and crystallisation was conducted in methanol as both solvent for the
reaction and solvent for the commercial choline hydroxide solution. A true crystallisation was
achieved, with seeding and use of isopropanol as antiꢀsolvent to deliver the substance 1 in
excellent purity (99.9 area%) albeit at the cost of a lower yield than in past campaigns.
In conclusion the experiences gained on kilogram scale confirmed that our choice of synthetic
route was scalable and reproduceable, and that the optimisations could deliver higher quality of
compound 1. However an alternative sequence could be envisaged which might lead to still
higher selectivity and potentially be more costꢀeffective.
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Table 2. Comparison of yields and purities of the chemical steps in various campaigns
towards compound 1.
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Yield % (purity in area%)
Campaign
Step 1*
85ꢀ90 (NA)
84 (93.0)
91 (98.8)
94 (98.5)
Step 2
50ꢀ60 (NA)
69 (95.0)
81 (99.0)
85 (99.9)
Step 3
Step 4
Research
87 (NA)
97 (99.1)
74 (97.8)
98 (99.5)
53 (NA)
93 (99.0)
88 (98.5)
78 (99.9)
1st PreꢀClinical
2nd PreꢀClinical
Clinical
* Step numbers correspond to those in Figure 3
2.3 Alternative Sequence
Of the various impurities which had been characterised and tracked, the key impurity of all
campaigns to date was the bisꢀamide 9 formed during the first amination step with the aniline 3.
An alternative sequence was therefore investigated which not only avoided the formation of this
impurity completely, but could also lead to cost reduction since the aniline 3 would be used later
in the synthesis scheme and so employed in lesser quantities. In effect the reverse order of
addition of the two amine fragments 3 & 4 was envisaged, starting with the 3ꢀpentylamine 4
addition onto the squarate diester 2 followed by the aniline 3.
Choline hydroxide is highly basic (pKa 13.9)12 and so was used as both base during the addition
of aniline 3 to the new intermediate 11 as well as saltꢀforming agent to directly arrive at
compound 1 without need for the former saltꢀswitching. Initial experiments were so encouraging
that isolation of crystalline intermediate 11 was later considered only as optional, since a oneꢀpot
variant starting from the 3ꢀpentylamine 4 and obtaining the drugꢀsubstance 1 from the same
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reactor by simple sequential addition of amines after successful conversion by inꢀprocess control
proved to be highly feasible (Figure 5). Specifically compounds 2 and 4 were combined in THF
at 0oC, and when full conversion to 11 was achieved then aniline 3 as preꢀformed choline salt
was added as a solution in THF/methanol. The product 1 was precipitated from solution by
addition of nꢀheptane as antiꢀsolvent rendering the sequence highly streamlined.
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Figure 5. Alternative sequence to compound 1.
A further advantage of this sequence is that the bisꢀaddition product 10 (Figure 4) was formed in
small amounts (0.4 area% after IPC, not detected in isolated compound 1) and, in contrast to 11,
is highly soluble and easily purged during crystallisation. The chemistry of the new sequence
was developed in the laboratory on a 35 g scale of squarate diester 2, delivering 91 % overall
yield of compound 1 with a purity of 99.45 area% after a single recrystallisation. Preliminary
costꢀcalculations suggest a saving of approximately 35%, brought about by the combination of
using the more expensive amine 3 later in the synthesis and the higher yields. Future
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manufacturing options will certainly be carefully weighed against the clear benefits of this
alternative sequence.
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3.
Conclusion
The evolving manufacturing concept for a CXCR2 receptor antagonist has been described
starting from the original research route and continuing through to clinical supply. Progress was
outlined in terms of yield improvements as well as purgeingꢀstrategies for the key impurities. An
alternative sequence has been explored on lab scale which avoids the switching of phenolate salt
from triethylammonium to freeꢀacid to choline salt, as well as providing an overall yield gain and
estimated cost reduction.
4.
Experimental Section
Materials. All commercially available materials and solvents were used as received without any
further purification. Purity was measured by HPLC using the following setup: Agilent 1200, with
Phenomenex Gemini C18 column (150 x 3mm, particle size 3.0 µm), operating at 40oC with
acetonitrile and 0.1 % phosphoric acid/water as eluent system, 215nm. NMR spectra were
measured on a Bruker 400MHz spectrometer. Melting points were determined by interpretation
of the endothermic event in DSC profiles. DSCs were measured on either a Mettler Toledo
DSC821e (compounds 7, 8 and 1), or a Mettler Toledo DSC1 (compounds 2, 3 and 5) in a closed
goldꢀplated highꢀpressure steel crucible (40 ꢁL), with a 4 °C / min heating ramp.
