Discovery of 2-(((1r,4r)-4-(((4-Chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetate (Ralinepag): An Orally Active Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.6b00871

The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in

Full text of DOI:10.1021/acs.jmedchem.6b00871

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Article
Discovery of 2-(((1r,4r)-4-(((4-chlorophenyl)
phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetate
(
(Ralinepag) :An Orally Active Prostacyclin Receptor Agonist
for the Treatment of Pulmonary Arterial Hypertension
Thuy-Anh Tran, Bryan Kramer, Young-jun Shin, Pureza Vallar, P. Douglas Boatman, Ning Zou,
Carleton R. Sage, Tawfik Gharbaoui, Ashwin Krishnan, Biman Pal, Sagar R Shakya, Antonio Garrido
Montalban, John Wesley Adams, Juan Ramirez, Dominic Peter Behan, Anna Shifrina, Anthony Blackburn,
Tina Leakakos, Yunqing Shi, Michael Morgan, Abu Sadeque, Weichao Chen, David John Unett,
Ibragim Gaidarov, Xiaohua Chen, Steve Chang, Hsin-Hui Shu, Shiu-feng Tung, and Graeme Semple
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00871 • Publication Date (Web): 10 Jan 2017
Downloaded from http://pubs.acs.org on January 10, 2017
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Discovery of 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl)(phenyl)carbamoyl)oxy)methyl)
cyclohexyl)methoxy)acetate (Ralinepag) : An Orally Active Prostacyclin
Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension
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ThuyꢀAnh Tran, Bryan Kramer, YoungꢀJun Shin, Pureza Vallar, P. Douglas Boatman, Ning Zou,
Carleton R. Sage, Tawfik Gharbaoui, Ashwin Krishnan, Biman Pal, Sagar R. Shakya, Antonio Garrido
Montalban, John W. Adams, Juan Ramirez, Dominic P. Behan, Anna Shifrina, Anthony Blackburn, Tina
Leakakos, Yunqing Shi, Michael Morgan, Abu Sadeque, Weichao Chen, David J. Unett, Ibragim
*
Gaidarov, Xiaohua Chen, Steve Chang, HsinꢀHui Shu, ShiuꢀFeng Tung and Graeme Semple
Arena Pharmaceuticals, 6154 Nancy Ridge Drive, San Diego, CA, 92121, USA.
Abstract
The design and synthesis of a new series of potent nonꢀprostanoid IP receptor agonists that showed oral
efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) is described. Detailed
profiling of a number of analogues resulted in the identification of 5c (Ralinepag) that has good
selectivity in both binding and functional assays with respect to most members of the prostanoid receptor
family, and a more modest 30ꢀ50ꢀfold selectivity over the EP3 receptor. In our hands, its potency and
efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NSꢀ304)
with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile
across species. Enterohepatic recirculation most probably contributes to a concentrationꢀtime profile after
oral administration in the cynomolgus monkey that showed a very low peakꢀtoꢀtrough ratio. Following
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the identification of an acceptable solid form, 5c was selected for further development for the treatment of
PAH.
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INTRODUCTION
Pulmonary arterial hypertension (PAH) is a rare but lifeꢀthreatening disease. The pathogenesis, which is
thought to result in part from an imbalance in the production of two cyclooxygenase metabolites of
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arachidonic acid, prostacyclin (PGI2) and thromboxane A2, includes pulmonary vasoconstriction,
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endothelial and smooth muscle cell proliferation, tissue remodeling and platelet mediated thrombosis.
The protective effects of PGI2 arise via activation of the prostacyclin receptor (IP) which leads to
stimulation of adenylate cyclase, with a resulting increase in intracellular cAMP levels, in platelets,
smooth muscle cells and immune cells. Therapeutic substitution of PGI2 or its analogues in PAH patients
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can reduce pulmonary artery pressure and slow disease progression. This class of compounds generally
has short halfꢀlives in vivo due to chemical and metabolic instability and has to be dosed by repeated
inhalation or continuous intravenous infusion. As a result, many of these drugs have limited and
sometimes problematic clinical application. Other drug classes have also been investigated for this
indication and several orally available endothelin antagonists (that act by blocking the vasoconstrictive
effects of endothelin peptides) and PDE5 inhibitors (that act by blocking the degradation of cGMP which
is vasodilatory) have been approved creating a multiꢀbillion dollar market and an improvement in patient
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care in this disease area. To improve upon the currently approved prostacyclin based drugs and to be
more competitive with the other classes in terms of compliance, stable, orally bioavailable PGI2 analogs
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and nonꢀprostanoid IP receptor agonists have been investigated. One such compound, NSꢀ304 or
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Selexipag, 1 which is a prodrug of the active species MRE269 (2, Figure 1), was recently approved for
the treatment of PAH.
The larger prostanoid ligand family evokes diverse biological actions in many tissues and cell types by
binding to multiple specific cell surface Gꢀprotein coupled receptors including prostaglandin D receptors
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DP1, DP2), prostaglandin E receptors (EP1, EP2, EP3, EP4), prostaglandin F2a receptor (FP) and the
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thromboxane receptor (TP) in addition to IP. Thus we sought to design new prostacyclin receptor agonists
with significantly improved pharmacokinetic profiles that like selexipag might be dosed orally but that
would not require metabolic activation, while staying mindful of the requirement for good receptor
selectivity to avoid other prostaglandin mediated activities. Although the majority of clinical side effects
of current prostacyclin agonists are thought to be on target, activation of other prostaglandin receptors,
such as EP3 in the gut for example, may contribute to some of the GI symptoms observed with previous
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agents.
RESULTS AND DISCUSSION
Designing and synthesis of a new series of IP agonists
For our earlier approaches to new IP receptor agonists, we surveyed multiple series of compounds
designed from an understanding of the structural requirements for activity based on previously known
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nonꢀprostanoid IP receptor agonists. Compounds of this type were shown to be potent agonists of the IP
receptor with good selectivity over most prostanoid receptor family members, but only very modest
selectivity over the DP1 receptor. 3 (Figure 2) was optimal in this series but had an IP to DP1 selectivity
ratio of just 8ꢀfold. Analogues from this series did demonstrate in vivo activity after SC dosing in the
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widely used rat monocrotaline model of PAH. However, this early design process failed to provide
compounds with significant oral activity in the same model. As a result, our reꢀdesign of these initial
prostacyclin agonist series focused on three key objectives. The first was to improve selectivity over DP1.
Secondly, we aimed to reduce aromatic ring count and lipophilicity in an effort to improve the in vivo,
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and eventually the clinical, profile. It has been suggested that either an increase in the sp character, or
the reduction in the number of aromatic rings in candidate drugs may correlate with improved clinical
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success. Finally, we were interested in significantly reducing the cycle times for synthesis and testing by
trying to target compounds with a shorter synthetic route compared to the 10ꢀ14 step syntheses required to
prepare our first series. We achieved the second of these objectives by deleting the fused aromatic ring in
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our first series (Figure 2. F(sp ) was increased by 2ꢀfold, albeit without any significant change in clogP).
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This approach had the added advantage of removing a chiral centre which helped to simplify the synthesis,
although two alternative relative stereochemical arrangements are possible. In addition, as this type of
scaffold was new in the prostaglandin space, it also opened up a number of additional possibilities for the
linker group that serves to orientate the two required aromatic groups, giving us a further opportunity to
reduce synthetic complexity. After briefly investigating a number of such linkerꢀaromatic combinations,
including using both of the pyridazinone building blocks as well as some related heterocycles we
explored previously, we quickly settled on the carbamate series 5, which was relatively simple to prepare
in only a few steps.
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For our initial investigation, we prepared both the cis- and trans-analogues for a number of biaryl
carbamates by using either the cis- or trans-cyclohexanedimethanol building block (6, Scheme 1) and
reacting it with the appropriate isocyanate (e.g. R = H) to provide 7. The alcohol was then elongated
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using a rhodium acetate catalysed insertion reaction with tꢀbutyl diazoacetate to provide the tꢀbutyl ester 8
in good yield. The target compounds were then prepared in parallel format by direct Ullmanꢀlike arylation
of the carbamate to give 9 followed by simple acid catalysed deprotection of the ester group to provide
the desired compounds 5.
In Vitro SAR of New Series
The new compounds were tested for functional agonist activity in a standard (Cisbio) cAMP assay using
recombinant IP or DP1 receptors stably expressed in CHOꢀK1 cells. Clonal cell lines were derived
following standard protocols but receptor expression levels were kept to a minimum to preclude receptor
reserve effects. To streamline our screening process, we focused on the DP1 receptor as the most likely
offꢀtarget issue based on the experience with our earlier series as discussed above, and that the new
compounds had been designed using modifications to that series. In addition, the first compound in the
series 5a, showed no activity at prostaglandin receptors other than DP1 based on a panel assays in
melanophores. More extensive profiling with other family receptors was therefore performed only on
potential preꢀclinical candidate compounds. This approach was later shown to be flawed when we noted
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some previously unseen offꢀtarget activity with compounds that were more potent than 5a on the IP
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receptor. Our new compounds were compared to 2 (the active metabolite of the approved compound 1 )
for receptor potency and intrinsic activity at the IP receptor as we felt this to be the most appropriate
comparator compound for our program, as at the time 1 was the only orally acting nonꢀprostanoid IP
agonist drug in development. In all cases (Table 1) the transꢀrelative stereochemical arrangement
provided compounds with significantly greater potency in the human IP receptor cAMP assay than their
cisꢀcounterparts (5aꢀh), with the biggest difference being observed between the 4ꢀmethoxy analogues 5e
and 5f. From this point onwards, only the transꢀisomers were prepared as we attempted to further
optimize the series.
