Design and optimization of pyrazinecarboxamide-based inhibitors of diacylglycerol acyltransferase 1 ((DGAT1) leading to a clinical candidate dimethylpyrazinecarboxamide phenylcyclohexylacetic acid (AZD7687)

Article abstract of DOI:10.1021/jm301296t

A new series of pyrazinecarboxamide DGAT1 inhibitors was designed to address the need for a candidate drug with good potency, selectivity, and physical and DMPK properties combined with a low predicted dose in man. Rational design and optimization of this series led to the discovery of compound 30 (AZD7687), which met the project objectives for potency, selectivity, in particular over ACAT1, solubility, and preclinical PK profiles. This compound showed the anticipated excellent pharmacokinetic properties in human volunteers.

Full text of DOI:10.1021/jm301296t

Article
pubs.acs.org/jmc
Design and Optimization of Pyrazinecarboxamide-Based Inhibitors
of Diacylglycerol Acyltransferase 1 (DGAT1) Leading to a Clinical
Candidate Dimethylpyrazinecarboxamide Phenylcyclohexylacetic
Acid (AZD7687)
Jonas G. Barlind,† Udo A. Bauer,† Alan M. Birch,‡ Susan Birtles,‡ Linda K. Buckett,‡ Roger J. Butlin,‡
Robert D. M. Davies,‡ Jan W. Eriksson,†,§ Clare D. Hammond,‡ Ragnar Hovland,† Petra Johannesson,*,†
Magnus J. Johansson,† Paul D. Kemmitt,‡ Bo T. Lindmark,† Pablo Morentin Gutierrez,‡
Tobias A. Noeske,† Andreas Nordin,† Charles J. O’Donnell,‡ Annika U. Petersson,† Alma Redzic,†
Andrew V. Turnbull,‡ and Johanna Vinblad†
†Cardiovascular and Gastrointestinal Innovative Medicines Unit Molndal, AstraZeneca R&D, S-431 83 Molndal, Sweden
̈
̈
‡AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire, SK10 4TG, U.K.
§Department of Molecular and Clinical Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden
S
* Supporting Information
ABSTRACT: A new series of pyrazinecarboxamide DGAT1 inhibitors was designed to address
the need for a candidate drug with good potency, selectivity, and physical and DMPK properties
combined with a low predicted dose in man. Rational design and optimization of this series led to
the discovery of compound 30 (AZD7687), which met the project objectives for potency, selectivity, in
particular over ACAT1, solubility, and preclinical PK profiles. This compound showed the anticipated
excellent pharmacokinetic properties in human volunteers.
acyltransferase 1 (ACAT1) for toxicological (adrenocortical de-
INTRODUCTION
■
generation) reasons, full details of which will be published elsewhere.
In addition, we sought to discover a high quality candidate drug, i.e.,
in terms of current medicinal chemistry thinking it should have
relatively low lipophilicity19 and molecular weight together with
high permeability,20 solubility, and general selectivity. A low ther-
apeutic dose was also a key target given the established correlation
between this and reduced risk of toxicity in man.21 To achieve these
goals, we sought to design a compact novel heterocyclic core struc-
ture with inherently superior properties on which to attach a suitable
lipophilic carboxylic acid appendage.
At the time of this work there were two alternative lead structures
known. Bayer had published their lipophilic keto acids, for example,
1,22 and Japan Tobacco/Tularik had together disclosed aminopyri-
midinooxazine-based lipophilic acids including the phenylcyclohexyl-
acetic acid 2.23 The latter, being less lipophilic, was more interesting to
us, and crude overlays were attempted with our oxadiazoles in order
to try to understand which were the key elements of the pharma-
cophore. Our early efforts included use of intermediates including aryl
Inhibitors of diacylglycerol acyltransferase 1 (DGAT1) have come to
the fore in recent years as potential therapies for diabetes, obesity, and
other elements of metabolic syndrome.1 Several accounts of medicinal
chemistry optimization programs have been published,2−16 and a
number of candidate drugs have entered clinical trials.1
The DGAT1 drug discovery program at AstraZeneca focused
initially on the development of a series of oxadiazole substituted
phenylcyclohexylacetic acids.14 Our experience during this work
reinforced the significance of the phenylcyclohexylacetic acid side
chain in delivering single digit nanomolar potency in conjunction
with excellent oral PK properties. This phenylcyclohexylacetic acid
side chain is also contained within several other DGAT1 inhibitors,
including trans-4-[4-[5-[[6-(trifluoromethyl)-3-pyridinyl]amino]-2-
pyridinyl]phenyl]cyclohexylacetic acid (LCQ-908),17 trans-4-[4-(4-
amino-5-oxo-7,8-dihydropyrimido[5,4-f ][1,4]oxazepin-6(5H)-yl)-
phenyl]cyclohexylacetic acid (PF-04620110),8 and trans-4-[4-(7-
aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl]cyclohexylacetid acid
(ABT-046),15 which have all been selected as clinical candi-
dates.8,15,18 Our earlier work on oxadiazole substituted phenylcyclo-
hexylacetic acids furthermore informed us of the need for high selec-
tivity for inhibition of DGAT1 over acyl-coenzyme A:cholesterol
Received: September 7, 2012
© XXXX American Chemical Society
A
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halide, triflates, phenacyl halide, and alkynes in multiparallel syntheses,
coupling, or cyclocondensing with diverse functionalized/substituted
heterocycles or thioamides containing pharmacophoric elements that
might be relevant for enzyme inhibition. However, none of these
efforts yielded significant inhibitors of DGAT1 (all with IC50 of
>10 μM or with <20% inhibition at 10 μM). It became clear that the
precise arrangement of the pharmacophoric elements in the
heterocycle was just as important as the lipophilic acid SAR in
delivering enzyme inhibition such that library synthesis from available
intermediates was not sufficient in this instance to aid discovery of a
new chemical series. A consideration of the low energy conformations
of 2 alongside SAR in that series led to the suggestions that (a) the
gem-dimethyl in 2 serves to destabilize the coplanar array and hence
allow a degree of twisting of the aryl substituent out of the plane of the
CN bond in the oxazine ring (and/or there is a beneficial
interaction of this lipophilic region with the enzyme) and (b) the
precise H-bonding array of 2 needs to be mimicked in some way. We
then hypothesized that a pyrazine ring substituted at C-5 (ortho to the
aryl attachment position, C-6) and with a pendent primary
carboxamide at C-2 could achieve the key interactions as illustrated
in Scheme 1. “L” is a lipophilic group such as methyl or bigger to allow
We herein describe the synthesis, structure−activity relation-
ships, and optimization of DGAT1 inhibitors based on the
pyrazine carboxamide core structure 4. This work resulted in the
identification of compound 30 as a clinical candidate for the
treatment of obesity and metabolic syndrome.
CHEMISTRY
■
Synthesis of the pyrazine ring was initially achieved via con-
densation of a diamine with a diketone as shown in Scheme 2. Acid-
catalyzed esterification of trans-4-phenylcyclohexanecarboxylic acid
(5) followed by Friedel−Crafts condensation with 2-bromopro-
pionyl chloride furnished bromo ketone 7. Hydrolysis of the
bromide followed by oxidation to diketone 9 and condensation
with 2,3-diaminopropionic acid24 gave the pyrazine 10 as a single
regioisomer. Conversion to the amide via the mixed anhydride
and subsequent ester hydrolysis gave the required pyrazinecar-
boxamido acid 12. The same method was initially applied in the
synthesis of the homologue 13 with the acetic acid side chain
though the yield was poor; hence, we report below only the im-
proved synthesis of this compound (Schemes 5 and 6). In an NMR
experiment the proton on the pyrazine ring showed a heteronuclear
multiple bond correlation to the pyrazine carbon bearing the methyl
in compound 12, which provided proof that the H and Me are
indeed meta-related rather than para as in the unwanted isomer
(data not shown).
Scheme 1
In the optimization toward new DGAT1 drug candidates we
required a convergent synthetic route without any regiochemical
ambiguity in the preparation of the substituted pyrazine. Having
a lead series consisting of a heterocyclic moiety linked with a
phenylcyclohexylacetic acid part, we realized that useful dis-
connection would be the Csp2−Csp2 between the heterocycle
and the phenyl ring. Accordingly, all subsequent efforts were
directed toward cross-coupling between these partners, which
furthermore allowed for easy variation of both sides. The halogen
component was always situated on the heterocycle.
Methyl 6-bromo-5-methylpyrazine-2-carboxylate (18)
(Scheme 3) was synthesized using a slightly modified procedure
to the one described by Pappo et al.25 Commercially available
Cbz-L-serine methyl ester (14) was subjected to a Swern-type
oxidation protocol, followed by trapping of the enolate with tosyl
chloride. The resulting Michael acceptor 15 was formally sub-
stituted with L-alanine ethyl ester hydrochloride to give inter-
mediate 16. Upon removal of the benzylcarbamate, spontaneous
ring-closure occurred. The reported25 air oxidation (aromatization)
of a similar 1,4,5,6-tetrahydropyrazinone failed in our hands. This
prompted us to screen other oxidation methods, of which the most
the aryl ring to be noncoplanar with the oxazine (2)orpyrazine(3, 4)
core. “A” is a H-bond acceptor (HBA). The carbonyl oxygen in 4
mimics one pyrimidine N; hence, we need coplanarity of the
carboxamide with the pyrazine, and R was chosen to be H in
the first iteration because incorporation of a HBA to mimic the other
pyrimidine N would likely have an adverse effect on the
pyrazinecarboxamide torsion, twisting the CONH2 group out of
the plane of the pyrazine. Bicyclic core 3 was less favored on account
of anticipated problems with poor solubility.
a
Scheme 2
a
Reagents and conditions: (a) Me3SiCHN2, MeOH, toluene; (b) MeCHBrCOCl, AlCl3, DCM, 0 °C; (c) NaOH, DMF, H2O, rt; (d) 1,1,
1-tris(acetoxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (“Dess−Martin periodinane”), DCM, rt; (e) 2,3-diaminopropionic acid hydrochloride,
Et3N, MeOH, rt; (f) (i) EtOCOCl, 4-methylmorpholine, DCM, 0 °C, (ii) NH3/MeOH; (g) LiOH, MeOH, THF, H2O, rt.
B
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a
(Scheme 6). Pyridine derivatives were prepared in an analogous
way. The resulting coupling products were further modified to
the final compounds (Tables 1−5) using standard chemistry
procedures (see Experimental Section).
Scheme 3
The isomeric methylpyrazine compound 31 was prepared
analogously (Scheme 7).
Preparation of the propionic acid analogue 61 proceeded by
standard homologation chemistry (Scheme 8).
Preparation of the 2-fluoro- and 2-chlorophenylcyclohexane
analogues (cis and trans) (Table 5) proceeded via the alkenyl-
borane 88 and 4-hydroxyphenylcyclohexyl intermediates 90 and
92 shown in Schemes 9 and 10. This methodology was also
employed as an alternative in the synthesis of the unsubstituted
phenyl compound 30 and its pyrazine acid analogue 37.
Spirocyclic acetic acid and tetrazole derivatives23 having a
1,4-trans relationship between the acidic group and the direct
aryl attachment on the cyclohexane ring were prepared by the
annulation method shown in Schemes 11 and 12.
