Discovery of trifluoromethyl(pyrimidin-2-yl)azetidine-2-carboxamides as potent, orally bioavailable TGR5 (GPBAR1) agonists: Structure-activity relationships, lead optimization, and chronic in vivo efficacy

Article abstract of DOI:10.1021/jm401731q

Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.

Full text of DOI:10.1021/jm401731q

Article
pubs.acs.org/jmc
Discovery of Trifluoromethyl(pyrimidin-2-yl)azetidine-2-
carboxamides as Potent, Orally Bioavailable TGR5 (GPBAR1)
Agonists: Structure−Activity Relationships, Lead Optimization, and
Chronic In Vivo Efficacy
Dean P. Phillips,*,† Wenqi Gao,† Yang Yang,† Guobao Zhang,† Isabelle K. Lerario,† Thomas L. Lau,†
Jiqing Jiang,† Xia Wang,† Deborah G. Nguyen,‡ B. Ganesh Bhat,§ Carol Trotter,‡ Heather Sullivan,§
Gustav Welzel,§ Jannine Landry,§ Yali Chen,‡ Sean B. Joseph,§ Chun Li,∥ W. Perry Gordon,∥
Wendy Richmond,∥ Kevin Johnson,∥ Angela Bretz,∥ Badry Bursulaya,⊥ Shifeng Pan,† Peter McNamara,§
and H. Martin Seidel
†
‡
§
∥
⊥
Departments of Medicinal Chemistry, Drug Discovery Biology, Pharmacology, Pharmacokinetics, and Structural Biology,
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
ABSTRACT: Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-
protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic
syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen
(HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and
bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective
as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but
this effect was lost upon chronic dosing.
Herein, we report on our efforts to identify systemically
bioavailable agonists and their effects in a chronic disease
model.
INTRODUCTION
■
TGR5 is a Gs-coupled GPCR activated by both primary and
secondary bile acids, particularly taurolithocholic acid. The
pattern of expression and ability to increase cAMP levels are
key determinants of its biological role.1 Activation of TGR5
leads to secretion of glucagon-like peptide-1 (GLP-1) and other
related incretins in gut enteroendocrine cells2 and to decreases
in LPS Th1 cytokines like TNF-α and interleukin-12 (IL-12) in
monocytes/macrophages3 and dendritic cells.4 Therefore,
TGR5 appears to be an attractive target in the treatment of
type 2 diabetes, where strategies to enhance GLP-15 and
mitigate chronic inflammation6 show clinical benefit. The
efforts of several pharmaceutical and biotech companies toward
identifying TGR5 agonists has recently been reviewed,7 with no
known compounds yet reaching the clinic. More recently,
several new compounds have been reported from Hoffman-
LaRoche,8 SIMM,9 and Pfizer10 (Figure 1, compounds 1−4).
CHEMISTRY
■
The goal of the research program was to identify orally
bioavailable TGR5 agonists with activity on the human and
mouse receptors to enable testing in disease-relevant preclinical
animal models. An HTS of 1.5 million compounds was
performed to identify starting points that were active on both
human and mouse TGR5, as demonstrated by their ability to
increase cellular cAMP in stable overexpressed cell lines. The
majority of hits showed preferential activation of the human
receptor, suggesting that the 83% sequence homology between
Received: November 8, 2013
Published: March 25, 2014
© 2014 American Chemical Society
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weight and clogP compared to other hit classes, and its
potential synthetic tractability.
Because 5 was racemic, the initial objective of the hit-to-lead
effort was to determine if the series exhibited any stereo-
chemical preference. The syntheses began with combining the
commercially available acids 6-R and 6-S with phenethylamine
8, followed by Boc-deprotection to give intermediates 9-R and
9-S. Addition to 2-chloropyrimidines 10a and 10b gave 13a−c.
Phenethylamine structure−activity relationships (SAR) were
further expanded by treating 7-R with 10a or 10b, then
activating the acid as the N-hydroxysuccinimide ester (11a or
11b), followed by treating with various phenyethylamines to
yield 13d−g (Scheme 1).
Synthesis of N-Heterocycles. After identifying the