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The SAR of the substituents on the aromatic ring groups was broadly similar to that observed in our
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earlier series. Substitution of one of the aromatic rings with a 3ꢀ or 4ꢀhalo substituent provided
compounds with single digit nanomolar potency and high intrinsic activity (agonist efficacy relative to
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ꢀM iloprost as a positive control) in the IP cAMP assay and in some cases >100ꢀfold selectivity over
DP1 in the same assay platform. A 3ꢀ or 4ꢀmethoxy group (5e and 5j) on one of the phenyl rings, again
provided analogues with single or double digit nanomolar potency and reasonable selectivity and was the
most polar substituent that was tolerated. The in vitro potency and intrinsic activity of these monoꢀ
substituted analogues were comparable to data for 2 in our hands (Table 1) with a marginal improvement
in functional selectivity with respect to DP1. An additional halogen substituent on either the same ring or
the second aromatic ring did not improve receptor potency significantly and for a number of examples
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5qꢀu) appeared to decrease efficacy. We were speculating at this stage that greater receptor efficacy may
eventually offer an advantage for our series in further pharmacological measurements or in clinical testing,
and so only compounds where there was a statistically significant improvement in intrinsic activity over 2
were considered for further studies.
Early Compound Profiling
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In addition to its function as a direct vasodilator, prostacyclin can also inhibit ADPꢀinduced platelet
aggregation. Such aggregated and activated platelets produce vasoactive substances such as thromboxane
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and serotonin that may cause harmful vasoconstriction and vascular remodeling. We thus measured the
activity of our compounds to confirm the data from the recombinant receptor assay by testing in a primary
human platelet aggregation assay as a potentially diseaseꢀrelevant, physiological readout. This secondary
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assay was carried out as previously described, with aggregation induced by 2.5 ꢀM ADP in the presence
or absence of IP receptor agonists. The activity of all compounds in this assay was broadly reflective of
their agonist effect in the cloned IP receptor assay with an approximate 2ꢀ10ꢀfold difference in the
observed potency. Again the potency of our compounds compared favourably to the inhibition IC50
observed for 2 (Table 1), although all compounds, regardless of efficacy in the cloned IP receptor assay,
were able to achieve 100 % inhibition of the aggregation response if tested at a high enough concentration.
We did note however, that all of the compounds in our series had a rather poor effect when tested against
the rat IP receptor, with EC values typically 10ꢀ30 fold weaker than for the human receptor, and we thus
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had some concerns about being able to demonstrate in vivo pharmacological activity in the rat. Despite
this, several compounds with good human receptor potency and selectivity, that in general all had
excellent stability in microsomes from both human and rat (Table 1), were surveyed for their PK
properties in rat (Table 2).
Based on the combination of the in vitro activity/selectivity and the PK data obtained from this selection
of compounds in particular the high Cmax values, 5c, 5g and 5m were identified as three potential
candidates for testing in our rat in vivo monocrotaline (MCT) model. The two methoxy substituted
compounds (5e and 5j) were discarded when we observed high clearance and a moderate Cmax after oral
administration in vivo. As the in vivo assay required chronic administration of compounds and was this
time and resource intensive, we were limited to the selection of two compounds to progress at this stage.
Of the three compounds of interest remaining, 5c had the highest total exposure (AUC), as well as longest
halfꢀlife in the rat, which we felt provided the best opportunity to observe in vivo pharmacological
activity. For the second selection we preferred 5g over 5m. Although both were more potent than 5c at the
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rat receptor which we thought may also be important, and each had excellent in vitro selectivity for the
human IP receptor over the DP1 receptor, 5g had significantly higher total exposure (AUC) in the rat PK
study.
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Scaleꢀup and In Vivo Testing of Selected Compounds
For testing in the 21ꢀday in vivo MCT model, the two compounds selected needed to be resynthesized on
a multiꢀgram scale. Having already narrowed down the range of biaryl carbamate groups of interest to just
two, and in view of the unpredictable yields from the copper catalysed arylation of 8, we elected to start
from the requisite biaryl amine 10 (Scheme 2, method 1). For 5c, the starting material 4ꢀchloroꢀNꢀ
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phenylaniline was commercially available and 11 (R = H, R = 3ꢀF), the starting material for the
preparation of 5g which was not then readily available was prepared by Buchwald coupling of 3ꢀ
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fluoroaniline (10, R = 3ꢀF) with bromobenzene. Treatment of the biaryl amines with triphosgene at 0°C
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in pyridine provided the intermediates 12 (R = H, R = 4ꢀCl) and 12 (R = H, R = 3ꢀF) that could be
isolated. For the 3ꢀfluoro analogue, the yield in the next reaction was enhanced by activation of this
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intermediate with DMAP but in the case of 12 (R = H, R = 4ꢀCl) the chlorocarbamate was reacted
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directly with transꢀ1,4–cyclohexanedimethanol to provide 13 (R = H, R = 4ꢀCl). The sequence was
completed with the carbene insertion reaction using tꢀbutyl diazoacetate followed by ester hydrolysis with
HCl/dioxane.
When dosed in the MCT model in a preventative mode, both compounds demonstrated a doseꢀdependent
inhibition of the increase in the ratio of right ventricular weight to the combined weight of left ventricle
and septum (RV/LV+S) induced by administration of monocrotaline (data for 5c is shown in Figure 3,
data for 5g is included in the supporting information), indicative of an inhibitory effect on the
development of right ventricular hypertrophy. At the highest dose of 30 mg/kg twice daily for 21
consecutive days after the initiation of the insult with monocrotaline, RV/LV+S values were similar to
those in sham controls. Supporting the observed effect on hypertrophy, 5c was able to significantly reduce
the MCTꢀinduced increase in pulmonary arterial pressure in a subꢀset of the test group (Figure 3) and
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pulmonary vessel wall thickness in 5 animals (see supporting information), at the highest dose of 30
mg/kg.
As might be expected from such highly lipophilic carboxylic acid compounds (cLogP for 5c = 5.6; cLogP
for 5g = 5.0), plasma protein binding was very high (~99%, i.e. 1% free fraction) for each across all
species. The unbound Cmax plasma concentrations for 5c and 5g after a single oral dose at 30 mg/kg were
estimated to be 300 nM (based on a 1% free fraction of the measured Cmax of a 30 mg/kg dose in a
separate experiment designed to measure dose proportionality). Although agonists of some GPCRs with
very efficient coupling can achieve full activation of the receptor at very low receptor occupancy, it is
worth noting in this case that the unbound plasma concentrations in vivo were in the same range as the in
vitro EC values for the two compounds at the recombinant rat IP receptor cAMP assay. In addition, the
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unbound Cmax for 5c following the ineffective 10mg/kg dose was around 100 nM (as calculated from the
PK experiment) which is 5ꢀfold lower than the EC50 at the rat IP receptor. This provided a possible
explanation for the lack of effect at this dose and allowed for a hypothesis as to the required efficacious
human exposure requirements. It remains unclear however that the same correlation can be made in
human as the cAMP assay may not be the most relevant readout.
Further Profiling
Encouraged by these data, we carried out more extensive profiling of 5c and 5g. In radioligand binding
assays of affinity for the IP receptor in multiple species, there was little to choose between the two
compounds (Table 3). In addition, 5c and 5g showed good binding selectivity over almost all the related
prostaglandin receptor subtypes tested, with the exception of EP3, and in a wider screen no additional offꢀ
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target liabilities were observed. Again, we noted clear species differences for the IP receptor affinities
and the difference between the human and rat receptor were similar to that observed in the IP cAMP
functional assays. In HTRF cAMP functional assays for other family members, no significant agonist
activity was observed at the other receptors tested (EP2, EP4), suggesting that any functional activity
arising from the modest binding affinity to these prostanoid receptors would most likely be in the
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antagonist direction. In contrast to our earlier series, the prostaglandin receptor for which 5c and 5g had
the most significant receptor binding affinity was human EP3 (the EP3v6 form of the receptor was used
for the study) and an EP3/IP binding selectivity ratio of 30ꢀ to 50ꢀfold was observed. This was a surprise
to us as no previous series of IP agonists we had tested (including 2) had any affinity for this receptor.
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Unlike IP, EP2 and EP4, EP3 is a G coupled receptor and as a result functional selectivity is not directly
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comparable in a cAMP assay as forskolin activation is required making a functional selectivity
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comparison difficult to quantify. In our melanophore platform however, both compounds behaved as
EP3 agonists with potencies similar to their binding affinities (see supporting data). Again, a true
functional selectivity is difficult to deduce but the data were similar to the agonist effect seen for iloprost
in the EP3 melanophore assay. We were satisfied though, based on the binding (K ) data, that the receptor
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selectivity for these two compounds was adequate for us to proceed but with a requirement to monitor
potential EP3 mediated effects.
Since GPCR functional assays performed in cells expressing high levels of recombinant receptors can
often be influenced by receptor reserve effects, which may exaggerate potency and efficacy, further
studies at the human IP receptor were performed to carefully characterize the in vitro potency and
efficacy of 5c and 5g. Thus, a series of assays were performed in which receptor expression levels were
systematically reduced. In addition, cAMP assays were performed in primary human pulmonary arterial
smooth muscle cells. When we reached a level of no receptor reserve in the recombinant assay, the
potency and efficacy data were essentially identical in each system (supporting information). In both of
these assay systems, as well as the screening assay, 5c showed significantly greater potency and efficacy
than 2. Further assays in pulmonary arterial smooth muscle cells (PASMC) from PAH patients have
recently been reported that also showed a greater efficacy for 5c in increasing cAMP levels compared to 2
(an effect that was blocked for both compounds by the IP antagonist ROꢀ1138452) although there was no
17
difference in EC .