Analogous cyclohexanecarboxylic acids, with both cis and trans
geometries, were obtained as described in Scheme 13.
Tetrazole analogue 82 was prepared by standard methodology
from intermediate 110 (Scheme 14)
Similar boronation and coupling protocols (Scheme 15) gave
access to the bicyclooctane acids 83 and 84.
a
Reagents and conditions: (a) Et3N, TsCl, DMSO, DMF, −40 to 0 °C
in 2 h; (b) L-Ala-OEt·HCl, Et3N, MeOH, rt, overnight; (c) (i) Pd/C,
NH4COOH, EtOH, rt, 3 h; (ii) PySO3 (polymer-bound), DCM, rt, 4
days; (d) POBr3, toluene, reflux, 8 h.
efficient protocol was the use of polymer-bound pyridinesulfur
trioxide. Treatment of 17 with POBr3 gave the required 6-bromo-5-
methylpyrazine-2-carboxylate building block 18.
The route described above did not allow easy variation in the
3-position. We then relied on the elegant Rh-catalyzed pyrazinone
synthesis reported by the Clapham and Janda groups at Scripps.26
Variation of the α-diazo-β-keto esters allowed for the easy access to
3-substituted pyrazinones. The desired α-diazo-β-keto esters were
synthesized in a safe manner by the use of polystyrene-supported
tosyl azide.27 This synthesis is illustrated in Scheme 4 for the
3,5-dimethyl substituted building block 22.
The boronic ester intermediate 28 was synthesized start-
ing from commercially available 4-(4′-hydroxyphenyl)cyclo-
hexanone (24) (Scheme 5). A Horner−Wadsworth−Emmons
reaction between tert-butyl (dimethoxyphosphoryl)acetate and
the starting ketone provided alkene 25 which was hydrogenated
to give a cis/trans mixture of phenol 26, predominantly trans
(70%). The trans-isomer was isolated by chromatography and
converted to the triflate 27. Subsequent palladium-catalyzed
coupling with pinacolborane gave the desired boronic ester
precursor 28.
Tetrazole derivative 85 was obtained via the boronate
intermediate 117 (Scheme 16).
The acylglucuronide derivative 120 of clinical candidate 30
was prepared following the method of Stachulski28 (Scheme 17).
In summary herein is described the efficient syntheses of both
mono- and disubstituted pyrazinecarboxamide structures
coupled to various phenylcyclohexylacetic acid side chains, in-
cluding substituted and spirocyclic variants, as well as those
containing a carboxylic acid bioisostere replacing the acetic acid
moiety.
RESULTS AND DISCUSSION
■
Compounds 12 and 13 (Table 1) were the first two analogues
synthesized within the pyrazinecarboxamide series. Although
modest in potency, they were on a par in terms of ligand lipo-
philicity efficiency (LLE) with the best oxadiazoles14 and achieved
high selectivity over ACAT1 (Table 6). Furthermore, the com-
pounds displayed excellent physicochemical properties as well as
Chloro- or bromopyrazine esters were cross-coupled with
boronic ester intermediate 28 using standard Suzuki conditions
a
Scheme 4
a
Reagents and conditions: (a) Rh2Oct4, toluene, 80 °C, Boc-Ala-NH2; (b) TFA, DCE, reflux, 4 h; (c) POCl3, BuCN, 150 °C, 10 min, microwave;
(d) NH3/MeOH.
C
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a
Scheme 5
a
Reagents and conditions: (a) NaH, tert-butyl (dimethoxyphosphoryl)acetate, THF, rt, overnight; (b) Pd/C, H2, EtOAc, 5 bar, 3 h; (c) TFAA,
pyridine, DCM, 0 °C, 30 min; (d) pinacolborane, Et3N, Pd(dppf)Cl2·DCM, reflux, 7 h.
Scheme 6
a
Scheme 7
a
Reagents and conditions: (a) pyridine, 60 °C; (b) POCl3, 90 °C; (c) 7 M NH3 in MeOH, rt; (d) Pd(dppf)Cl2·DCM, K3PO4, DME, EtOH, H2O,
80 °C; (e) KOH, t-BuOH, 45 °C.
good DMPK properties (for 13) as described in Table 6. On the
basis of these encouraging data, compounds 12 and 13 were selected
as the starting point for a program to explore the structure−activity
relationship within this series with the aim of improving potency
while keeping good physicochemical and DMPK properties.
Removing a methyl group from 13 to give 29 or adding a
second to give 30 (Table 1) gave significant shifts in potency,
discussed further in the next section. Substitution of the left-hand
side primary amide (Table 1, compounds 32−35) resulted in loss
of potency, indicating the importance of both primary amide
hydrogens for DGAT1 inhibition. Replacement of the primary
amide with a carboxylic acid, primary amine, amidine, or cyano-
amidine (36−40) was not tolerated. Thioamide 41 was the only
attempted variation with retained potency in this part of the
molecule, consistent with the supposition that the H-bond donor
and acceptor are simultaneous requirements and must be presented
to the DGAT1 protein syn to each other. Thioamide 41 displayed
reduced lipophilicity ligand efficiency (LLE, Table 1) and reduced
selectivity over ACAT1 (IC50 = 16 μM) compared to 13. Thus,
apart from generating interesting SAR information, thioamide 41
was not regarded as delivering any benefits over the corresponding
amide 13.
D
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a
Scheme 8
a
Reagents and conditions: (a) 1 M LiAlH4, THF, 0 °C; (b) MsCl, Et3N, DCM; (c) NaCN, DMF, 80 °C; (d) NaOH, 1,2-propanediol, 140 °C;
(e) MeOH, H2SO4; (f) TFAA, Et3N, DCM; (g) pinacolborane, Pd(dppf) Cl2·DCM, dppf, KOAc, dioxane, 85 °C; (h) 23, Pd(dtbpf)Cl2, K2CO3,
MeCN, 80 °C; (i) KOH, t-BuOH, 40 °C.
a
Scheme 9
a
Reagents and conditions: (a) TFAA, 2,6-di-tert-butyl-4-methylpyridine, DCM, rt; (b) pinacolborane, KOAc, dppf, Pd(dppf)Cl2·DCM, dioxane,
80 °C; (c) 4-bromo-3-fluorophenol or 4-bromo-3-chlorophenol, K2CO3, Pd(dppf)Cl2·DCM, DMF, 100 °C; (d) 10% Pd/C, H2, EtOH, rt.
Pyridine analogues 42−44 (Table 2) show that both of the
pyrazine nitrogens are important for DGAT1 inhibition.
instead of methyl was not tolerated for R2 (50) and resulted in
∼20-fold reduced potency at R1 (46). Addition of the methyl
group in R1 turned out to be very successful, giving a 4-fold boost
in potency for 30 compared to 13. The added lipophilicity
contribution from the R1 methyl was also seen to give a benefit
on permeability and cell potency, without compromising the
good physicochemical properties (Table 6). The R1 amino
As a next step we turned to investigate further the SAR for sub-
stituents on the pyrazine ring. The results from this investigation
(Table 3) showed that for both R1 and R2 only small sub-
stituents were beneficial, particularly for R2, and methyl in both
R1 and R2 (30) was the best of the combinations tried. Ethyl
E
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a
Scheme 10
a
Reagents and conditions: (a) TFAA, Et3N, DCM, 0 °C; (b) pinacolborane, KOAc, dppf, Pd(dppf)Cl2·DCM, dioxane, 80 °C; (c) 23, K3PO4,
Pd(dppf) Cl2·DCM, DME, EtOH, H2O, 80 °C; (d) KOH, t-BuOH, 40 °C; (e) 6 M HCl, dioxane, 100 °C.
a
Scheme 11
a
Reagents and conditions: (a) (methoxymethyl)triphenylphosphonium chloride, KO-t-Bu, dioxane, rt; (b) methyl vinyl ketone, TsOH, toluene,
100 °C, 6 h; (c) 10% Pd/C, AcOH, H2, rt; (d) tert-butyl diethylphosphonoacetate, NaH, THF, rt; (e) pinacolborane, KOAc, dppf,
Pd Pd(dppf)Cl2·DCM, dioxane, 80 °C; (f) 10% Pd/C, THF, H2, rt.
F
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a
Scheme 12
a
Reagents and conditions: (a) 23, K2CO3, Pd(dtbpf)Cl2, MeCN, 80 °C; (b) TFA, DCM, rt; (c) TFA, DCM 0 °C; (d) 3-aminopropionitrile,
PyBroP, DIPEA, DCM, rt; (e) TMSN3, PPh3, DIAD, THF, rt; (f) 23, K3PO4, Pd(dppf) Cl2·DCM, DME, EtOH, H2O, 80 °C.
a
Scheme 13
a
Reagents and conditions: (a) (methoxymethyl)triphenylphosphonium chloride, KO-t-Bu, dioxane, 70 °C, 16 h; (b) Oxone, DMF, rt; (c) (Boc)2O,
DMAP, t-BuOH, rt to 70 °C; (d) pinacolborane, Pd(dppf)Cl2·DCM, dppf, KOAc, dioxane, °C; (e) 23, Pd(dppf)Cl2·DCM, K3PO4, DME (8 mL),
EtOH, H2O; (f) 4 M HCl in dioxane, rt.
substituent in 49 was an attempt to lock the conformation of
the primary amide via an internal hydrogen bond. Compound
49, however, was inactive, suggesting that the polar NH not
involved in the H-bond to the carbonyl is not tolerated in
a region in which the potent compound 2 has an acceptor
(a pyrimidine N).
In contrast to the so far relatively steep SAR found for the
left and middle parts of the molecules, variations to the right-
hand side carboxylic acid were all found to result in highly
potent compounds as outlined in Table 4. The 3,5-dimethyl-
pyrazine group continued to deliver good potency in these
analogues. The higher log D of the neutral alcohols (53, 54)
G
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a
Scheme 14
a
Reagents and conditions: (a) TFA, DCM, 0 °C; (b) 3-aminopropionitrile, PyBroP, DIPEA, DCM, rt; (c) TMSN3, PPh3, DIAD, THF, rt; (d) 23,
K3PO4, Pd(dppf)Cl2, DME, EtOH, H2O, 80 °C.
a
Scheme 15
a
Reagents and conditions: (a) PhI(OCCF3)2, I2, CHCl3; (b) pinacolborane, Pd(dppf)Cl2.DCM, KOAc, DMSO; (c) 23, Pd(dppf)Cl2·DCM, K3PO4,
DME, EtOH, H2O; (d) KOH, t-BuOH, 45 °C.
a
Scheme 16
a
Reagents and conditions: (a) NaOH, MeOH, H2O, 120 °C, microwave; (b) 3-aminopropionitrile, PyBroP, DIPEA, DCM, rt; (c) TMSN3, PPh3,
DIAD, THF, rt; (d) 23, Pd(dppf)Cl2·DCM, K3PO4, DME, EtOH, H2O, 80 °C.
and amides (55, 56), however, resulted in reduced aqueous
solubility (Table 6).
Elongation with an amino acid as in 57 and 58 and intro-
duction of carboxylic acid bioisosteres as the acylated sulfon-
amide 59 and tetrazole 60 all resulted in compounds with good
potency and good physicochemical properties (Table 6). Similar
right-hand side SAR has been described for the Japan Tobacco/
Tularic series.23
Variation of the phenylcyclohexane core was next examined.