Highꢀresolution massꢀspectra were performed by using electrospray ionization in positive ion
modus after separation by liquid chromatography. The elemental composition was derived from
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the mass spectra acquired at the high resolution of about 30’000 on a Q Exactive Plus mass
spectrometer (Thermo Scientific) coupled to an Ultimate 3000 UHPLC (Thermo Scientific). The
high mass accuracy below 1.5 ppm was obtained by using a lock mass.
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3-Amino-6-chloro-2-hydroxy-N-methoxy-N-methylbenzene-1-sulfonamide (3)
Concentrated sulfuric acid (64.5 kg) was carefully diluted with cold water (35 kg) and then
slowly added to a cold (0 oC) solution of compound 5 (27.3 kg, 1.38 mol) in 1,4ꢀdioxane (102.4
kg) in order to keep the internal temperature below 45 oC. The reaction mixture was purged with
nitrogen three times and then heated to 85 oC for 17 hours (or until IPC (HPLC) indicated no
starting material or amide intermediate was detectable). The solution was cooled to 20 oC before
dilution with water (581 kg) and then dioxane was then distilled off (216 kg) under vacuum
while maintaining the jacket temperature under 50 oC. 30% NaOH solution was added slowly to
the solution, so that the pH value could be adjusted to 5.5, maintaining the temperature below 40
oC. During the neutralization compound 3 was precipitated as a brown solid. After filtration,
washing with water (34.2 kg) and drying under vacuum to constant weight, compound 3 was
isolated (20.5 kg, 94 % yield).
m.p. 131.0 ꢀ 134.0 oC
HPLC retention time: 9.08 min
DSC: onset 95 oC, ꢀ1014 J/g
1H NMR (300 MHz, CDCl3) δ 9.98ꢀ9.88 (br s, 1H), 6.93ꢀ6.88 (d, 1H, J = 8.3 Hz), 6.86ꢀ6.80 (d,
1H, J = 8.5 Hz), 4.18ꢀ3.90 (br s, 2H), 3.67 (s, 3H), 3.08 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
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147.6, 136.2, 122.9, 120.8, 119.0, 113.7, 63.2, 37.5; HRMS: C8H11ClN2O4S, M = 266.01,
calculated [M+H]+ = 267.02008, measured [M+H]+ = 267.02011.
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6-chloro-3-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxy-N-methoxy-N-
methylbenzenesulfonamide (7)
Triethylamine (13.2 kg, 279.21 mmol) was added to a solution of compound 2 (23.4 kg, 137.52
mol) in ethanol (174 kg) at 20 oC. To this solution was added compound 3 (28.0 kg, 104.99 mol)
as a THF (101 kg) solution over a 2 hr addition time in order to maintain the internal temperature
of 20oC(+/ꢀ 3 oC). The mixture was further stirred at 20 oC for 5 hrs upon which a sample for inꢀ
process control (HPLC) was taken. The reaction mixture was heated to 62 oC to ensure complete
dissolution upon which it was cooled back to 20 oC over a period of not less than 2.5 hrs. The
resulting suspension was stirred for a further 3 hrs and then filtered. The wetꢀcake was washed
three times with ethanol (56 kg each) and the solid dried under vacuum at 50 oC overnight to
yield compound 7 as an ethanol solvate of the triethylammonium salt (43.4 kg, 85 % uncorrected
yield).
m.p. broad, begins at 130.0oC
HPLC retention time: 10.74 min
DSC: onset 145 oC, ꢀ1243 J/g
1H NMR (300 MHz, CDCl3): Tautomerism observed δ 7.65−7.15 (br s, 1H), 6.70−6.64 (br s,
1H), 4.96ꢀ4.85 (q, 2H, J = 7.1 Hz), 3.68 (s, 3H). 3.07ꢀ2.93 (m, 9H), 1.58ꢀ1.51 (t, H, J = 7.0 Hz),
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1.27ꢀ1.20 (t, 9H, J = 7.1 Hz); C NMR (100 MHz, CDCl3): δ 129.7, 114.2, 70.1, 62.9, 45.7,
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37.6, 15.9, 8.9; HRMS: C14H15O7N2ClS, M = 390.04, calculated [M+H]+ = 391.03613, measured
[M+H]+ = 391.03598.