5
0
Finally, as part of our in vitro evaluations, neither 5c nor 5g showed any measurable inhibition of the
most highly expressed cytochrome P450 enzymes (IC > 50 ꢀM for CYPs 1A2, 2D6, 3A4 2C8, 2C9 and
5
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C19) and no inhibition of hERG channel functional activity was observed in a patch clamp assay (IC >
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0 ꢀM).
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Selection of a Development Candidate
The first meaningful differentiation between the two compounds was observed during our early solid state
characterization. Surprisingly, although the structures of the two compounds were very similar, both the
free acid (m.p. ~128°C) and the sodium salt (m.p. ~243°C) of 5c were crystalline and nonꢀhygroscopic
solids, whereas the free acid of 5g was an oil and the sodium salt was a low melting monohydrate (see
supporting information). It was on the basis of these data that the 4ꢀchloroꢀanalogue 5c was selected for
further investigation as it had two potential solid state forms that met our inꢀhouse solid state screening
developability criteria. 5g on the other hand had only a single acceptable solid form which being a hydrate,
may have had a higher likelihood of running into issues in development. In addition, when we
investigated further scale up synthesis methods, all key synthetic intermediates for 5c could be isolated as
solids. This was not the case for 5g which might also have presented significant development challenges.
5c was subsequently prepared in kilogram amounts using some modifications to the first scaleꢀup route,
primarily directed towards improving early process safety by the replacement of phosgene and the
carbene insertion reactions with more benign alternatives (Scheme 2, method 2) and at shortening the
1
2
overall procedure by telescoping some steps. Hence, 11 (R = H, R = 4ꢀCl) was treated with 1,1'ꢀ
carbonyldiimidazole (CDI) in acetonitrile in the presence of potassium phosphate. After this first reaction
1
2
was complete, the intermediate 12 (R = H, R = 4ꢀCl) was not isolated but rather excess 1,4ꢀtransꢀ
1
2
cyclohexane dimethanol was added to the reaction mixture to provide 13 (R = H, R = 4ꢀCl) which was
1
2
isolated by removal of acetonitrile and precipitation from water. 13 (R = H, R = 4ꢀCl) was alkylated with
tertꢀbutyl bromoacetate utilizing 50% aqueous sodium hydroxide and tetrabutyl ammonium bromide in
toluene. On reaction completion, the intermediate tertꢀbutyl ester was simply heated to hydrolyze the ester.
The resultant acid was isolated as the sodium salt which crystallized from aqueous acetone. The sodium
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salt could then be acidified with hydrochloric acid and the free acid of 5c isolated by filtration with purity
18
in excess of 99%.
With now a significant amount of material in hand we were able to collect additional data to confirm the
nomination of 5c for further development. In PK studies across a number of preclinical species 5c
exhibited doseꢀdependent exposure with good bioavailability after oral administration in all species tested
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(
Table 4 and supporting information). Particularly noteworthy was the concentrationꢀtime profile and
long elimination halfꢀlife in the monkey. 5c appeared to have a very low peakꢀtoꢀtrough ratio in the range
of 3ꢀ5 following oral administration, suggesting that this compound might have an advantageous profile
in the clinic. As the majority of the side effects observed with prostacyclin therapies, such as flushing,
systemic hypotension and jaw pain, are believed to be largely on target, they may be greatly ameliorated
by such minimal fluctuations in plasma exposure if these can be confirmed in human studies. Such tight
control of plasma levels may therefore be expected to provide improved tolerability compared to existing
prostacyclin treatments. In addition, the long halfꢀlife in preclinical species also strongly suggested the
possibility for once daily oral formulation.
One explanation for the prolonged exposure was the possibility of enterohepatic recirculation. This
suggestion was inferred from concentrationꢀtime profiles in PK studies across species in which a second,
delayed absorption phase was routinely observed. A more definitive study was therefore conducted in
bileꢀduct cannulated (BDC) rats. After an intravenous administration of 5c to either normal or BDC rats,
the second absorption phase was eliminated in the BDC rats with an accompanying 3ꢀfold reduction in the
apparent plasma halfꢀlife, supporting the suggestion of recycling of 5c back to the plasma via bile
excretion into the gut in normal rats. Concentrations of 5c were negligible in bile and urine compared to
plasma indicating that the major path of elimination was hepatic metabolism and that a conjugated species
may be involved.
A taurineꢀconjugated metabolite was subsequently identified in bile (M1, Figure 4), which was also
observed in circulating plasma, although at much lower concentrations (~5%) compared to parent. Further,
two oxidized products, one of which was also a taurineꢀconjugate were identified (M2 and M3), in plasma.
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Both conjugated metabolites of 5c had weak activity at the IP receptor (Figure 4), whereas the low level
metabolite M2 unexpectedly had doubleꢀdigit nanomolar functional activity at the human receptor. It is
known that taurine can be conjugated to bile acids and these conjugates may be hydrolyzed in the gut
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which allows for bile acid reabsorbtion. Having observed a taurine conjugate metabolite, we
hypothesized that 5c, which like bile acids is also a highly lipophilic carboxylic acid, could also be a
substrate for the same recirculation mechanism. To confirm that the 5c taurine conjugate was a potential
source of the observed recirculation, we synthesized M1 (Scheme 3). When dosed orally to rats
essentially identical plasma levels of 5c were obtained compared to dosing of the parent compound. To
eliminate the potential for a simple acidic hydrolysis of the conjugate in the GI tract to produce 5c which
could be absorbed directly, we incubated M1 in simulated gastric fluid at room temperature for 3h with
little chemical degradation to the parent. It is therefore highly likely that this conjugation and recirculation
process contributes significantly to the favorable pharmacokinetic profile. Interestingly, no analogous
conjugate was observed in vivo following dosing of 5g, further highlighting the differences in behavior of
the two compounds despite their very similar structures.
CONCLUSIONS
5c is an orally bioavailable, nonꢀprostanoid IP receptor agonist that is efficacious in the rat MCT model of
PAH. It has good selectivity in both binding and functional assays with respect to most members of the
prostanoid receptor family, but a more modest 30ꢀ50ꢀfold selectivity over the EP3 receptor. In our hands,
its potency and efficacy are superior in vitro to 2 (the active metabolite of the clinical compound 1) in in
vitro measures of IP receptor mediated cAMP signaling and it is more potent in inhibiting ADPꢀinduced
aggregation in ex vivo platelet assays. 5c is rapidly absorbed after oral administration in preclinical
species and its systemic clearance is primarily due to hepatic metabolism with probable enterohepatic
recirculation. The elimination halfꢀlife ranges from 5 to 8 hours in rodents to 10 and 18 hours in dog and
monkey, respectively. Exposure was so prolonged in the monkey that it may provide very low peak to
trough plasma concentration ratios with repeated dosing in patients that could closely mimic continuous
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intravenous infusion. Based on these data, as well as an acceptable safety profile in INDꢀenabling studies,
5
c as its free acid solid form (which was subsequently given the USAN name ralinepag) was selected for
further development for the treatment of PAH. Clinical studies are underway to determine if the excellent
in vitro and PK profile can provide improvements in clinical efficacy over current therapies.
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EXPERIMENTAL
Chemistry
1
Proton nuclear magnetic resonance ( HꢀNMR) spectra were recorded on a Bruker Avanceꢀ400 equipped
with a QNP (Quad Nucleus Probe) or a BBI (Broad Band Inverse) and zꢀgradient. Chemical shifts are
given in parts per million (ppm) with the residual solvent signal used as reference. NMR abbreviations
are used as follows: s = singlet, d = doublet, dd = doublet of doublet, ddd = doublet of doublet of
doublets, dt = doublet of triplet, t = triplet, tt = triplet of triplets, q = quartet, m = multiplet, br = broad.
Microwave irradiations were carried out using either an Initiator or Initiator+ machine (Biotage). Thinꢀ
layer chromatography (TLC) was performed on silica gel 60 F254 (Merck), preparatory thinꢀlayer
chromatography (prep TLC) was preꢀformed on PK6F silica gel 60 A 1 mm plates (Whatman), and
column chromatography was carried out either manually on a silica gel column using Kieselgel 60, 0.063ꢀ
0
.200 mm (Merck) or using preꢀpacked columns for Isolera 1 (Biotage). Evaporation was done in vacuo
on a Buchi rotary evaporator.
Test compound purity was measured by UV (254 nm) peak area over a 5 min 0ꢀ100% Acetonitrile
gradient elution. LCMS specs: 1) PC: HPLCꢀpumps: LCꢀ10AD VP, Shimadzu Inc.; HPLC system
controller: SCLꢀ10A VP, Shimadzu Inc; UVꢀDetector: SPDꢀ10A VP, Shimadzu Inc; Autosampler: CTC
HTS, PAL, Leap Scientific; Mass spectrometer: API 150EX with Turbo Ion Spray source, AB/MDS
Sciex; Software: Analyst 1.2. 2) Mac: HPLCꢀpumps: LCꢀ8A VP, Shimadzu Inc; HPLC system
controller: SCLꢀ10A VP, Shimadzu Inc. UVꢀDetector: SPDꢀ10A VP, Shimadzu Inc; Autosampler: 215
Liquid Handler, Gilson Inc; Mass spectrometer: API 150EX with Turbo Ion Spray source, AB/MDS
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Sciex Software: Masschrom 1.5.2. All test compounds were of greater than 95% purity by one or more of
the above methods and LCꢀMS purity for individual compounds is noted in the experimental.
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Small scale Synthesis Methods
Methods for the preparation of trans isomers are described. Identical methods were used for the cis
isomers where prepared using (1s,4s)ꢀcyclohexaneꢀ1,4ꢀdiyldimethanol in place of (1r,4r)ꢀcyclohexaneꢀ
1
,4ꢀdiyldimethanol.