Simple fluoro or chloro substitution ortho to the cyclohexyl
H
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a
Scheme 17
a
Reagents and conditions: (a) HATU, DABCO, MeCN, rt; (b) Pd(PPh3)4, pyrrolidine, THF, rt.
group23 (compounds 73 and 74) maintained DGAT1 activity.
1,4-Cis substitution was investigated for a small number of
analogues (75−77). Potency was maintained, but no advantage
over the more readily accessible trans isomers was identified.
Various spirocyclic analogues23 (78−82) were investigated,
seeking potency enhancement from a locked conformation
between the phenyl and cyclohexyl rings. Although these proved
to be some of the most potent compounds, their intrinsic higher
lipophilicities led to no advantage in overall properties to offset
their synthetic complexity. In a previous series of DGAT1 in-
hibitors we had utilized the bicyclooctane system as an alterna-
tive to cyclohexyl.3 This system maintained potency in the present
series (compounds 83−85) but again with the disadvantage of
increased lipophilicity.
Wider selectivity and physical and DMPK properties for re-
presentative compounds are given in Table 6. In general potency
against ACAT1 was low, the good selectivity for DGAT1, which
was a prerequisite of the program, thus being achieved. Erosion
of the margin to less than 100-fold for the spirocyclic compound
78 precluded its further development. Aqueous solubility was
generally good as might be expected from carboxylic acids of
relatively low lipophilicity. This was combined with good per-
meability for the simple carboxylic acids, as measured in the
Caco-2 or MDCK assay, which was reassuring because low
permeability can be an issue for compounds that have log D < 1.7
with molecular weight in the 350−400 range.20 Low intrinsic
clearance was seen generally with the acids in Table 6. These
properties combined to give very good rat PK profiles with long
oral half-lives and high bioavailability seen across a range of core
structures. The short half-life of primary alcohol 53 presumably
reflects the metabolic instability of this functionality. The elements
in Table 6 were considered alongside synthetic accessibility, and
compound 30 was chosen for extensive in vitro and in vivo profiling
as a potential candidate drug. Full data are presented in Table 7.
Compound 30 showed similar inhibitory potency against
recombinant human protein and against that expressed in human
liver microsomes (Table 7). Potency was found to be signi-
ficantly higher when assessed in human adipose tissue explants
and in an intestinal cell line (HuTu80). Little difference in
DGAT1 activity was encountered across species. No activity was
seen against human DGAT2 and ACAT2. The compound was
free of activity against the hERG encoded ion channel and against
a panel of common CYP P450 enzymes. In a panel of 339 in vitro
radioligand binding and enzyme assays covering a diverse range
of enzymes, receptors, ion channels, and transporters, compound
30 showed inhibition of acyl-CoA:cholesterol acetyltransferase
(79% at 10 μM), fatty acid amide hydrolase (IC50 = 3.7 μM),
muscarinic M2 receptor (IC50 = 80.5 μM), and phosphodiester-
ase PDE10A1 (IC50 = 5.5 μM). Plasma protein binding was
relatively consistent across species.
This compound also proved to have excellent pharmaceutical
properties with high solubility and permeability from a crystalline
nonhygroscopic single polymorph. No issues were encountered
with chemical or light stability.
Compound 30 pharmacokinetics were determined in mice,
rats, and dogs (Table 8). Low metabolic clearance and volume of
distribution were observed in each species with good to excellent
bioavailability. These data in combination with the very low turn-
over seen in human hepatocytes predicted an attractive PK
profile in man.
The most significant metabolite identified in human, rat, and
dog hepatocytes was the acyl glucuronide 120. It is believed that
in some circumstances the initially formed 1β-isomer of an acyl
glucuronide metabolite can directly lead to drug−protein co-
valent conjugates through acylation, while the 2- and 4-O-acyl
rearrangement products can give rise to adducts through Schiff
base formation with protein amino groups.28 The in vitro stability
of acyl glucuronide 120 was assessed in pH 7.8 buffer: no mea-
surable degradation or acyl migration was observed over 45 min,
giving confidence that this metabolite would be unlikely to give
rise to safety issues in man.
The pharmacodynamic effects of compound 30 were
determined in the rat oral lipid tolerance test (OLTT) and the
I
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Table 1. Initial Pyrazine Primary Amide and N-Substituted
Amide SAR
Table 2. Pyrazine Replaced by Pyridines
a
b
Mean values from a minimum of three experiments. Chromatog-
rapic log D, as in Table 1.
Table 3. Pyrazine Substitution
a
c
compd
R1
Me
R2
hDGAT1 IC50 (μM)
log D
LLE
d
30
45
46
47
48
49
50
51
52
Me
Me
Me
Me
Me
H
0.08
0.9
6.2
b
CHF2
Et
0.41
1.4
1.5
2.6
2.0
0.5
1.7
2.6
5.0
1.8
4.2
i-Pr
Ph
>10
>10
>10
>9.1
>10
>10
<2.4
<3.1
<4.5
<3.3
<2.4
<2.5
NH2
Me
Me
Me
Et
i-Pr
Ph
2.5
a
b
Mean values from a minimum of three experiments. Mean value
c
from two experiments. Chromatographic log D method, as in Table 1.
d
Octanol/water partitioning, pH 7.4.
rat TAG synthesis test. Figure 1 shows the relationship between
plasma triglycerides and free compound levels in plasma for
compound 30 in the rat OLTT assay. Compound 30 was able to
inhibit appearance of TAG in plasma following the lipid challenge
in an exposure dependent manner. A direct response PK/PD
model (Emax sigmoidal model) was used to successfully fit the PK/
PD data showing a clear correlation between free compound levels
in plasma and an effect in the OLTT. PK/PD parameters for the
model fit are shown in Table 9. The in vivo IC50 (0.042 μM) is in
very good agreement with the in vitro one (0.080 μM), suggest-
ing that free concentrations in plasma are a good surrogate for
pharmacological active drug levels in the gut cells in the OLTT
assay.
a
b
Mean values from a minimum of three experiments. Chromato-
graphic log D, pH 7.4, unless otherwise indicated: Waters ACQUITY
UPLC BEH C18 1.7 μm, 50 mm × 2.1 mm column; mobile phase 10
mM ammonium acetate in CH3CN/H2O, 95/5 to 5/95 gradient
elution; log D calculated from retention time vs standard curve.
Figure 2 shows the relationship between adipose TAG
synthesis (expressed as TAG/DAG ratio in adipose tissue) and
free compound levels in plasma for compound 30 in the rat.
c
Octanol/water partitioning, pH 7.4.
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Table 4. Carboxylic Acid Replacements and Variations
a
b
c
IC50: mean values from a minimum of three experiments. Chromatographic method as in Table 1. Octanol/water partitioning as in Table 1.
Compound 30 was able to reduce the formation of TAG in
adipose tissue (measured as TAG/DAG ratio) in an exposure
dependent manner. A direct response PK/PD model (Emax
model) was used to successfully fit the PK/PD data showing a
clear correlation between free compound levels in plasma and an
effect in the adipose TAG/DAG ratio. PK/PD parameters for the
model fit are shown in Table 10. The in vivo IC50 (0.132 μM) in
the adipose tissue assay also is in very good agreement with the in
vitro IC50 (0.080 μM), showing once again that free plasma
concentrations are a good surrogate for pharmacological active
drug levels in the adipose tissue cells in the TAG synthesis assay.
In addition compound 30 was shown to have beneficial effects in
several preclinical metabolic disease models (e.g., enhanced
GLP-1 secretion, delayed gastric emptying, reduced food intake,
improved insulin sensitivity, reduced atherosclerosis), and these
will be described in detail elsewhere.
On the basis of the attractive profile described above
Compound 30 was selected as a clinical candidate drug. Figure 3
gives the rising dose PK profile from human volunteer studies.
At all dose levels studied, compound 30 was rapidly absorbed
with the maximal plasma concentration reached within 1 h with
exposure increasing proportionally with dose (Figure 3). The
geometric mean terminal half-life estimated at all dose levels
ranged between 9 and 14 h. These data are consistent with the
preclinical DMPK and physicochemical data presented above.
Pharmacodynamic data, to be published elsewhere, indicated
5 mg u.i.d. as an effective dose for lowering serum triglyceride
following a standardized mixed meal.
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Table 5. Phenylcyclohexylacetic Acid Variations
a
b
IC50: mean values from a minimum of three experiments. Octanol/water partitioning as in Table 1.
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Table 6. Wider Selectivity and Physical and DMPK Properties for Representative Compounds
ACAT1
IC50
(μM)
rat hepatocyte intrinsic
permeability
rat oral
f
rat
bioavailability
(%)
a
c
d
DGAT1
compd MW IC50 (μM)
solubility,
human plasma protein
clearance
Papp
half-life
(h)
b
pH 7.4 (μM)
binding (% free)
((μL/min)/106 cells)
(×10−6 cm/s)
12
13
29
30
53
54
55
56
57
58
59
60
78
83
339
353
339
367
353
325
366
338
452
424
444
391
393
393
1.1
79
1200
>100
NT
700
4
NT
5.7
3.5
4.7
3.0
2.8
13
72
NT
NT
5.7
11
NT
80
e
0.34
1.9
>33
>100
34
<6
NT
<2
NT
36
35
e
29
62
g
0.08
0.69
1.2
12
14
93
e
75
58
0.26
NT
NT
NT
1.9
3
e
NT
25
3
38
NT
NT
NT
19
0.52
1.9
NT
<1
77
18
e
NT
NT
NT
NT
44
NT
7.8
NT
11
NT
NT
<2
17
13
e
0.11
1.1
86
0.71
>100
100
270
562
113
NT
NT
0.43
NT
32
e
0.10
0.06
0.03
0.08
2.0
g
0.9
1.3
0.6
<2
<2
<2
3.7
12
13
9.0
18
g
2.4
15
7.5
65
g
7.3
55
a
b
Solubility determined in 0.1 M phosphate buffer, pH 7.4, at 25 °C for 24 h. PPB was assessed by equilibrium dialysis in the appropriate species plasma
c
at 37 °C. Free and bound concentrations were quantified by LC−UVMS. Compounds were incubated at 1 μM with suspended rat hepatocytes at
d
1 × 106 cells/mL, and intrinsic clearance was calculated from rate of disappearance of parent. Permeability measured in MDCK cells in the A to B
e
f
direction, pH 7.4. Permeability measured in Caco2 cells in the A to B direction, pH 7.4. Oral dosing in female Sprague−Dawley rats at 4.0 μM/kg
g
in DMA/tetraethylene glycol/water, 1:1:1, solution. Oral dosing in male Han−Wistar rats at 2.54−5.4 μM/kg in water/(5% DMSO/95%
hydroxypropyl β-cyclodextrin), 25% w/v.