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6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxy-N-
methoxy-N-methylbenzenesulfonamide (8)
A solution of 3ꢀpentylamine 4 (8.6 kg, 98.6 mol) in methanol (16.9 kg) was added to a hot
solution (55 oC) of compound 7 (35.0 kg, 71.14 mol) in methanol (70 kg), rinsing the addition
apparatus with methanol (28 kg). The suspension was left to stir for 2 hrs upon which an inꢀ
process control sample was taken. The reaction solution was carefully acidified to pH 3.4 ꢀ 4.2
by slow addition of aqueous HCl (2N, maximum 65 kg) during which precipitation of compound
8 occured. On obtaining the correct pH the suspension was cooled to 22 oC over 2 hrs and stirred
for a further 2 hrs at this temperature. The suspension was filtered, with washing of the filterꢀ
cake with methanol (81 kg) followed by drying under vacuum at 50 oC to gives compound 8 as a
light yellow solid (32.3 kg, 98 % when calculated as the methanol solvate).
HPLC retention time: 12.62 min
DSC: onset 185 oC, ꢀ1399 J/g
1H NMR (300 MHz, DMSOꢀD6): δ 10.17 (s, 1H), 9.40 (s, 1H), 8.29ꢀ8.24 (d, 1H, J = 9.0 Hz),
8.15ꢀ8.10 (d, 1H, J = 8.8 Hz), 7.30ꢀ7.25 (d, 1H, J = 8.8 Hz), 3.94ꢀ3.84 (m,1H), 3.63 (s, 3H), 3.04
(s, 3H), 1.70ꢀ1.57 (m, 2H), 1.54ꢀ1.41 (m, 2H), 0.94ꢀ0.86 (t, 6H, J = 7.4 Hz); 13C NMR (100
MHz, CDCl3): δ 184.6, 179.8, 169.7, 162.1, 149.1, 128.8, 125.3, 124.9, 122.8, 114.5, 62.9, 57.2,
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37.3, 27.9, 10.0; HRMS: C17H22O6N3ClS, M = 431.09, calculated [M+H]+ = 432.09906,
measured [M+H]+ = 432.09927.
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2-Hydroxy-N,N,N-trimethylethanaminium 2-(N-methoxy-N-methylsulfamoyl)-6-({3,4-
dioxo-2-[(pentan-2-yl)amino]cyclobut-1-en-1-yl}amino)phenolate (1)
Commercial 50 w/w% choline hydroxide (6) in methanol (14.4 kg, 59.4 mol) was added over 30
mins to a stirred solution of compound 8 (25.0 kg, 57.9 mol) in methanol (81.3 kg) at 65 oC,
rinsing the addition apparatus with methanol (22.9 kg). The solution was left to stir for 1 hr, upon
which it was hot filtered to remove particles. Isopropanol (64.1 kg) was slowly added, prior to
cooling of the solution to 55 oC, at which point the solution was seeded with compound 1 (16 g)
and left to stir for a further 15 mins. The stirred suspension was cooled to 0 oC over 2 hrs, then
left to stir for a further 2 hrs. After filtration the wetꢀcake was washed with isopropanol (52.1 kg)
and the crystals dried under vacuum at 50 oC to give compound 1 as a light yellow solid (25.4 kg,
82 %).
HPLC retention time: 12.62 min
DSC: onset 185 oC, ꢀ1065 J/g
1H NMR (300 MHz, DMSOꢀD6): δ 9.61ꢀ9.42 (br s, 1H), 8.47ꢀ8.36 (d, 1H, J = 9.0 Hz), 7.74ꢀ7.65
(d, 1H, J = 8.0 Hz), 5.99ꢀ5.92 (d, 1H, J = 8.3 Hz), 5.20ꢀ5.36 (br s, 1H), 3.91ꢀ3.77 (m, 3H), 3.42ꢀ
3.36 (m, 2H), 3.16ꢀ3.04 (s, 9H), 2.94ꢀ2.91 (s, 3H), 1.64ꢀ1.51 (m, 2H), 1.50ꢀ1.36 (m, 2H), 0.93ꢀ
0.81 (t, 6H, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3): δ 183.0, 180.4, 169.1, 164.5, 162.7,
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132.6, 127.4, 117.0, 112.3, 108.1, 66.9, 62.4, 58.9, 55.1, 53.1, 53.1, 27.9, 10.1; HRMS:
C17H22O6N3ClS, M = 431.09, calculated [M+H]+ = 432.09906, measured [M+H]+ = 432.09857.