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ ((Diphenylcarbamoyloxy)methyl)cyclohexyl)methoxy)acetic Acid. (5a)
The title compound was obtained from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy
)methyl)cyclohexyl)methoxy) acetate and iodoꢀbenzene, using a similar method to the one described for
+
1
Compound 5e. LCMS m/z = 398.10 [M+H] ; Purity : 98%; HꢀNMR (400MHz, DMSOꢀd6) δ 1.10ꢀ
.42 (m, 8H), 1.53ꢀ1.60 (m, 2H), 1.70ꢀ1.87 (m, 2H), 3.91ꢀ3.93 (d, J= 4.2 Hz, 2H), 3.98 (s, 2H), 7.20ꢀ7.31
m, 6H), 7.30ꢀ7.50 (m, 4H).
1
(
Preparation of 2ꢀ(((1s,4s)ꢀ4ꢀ((diphenylcarbamoyloxy)methyl)cyclohexyl)methoxy)acetic acid. (5b)
The title compound was obtained from tertꢀbutyl 2ꢀ(((1s,4s)ꢀ4ꢀ((phenylcarbamoyloxy)
methyl)cyclohexyl)methoxy) acetate and iodoꢀbenzene, using a similar method to the one described for
+
1
Compound 5e. LCMS m/z = 398.45 [M+H] ; Purity : 99% ; H NMR (400MHz, DMSOꢀd ) δ 1.15ꢀ1.40
6
(
m, 8H), 1.50ꢀ1.62 (m, 2H), 1.75ꢀ1.81 (m, 2H), 3.90ꢀ3.92 (d, J = 4.3Hz, 2H), 3.96 (s, 2H), 7.21ꢀ7.32 (m,
H), 7.35ꢀ7.39 (m, 4H).
6
Preparation of 2ꢀ(((1s,4s)ꢀ4ꢀ(((4ꢀchlorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic
acid (5d)
The title compound was obtained from tertꢀbutyl 2ꢀ(((1s,4s)ꢀ4ꢀ((phenylcarbamoyloxy)
methyl)cyclohexyl)methoxy) acetate and 1ꢀchloroꢀ4ꢀiodobenzene, using a similar method to the one
+
1
described for Compound 5e. LCMS m/z = 432.1 [M+H] ; Purity : 99% ; H NMR(400 MHz, DMSOꢀd ) δ
6
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ppm 1.28ꢀ 1.53 (m, 8H), 1.60ꢀ1.81 (m, 2H), 3.29 (d, J = 7.07 Hz, 2H), 3.96 (s, 2H), 3.98 (d, J = 6.69 Hz,
2
H), 7.24ꢀ7.33 (m, 5H), 7.36ꢀ7.48 (m, 4H).
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Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀMethoxyphenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic Acid (5e)
Step A: Preparation of ((1r,4r)ꢀ4ꢀ(Hydroxymethyl)cyclohexyl)methyl phenylcarbamate.
To a solution of (1r,4r)ꢀcyclohexaneꢀ1,4ꢀdiyldimethanol (5 g, 34.7 mmol) in pyridine at r.t., was added
phenyl isocyanate (4.13 g, 34.7 mmol). The reaction mixture was stirred for 5 h, concentrated and
extracted with ethyl acetate. The extract was dried over MgSO and concentrated in vacuo. The residue
4
was purified by silica gel column chromatography to give the title compound (4.69 g, 51.5%). LCMS m/z
+
1
=
264.43 [M+H] ; HꢀNMR (400MHz, DMSOꢀd6) δ ppm 0.81ꢀ 1.09 (m, 4H), 1.30ꢀ1.39 (m, 1H), 1.51ꢀ
1
.62 (m, 1H), 1.75ꢀ1.88 (m, 4H), 3.15ꢀ3.25 (d, J= 5.8Hz, 2H), 3.82ꢀ3.95 (d, J = 6.56 Hz, 2H), 4.52 (t, J =
.31 Hz, 1H), 6.29 (m, 1H), 7.30 (m, 2H), 7.48 (m, 2H), 9.62 (s, 1H).
5
Step B: Preparation of tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy)methyl) cyclohexyl)methoxy)
Acetate:
To a solution of ((1r,4r)ꢀ4ꢀ(hydroxymethyl)cyclohexyl)methyl phenylcarbamate (2.5 g, 9.49 mmol) and
diacetoxyrhodium (0.210 g, 0.475 mmol) in dichloromethane (50 mL) at 0°C was added dropwise a
solution of tertꢀbutyl 2ꢀdiazoacetate (1.350 g, 9.49 mmol) in dichloromethane (5mL) over 20 min. After
stirring for 30 min at r.t., the solid was removed by filtration and the filtrate concentrated under reduced
pressure. The residue was purified by silica gel column chromatography to give the title compound (3.32
+
1
g, 93%). LCMS m/z = 378.43 [M+H] ; HꢀNMR (400MHz, DMSOꢀd6) δ ppm 0.85ꢀ1.08(m, 4H), 1.42 (s,
9H), 1.41ꢀ1.62 (m, 2H), 1.78ꢀ1.81 (m, 4H), 3.25 (d, J= 6.3 Hz, 2H), 3.92 (d, J= 4.6 Hz, 2H), 6.29 (m, 1H),
7
.31 (m, 2H), 7.48 (m, 2H), 9.62 (s, 1H).
Step C: Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀMethoxyphenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic Acid
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tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy)methyl) cyclohexyl)methoxy) acetate: (50.0 mg, 0.132
mmol), copper(1) iodide (12.61 mg, 0.066 mmol), K PO (56.2 mg, 0.265 mmol), 4ꢀmethoxyphenyl
3
4
iodide (31.0 mg, 0.132 mmol) and dioxane (1.6 mL) were added to a microwave vial. The reaction
mixture was heated under microwave irradiation at 150°C for 5 h. The cooled mixture was filtered
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through a plug of MgSO . The solvent was evaporated and the resulting oil was redissolved in HCl (4 M
4
in dioxane, 1.987 mmol) and the mixture stirred overnight at r.t. After removal of the solvent, the residue
was purified by preparative LCMS to provide the title compound as a white solid (12.2 mg, 21.6%).
LCMS m/z = 428.4 [M+H]+, Purity : 99%; 1H NMR (400 MHz, CD3OD) δ ppm 0.92ꢀ0.97(m, 4H),
1
.22ꢀ1.25 (m, 2H), 1.47ꢀ1.79 (m, 4H), 3.15 (d, J= 6.5Hz, 2H), 3.79 (s, 3H), 3.95 (d, J= 6.0Hz, 2H), 4.02
(s, 2H), 6.90ꢀ7.33 (m, 9H).
Preparation of 2ꢀ(((1s,4s)ꢀ4ꢀ(((4ꢀmethoxyphenyl)(phenyl)carbamoyloxy)methyl) cyclohexyl)methoxy)
acetic acid (5f)
The title compound was obtained from tertꢀbutyl 2ꢀ(((1s,4s)ꢀ4ꢀ((phenylcarbamoyloxy
)
methyl)cyclohexyl)methoxy) acetate and 1ꢀiodoꢀ4ꢀmethoxybenzene, using a similar method to the one
+
1
described for Compound 5e. LCMS m/z = 428.2 [M+H] ; Purity : 99%; H NMR(400 MHZ, DMSOꢀd )
6
δ ppm 1.34ꢀ1.56 (m, 8H), 1.70ꢀ1.88 (m, 2H), 3.31 (s, 3H), 3.39 (d, J = 7.07 Hz, 2H), 4.00 (d, J = 7.20 Hz,
2H), 4.11 (s, 2H), 6.94ꢀ7.01 (m, 2H), 7.23ꢀ7.31 (m, 4H), 7.42ꢀ7.49 (m, 3H).
Preparation of 2ꢀ(((1s,4s)ꢀ4ꢀ(((3ꢀfluorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic
acid (5h)
The
title
compound
was
obtained
from
tertꢀbutyl
2ꢀ(((1s,4s)ꢀ4ꢀ
(
(phenylcarbamoyloxy)methyl)cyclohexyl)methoxy) acetate and 1ꢀfluoroꢀ3ꢀiodobenzene, using a similar
+
1
method to the one described for Compound 5e. LCMS m/z = 416.4 [M+H] ; Purity : 99%; H NMR(400
MHZ, DMSOꢀd ) δ ppm 1.28ꢀ 1.42 (m, 8H), 1.71 (s, 2H), 3.28 (d, J = 7.07 Hz, 2H), 3.96 (s, 2H), 3.99 (d,
6
J = 6.44 Hz, 2H), 7.03ꢀ 7.11 (m, 2H), 7.20ꢀ7.33 (m, 4H), 7.37ꢀ7.43 (m, 3H).
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Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀchlorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic
acid (5i)
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The
title
compound
was
obtained
from
tertꢀbutylꢀ2ꢀ(((1r,4r)ꢀ4ꢀ
((phenylcarbamoyloxy)methyl)cyclohexyl)methoxy) acetate and 1ꢀchloroꢀ3ꢀiodobenzene, using a similar
+
method to the one described for Compound 5e. LCMS m/z = 432.6 [M+H] ; Purity : 99%.
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀmethoxyphenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)
acetic acid (5j)
The
title
compound
was
obtained
from
tertꢀbutylꢀ2ꢀ(((1r,4r)ꢀ4ꢀ
((phenylcarbamoyloxy)methyl)cyclohexyl)methoxy) acetate and 1ꢀbromoꢀ3ꢀmethoxybenzene, using a
+
similar method to the one described for Compound 5e. LCMS m/z = 428.3 [M+H] ; Purity : 98%; 1H
NMR (400 MHz, DMSOꢀd6) δ ppm 0.81ꢀ0.98(m, 4H), 1.35ꢀ1.55 (m, 2H), 1.60ꢀ1.75 (m, 4H), 3.15 (d, J=
6
.5Hz, 2H), 3.50 (S, 2H), 3.75 (S, 3H), 3.95 (d, J= 6.0Hz, 2H),6.80ꢀ6.95 (m, 4H), 7.25ꢀ7.45 (m, 5H).