Table 7. Full Potency, Selectivity, DMPK, and Physical Properties for Compound 30
human DGAT1
a
human DGAT1 liver
human adipose
ab
human intestinal cell
human
human
human
a
ab
,
a
a
a
,
recombinant
microsome
tissue
0.01
dog DGAT1 liver
HuTu80
DGAT2
ACAT1
ACAT2
>10
0.08
0.07
0.01
>300
34
rat DGAT1 liver
rat adipose
ab
mouse DGAT1 liver
human plasma protein rat plasma protein mouse plasma protein
ab
,
ab
,
ab
,
c c c
,
microsome
tissue
0.09
microsome
microsome
binding
5.5
binding
3
binding
10
0.15
0.10
0.06
dog plasma protein
crystalline solubility,
photostability Hannau
sun test
c
a
a
binding
hERG
CYP P450
pH 7.4 (μM)
chemical stability (25 °C across pH 1−10)
t1/2 > 100 days
6
>32 >30 vs 1A2, 2C9, 2C19, 2D6, 3A4
700
t1/2 = 7.6 h
human hepatocyte CLint
mouse hepatocyte CLint
rat hepatocyte CLint
dog hepatocyte CLint
permeability Caco-2
permeability MDCK
((μL/min)/106 cells)
((μL/min)/106 cells)
((μL/min)/106 cells)
((μL/min)/106 cells)
(×10−6 cm·s−1
)
(×10−6 cm·s−1
)
<2
7.3
<2
5.5
11
12
a
b
c
IC50, μM. See Experimental Section for assay details. % free.
Table 8. PK Parameters for Compound 30
a
b
c
mouse
rat
dog
clearance ((mL/min)/kg)
volume of distribution (L/kg)
half-life (h)
0.77
0.9
0.50
0.35
14
1.2
1.0
11
13
bioavailability (%)
>100
93
∼100
a
Oral dosing at 8.16 μM/kg as an aqueous suspension with hydroxypropyl
methylcellulose/Tween, iv dosing as a solution at 5.4 μM/kg in male
b
C57 BL/6 mice. Oral dosing as a solution at 5.4 μM/kg, iv dosing as
c
a solution at 2.7 μM/kg in male Han−Wistar rats. Oral dosing at
27.2 μM/kg as a suspension (hydroxypropyl β-cyclodextrin/Tween),
iv dosing at 1.4 μM/kg as a solution in Beagle dogs. All solutions in
water/(5% DMSO/95% hydroxypropyl β-cyclodextrin), 25% w/v.
CONCLUSION
■
The herein described rational design and optimization of a series
of compounds based on a pyrazinecarboxamide core structure
culminated in the identification of the clinical candidate drug
compound 30 (AZD7687). This compound fulfilled the design
objectives set at the start of the program, as it showed high selec-
tivity for DGAT1 over ACAT1 and had very good physico-
chemical and preclinical DMPK properties that translated into a
Figure 1. PK/PD analysis of rat OLTT (plasma triglycerides (mean
SEM) vs free compound concentrations in plasma) for compound 30. A
direct response (Emax sigmoidal) model was used to fit the PK/PD data.
desirable clinical PK profile consistent with a low efficacious dose
in man.
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Table 9. PK/PD Parameters for Model Fit for Compound 30
in the Rat OLTT
E = E0 − [(ImaxCγ)/(IC50γ + Cγ)]
parameter
estimate
units
Stderr, %
IC50
γ
0.042
1.6
μM
10
19
E0
3.2
mM
mM
2.7
5.1
Imax
2.3
Figure 3. Mean plasma concentration from ascending doses of
compound 30 in human volunteers.
triacylglycerol synthesis tests in the rat protocols were again as pre-
viously described.3
Human liver microsomes were purchased from the UK Human
Tissue Bank. Rat, mouse, and dog microsomes were prepared from fresh
liver according to the method described by Coleman.29 DGAT activity
was then assayed as previously described.3
For the adipose TAG synthesis assay, measurement of TAG and DAG
synthesis in rat epididymal and human subcutaneous adipose tissue was
performed ex vivo by measurement of 14C palmitate incorporation into
TAG and DAG, and this was used to obtain an index of DGAT1 activity.
The human adipose tissue samples were obtained from volunteers by
needle biopsies of abdominal subcutaneous tissue following local
anesthesia. This procedure was approved by the Regional Ethical Review
Boards in Gothenburg and Umeå, respectively, and the South Cheshire
Local Research Ethics Committee. Written informed consent was
obtained from all subjects prior to initiation of any study-related pro-
cedures. The human adipose tissue biopsy was washed in PBS (Gibco,
Paisley, U.K.). To remove excess medium and blood clots, the biopsy
was put on a cell strainer with a paper napkin underneath. Whole rat
epididymal fat pads were removed. Approximately 3 × 50 mg of adipose
tissue from each species was placed into a 48-well tissue culture plate.
The adipose tissue was incubated with 200 μL of DMEM (6 mM
glucose, 5% FCS (Gibco, Paisley, U.K.), 0.5% penicillin/streptomycin
(Gibco, Paisley, U.K.) containing compound dissolved in DMSO (0.1% final
concentration) for 30 min at 37 °C, 5% CO2. 14C palmitic acid (2.5μCi/mL)
and a mixture of sodium palmitate complexed to BSA (0.12 mM sodium
palmitate, 0.03 mM BSA final concentration) were added to the wells and the
samples incubated for a further 2 h for the human adipose tissue and 3 h for
the rat adipose tissue at 37 °C, 5% CO2. The culture medium was removed,
and the biopsies were transferred to lysis matrix D tubes.
Figure 2. PK/PD analysis of rat adipose TAG synthesis assay (adipose
tissue TAG/DAG ratio vs free compound concentrations in plasma) for
compound 30. A direct response (Emax model) was used to fit the PK/
PD data.
Table 10. PK/PD Parameters for Model Fit for Compound 30
in the Rat Adipose TAG Synthesis Assay
E = E0{1 − [C/(IC50 + C)}
parameter
estimate
units
Stderr, %
IC50
0.132
5.0
μM
13
E0
4.9
EXPERIMENTAL SECTION
■
All solvents and chemicals used were reagent grade. Anhydrous solvents
tetrahydrofuran (THF) and dimethoxyethane (DME) were purchased
from Aldrich and used as such. Flash column chromatography was
carried out using prepacked silica cartridges (from 4 to 330 g) from
Redisep, Biotage, or Crawford and with elution using an Isco Com-
panion system. Purity and characterization of compounds were estab-
lished by a combination of liquid chromatography−mass spectrometry
(LC−MS), gas chromatography−mass spectrometry (GC−MS), and
NMR analytical techniques. 1H NMR spectra were recorded on Bruker
Avance DPX400 (400 MHz), DPX300 (300 MHz), and AV700 (700
MHz) instruments and were determined in CHCl3-d, DMSO-d6, and
MeOH-d with trimethylsilane (TMS) (0.00 ppm) or solvent peaks as
the internal reference. Chemical shifts are reported in ppm relative to
solvent signal, and coupling constants (J) are reported in hertz (Hz).
Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet; br, broad peak. Merck precoated thin layer chromatography
(TLC) plates (silica gel 60 F254, 0.25 mm, article 5715) were used for TLC
analysis. Solutions were dried over anhydrous magnesium sulfate, and the
solvent was removed by rotary evaporation under reduced pressure.
DGAT1, DGAT2, ACAT1, and ACAT2 in vitro assay protocols and
measurement of triacylglycerol synthesis in HuTu 80 cells were as
previously described.3 Oral lipid tolerance test (OLTT) and adipose
The lipids were extracted using the Folch extraction method. An
3
amount of 1 mL of methanol plus H-oleate (to account for losses
during the extraction phase) was added to each sample. The tissue was
homogenized (FastPrep), and the contents were transferred to glass
Pyrex tubes. The lysis matrix D tubes were washed with 1 mL of methanol,
and the contents were transferred to the glass Pyrex tubes. An amount of
4 mL of chloroform was added before the tubes were vortexed and
incubated at 40 °C for 1 h. Then 1.2 mL of saline (0.73%) was added and
the glass Pyrex tubes were vortexed and left in a fridge overnight to separate.
The bottom solvent layer was transferred into a fresh tube and solvent was
evaporated under a stream of nitrogen. The extract was solubilized in
isohexane/acetic acid (99:1). Lipids were separated via normal phase high
performance liquid chromatography (HPLC) using a Lichrospher diol-5,
4 mm × 250 mm column and a gradient solvent system of isohexane/acetic
acid (99:1) and isohexane/propan-2-ol/acetic acid (85:15:1), flow rate of
1 mL/min. Incorporation of radiolabel into TAG and DAG was analyzed
N
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using a Radiomatic Flo-one detector (Packard, Meriden, CT) connected
to the HPLC machine.
Purity of test compounds was >95%, unless otherwise indicated, as
determined by HPLC methods and supported by high resolution mass
spectrometry data where possible.
Synthesis methods for key compounds are given below. Experimental
details for the synthesis of the rest of the compounds are available in
Supporting Information.
hydride in mineral oil (3.28 g, 79.3 mmol) to give a thick suspension. In a
separate flask 60% sodium hydride in mineral oil (4.06 g, 98.0 mmol)
was suspended in dry THF (375 mL) under nitrogen. The suspen-
sion was cooled to 0 °C, and tert-butyl P,P-dimethylphosphonoacetate
(16.8 mL, 82.3 mmol, CAS 62327-21-3) was added carefully. Both
mixtures were stirred for ∼15 min before the ice baths were removed.
After 2 h at room temperature the ketone solution was transferred to the
ylide solution via a syringe. The combined mixture was stirred under
nitrogen overnight. The reaction was quenched by the addition of water
(100 mL), and THF was then removed under vacuum. More water was
added (150 mL), and the aqueous solution was extracted with EtOAc
(3 × 200 mL). The combined organic layers were washed with brine
(2 × 200 mL) and dried through a phase separator. Concentration gave
25 as a colorless oil (24.6 g, 99%). 1H NMR (500 MHz, CDCl3) δ 1.49
(s, 9H), 1.55−1.63 (m, 2H), 1.94−2.07 (m, 3H), 2.26−2.39 (m, 2H),
2.66−2.76 (m, 1H), 3.85−3.96 (m, 1H), 4.58 (s, 1H), 5.60 (s, 1H), 6.76 (d,
J = 8.52 Hz, 2H), 7.07 (d, J = 8.49 Hz, 2H). ESI-MS (m/z): 287 (MH−).
Step 2: tert-Butyl [trans-4-(4-Hydroxyphenyl)cyclohexyl]acetate
(26). To a solution of 25 (4.2 g, 14.56 mmol) in EtOAc (120 mL) was
added 10% palladium on carbon (390 mg). The reaction was hydro-
genated for 3 h (5 bar). The catalyst was filtered off through Celite, and
the organic filtrate was washed with water, dried through a phase separator,
and concentrated to a colorless oil (4.02 g of cis/trans mixture). The crude
mixture was purified by chiral HPLC (neutral) using a Chiralpak AD
(250 mm × 50 mm) column and 100% ACN as mobile phase to afford
the desired pure trans-phenol 26 (2.88 g, 68%) as a white solid and also
pure cis-phenol. NMR analysis of both isomers confirmed that the major
isomer was trans. 1H NMR (400 MHz, CDCl3) δ 1.04−1.22 (m, 2H),
1.35−1.55 (m, 11H), 1.71−1.95 (m, 5H), 2.15 (d, J = 6.82 Hz, 2H),
2.33−2.48 (m, 1H), 4.86 (s, 1H), 6.76 (d, J = 8.08 Hz, 2H), 7.07 (d,
J = 8.24 Hz, 2H). ESI-MS (m/z): 289 (MH−).