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3-Ethoxy-4-[(pentan-2-yl)amino]cyclobut-3-ene-1,2-dione (11)
Compound 4 (12.20 g, 141.63 mmol) in methanol (50 ml) was added over 40 mins to a stirred
solution of compound 2 (24.10 g, 141.63 mmol) in methanol (150 ml) at room temperature. After
4 hrs the solution was heated to 50 oC and distilled down to 80ml total volume by reduction of
the pressure. To the suspension was added heptane fraction (200 ml) and the volume again
reduced to 80 ml by distillation. This was repeated once more, upon which heptane fraction (100
ml) and ethylacetate (21 ml) were added and the mixture cooled to 0 oC over 16 hrs. The crystals
were filtered and washed with heptane fraction (40 ml), and then dried under vacuum at 50 oC to
give compound 11 as a white solid (25.2 g, 84 %).
m.p. 89.0ꢀ90.0 oC
HPLC retention time: 6.84 min
DSC: onset 255 oC, ꢀ497 J/g
1H NMR (400 MHz, DMSOꢀD6): Tautomerism observed, δ 8.64 (d, 0.5H, J = 9.0 Hz), 8.43 (d,
0.5H, J = 9.3 Hz), 4.67 (q, 2H, J = 7.0 Hz), 3.80ꢀ3.70 (m, 0.5H), 3.37ꢀ3.27 (m, 0.5H), 1.60ꢀ1.33
(m, 6H), 0.84 (dt, 6H, J = 7.3, 2.9 Hz); 13C NMR (100 MHz, DMSOꢀD6): Tautomerism
observed, δ 189.3, 189.2, 181.8, 181.6, 176.6, 175.8, 172.6, 172.3, 68.7, 58.4, 57.6, 27.7, 27.5,
15.7, 10.3, 10.3; HRMS: C11H17NO3, M = 211.10, calculated [M+H]+ = 212.12812, measured
[M+H]+ = 212.12819.
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Alternative one-pot sequence to 2-Hydroxy-N,N,N-trimethylethanaminium 2-(N-methoxy-
N-methylsulfamoyl)-6-({3,4-dioxo-2-[(pentan-2-yl)amino]cyclobut-1-en-1-
yl}amino)phenolate (1)
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Compound 4 (18.14 g, 201.84 mmol) was added over 25mins to a stirred solution of compound 2
(34.70 g, 199.84 mmol) in THF (71 ml) at 0 oC. After 30 mins an inꢀprocess control (HPLC)
confirmed full conversion to the intermediate compound 11. In a separate vessel compound 3
(42.64 g, 159.88 mmol) was suspended in THF (100 ml) and choline hydroxide in water (46
w/w%, 43.18 ml, 175.86 mmol) was added to obtain a solution. This solution was then added to
the reaction mixture containing compound 11 which was maintained at 0 oC, flushing the
addition funnel with THF (10 ml). The reaction mixture was heated to 56 oC over 1 hr upon
which it was stirred for a further 2.5 hr with inꢀprocess control confirming conversion to
compound 1. A mixture of THF/heptane (4:1, 500 ml) was added to the solution over 40 min,
followed by a further amount of THF (500 ml). The suspension was then cooled to room
temperature over 1 hr, whereupon a further amount of heptane (100 ml) was added followed by
THF/heptane (4:1, 500 ml). The suspension was filtered and the wetꢀcake washed with THF (300
ml) before drying under vacuum at 50 oC to provide compound 1 as light yellow crystals (77.98
g, 91 %).
Analytics consistent with compound 1 above prepared by stepwise linear method.
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ACKNOWLEDGMENT
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We are grateful to Isabelle S. Gallou for continual support during the development phase of this
project, as well as to Francesco Venturoni for proof reading the manuscript.
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SUPPORTING INFORMATION
Experimental procedures and full characterization (1H, 13CꢀNMR, HRꢀMS, HPLC retention time)
for all compounds, as well as DSC data. This information is available free of charge via the
Internet at http://pubs.acs.org.
ABBREVIATIONS
CXCR1/CXCR2, Cxc Chemokine Receptor 1 and 2; TEA, triethylamine; THF, tetrahydrofuran;
EtOAc, ethylacetate; NMR, nuclear magnetic resonance; GC, gas chromatography; IPC, inꢀ
process control; DSC, dynamic scanning calorimetry.
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Choline salt of an antiꢀinflammatory substituted cyclobutenedione compound. WO
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9. For a review on the topic see: Teasdale, A.; Delaney, E. J.; Eyley, S. C.; Jacq, K.; Taylorꢀ
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10. Within this manuscript area% is defined as the area of the peak of interest compared to
the sum area of all peaks in the spectrum, with the exception of solventꢀrelated peaks.
11. Anslyn, E. V. and Dougherty, D. A., Modern Physical Organic Chemistry, Palgrave
Macmillan, 2005.
12. Dawson, R. M. C., Data for Biochemical Research, Oxford, Clarendon Press, 1959.
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