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ((phenyl(pꢀtolyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic acid (5k)
The title compound was obtained as a white solid from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy)
methyl)cyclohexyl)methoxy acetate and 1ꢀiodoꢀ4ꢀmethylbenzene using a similar method to the one
+
described for Compound 5e. LCMS m/z = 412.2 [M+H] Purity : 99%.
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀfluorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)methoxy)acetic
acid (5l)
The title compound was obtained as
a
white solid from tertꢀbutylꢀ2ꢀ(((1r,4r)ꢀ4ꢀ
(
(phenylcarbamoyloxy)methyl)cyclohexyl)methoxy) acetate and 1ꢀfluoroꢀ4ꢀiodobenzene, using a similar
+
1
method to the one described for compound 5e, LCMS m/z = 416.5 [M+H] ; Purity : 99%.; HꢀNMR (400
MHz, Methanolꢀd4) δ ppm 0.79ꢀ1.08 (m, 4H), 1.45ꢀ1.60 (m, 2H), 1.61ꢀ1.73 (m, 2H), 1.77ꢀ1.89 (m, 2H),
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.29 (d, J = 6.57 Hz, 2H), 15 3.83 (s, 2H), 3.97 (d, J= 6.06 Hz, 2H), 7.07ꢀ7.14 (m, 2H), 7.21ꢀ7.34 (m, 5H),
.35ꢀ7.41 (m, 2H).
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Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchloroꢀ3ꢀfluorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy) acetic acid (5m)
The title compound was obtained as a white solid from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy
)
methyl)cyclohexyl)methoxy)acetate and 1ꢀchloroꢀ2ꢀfluoroꢀ4ꢀiodobenzene, using a similar method to the
+
1
one described in Compound 5e. LCMS m/z = 450.1 [M+H] ; Purity : 99%; HꢀNMR (400 MHz,
Methanolꢀd4) δ ppm 0.69ꢀ0.91 (m, 4H), 1.08ꢀ1.31 (m, 1H), 1.32ꢀ1.48 (m, 1H), 1.48ꢀ1.64 (m, 2H), 1.64ꢀ
1.81 (m, 2H), 3.17 (d, J= 6.57 Hz, 2H), 3.71 (s, 2H), 3.87 (d, J= 6.06 Hz, 2H), 6.94 (ddd, J= 8.75, 2.43,
1.20 Hz, 1H), 7.14ꢀ7.24 (m, 4H), 7.27ꢀ7.34 (m, 3H).
Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀchloroꢀ4ꢀfluorophenyl)(phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic acid (5n)
The title compound was obtained as a white solid from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy
)methyl)cyclohexyl)methoxy)acetate and 2ꢀchloroꢀ1ꢀfluoroꢀ4ꢀiodobenzene, using a similar method to the
+
one described in Compound 5e. LCMS m/z = 450.2 [M+H] ; Purity : 99%.
Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((3,4ꢀdifluorophenyl)(phenyl)carbamoyloxy)
methyl)cyclohexyl)
methoxy)acetic acid (5o)
The title compound was obtained as a white solid from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((phenylcarbamoyloxy)
methyl)cyclohexyl)methoxy)acetate and 1,2ꢀdifluoroꢀ4ꢀiodobenzene, using a similar method to the one
+
1
described in Compound 5e. LCMS m/z = 434.5 [M+H] ; Purity : 99%; H NMR (400 MHz, Methanolꢀd )
4
δ ppm 0.93ꢀ 1.11 (m, 4H), 1.52ꢀ1.68 (m,2H), 1.67ꢀ1.81 (m, 2H), 1.84ꢀ1.98 (m, 2H), 3.36 (d, J = 6.44 Hz,
2
H), 3.90 (s, 2H), 4.05 (d, J = 6.06 Hz, 2H), 7.10ꢀ7.18 (m, 1H), 7.27ꢀ7.42 (m, 5H), 7.45ꢀ7.53 (m, 2H).
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Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀFluorophenyl)(4ꢀmethoxyphenyl)carbamoyloxy)methyl) cyclohexyl)
methoxy)acetic Acid (5p)
Step A: Preparation of tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ (Hydroxymethyl)cyclohexyl)methoxy) acetate.
To a solution of (1r,4r)ꢀcyclohexaneꢀ1,4ꢀdiyldimethanol (5.0 g, 34.7 mmol) in benzene (20 mL) at r.t.,
was added tetrabutylammonium iodide (6.40 g, 17.34 mmol) and 50% aqueous NaOH (10 mL, 34.7
mmol). The mixture was stirred vigorously for 5 min and then tertꢀbutyl 2ꢀbromoacetate (5.63 mL, 38.1
mmol) was added. The reaction was stirred vigorously for a further 2 h. The mixture was partitioned
between 50% aqueous NaOH (100 mL) and EtOAc (100 mL). The aqueous layer was extracted again
with EtOAc (100 mL). The combined organic layer was dried and concentrated. The residue was purified
by silica gel column chromatography to provide the title compound as a colorless oil (3.96g, 44%).
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+
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LCMS m/z = 259.3 [M+H] ; HꢀNMR (400 MHz, CDCl3) δ ppm 0.89ꢀ1.06 (m, 4H), 1.47 (s, 9H), 1.55ꢀ
1
.68 (m, 2H), 1.76ꢀ1.98 (m, 4H), 3.32 (d, J= 6.57 Hz, 2H), 3.45 (d, J= 6.32 Hz, 2H), 3.93 (s, 2H).
Step B: Preparation of tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ((3ꢀFluorophenylcarbamoyloxy)methyl)cyclohexyl)
methoxy) acetate.
To a solution of tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ(hydroxymethyl) cyclohexyl)methoxy)acetate (1.0 g, 3.87 mmol)
and pyridine (0.438 mL, 5.42 mmol) in CH Cl (10 mL) was added 3ꢀfluorophenyl isocyanate (0.480 mL,
2
2
4
.26 mmol) and the reaction was stirred at r.t. overnight. The reaction was then heated to reflux for 5 h.
After removal of the solvent, the residue was purified by silica gel column chromatography to yield the
+
1
title compound as a white solid (1.12 g). LCMS m/z = 340.4 [Mꢀ tertꢀbutyl +H] ; HꢀNMR (400 MHz,
CDCl3) δ ppm 0.85ꢀ1.04 (m, 4H), 1.41 (s, 9H), 1.51ꢀ1.64 (m, 2H), 1.69ꢀ1.87 (m, 4H), 3.26 (d, J= 6.32
Hz, 2H), 3.87 (s, 2H), 3.92 (d, J= 6.57 Hz, 2H), 6.57 (s, 1H), 6.68 (dt, J=8.34, 2.53 Hz, 1H), 6.94 (d, J=
8.59 Hz, 1H), 7.13ꢀ7.18 (m, lH), 7.20ꢀ7.28 (m, 1H).
Step C: Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀFluorophenyl)(4ꢀmethoxyphenyl)carbamoyloxy)methyl)
cyclohexyl)methoxy)acetic Acid.
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The title compound was obtained as a white solid from 1ꢀiodoꢀ4ꢀmethoxybenzene and tertꢀbutyl 2ꢀ
((1r,4r)ꢀ4ꢀ((3ꢀfluorophenylcarbamoyloxy) methyl) cyclohexyl) methoxy)acetate, using a similar method
(
+.
1
to the one described for compound 5e, LCMS m/z = 446.5 [M+H] ; Purity : 99%; HꢀNMR (400 MHz,
DMSOꢀd6) δ ppm 0.86 (t, J= 11 Hz, 4H), 1.39 (s, 2H), 1.54ꢀ1.62 (m, 2H), 1.64ꢀ1.74 (m, 2H), 3.23 (d, J=
6.32 Hz, 2H), 3.76 (s, 3H), 3.89 (d, J= 6.19 Hz, 2H), 3.94 (s, 2H), 6.91ꢀ6.98 (m, 2H), 6.99ꢀ7.07 (m, 2H),
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.17ꢀ7.26 (m, 3H), 7.36 (dt, J = 8.18, 6.88 Hz, 1H), 12.52 (br s, 1H).
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀChlorophenyl)(3ꢀfluorophenyl)carbamoyloxy) methyl)cyclohexyl)
methoxy) acetic Acid. (5q)
The title compound was obtained as a white solid from 1ꢀchloroꢀ4ꢀiodobenzene and tertꢀbutyl 2ꢀ(((1r,4r)ꢀ
4ꢀ((3ꢀfluorophenylcarbamoyloxy) methyl) cyclohexyl)methoxy)acetate, using a similar method to the one
+
1
described for Compound 5e. LCMS m/z = 450.5 [M+H] . Purity : 99%; HꢀNMR (400 MHz, DMSOꢀd6)
δ ppm 0.77ꢀ0.98 (m, 4H), 1.34ꢀ1.42 (m, 1H), 1.42ꢀ1.52 (m, 1H), 1.53ꢀ1.63 (m, 2H), 1.63ꢀ1.75 (m, 2H),
3
.23 (d, J = 6.44 Hz, 2H), 3.91 (d, J = 6.06 Hz, 2H), 3.94 (s, 2H), 7.04ꢀ7.14 (m, 2H), 7.23ꢀ7.28 (m, 1H),
.29ꢀ7.36 (m, 2H), 7.36ꢀ7.42 (m, 1H), 7.42ꢀ7.49 (m, 2H), 12.52 (br s, 1H).