Step 3: tert-Butyl [trans-4-(4-{[(Trifluoromethyl)sulfonyl]oxy}-
phenyl)cyclohexyl]acetate (27). To a solution of 26 (2.86 g, 9.9 mmol)
in DCM (75 mL) was added pyridine (1.59 mL, 19.7 mmol). The solu-
tion was cooled on an ice bath, and trifluoromethanesulfonic anhydride
(1.99 mL, 11.8 mmol) was added dropwise. The ice bath was removed,
and the reaction mixture was stirred for 10 min. The reaction was
quenched with 1 M HCl (aq), diluted with DCM, and washed with
saturated NaHCO3 (aq) and brine. The organic layer was dried through
a phase separator and concentrated. The crude product was purified by
flash chromatography (8% EtOAc in heptanes) to afford 27 (4.06 g,
98%) as a white crystalline solid. 1H NMR (500 MHz, CDCl3) δ 1.11−
1.23 (m, 2H), 1.44−1.55 (m, 11H), 1.77−1.98 (m, 5H), 2.17 (d, J =
7.02 Hz, 2H), 2.47−2.57 (m, 1H), 7.20 (d, J = 8.67 Hz, 2H), 7.28 (d, J =
8.67 Hz, 2H). ESI-MS (m/z): 421 (MH−).
Ethyl 6-Chloro-3,5-dimethylpyrazine-2-carboxylate (22). Step
1. Ethyl 2-Diazo-3-oxobutanoate (19). Polymer-bound tosyl azide
(11 g, 15.4 mmol) (typical loading 1.4 mmol/g, prepared according to
Green et al.)27 was swollen in dry DCM (40 mL). Ethyl acetoacetate
(1.0 g, 7.7 mmol, CAS 141-97-9) and triethylamine (3.2 mL, 23.1 mmol)
were dissolved in DCM (10 mL) and added to the polymer containing
solution. The resulting mixture was then shaken at room temperature under
nitrogen until the reaction was judged completed by TLC, typically 6 h. The
supernatant was filtered off. Then the resin was washed with DCM (3 ×
30 mL) to rinse out residual product. The reaction mixture was evaporated
to dryness to afford 19 (1.1 g, 92%) as a yellow oil. 1H NMR (400 MHz,
CDCl3) δ 1.33 (t, J = 7.13 Hz, 3H), 2.48 (s, 3H), 4.30 (q, J = 7.13 Hz, 2H).
Typically these intermediates were not characterized because of their
high energetic properties30 but carried through to the following step as
crude products.
Step 2. Ethyl 2-{[N-(tert-Butoxycarbonyl)-L-alanyl]amino}-3-oxo-
butanoate (20). Intermediate 19 (500 mg, 3.2 mmol) and Boc-L-Ala-
NH2 (843.8 mg, 4.5 mmol, CAS 85642-13-3) were added to a round
bottomed flask, sealed, and back-filled with argon. Dry toluene (30 mL)
was added via syringe, and the resulting heterogeneous mixture was
stirred at 90 °C for 10 min to get a homogeneous solution. Meanwhile,
the rhodium(II) octanoate dimer (62.3 mg, 0.080 mmol, CAS 73482-
96-9) was dissolved in toluene (5 mL) and put on an ultrasound bath for
5 min to get a fine Rh dispersion. The dispersion was then added drop-
wise to the reaction mixture at 80 °C (a violent nitrogen effervescence
was observed). After being stirred for another 20 min at elevated tem-
perature, the reaction mixture was concentrated under reduced pressure
to give a black pasty solid. The crude was purified by flash chromatography
(20−80% EtOAc in heptanes) to afford 20 (850 mg, 84%) (diastereomeric
mixture) as a yellow oil. ESI-MS (m/z): 317 (MH+).
Step 3. Ethyl 3,5-Dimethyl-6-oxo-1,6-dihydropyrazine-2-carbox-
ylate (21). To a solution of 20 (800 mg, 2.53 mmol) in dry DCE
(40 mL) was added TFA (1.95 mL, 25.3 mmol). The reaction mixture
was heated to reflux for 4 h, and the solvent was removed under vacuum.
The crude product was purified by flash chromatography (20−80%
EtOAc in heptanes) to afford 21 (160 mg, 32%) as a white-yellow powder.
The crude product from this reaction can optionally be used directly in
the next step without purification. 1H NMR (400 MHz, CDCl3) δ 1.42 (t,
J = 7.15 Hz, 3H), 2.53 (s, 3H), 2.63 (s, 3H), 4.44 (q, J = 7.15 Hz, 2H).
ESI-MS (m/z): 197 (MH+).
Step 4. Ethyl 6-Chloro-3,5-dimethylpyrazine-2-carboxylate (22).
To a suspension of 21 (0.23 g, 1.17 mmol) in butyronitrile (4 mL) was
added POCl3 (0.27 mL, 2.93 mmol). The mixture was heated to 150 °C
for 10 min in the microwave oven and then allowed to cool to room tem-
perature. To the reaction mixture was added water (2 mL). The phases
were separated, and the organic layer was concentrated to dryness. The
crude product was purified by flash chromatography (0.5% acetic acid in
DCM) to afford 22 as a yellow oil (0.18 g, 73%). 1H NMR (500 MHz,
CDCl3) δ 1.43 (t, J = 7.13 Hz, 3H), 2.68 (s, 3H), 2.78 (s, 3H), 4.45
(q, J = 7.13 Hz, 2H). ESI-MS (m/z): 215 (MH+).
Step 4: tert-Butyl {trans-4-[4-(4,4,5,5-Tetramethyl-1,3,2-dioxabor-
olan-2-yl)phenyl]cyclohexyl}acetate (28). To a suspension of
PdCl2(dppf) (0.93 g, 1.27 mmol, CAS 72287-26-4) in dioxane (135 mL)
under nitrogen was added 27 (17.9 g, 42.4 mmol), triethylamine (17.7 mL,
127 mmol), and finally a 1 M solution of 4,4,5,5-tetramethyl-1,3,2-
dioxaborolane in THF (72 mL, 72 mmol, CAS 25015-63-8). The mix-
ture was refluxed for 7 h, cooled to 10 °C, and carefully quenched with
water. The aqueous solution was extracted with two portions of DCM,
and the combined organic layers were dried through a phase separator
and concentrated to give a black oil. The crude residue was purified by
flash chromatography (6% EtOAc in heptanes) to afford 28 (12.1 g,
1
71%) as a white powder. H NMR (500 MHz, CDCl3) δ 1.09−1.23
(m, 2H), 1.33 (s, 12H), 1.47 (s, 9H), 1.48−1.6 (m, 2H), 1.76−1.95
(m, 5H), 2.15 (d, J = 6.98 Hz, 2H), 2.4−2.58 (m, 1H), 7.23 (d, J = 7.92 Hz,
2H), 7.75 (d, J = 7.94 Hz, 2H).
6-Chloro-3,5-dimethylpyrazine-2-carboxamide (23). A 7 N
solution of ammonia in MeOH (113 mL, 791.99 mmol) was added to
intermediate 22 (3.4 g, 15.84 mmol), and the resulting suspension was
stirred at room temperature for 20 h. The reaction mixture was filtered
and air-dried to afford 23 as a cream solid (2.1 g, 72%). 1H NMR (400 MHz,
DMSO-d6) δ 2.59 (s, 3H), 2.67 (s, 3H), 7.70 (s, 1H), 7.99 (s, 1H). ESI-MS
(m/z): 186 (MH+).
tert-Butyl {trans-4-[4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenyl]cyclohexyl}acetate (28). Step 1: tert-Butyl [4-(4-
Hydroxyphenyl)cyclohexylidene]acetate (25). To an ice-cold solution
of 4-(4-hydroxyphenyl)cyclohexanone 24 (15.22 g, 78.4 mmol, CAS
105640-07-1) in THF (375 mL) under nitrogen was added 60% sodium
{trans-4-[4-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)phenyl]
cyclohexyl}acetic Acid (30). Method 1. Step 1: Ethyl 6-{4-[trans-4-
(2-tert-Butoxy-2-oxoethyl)cyclohexyl] phenyl}-3,5-dimethylpyra-
zine-2-carboxylate. To a solution of ethyl 6-chloro-3,5-dimethylpyr-
azine-2-carboxylate (22) (0.580 g, 2.70 mmol) in DME (22 mL), EtOH
(12 mL), and water (5 mL) were added boronic ester 28 (1.244 g,
3.11 mmol), potassium phosphate (tribasic) (0.688 g, 3.24 mmol), and
PdCl2(dppf) (0.119 g, 0.16 mmol). The solution was degassed with
nitrogen for 10 min and then heated in the microwave oven at 140 °C for
20 min. Additional amounts of boronic ester and PdCl2(dppf) were
O
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Article
added to drive the reaction. The microwave heating was continued at
140 °C for an additional 30 min. The mixtures were combined, filtered
through a glass filter funnel, and evaporated. The remaining black
residue was dissolved in EtOAc, washed with 0.5 M HCl (aq), and
extracted with two portions of EtOAc. The combined organic layers
were dried through a phase separator and concentrated to dryness. The
crude compound was purified by flash chromatography (0−50% EtOAc
(s, 1H), 7.64 (d, J = 8.25 Hz, 2H), 7.97 (s, 1H). ESI-MS (m/z, %): 392
(MH+, 100). HPLC purity 100%.
Step 2. {trans-4-[4-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)-
phenyl]cyclohexyl}acetic Acid (30). 30 was prepared from intermediate
93a (5.2 g, 13.67 mmol) according to the procedure described for
compound 31. The crude product was recrystallized from hot EtOH
(250 mL) to give a yellow solid which was washed with diethyl ether
(100 mL) and dried under vacuum at room temperature to give 30 (4.51 g,
1
in heptane) to afford the title compound (1.063 g, 80%) as an oil. H
1
90%) as a white solid. H NMR (400 MHz, DMSO-d6) δ 1.07−1.2
NMR (500 MHz, CDCl3) δ 1.12−1.22 (m, 2H), 1.42 (t, J = 7.13 Hz,
3H), 1.47 (s, 9H), 1.49−1.59 (m, 2H), 1.76−1.97 (m, 5H), 2.16 (d, J =
6.99 Hz, 2H), 2.48−2.59 (m, 1H), 2.66 (s, 3H), 2.80 (s, 3H), 4.45 (q, J =
7.12 Hz, 2H), 7.30 (d, J = 8.10 Hz, 2H), 7.52 (d, J = 8.13 Hz, 2H).
Step 2: 6-{4-[trans-4-(2-tert-Butoxy-2-oxoethyl)cyclohexyl]-
phenyl}-3,5-dimethylpyrazine-2-carboxylic Acid. To a solution of
ethyl 6-{4-[trans-4-(2-tert-butoxy-2-oxoethyl)cyclohexyl]phenyl}-3,5-
dimethylpyrazine-2-carboxylate (0.530 g, 1.17 mmol) in THF (10 mL)
was added LiOH (1M, 15 mL). The mixture was stirred at room tem-
perature for 5 h. THF was removed under vacuum, and the aqueous
residue was acidified with 1 M HCl (aq) and extracted with EtOAc. The
organic layer was dried through a phase separator and concentrated to
afford the title compound (0.5 g, 100%). 1H NMR (400 MHz, CDCl3) δ
1.12−1.3 (m, 2H), 1.47 (s, 9H), 1.52−1.63 (m, 2H), 1.8−2.02 (m, 5H),
2.17 (d, J = 6.89 Hz, 2H), 2.51−2.63 (m, 1H), 2.72 (s, 3H), 3.01 (s, 3H),
7.36 (d, J = 8.06 Hz, 2H), 7.51 (d, J = 8.08 Hz, 2H). ESI-MS (m/z): 425
(MH+).