7
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ((Bis(4ꢀfluorophenyl)carbamoyloxy)methyl)cyclohexyl) methoxy)acetate (5r)
Step A: Preparation of Methyl 4ꢀFluoroꢀ2ꢀ((1r,4r)ꢀ4ꢀ (hydroxymethyl)cyclohexyl)phenylcarbamate.
4
ꢀFluorophenyl isocyanate (4.75 g, 34.7 mmol), (1r,4r)ꢀcyclohexaneꢀ1,4ꢀdiyldimethanol (5.0 g, 34.7
mmol), and pyridine (3.93 mL, 48.5 mmol) were dissolved in CH Cl (30 mL). The reaction mixture was
2
2
stirred at r.t. overnight. After removal of the solvent, the residue was purified by silica gel column
+
chromatography to yield the title compound as a white solid (4.92 g, 50%). LCMS m/z = 282.4 [M+H] ;
1HꢀNMR (400 MHz, DMSOꢀd6) δ ppm 0.81ꢀ1.07 (m, 4H), 1.25ꢀ1.38 (m, 1H), 1.49ꢀ1.64 (m, 1H), 1.72ꢀ
1
.82 (m, 4H), 3.19ꢀ3.24 (m, 2H), 3.89 (d, J = 6.57 Hz, 2H), 4.34 (t, J= 5.31 Hz, 1H), 7.06ꢀ7.15 (m, 2H),
.46 (dd, J= 8.97, 4.93 Hz, 2H), 10 9.61 (s, 1H).
7
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Step B: Preparation of tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ((4ꢀFluorophenylcarbamoyloxy)methyl)cyclohexyl)
methoxy) acetate.
To a solution of methyl 4ꢀfluoroꢀ2ꢀ((1r,4r)ꢀ4ꢀ (hydroxymethyl)cyclohexyl)phenylcarbamate (2.0 g, 7.11
mmol) and rhodium(II) acetate (0.157 g, 0.355 mmol) in CH Cl (10 mL) at 0 °C was slowly added tertꢀ
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2
2
butyl 2ꢀdiazoacetate (1.084 mL, 7.82 mmol) preꢀdissolved in CH Cl (5 mL) via an addition funnel. The
2
2
reaction was stirred at 0 °C for 1 h and then at r.t. for another 1 h. After removal of the solvent, the
residue was purified by silica gel column chromatography to yield the title compound as a tan solid (1.9 g,
+
1
6
1%). LCMS m/z = 340.4 [Mꢀ tertꢀbutyl +H] ; HꢀNMR (400 MHz, DMSOꢀd6) δ ppm 0.97 (d, J= 10.36
Hz, 4H), 1.42 (s, 9H), 1.44ꢀ1.53 (m, 1H), 1.54ꢀ1.64 (m, 1H), 1.72ꢀ1.82 (m, 4H), 3.26 (d, J = 6.32 Hz, 2H),
3.90 (d, J = 6.57 Hz, 2H), 3.92 (s, 2H), 7.06ꢀ7.15 (m, 2H), 7.46 (dd, J = 8.84, 4.93 Hz, 2H), 9.61 (s, 1H).
Step
C
:
Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ((Bis(4ꢀfluorophenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic Acid
The title compound was obtained as
a
white solid from tertꢀbutyl 2ꢀ(((1r,4r)ꢀ4ꢀ((4ꢀ
fluorophenylcarbamoyloxy)methyl)cyclohexyl)methoxy) acetate and 1ꢀfluoroꢀ4ꢀiodobenzene, using a
+
similar method to the one described for Compound 5e, LCMS m/z = 434.5 [M+H] ; Purity : 99%.
Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀfluorophenyl)(3ꢀfluorophenyl)carbamoyloxy)
methyl)cyclohexyl)methoxy) acetic Acid. (5s)
The title compound was obtained as a white solid from 1ꢀchloroꢀ4ꢀiodobenzene and tertꢀbutyl 2ꢀ(((1r,4r)ꢀ
4
ꢀ((3ꢀfluorophenylcarbamoyloxy) methyl) cyclohexyl)methoxy)acetate, using a similar method to the one
+
described for Compound 5e. LCMS m/z = 434.4 . [M+H] ; Purity : 99%.
Preparation
of
2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchloroꢀ3ꢀfluorophenyl)(3ꢀfluorophenyl)carbamoyloxy)methyl)
cyclohexyl) methoxy) acetic acid (5t)
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The title compound was obtained as a white solid from 1ꢀchloroꢀ2ꢀfluoroꢀ4ꢀiodobenzene and tertꢀbutyl 2ꢀ
(
((1r,4r)ꢀ4ꢀ((3ꢀfluorophenylcarbamoyloxy) methyl) cyclohexyl)methoxy)acetate, using a similar method
+
to the one described for Compound 5e. LCMS m/z = 468.5 . [M+H] ; Purity : 99%.
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First Scaleꢀup Route
Preparation of Sodium 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl) (phenyl) carbamoyloxy) methyl)
cyclohexyl)methoxy)acetate (5c)
1
2
Step A Preparation of 4ꢀchlorophenyl(phenyl)carbamic chloride (12c, X=Cl, R = H, R = 4ꢀCl):
4ꢀChloroꢀNꢀphenylaniline (10 g, 49.1 mmol) was dissolved in CH Cl (30 mL). The solution was cooled
2
2
on an ice bath (0°C) and then triphosgene (16.03 g, 54.0 mmol) was added. Pyridine (5.56 ml, 68.7 mmol)
preꢀdissolved in CH Cl (10 mL) was added slowly via addition funnel to the reaction mixture
2
2
(
exothermic; the solution went from a green color to an orangeꢀyellow color). Upon complete addition,
the reaction was stirred on the ice bath another 15 min and then warmed to r.t. and stirred for an hour.
After this time, the reaction was complete as judged by TLC. The reaction was again cooled on an ice
bath and quenched by the slow addition of H O (20 mL; exothermic; reaction bubbled vigorously). The
2
reaction was extracted (100 mL each of H O and CH Cl ). The aqueous layer was extracted again with
2
2
2
CH Cl (100 mL). The combined organic layers were dried, concentrated, and the product purified by
2
2
chromatography (0, 10, 20, 30, 40% CH Cl /Hexanes step gradient) to provide 4ꢀchlorophenyl (phenyl)
2
2
1
carbamic chloride (12.34 g, 45.9 mmol, 93 % yield), as a light yellow oil. HꢀNMR (400MHz, DMSOꢀd6)
δ ppm 7.31ꢀ7.81 (m).
Step B: Preparation of ((1r,4r)ꢀ4ꢀ(Hydroxymethyl)cyclohexyl)methyl 4ꢀchlorophenyl(phenyl)carbamate
1
2
(11, R = H, R = 4ꢀCl)
4
ꢀChlorophenyl(phenyl)carbamic chloride (12.34 g, 46.4 mmol) and (1r,4r)cyclohexaneꢀ1,4ꢀdiyl
dimethanol (6.69 g, 46.4 mmol) were dissolved in pyridine (50 mL, 618mmol). The reaction mixture was
heated to reflux overnight then cooled and concentrated under reduced pressure. The residue was
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resuspended in Et O/EtOAc (50:50), filtered and the filtered solid washed with Et O/EtOAc (50:50). The
2
2
filtrate was partitioned between 1M HCl (200 mL) and EtOAc (200 mL). The aqueous layer was
extracted again with EtOAc (100 mL). The organic layers were combined and washed with H O (200
2
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mL), dried, and concentrated. The residue was purified by silica gel column chromatography to provide
+
1
the title compound as a light pink colored solid (10.4 g, 60%). LCMS m/z = 374.1 [M+H] ; HꢀNMR
(
400 MHz, DMSOꢀd6) δ ppm 0.73ꢀ0.92 (m, 4H), 1.13ꢀ1.27(m, 1H), 1.36ꢀl.48 (m, 1H), 1.53ꢀ1.62(m, 2H),
1
.62ꢀ1.73 (m, 2H),3.17(d, J = 6.19Hz, 2H), 3.89 (d, J= 6.06 Hz, 2H), 4.29 (br s, 1H), 7.23ꢀ7.32 (m, 5H),
.34ꢀ7.45 (m, 4H).
7
Step C: Preparation of tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl)(phenyl) carbamoyloxy)methyl)
t
1
2
cyclohexyl) methoxy)acetate (9, R = Bu, R = H, R = 4ꢀCl)
((1r,4r)ꢀ4ꢀ(Hydroxymethyl)cyclohexyl)methyl4ꢀchlorophenyl(phenyl)carbamate (8.9 g, 23.80 mmol) was
dissolved in CH Cl (30 mL). Diacetoxyrhodium (0.526 g, 1.190 mmol) was added and the reaction was
2
2
cooled on an ice bath. tertꢀButyl 2ꢀdiazoacetate (3.63 mL, 26.2mmol) preꢀdissolved in CH Cl (10 mL)
2
2
was added slowly to the reaction via an addition funnel. The reaction was stirred in an ice bath for 1 h,
warmed to r. t. and stirred for an additional 1 h. After removal of the solvent, the residue was purified by
silica gel column chromatography to provide the title compound as a colorless oil (8.8g, 75%). LCMS
+
1
m/z = 432.6 [M ꢀtertꢀbutyl group +H] ; HꢀNMR (400 MHz, DMSOꢀd6) δ ppm 0.77ꢀ0.95 (m, 4H), 1.33ꢀ
1
.50 (m, 2H), 1.42 (s, 9H), 1.52ꢀ1.62 (m, 2H), 1.63ꢀ1.75 (m, 2H), 3.22 (d, J= 6.32 Hz, 2H), 3.83ꢀ3.93 (m,
H), 7.23ꢀ7.32 (rn, 5H), 7.35ꢀ7.44 (m, 4H).
4
Step D: Preparation of Sodium 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl)(phenyl)carbamoyloxy) methyl)
cyclohexyl)methoxy) acetate (5c, sodium salt).
tertꢀButyl 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl)(phenyl) carbamoyloxy)methyl) cyclohexyl) methoxy)acetate
(8.8 g, 18.03 mmol) was dissolved in HCl (4 M in dioxane, 100mL, 400 mmol). The reaction was stirred
at r.t. overnight and concentrated under reduced pressure to provide an oil. The oil was partitioned
between H O (100 mL) and EtOAc (100 mL). The aqueous layer was extracted again with EtOAc (100
2
mL). The combined organic layer was washed with H O (150 mL), dried, and concentrated to yield a light
2
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yellow oil. The oil was dissolved in a minimal amount of MeOH (10ꢀ20 mL) and cooled in an ice bath.
NaOH (1M, 27.0 mL, 27.0 mmol) was added with stirring during which time a white solid precipitate was
formed. The mixture was diluted with H O (20 mL). The solid was collected by filtration and washed
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with cold H O (20 mL). The resulting white solid was dried in a vacuum oven (60 °C overnight) to
2
+
1
provide the title compound (7.7 g, 98%). LCMS m/z = 432.5 [M+H] ; Purity : 99%. ; HꢀNMR (400
MHz, DMSOꢀd6) δ ppm 0.73ꢀ0.93 (m, 4H), 1.28ꢀ1.40 (m, 1H), 1.40ꢀ1.50 (m, 1H), 1.50ꢀ1.61 (m, 2H),
1
7
.63ꢀ1.77 (m, 2H), 3.16 (d, J= 6.57 Hz, 2H), 3.47 (s, 2H), 3.89 (d, J= 6.06 Hz, 2H), 7.23ꢀ7.32 (m, 5H),
.35ꢀ7.44 (m, 4H).
Preparation of Sodium 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀFluorophenyl)(phenyl)carbamoyloxy)methyl) cyclohexyl)
methoxy)acetate. (5g)
1
2
Step A: Preparation of 3ꢀFluoroꢀNꢀphenylaniline (11, R = H, R = 3ꢀF)
In a 3 liter, threeꢀneck flask equipped with mechanical stirring, a solution of 3ꢀfluoroaniline (75 g, 675
mmol), bromobenzene (73 mL, 690 mmol), and dichloro[1,1'ꢀ20 bis(diphenylphosphino)ꢀferrocene]
palladium(II) dichloromethane adduct (15 g, 18 mmol) in anhydrous toluene (1.3 L) containing sodium
tertꢀbutoxide (130 g, 1.35 mol) was heated at 105°C for 3 h. The reaction mixture was then cooled to 80
°C, and quenched by gradually pouring the reaction mixture into ice/water (1 L). The aqueous layer was
removed, and was then extracted with an additional volume of toluene (300 mL). The organic extracts
were combined, rinsed with brine, dried over MgSO , and passed through a silica plug (1.3 kg), eluting
4
+
with toluene. The solvent was removed to give a dark amber oil (86 g, 68%). LCMS m/z = 188 [M+H] ;
1
HꢀNMR (400 MHz, CDCl ) δ 6.45 (t, J= 8.5 Hz, 1H), 6.62ꢀ6.66 (m, 2H), 6.87 (t, J=7.2 Hz, 1H), 6.98 (d,
3
J= 7.6 Hz, 2H), 7.05 (q, J= 7.5 Hz ,1H), 7.17 (t, J= 8.6 Hz, 2H).
1
2
Step B: Preparation of 3ꢀFluorophenyl(phenyl)carbamic chloride (12, X=Cl, R = H, R = 3ꢀF)
A 3 liter threeꢀneck mechanically stirred flask under N2 containing a solution of 3ꢀfluoroꢀNꢀphenylaniline
(
86 g, 460 mmol) in 1.2 L dichloromethane was cooled in an ice bath to 0 °C, and then triphosgene (150
g, 505 mmol) was added. A solution of pyridine (52 mL, 640 mmol) in dichloromethane (200 mL) was
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added in a dropwise fashion. Initial addition resulted in a temperature spike to 25 °C after the first 10 mL
had been added over 10 min. The addition was paused, and the reaction mixture was stirred for 1 h while
reꢀcooling to 5 °C. Addition of the pyridine solution was again commenced at a rate of 5 mL/min, at
which an internal reaction temperature of 5ꢀ10 °C could be maintained. After addition was complete
(about 1 h), the reaction had proceeded to completion, and was quenched by the slow addition of ice
water (500 g). Gas was passed through a 20% sodium hydroxide trap, until all gas evolution had ceased
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(
about 3 h). The aqueous layer was removed, and was then extracted with an additional 300 mL of
dichloromethane. The organic extracts were combined, dried over MgSO , and the solvent was removed.
4
Clean product was readily isolated as a viscous, pink oil, which gradually formed a pale pink solid upon
+
1
seeding with crystals. LCMS m/z = 250.0 [M+H] ; HꢀNMR (CDCl , 400 MHz) δ 7.00ꢀ7.07 (m, 1H),
3
7.10 (d, J= 9.6 Hz, 1H), 7.15 (d, J= 8.1 Hz, 1H), 7.35 (d, J = 7.7 Hz, 2H), 7.35ꢀ7.41 (m, 2H), 7.42ꢀ7.48
(
m, 2H).
Step C: Preparation of 4ꢀ(Dimethylamino)ꢀ1ꢀ((3ꢀfluorophenyl)(phenyl)carbamoyl)pyridinium chloride
1
2
(
12, X=DMAP, R = H, R = 3ꢀF)
To a solution of crude 3ꢀfluorophenyl(phenyl)carbamic chloride (62.4 g, 250 mmol) in acetonitrile (500
mL) in a 2 liter mechanically stirred threeꢀneck flask was added a solution of 4ꢀ dimethylaminopyridine
(
30.5 g, 250 mmol) in 500 mL acetonitrile. The flask warmed slightly as crystallization began to occur,
and then cooled again to ambient temperature. The resulting suspension was stirred overnight, cooled to
0 °C in an ice bath and the precipitate filtered, rinsing with cold acetonitrile (100 mL) to provide the title
1
compound as a fine, white solid (88.27 g). LCMS m/z = 336. [M+H]+; 1HꢀNMR (400 MHz, Methanolꢀ
d4) δ 3.29 (s, 6H), 6.92 (d, J = 8.1 Hz, 2H), 7.11 (t, J= 8.6 Hz, lH), 7.16 (d , J= 8.8 Hz, lH), 7.21 (d, J= 9.5
Hz, lH), 7.33ꢀ7.38 (m, 3H), 7.41ꢀ7.47 (m, 3H), 8.37 (d, J = 8.1 Hz, 2H).
Step D: Preparation of ((1r,4r)ꢀ4ꢀ(Hydroxymethyl)cyclohexyl)methylꢀ3ꢀfluorophenyl(phenyl)carbamate
1
2
(
13, R = H, R = 3ꢀF)
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A suspension of 4ꢀ(dimethylamino)ꢀ1ꢀ((3ꢀfluorophenyl)(phenyl)carbamoyl)ꢀpyridinium chloride (88.25 g,
37 mmol), (1r,4r)ꢀcyclohexaneꢀ1,4ꢀdiyldimethanol (137 g, 950 mmol) and 4ꢀdimethylaminopyridine
29.0 g, 237 mmol) in acetonitrile (1 L) was heated at 53 °C for 18 h. Upon cooling, the solvent was
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removed, and the residue was taken up in isopropyl acetate (500mL) and 1M HCl (500 mL), heated to
suspend all solids, and then filtered through glass fiber filter paper to attempt to remove a sparingly
soluble bisꢀcarbamate impurity. The aqueous filtrate was discarded, and the organic filtrate was washed
with an additional 500 mL of 1 N HCl, followed by water (5 x 500 mL). Heptane (100 mL) was added to
the organic phase; which was further washed with water (2 x 500 mL) and brine (100 mL), dried over
MgSO , and concentrated to dryness. The residue was taken up in isopropyl acetate (100 mL) and heptane
4
(300 mL) was added. Crystals gradually formed over 1 h, providing a white precipitate, which was
collected by filtration, and washed with 25% isopropyl acetate/heptane (100 mL). The filtrate was
concentrated to dryness, and the residue was taken up in hot 25% isopropyl acetate/heptane (100 mL) and
filtered hot. As the filtrate cooled, more solids precipitated, which were collected by filtration and
combined with the first crop. This material still contained about 5% bisꢀcarbamate byꢀproduct, which
could not be readily removed by filtration. The solid was then taken up in dichloromethane (200 mL) and
subjected to plug filtration over 1.6 kg of silica gel, eluting the product with 20% ethyl
acetate/dichloromethane to provide the title compound as a white solid (71 g, 83%) and the remaining
+
1
bisꢀcarbamate with dichloromethane. LCMS m/z = 358. [M+H] ; HꢀNMR (CDCl , 400 MHz) δ 0.91ꢀ
3
0
.98 (m, 4H), 1.35ꢀ1.44 (m, 1H), 1.54ꢀ1.60 (m, 1H), 1.68ꢀ1.73 (m, 2H), 1.79ꢀ1.83 (m, 2H), 3.45 (d,J= 6.4
Hz, 2H), 4.01 (d,J= 6.4 Hz, 2H), 6.91 (t, J= 7.6 Hz, 1H), 7.04 (d,J= 8.6 Hz, 2H), 7.22ꢀ7.30 (m, 4H), 7.38
t, J= 7.8 Hz, 2H).