Step 3: tert-Butyl {trans-4-[4-(6-Carbamoyl-3,5-dimethylpyrazin-
2-yl)phenyl]cyclohexyl}acetate. To a solution of PyBOP (717 mg,
1.38 mmol) and NH4Cl (196.6 mg, 3.68 mmol) in DMF (50 mL) were
added 6-{4-[trans-4-(2-tert-butoxy-2-oxoethyl)cyclohexyl]phenyl}-3,5-
dimethylpyrazine-2-carboxylic acid (390.0 mg, 0.92 mmol) and finally
DIPEA (0.80 mL, 4.59 mmol) to initiate the reaction. The solution was
stirred for 5 h at room temperature. The reaction was quenched
by the addition of saturated Na2CO3 (aq) and extracted with toluene
(3 × 100 mL). The combined organic layers were washed with brine and
water, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The crude compound was purified by flash chromatography
(20−60% EtOAc in petroleum ether) to afford the title compound as a
white solid (376 mg, 96%). 1H NMR (400 MHz, CDCl3) δ 1.12−1.25
(m, 2H), 1.47 (s, 9H), 1.51−1.63 (m, 2H), 1.78−2.04 (m, 5H), 2.17
(d, J = 6.90 Hz, 2H), 2.5−2.6 (m, 1H), 2.67 (s, 3H), 2.98 (s, 3H), 7.33
(d, J = 8.13 Hz, 2H), 7.52 (d, J = 8.11 Hz, 2H), 7.80 (br s, 2H). ESI-MS
(m/z): 424 (MH+).
Step 4: {trans-4-[4-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)-
phenyl]cyclohexyl}acetic Acid (30). To a solution of tert-butyl {trans-
4-[4-(6-carbamoyl-3,5-dimethylpyrazin-2-yl)phenyl]cyclohexyl}acetate
(363 mg, 0.86 mmol) in DCM (30 mL) was added TFA (3 mL)
dropwise. The colorless solution gradually turned into a pale yellow clear
solution. The reaction mixture was stirred under nitrogen at room tem-
perature overnight. The solvent was evaporated to dryness. The crude
compound was purified by flash chromatography (20−50% EtOAc in
heptane + 3% formic acid) to afford 30 as a white solid, mp 220−223 °C
(220 mg, 69%). 1H NMR (500 MHz, DMSO-d6) δ 1.06−1.22 (m, 2H),
1.42−1.59 (m, 2H), 1.68−1.89 (m, 5H), 2.15 (d, J = 6.93 Hz, 2H),
2.51−2.56 (m, 1H), 2.59 (s, 3H), 2.74 (s, 3H), 7.36 (d, J = 8.13 Hz, 2H),
7.61 (br s, 1H), 7.65 (d, J = 8.12 Hz, 2H), 8.00 (br s, 1H). ESI-HRMS
calculated for C21H26N3O3 (MH+) 368.1974. Found: 368.1973.
{trans-4-[4-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)phenyl]-
cyclohexyl}acetic Acid (30). Method 2. Step 1. Methyl {trans-4-[4-
(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)phenyl]cyclohexyl}acetate
(93a). 93a was prepared according to the procedure described for
methyl {trans-4-[4-(6-carbamoyl-5-methylpyrazin-2-yl)phenyl]cyclo-
hexyl}acetate (compound 31 step 8) from 23 (3.15 g, 16.97 mmol)
and intermediate 66 (6.08 g, 16.97 mmol). The reaction mixture was
heated to 80 °C, under nitrogen, and left to stir overnight for 16 h. The
crude product was purified by flash silica chromatography (5−90%
EtOAc in isohexane) to afford 93a (5.21 g, 81%) as a yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 1.07−1.23 (m, 2H), 1.42−1.59 (m, 2H),
1.69−1.89 (m, 5H), 2.25 (d, J = 6.67 Hz, 2H), 2.49−2.56 (m, 1H), 2.58
(s, 3H), 2.73 (s, 3H), 3.60 (s, 3H), 7.35 (d, J = 8.23 Hz, 2H), 7.57
(m, 2H), 1.44−1.6 (m, 2H), 1.7−1.8 (m, 1H), 1.81−1.89 (m, 4H), 2.15
(d, J = 6.91 Hz, 2H), 2.53−2.55 (m, 1H), 2.58 (s, 3H), 2.73 (s, 3H), 7.36
(d, J = 8.24 Hz, 2H), 7.57 (s, 1H), 7.64 (d, J = 8.25 Hz, 2H), 7.97 (s, 1H),
11.99 (s, 1H). ESI-MS (m/z, %): 368 (MH+, 100).
{trans-4-[4-(6-Carbamoyl-5-methylpyrazin-2-yl)phenyl]-
cyclohexyl}acetic Acid (31). Step 1. Methyl 6-Hydroxy-3-methyl-
pyrazine-2-carboxylate (63). A solution of (S)-methyl 2-(2-amino-
acetamido)-3-oxobutanoate hydrochloride31 (62) (1.3 g, 5.79 mmol) in
pyridine (58 mL) was stirred at 60 °C for 90 min. The reaction mixture
was evaporated to afford crude product which was purified by flash silica
chromatography (50−80% EtOAc in isohexane) to afford 63 as a yellow
solid (0.340 g, 35%). 1H NMR (400 MHz, CDCl3) δ 2.59 (s, 3H), 3.94
(s, 3H), 8.27 (s, 1H). ESI-MS (m/z): 169 (MH+).
Step 2. Methyl 6-Chloro-3-methylpyrazine-2-carboxylate (64). A
suspension of intermediate 63 (314 mg, 1.87 mmol) and phosphorus
oxychloride (2.1 mL, 22.40 mmol) was stirred at 90 °C for 70 min. The
reaction mixture was added dropwise to water (20 mL) while main-
taining the temperature below 40 °C. The mixture was extracted with
DCM (5 × 50 mL). The organic layer was dried over MgSO4, filtered,
and evaporated to afford a yellow oil which was purified by flash silica
chromatography (0−20% EtOAc in isohexane) to afford 64 as a white
solid (142 mg, 41%). 1H NMR (400 MHz, CDCl3) δ 2.76 (s, 3H), 3.93
(s, 3H), 8.56 (s, 1H). ESI-MS (m/z): 187 (MH+).
Step 3. 6-Chloro-3-methylpyrazine-2-carboxamide (65). A 7 N
solution of ammonia in MeOH (5.5 mL, 38.05 mmol) was added to
intermediate 64 (142 mg, 0.76 mmol), and the resulting suspension was
stirred at room temperature for 4 h. The reaction mixture was evap-
orated to afford 65 as a white solid (109 mg, 83%). 1H NMR (400 MHz,
CDCl3) δ 2.89 (s, 3H), 5.49 (s, 1H), 7.48 (s, 1H), 8.56 (s, 1H). ESI-MS
(m/z): 170 (MH−).
Step 4. Methyl 2-(4-(4-Hydroxyphenyl)cyclohexylidene)acetate.
Trimethyl phosphonoacetate (10.2 mL, 63.08 mmol) was added por-
tionwise to NaH (3.15 g, 78.85 mmol, 60% mineral oil dispersion) in
THF (250 mL) at 0 °C under nitrogen. The resulting suspension was
stirred at room temperature for 30 min. In a separate flask, 4-(4-hydroxy-
phenyl)cyclohexanone (10 g, 52.57 mmol) was added portionwise to
NaH (2.1 g, 52.57 mmol, 60% mineral oil dispersion) in THF (200 mL)
at room temperature, and the resulting suspension was stirred at room
temperature for 25 min. This was added to the mixture containing the
phosphonate, and the resulting white suspension was stirred at room
temperature for 18 h. The reaction mixture was quenched with water
(250 mL), extracted with EtOAc (3 × 200 mL), and the organic layer
was dried over MgSO4, filtered, and evaporated to afford the title com-
1
pound as a colorless waxy solid (12.9 g, 100%). H NMR (400 MHz,
DMSO-d6) δ 1.31−1.57 (m, 2H), 1.86−2.06 (m, 3H), 2.25−2.41 (m,
2H), 2.69 (tt, J = 3.28, 11.94 Hz, 1H), 3.60 (s, 3H), 3.76−3.85 (m, 1H),
5.68 (s, 1H), 6.62−6.69 (m, 2H), 6.97−7.02 (m, 2H), 9.10 (s, 1H). ESI-
MS (m/z): 245 (MH−).
Step 5. Methyl [trans-4-(4-Hydroxyphenyl)cyclohexyl]acetate (67)
and Methyl [cis-4-(4-Hydroxyphenyl)cyclohexyl]acetate (90d). Methyl
2-(4-(4-hydroxyphenyl)cyclohexylidene)acetate (13.28 g, 53.92 mmol)
and 10% palladium on carbon (1.3 g, 5.39 mmol) in EtOAc (250 mL)
were stirred under an atmosphere of hydrogen at room temperature for
20 h. The reaction mixture was filtered and the filtrate concentrated
under reduced pressure to provide crude product as a mix of trans and
cis isomers (∼4:3 ratio). The crude material was recrystallized from
EtOAc (10 mL) to provide the title compound as colorless crystals
(4.173g, 31%, ratio trans/cis of ∼10:1).
Methyl [trans-4-(4-hydroxyphenyl)cyclohexyl]acetate (67): 1H
NMR (400 MHz, DMSO-d6) δ 1.02−1.18 (m, 2H), 1.32−1.48 (m,
2H), 1.66−1.84 (m, 5H), 2.23 (d, J = 6.79 Hz, 2H), 2.33 (tt, J = 2.95,
P
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Journal of Medicinal Chemistry
Article
11.92 Hz, 1H), 3.60 (s, 3H), 6.66 (d, J = 8.39 Hz, 2H), 7.00 (d,
J = 8.43 Hz, 2H), 9.04 (s, 1H).
2.43−2.54 (m, 1H), 2.68 (s, 3H), 7.31 (d, J = 8.36 Hz, 2H), 7.62 (s, 1H),
8.07 (d, J = 8.39 Hz, 2H), 8.14 (s, 1H), 9.11 (s, 1H), 11.89 (s, 1H). ESI-
MS (m/z): 352 (MH−). ESI-HRMS calculated for C20H24N3O3 (MH+)
354.181 22; found, 354.181 05. HPLC purity 100%.
Methyl [cis-4-(4-hydroxyphenyl)cyclohexyl]acetate (90d): 1H NMR
(400 MHz, DMSO-d6) δ 1.51−1.64 (m, 8H), 2.14−2.19 (m, 1H), 2.44
(m, 3H), 3.60 (s, 3H), 6.67 (d, J = 8.51 Hz, 2H), 7.04 (d, J = 8.48 Hz,
2H), 9.04 (s, 1H).
3,5-Dimethyl-6-{4-[trans-4-(1H-tetrazol-5-ylmethyl)-
cyclohexyl]phenyl}pyrazine-2-carboxamide (60). Step 1: Ethyl 6-
{4-[trans-4-(2-Cyanoethylcarbamoyl)cyclohexyl]phenyl}-3,5-dime-
thylpyrazine-2-carboxylate. Ethyl 6-{4-[trans-4-(2-tert-butoxy-2-
oxoethyl)cyclohexyl]phenyl}-3,5-dimethylpyrazine-2-carboxylate (see
compound 30, method 1, step 1) (300 mg, 0.66 mmol) was dissolved
in DCM (20 mL), and TFA (2 mL) was added dropwise. The mixture
was stirred at room temperature for 16 h and then concentrated to dryness
to give the liberated carboxylic acid. The acid (250 mg, 0.63 mmol) was
dissolved in DCM (20 mL), and 2-cyanoethylamine (66.3 mg, 0.95 mmol),
DIPEA (0.22 mL, 1.3 mmol), and PyBOP (492 mg, 0.95 mmol) were
added in one portion. The resulting mixture was stirred at room tem-
perature for 5 h. The crude compound was purified by flash chromatog-
raphy (95% EtOAc in pentane) to afford the title compound (267 mg,
0.60 mmol). ESI-MS (m/z): 449 (MH+).
Step 2: 6-{4-[trans-4-(1H-Tetrazol-5-ylmethyl)cyclohexyl]phenyl}-
3,5-dimethylpyrazine-2-carboxylic Acid. The 2-cyanoamide (240 mg,
0.54 mmol) from the previous step was dissolved in THF (10 mL). Then
DEAD (699 mg, 1.61 mmol), triphenylphosphine (421 mg, 1.61 mmol),
and trimethylsilyl azide (184.9 mg, 1.61 mmol) were added. The reaction
mixture was stirred at room temperature for 16 h under nitrogen atmo-
sphere. Then another equivalent of DEAD, triphenylphosphine, and
trimethylsilyl azide was added and the reaction mixture was stirred for
another 16 h and evaporated to dryness. The crude was dissolved in THF
(10 mL) and water (1 mL). Then LiOH (51 mg, 2.14 mmol) was added.
The mixture was stirred for 16 h, acidified to pH 1 using 3 M HCl (aq), and
extracted with DCM (3 × 50 mL). The organic layers were dried using
Na2SO4 and evaporated to dryness to afford the title compound. ESI-MS
(m/z): 392.9 (MH+).
Step 6. Methyl [trans-4-(4-{[(Trifluoromethyl)sulfonyl]oxy}-
phenyl)cyclohexyl]acetate (91a). Trifluoromethanesulfonic anhydride
(4.07 mL, 24.16 mmol) was added dropwise to a solution of inter-
mediate 67 (5.0 g, 20.14 mmol) in DCM (100 mL) at 0 °C, and the
mixture was allowed to stir at 0 °C for 20 min. Triethylamine (7.9 mL,
56.38 mmol) was added dropwise over a period of 40 min while main-
taining the internal temperature under 5 °C, and after complete addition
the reaction mixture was allowed to stir at 0 °C for a further 1.5 h under
nitrogen. The mixture was warmed to room temperature and then quen-
ched cautiously with water (100 mL). The organic layer was separated,
the aqueous layer was reextracted with DCM (100 mL), and the com-
bined organics were washed with saturated NaHCO3 (75 mL) before
being evaporated to afford crude product which was purified by flash
silica chromatography (5−30% EtOAc in isohexane) to afford the 91a as
1
a colorless oil which crystallized on standing (7.27 g, 95%). H NMR
(400 MHz, DMSO) δ 1.05−1.22 (m, 2H), 1.38−1.55 (m, 2H), 2.24 (d,
J = 6.72 Hz, 2H), 2.5−2.58 (m, 1H), 3.59 (s, 3H), 7.34−7.39 (m, 2H),
7.39−7.44 (m, 2H). ESI-MS (m/z, %): 379 (MH−).
Step 7. Methyl {trans-4-[4-(4,4,5,5-Tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenyl]cyclohexyl}acetate (66). A solution of intermediate
91a (10 g, 26.29 mmol), pinacoldiborane (7.34 g 28.92 mmol), KOAc
(7.74 g, 78.87 mmol), dppf (0.884 g, 1.58 mmol), and Pd(dppf)Cl2
(1.3 g, 1.58 mmol) was degassed and heated to 80 °C for 17 h. The reac-
tion mixture was allowed to cool and then evaporated to dryness. The
residue was suspended in EtOAc and filtered through a pad of silica (∼3 in.
diameter × 1 in. deep), washing through with EtOAc. The solution was
evaporated to afford crude product which was purified by flash silica
chromatography (0−20% EtOAc in isohexane) to afford 66 as a white
Step 3: 3,5-Dimethyl-6-{4-[trans-4-(1H-tetrazol-5-ylmethyl)-
cyclohexyl]phenyl}pyrazine-2-carboxamide (60). This compound
was synthesized from the carboxylic acid from a previous step using a
similar protocol for amide coupling as described for compound 30 (step
1
solid. (7.07 g, 75%). H NMR (400 MHz, DMSO-d6) δ 1.1−1.26
(m, 2H), 1.34 (s, 12H), 1.44−1.61 (m, 2H), 1.74−1.92 (m, 5H), 2.30 (d,
J = 6.73 Hz, 2H), 2.47−2.54 (m, 1H), 3.66 (s, 3H), 7.29 (d, J = 8.01 Hz,
2H), 7.65 (d, J = 8.07 Hz, 2H). ESI-MS (m/z, %): mass ion not observed.
HPLC purity 100%.
1
3) to afford 60 as a white solid, mp 208−209 °C. (32 mg, 35%). H
NMR (600 MHz, CD3OD) δ 1.23−1.36 (m, 2H), 1.51−1.67 (m, 2H),
1.87−2 (m, 5H), 2.56−2.67 (m, 4H), 2.85 (s, 3H), 2.92 (d, J = 6.97 Hz,
2H), 7.37 (d, J = 8.15 Hz, 2H), 7.58 (d, J = 8.16 Hz, 2H). ESI-HRMS
calculated for C21H25N7O (MH+), 392.2199. Found: 392.2188.
Step 8. Methyl {trans-4-[4-(6-Carbamoyl-5-methylpyrazin-2-yl)-
phenyl]cyclohexyl}acetate. A solution of intermediate 65 (109 mg,
0.64 mmol), intermediate 66 (228 mg, 0.64 mmol), and K3PO4 (162
mg, 0.76 mmol) in DME (10 mL), EtOH (2.5 mL), and water (2.5 mL)
was degassed before addition of Pd(dppf)Cl2·DCM (26.1 mg, 0.03
mmol). The reaction mixture was heated to 80 °C under nitrogen and
left to stir at 80 °C for 30 min. The reaction mixture was allowed to cool
to room temperature and then evaporated. The crude product was par-
titioned between EtOAc (70 mL) and water (30 mL), filtered through
Celite, and the organic phase was separated and washed with saturated
brine (50 mL). The organic layer was dried over MgSO4, filtered, and
evaporated to afford crude product which was purified by flash chro-
matography (0−70% EtOAc in isohexane) to afford the title compound
(194 mg, 83%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.07−
1.23 (m, 2H), 1.42−1.56 (m, 2H), 1.75−1.93 (m, 5H), 2.21 (d, J = 6.69
Hz, 2H), 2.44−2.55 (m, 1H), 2.94 (s, 3H), 3.62 (s, 3H), 5.47 (s, 1H),
7.29 (d, J = 8.22 Hz, 2H), 7.80 (s, 1H), 7.85 (d, J = 8.36 Hz, 2H), 8.95
(s, 1H). ESI-MS (m/z): 368 (MH+).
[trans-5′-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)-2′,3′-
dihydrospiro[cyclohexane-1,1′-inden]-4-yl]acetic Acid (78).
Step 1. (5-Bromo-2,3-dihydro-1H-inden-1-ylidene)methyl Methyl
Ether (95). (Methoxymethyl)triphenylphosphonium chloride (41.1 g,
120 mmol) was added to a stirred suspension of potassium tert-butoxide
(13.46 g, 120 mmol) in 1,4-dioxane (224 mL) at room temperature over
a period of 10 min under nitrogen. The resulting red solution was stirred
at room temperature for 2 h, and then a solution of 5-bromo-1-indanone
(11 g, 52.17 mmol) in 1,4-dioxane (126 mL) was added over a period of
10 min under nitrogen. The resulting solution was stirred at room
temperature for 3 h and then allowed to stand over the weekend. The
reaction mixture was poured into water (500 mL), extracted with EtOAc
(2 × 500 mL), and the organic layer was washed with saturated brine
(300 mL), dried over MgSO4, filtered, and evaporated to afford a black
oil. To the crude oil were added EtOAc and isohexane (1:2, 400 mL) to
give a solid which was collected by filtration and washed with EtOAc and
isohexane (200 mL). The filtrate was evaporated to afford crude product
which was purified by flash silica chromatography (0−5% EtOAc
Step 9. {trans-4-[4-(6-Carbamoyl-5-methylpyrazin-2-yl)phenyl]-
cyclohexyl}acetic Acid (31). Powdered KOH (89 mg, 1.58 mmol) was
added in one portion to methyl {trans-4-[4-(6-carbamoyl-5-methylpyr-
azin-2-yl)phenyl]cyclohexyl}acetate (194 mg, 0.53 mmol) in t-BuOH
(5 mL) at 45 °C . The resulting solution was stirred at 45 °C for 150 min,
and a thick white suspension slowly formed. Then 1 N citric acid (5 mL)
was added. The mixture was evaporated to remove the organic solvent,
and the suspension was filtered and dried to give a white solid. The crude
product was purified by preparative HPLC (Waters XBridge Prep C18
OBD column, 5 μm silica, 50 mm diameter, 150 mm length) (5−95%
MeCN in H2O (containing 0.1% formic acid)) to afford 31 as a white
1
in isohexane) to afford 95 as a yellow oil (9.96 g, 80%). H NMR
(400 MHz, CDCl3) δ 2.64−2.71 (m, 1H), 2.71−2.79 (m, 1H), 2.9−
2.99 (m, 2H), 3.72 (s, 3H), 6.62 (t, J = 2.57 Hz, 1H), 7.10 (d, J = 8.22 Hz,
1H), 7.27−7.35 (m, 2H).