Step E: Preparation
(
of
Ethylꢀ2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀfluorophenyl)phenyl)carbamoyloxy)methyl)
1
2
cyclohexyl)methoxy) acetate (7, R=Et, R = H, R = 3ꢀF)
In a 250 mL threeꢀneck reactor equipped with a stirrer, a thermocouple, a cooling bath, an addition funnel
and a nitrogen inlet was placed ((1r,4r)ꢀ4ꢀ(hydroxymethyl)cyclohexyl)methylꢀ3ꢀfluorophenyl(phenyl)
carbamate (8 g, 22.38 mmol). This was dissolved in dichloromethane (150 mL). The mixture was cooled
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and stirred well at 4 °C in an isopropanol/ice bath. Diacetoxyrhodium (0.5 g, 1.12 mmol) was added.
After the addition was complete, ethyl diazoacetate (3.69 g, 32.34 mmol) was dissolved in
dichloromethane (30mL) and added to the reaction mixture keeping the temperature below 10 °C. After
addition, the reaction mixture was warmed to 30 °C and the progress of the reaction was followed by
LCMS. Based on the LCMS inꢀprocess control, further batches of ethyl diazoacetate (0.63 g, 5.52 mmol,
followed by 0.710 g, 6.22 mmol dissolved in dichloromethane (15 mL)) were added separately at 25° C.
The reaction mixture was stirred at 30 °C until LCMS showed complete consumption of the starting
material. The reaction mixture was diluted with water (100 mL) and the mixture was filtered through a
bed of celite (35 g) to remove the catalyst. The organic layer was then separated and dried over
magnesium sulfate (15 g) and filtered. The solvent was removed to provide the title compound as an oil
(9.9 g), which still contained a small amount of ethyl diazoacetate and was used without further
10
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purification. LCMS m/z = 444.5 [M+H] ; HꢀNMR (400 MHz, DMSOꢀd6) δ ppm 0.82ꢀ0.96 (m, 4H), 1.22
t, J = 7.07 Hz, 3H), 1.27 (t, J = 7.14 Hz, 1H), 1.37ꢀ1.53 (m, 2H), 1.57ꢀ1.78 (m, 4H), 3.26 (d, J= 6.32 Hz,
H), 3.94 (d, J= 6.06 Hz, 2H), 4.06 (s, 2H), 4.14 (q, J= 7.07 Hz, 3H), 4.23 (q, J= 7.07 Hz, 1H), 7.05ꢀ7.11
m, 2H), 7.24 (dt, J = 10.64, 2.26 Hz, 1H), 7.28ꢀ7.35 (m, 3H), 7.36ꢀ7.45 (m, 3H).
(
2
(
Step F: Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀFluorophenyl) (phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic Acid (5g, free acid)
In a 500 mL, threeꢀneck reactor equipped with a stirrer, a thermocouple, a heating oil bath, an addition
funnel and a nitrogen inlet was placed ethylꢀ2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀfluorophenyl) (phenyl)carbamoyloxy)
methyl) cyclohexyl)methoxy)acetate (9.9 g, 22.32 mmol), which was dissolved in acetonitrile (150 mL).
To this mixture lithium bromide (19.58 g, 225.00 mmol) was added. After the addition was complete,
triethylamine (6.84 g, 67.6 mmol) was added and the reaction mixture was heated at 70 °C. The progress
of the reaction was followed by LCMS. Based on the LCMS the starting material was consumed in 2 h.
Solvent was removed and the reaction mixture was diluted with water (200 mL) and made acidic with
hydrochloric acid (3 M, 7.8 mL). The precipitated solids were filtered and the wet solid was dissolved in
isopropyl acetate (200 mL). The isopropyl acetate solution was dried over magnesium sulfate (15 g),
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filtered and the solvent was removed. The residue was dried in a vacuum oven to provide the title
+
1
compound (9.2 g, 95%). LCMS m/z = 416.4 [M+H] ; HꢀNMR (400 MHz, DMSOꢀd6) δ ppm 0.81ꢀ0.96
(
m, 4H), 1.36ꢀ1.53 (m, 2H), 1.55ꢀ1.77 (m, 4H), 3.25 (d, J= 6.44 Hz, 2H), 3.93 (d, J= 5.94 Hz, 2H), 3.97
0
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(s, 2H), 7.05ꢀ7.13 (m, 2H), 7.24 (dt, J= 10.64, 2.26 Hz, 1H), 7.28ꢀ7.36 (m, 3H), 7.37ꢀ7.46 (m, 3H), 12.53
(br s, 1 H)
Step G: Preparation of 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀFluorophenyl) (phenyl)carbamoyloxy)methyl)cyclohexyl)
methoxy)acetic Acid Sodium Salt (5g, sodium salt)
In a 500 mL, threeꢀneck reactor equipped with a stirrer, a thermocouple, a heating oil bath, an addition
funnel and a nitrogen inlet was placed 2ꢀ(((1r,4r)ꢀ4ꢀ(((3ꢀfluorophenyl) (phenyl)carbamoyloxy)
methyl)cyclohexyl)methoxy)acetic acid (9.2 g, 22.83 mmol) and 2ꢀpropanol (100 mL). The reaction
mixture was heated at 30 °C (bath temperature), until all of the acid was dissolved completely. To the
orange solution, sodium hydroxide (1 M, 22 mL, 22mmol) was added slowly keeping the internal
temperature around 25 °C. The sodium salt separated out as crystals. The thick slurry was stirred at 25 °C
for 2 h and then cooled in an ice water bath for 20 ꢀ 40 min. The solids were filtered and dried in a
vacuum oven at 40 °C overnight until most of the residual 2ꢀpropanol was removed to provide the title
+
1
compound (7.4 g, 74%). LCMS m/z = 416.5 [M+H] ; Purity : 99%. HꢀNMR (400 MHz, DMSOꢀd6) δ
ppm 0.77ꢀ0.95 (m, 4H), 1.34ꢀ1.53 (m, 2H), 1.55ꢀ1.75 (m, 4H), 3.19 (d, J= 6.44 Hz, 2H), 3.52 (s, 2H),
3
7
.93 (d, J= 5.94 Hz, 2H), 7.05ꢀ7.13 (m, 2H), 7.24 (dt, J= 10.64, 2.26 Hz, 1H), 7.28ꢀ7.35 (m, 3H), 7.37ꢀ
.46 (m, 3H).
Second Scaleꢀup Route
Step A: Preparation of ((1r,4r)ꢀ4ꢀ(Hydroxymethyl)cyclohexyl)methyl 4ꢀchlorophenyl(phenyl)carbamate
1
2
(13 R = H, R = 4ꢀCl)
A solution of (1r,4r)cyclohexaneꢀ1,4ꢀdiyldimethanol in acetonitrile was prepared by charging
(
1r,4r)cyclohexaneꢀ1,4ꢀdiyldimethanol (4.22 kg) and acetonitrile (13.37 kg) to a 50 L glass lined reactor
equipped with overhead agitation, jacket temperature control, and a nitrogen inlet. The reactor contents
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were stirred at 163 rpm and heated to 65°C for 1 hour to dissolve most of the (1r,4r)cyclohexaneꢀ1,4ꢀ
diyldimethanol. The mixture was cooled to 45°C and transferred to a 20 L fluorinated high density
1
2
polyethylene (HDPE) carboy for addition to 12 (X=CDI, R = H, R = 4ꢀCl) at a later time.
1
2
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11, (R = H, R = 4ꢀCl, 1.71 kg), K PO (0.53 kg), carbonyl diimidazole (CDI, 1.49 kg), and acetonitrile
3 4
(6.69 kg) were charged to a 50 L glass lined reactor equipped with overhead agitation, jacket temperature
control, and a nitrogen inlet. The reactor contents were stirred at 175 rpm and heated to 65°C to 70°C for
1
2
1
2
4
.5 hours, after which conversion of 11 (R = H, R = 4ꢀCl) to 12 (R = H, R = 4ꢀCl) was verified to be
greater than 98.0% by HPLC peak area. The reaction mixture was cooled to less than 40°C and the
1r,4r)cyclohexaneꢀ1,4ꢀdiyldimethanol solution prepared earlier added to the mixture. The reactor
(
contents were stirred at 178 rpm and heated at 65°C to 70°C for 5 hours, after which conversion was
99.6% by HPLC peak area
The 50 L reactor contents were filtered and the filter cake was rinsed with acetonitrile (1.10 kg). The
filtrate was transferred back to the reactor. Most of the acetonitrile (18.48 kg) was then removed at an
internal temperature of less than 40°C by vacuum distillation at 80 mm Hg. Water (5.67 kg) was added to
the 50 L reactor and approximately 1.55 kg of water/acetonitrile mixture was then removed by vacuum
distillation at an internal temperature less than 40°C and 70 mm Hg.
Further water (5.68 kg) was then added to the 50 L reactor and the product precipitated during the
addition. The resulting mixture was stirred at 20°C to 25°C for 4 hours. The precipitated product was
filtered and washed in two portions with aqueous acetonitrile (1.68 kg acetonitrile dissolved in 6.39 kg
1
2
water). The product was vacuum dried at ≤ 60°C to a loss on drying value of ≤ 2.0 wt%. 13 (R = H, R
= 4ꢀCl, 2.29 kg) was thus obtained in 73% yield (over 2 telescoped steps) and 98.1% purity by HPLC
peak area.
Step B: Preparation of Sodium 2ꢀ(((1r,4r)ꢀ4ꢀ(((4ꢀchlorophenyl) (phenyl) carbamoyloxy) methyl)
cyclohexyl)methoxy)acetate (5c)
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