Step 2. 5′-Bromo-2′,3′-dihydro-4H-spiro[cyclohex-2-ene-1,1′-
inden]-4-one (96). Methyl vinyl ketone (2.9 mL, 35.88 mmol) was
added to a stirred mixture of 95 (4.29 g, 17.94 mmol) and p-toluene-
sulfonic acid monohydrate (3.41 g, 17.94 mmol) in toluene (20 mL),
and the resulting mixture was stirred at 100 °C for 6 h and then allowed
to cool to ambient temperature overnight. The mixture was poured into
saturated NaHCO3 (75 mL), extracted with EtOAc (3 × 200 mL), and
1
solid (120 mg, 64%). H NMR (400 MHz, DMSO-d6) δ 0.99−1.14
(m, 2H), 1.37−1.52 (m, 2H), 1.64−1.8 (m, 4H), 2.07 (d, J = 6.90 Hz, 2H),
Q
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Journal of Medicinal Chemistry
Article
the combined organic extracts were dried over MgSO4, filtered, and
evaporated to afford crude product which was purified by flash silica
chromatography (10% EtOAc in isohexane) to afford 96 as a pale yellow
solid (2.78 g, 56%). 1H NMR (400 MHz, DMSO) δ 1.73−1.83 (m, 2H),
2.01 (td, J = 4.54, 13.37 Hz, 2H), 2.16−2.26 (m, 4H), 2.58 (td, J = 6.12,
14.30 Hz, 2H), 2.93 (t, J = 7.32 Hz, 2H), 7.20 (d, J = 8.10 Hz, 1H), 7.28−
7.35 (m, 1H), 7.39−7.43 (m, 1H). EI-MS (m/z) 278, 280 (M+•)
Step 3. 5′-Bromo-2′,3′-dihydro-4H-spiro[cyclohexane-1,1′-
inden]-4-one (97). 10% palladium on carbon (270 mg, 2.54 mmol)
was added to a solution of 96 (2.7 g, 9.74 mmol) in acetic acid (25 mL).
The mixture was evacuated with hydrogen and allowed to stir under an
atmosphere of hydrogen at room temperature for 4 h. The reaction
mixture was diluted with DCM (150 mL) and filtered through Celite,
washing with DCM (150 mL), and the filtrate was evaporated to afford
Step 7. tert-Butyl [trans-5′-(6-Carbamoyl-3,5-dimethylpyrazin-2-
yl)-2′,3′-dihydrospiro[cyclohexane-1,1′-inden]-4-yl]acetate (101).
Acetonitrile (7 mL) was added to 1,1-bis(di-tert-butylphosphino)-
ferrocenepalladium dichloride (0.093 g, 0.16 mmol), and the mixture
was stirred at room temperature for 5 min before addition of potassium
carbonate (0.871 g, 6.30 mmol), water (7 mL), and 100 (1.3 g,
3.15 mmol). After a further 5 min 23 (0.585 g, 3.15 mmol) was added
and the reaction mixture heated to 80 °C for 17 h. The acetonitrile was
evaporated and the resultant suspension partitioned between EtOAc
(50 mL) and brine (50 mL). The organic phase was dried (Na2SO4) and
evaporated to a dark brown oil, which was purified by flash silica
chromatography (elution gradient of 0−60% EtOAc in isohexane) to
afford 101 as a solid (1.26 g, 89%). 1H NMR (400 MHz, DMSO-d6) δ
1.15−1.29 (m, 2H), 1.41 (s, 9H), 1.49−1.58 (m, 2H), 1.6−1.7 (m, 4H),
1.73−1.82 (m, 1H), 1.97 (t, J = 7.36 Hz, 2H), 2.14 (d, J = 6.91 Hz, 2H),
2.57 (s, 3H), 2.73 (s, 3H), 2.89 (t, J = 7.30 Hz, 2H), 7.29 (d, J = 7.86 Hz,
1H), 7.48 (dd, J = 1.62, 7.85 Hz, 1H), 7.52−7.54 (m, 1H), 7.57 (s, 1H),
7.96 (s, 1H). ESI-MS (m/z, %): 450 (MH+, 100). HPLC purity 100%.
Step 8. [trans-5′-(6-Carbamoyl-3,5-dimethylpyrazin-2-yl)-2′,3′-
dihydrospiro[cyclohexane-1,1′-inden]-4-yl]acetic Acid (78). A solu-
tion of 101 (1.16 g, 2.58 mmol) in trifluoroacetic acid (12 mL) was
allowed to stand at room temperature for 30 min. The reaction mixture
was evaporated to a solid which was purified by recrystallization from
MeOH to afford 78 as a white crystalline solid (0.648 g, 64%). 1H NMR
(300 MHz, DMSO-d6) δ 1.12−1.31 (m, 2H), 1.47−1.59 (m, 2H), 1.59−
1.86 (m, 5H), 1.98 (t, J = 7.30 Hz, 2H), 2.17 (d, J = 6.79 Hz, 2H), 2.58 (s,
3H), 2.73 (s, 3H), 2.90 (t, J = 7.26 Hz, 2H), 7.30 (d, J = 7.85 Hz, 1H),
7.50 (d, J = 7.82 Hz, 1H), 7.54 (s, 1H), 7.62 (s, 1H), 8.01 (s, 1H), 12.05
(s, 1H). ESI-MS (m/z, %): 394 (MH+, 100). ESI-HRMS calculated for
C23H28N3O3 (MH+) 394.21252; found, 394.21225. HPLC purity 100%.
1
97 as a yellow solid (2.290 g, 84%). H NMR (400 MHz, CDCl3) δ
1.87−1.96 (m, 3H), 2.04 (td, J = 4.84, 13.39 Hz, 2H), 2.24 (t, J = 7.32 Hz,
2H), 2.4−2.6 (m, 6H), 2.99 (t, J = 7.32 Hz, 2H), 7.00 (d, J = 8.08 Hz, 1H),
7.29−7.34 (m, 2H), 7.38 (d, J = 1.39 Hz, 1H). HPLC purity 93.84%.
Step 4. tert-Butyl (5′-Bromo-2′,3′-dihydro-4H-spiro[cyclohexane-
1,1′-inden]-4-ylidene)acetate (98). To a solution of tert-butyl
diethylphosphonoacetate (8.8 mL, 37.47 mmol) in THF (100 mL)
was added 60% w/w sodium hydride (1.5 g, 37.47 mmol). The reaction
mixture was stirred at room temperature for 30 min. A solution of 97 in
THF (50 mL) was added, and stirring continued at room tempera-
ture for 1 h. The reaction mixture was quenched with saturated brine
(150 mL), extracted with EtOAc (2 × 200 mL), and the organic layer
was dried over Na2SO4, filtered, and evaporated to afford the crude
product as a brown oil (13.7 g). The crude oil was triturated with
isohexane to give a solid which was collected by filtration and dried
1
under vacuum to give 98 as a beige solid (7.26 g, 62%). H NMR
(400 MHz, DMSO-d6) δ 1.49 (s, 9H), 1.6−1.82 (m, 4H), 2.08−2.22 (m,
3H), 2.26−2.48 (m, 2H), 2.95 (t, J = 7.33 Hz, 2H), 3.73 (d, J = 14.39 Hz,
1H), 5.67 (s, 1H), 7.20 (d, J = 8.12 Hz, 1H), 7.32−7.38 (m, 1H), 7.44−
7.46 (m, 1H). HPLC purity 100%.
ASSOCIATED CONTENT
* Supporting Information
Experimental details for the synthesis of compounds 12, 13, 18,
29, 32−59, 61, 73−77, 79−85, 120. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
Step 5. tert-Butyl [5′-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-
yl)-2′,3′-dihydro-4H-spiro[cyclohexane-1,1′-inden]-4-ylidene]-
acetate (99). Bis(pinacolato)diboron (6.11 g, 24.05 mmol), potassium
acetate (5.67 g, 57.72 mmol), and 1,1′-bis(diphenylphosphino)ferro-
cene (0.539 g, 0.96 mmol) were added to a solution of 98 (7.26 g,
19.24 mmol) in dioxane (150 mL). The reaction mixture was degassed
and (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium(II)
(0.791 g, 0.96 mmol) added. The reaction micture was heated to
80 °C overnight. The reaction mixture was allowed to cool and then
evaporated. The residue was suspended in EtOAc and filtered through a
pad of silica gel, washing through with EtOAc. The filtrate was evap-
orated to a brown oil (12g) which was purified by flash silica chromato-
graphy (elution gradient of 0−10% EtOAc in isohexane) to afford 99 as
a solid (5.42 g, 66%). 1H NMR (400 MHz, DMSO-d6) δ 1.27 (s, 12H),
1.42 (s, 9H), 1.6−1.77 (m, 4H), 2.01−2.18 (m, 3H), 2.21−2.42 (m,
2H), 2.87 (t, J = 7.31 Hz, 2H), 3.66 (d, J = 14.43 Hz, 1H), 5.74 (s, 1H),
7.17 (d, J = 7.56 Hz, 1H), 7.46 (d, J = 7.51 Hz, 1H), 7.50 (s, 1H). ESI-MS
(m/z, %): 423 (MH−, 100). HPLC purity 100%.
AUTHOR INFORMATION
Corresponding Author
*Phone: +46-31-7064308. Fax: +46-31-7763700. E-mail: petra.
johannesson@astrazeneca.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Usha Chauhan is thanked for her skillful generation of enzyme
inhibition data. Martin Packer is thanked for computational input
into the design of the pyrazinecarboxamide system.
ABBREVIATIONS USED
■
Step 6. tert-Butyl [trans-5′-(4,4,5,5-Tetramethyl-1,3,2-dioxabor-
olan-2-yl)-2′,3′-dihydrospiro[cyclohexane-1,1′-inden]-4-yl]acetate
(100). To a solution of 99 (5.42 g, 12.77 mmol) in tetrahydrofuran
(125 mL), under nitrogen, was added 10% (w/w) palladium on carbon
(1.359 g, 1.28 mmol). The atmosphere was replaced with hydrogen and
the mixture stirred at room temperature for 17 h. The reaction mixture
was filtered and evaporated to a colorless oil. This was dissolved in
methanol (∼20 mL) and cooled in an dry ice/acetone bath to produce a
white solid. The suspension was allowed to warm and then evaporated
to give the crude product as a white solid. This was recrystallized by
dissolving in DCM (∼20 mL), adding methanol (∼20 mL), and cooling
in a dry ice/acetone bath. The resultant suspension was filtered and the
ACAT, acyl-coenzyme A:cholesterol acyltransferase; b.i.d., “bis
in die” latin for twice a day; CLint, intrinsic clearance; DAG,
diacylglycerol; DIAD, diisopropyl azodicarboxylate; DGAT,
diacylglycerol acyltransferase; DIPEA, N,N-diisopropylethyl-
amine; DMEM, Dulbecco's modified Eagle medium; Dppf,
1,1′-bis(diphenylphosphino)ferrocene; Dtbf, 1,1′-bis(di-tert-
butylphosphino)ferrocene; FCS, fetal calf serum; GLP-1,
glucagon-like peptide 1; HATU, O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; LLE, li-
gand lipophilicity efficiency; log D, logarithm of distribution
coefficient; MDCK, Madin−Darby canine kidney; OLTT, oral
lipid tolerance test; Oxone, potassium peroxomonosulfate;
(PinB)2, bis(pinacolato)diboron; PyBOP, benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate; PyBroP,
bromo-tris-pyrrolidinophosphonium hexafluorophosphate;
1
collected solid dried to give 100 as a white solid (2.290 g, 42%). H
NMR (400 MHz, DMSO-d6) δ 1.19−1.31 (m, 2H), 1.33 (s, 12H), 1.47
(s, 9H), 1.49−1.55 (m, 2H), 1.6−1.75 (m, 4H), 1.76−1.84 (m, 1H),
1.97 (t, J = 7.37 Hz, 2H), 2.19 (d, J = 6.91 Hz, 2H), 2.87 (t, J = 7.31 Hz,
2H), 7.24 (d, J = 7.48 Hz, 1H), 7.51−7.56 (m, 2H). HPLC purity 100%.
R
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