preferred stereochemistry of the piperidine-3-carboxamide
and the substitution of the phenethyl side chain, we turned
our attention toward investigating the SAR of the pyrimidine
ring. The syntheses shown in Scheme 2 began with 9-R or 15,
which was prepared via amide coupling of 14 with 6-R,
followed by Boc-deprotection. Compounds 19a,c−g,i−j were
then prepared via nucleophilic aromatic substitution with the
appropriate chloro-substituted heterocycle. Compound 19b
required a slightly different route that began with nucleophilic
substitution with 9-R on to 17, followed by subsequent
diazotization, conversion into the intermediate chloride, and
Negishi coupling with methylzinc(II) chloride.11 For com-
pound 19h, an alternative procedure was required due to the
incompatibility of the p-cyano group under the Negishi
conditions. Thus, 18 was treated with methyl magnesium-
bromide in the presence of Fe(acac)3, followed by nucleophilic
substitution with 15.12
Figure 1. Reported TGR5 agonists.
human and mouse TGR5 was enough to confer significant
ligand divergence. Furthermore, and perhaps not surprising
given the endogenous ligand preference for lipophilic secondary
bile acids, the majority of the hits were highly lipophilic
compounds that were not ideal to initiate hit-to-lead efforts.
However, nipecotamide 5 (Figure 2) was identified as an
In the next phase of the lead optimization effort, the N-
methylpiperazine moiety was added as a potential solubilizing
group to the methyl- and trifluoromethyl-substituted pyrimi-
dine leads. The syntheses for the target compounds shown in
Scheme 3 depended upon the desired position for the
piperazine moiety relative to the nipecotamide core. Com-
pounds 22a and 22b began with the nucleophilic addition of 20
Figure 2. Profile of screening hit 5.
interesting starting point given its potency on both human
(hTGR5) and mouse (mTGR5) isoforms, moderate molecular
a
Scheme 1. Synthesis of (Pyrimidin-2-yl)piperidine-2-carboxamides
a
Reagents and conditions: (a) w/6-R or 6-S, HATU, DIEA; (b) TFA; (c) DIEA, iPrOH or DMA, 150 °C (d) w/7-R, 10a or 10b, DIEA, iPrOH, 150
°C, EDCl, N-hydroxysuccinimide, DCM; (f) ACN, 25 °C. * denotes stereochemistry.
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a
Next, we turned our attention to the installation of
heterocycle replacements of the phenyl group. The synthesis
shown in Scheme 4 of the pyridine side chain was accomplished
in a one-pot procedure by nickel-assisted sodium borohydride
reduction of nitrile 24 and concomitant Boc-protection.13
Chloropyridine 25 was then subjected to palladium-catalyzed
cyanation and Boc-deprotection to give 26.14 Compound 31a
was prepared in an analogous manner to 23a, from 30 and 21e;
31a was then saponified to give 31b. Amine 26 was coupled to
31b and was followed by nucleophilic aromatic substitution
with 20 to yield 32a. Compound 32b was prepared in a similar
manner, 23a from 1-Boc-piperazine, followed by deprotection.
The synthesis of the pyrimidine isostere was accomplished via
palladium-catalyzed cross-coupling of bromide 27 with
potassium trifluoroborate 28,15 followed by Boc-deprotection
to yield amine 29. The 2-cyanopyrimidine was not stable to the
nucleophilic aromatic substitution conditions, so 31a was first
treated with 1-ethylpiperazine, which was then saponified to
yield the intermediate carboxylic acid and subjected to coupling
with 29 to yield 32c.
Scheme 2. Preparation of N-Heterocycle Analogues
Ring size and position of the amide moiety was also
investigated. Hence, the R and S piperidine-, azetidine-, and
pyrrolidine-2-carboxylic acids were converted into the
respective target compounds via HATU, EDCI, or T3P
coupling with 14 or 26, followed by Boc-deprotection,
nucleophilic substitution on to 21e, and then addition of a
substituted piperazine to yield 34a−f.
a
Reagents and conditions: (a) (6-R, EDCl, HOBt, DMF; (b) 4 M HCl
in dioxane; (c) 9-R or 15, solvent DIEA, heat; (d) solvent, DIEA, heat;
(e) tBuONO, CuCl2, ACN, 0−25 °C; (f) ClZnCH3, 5 mol %
Pd(PPh3)2Cl2, 25 °C; (g) CH3MgBr, Fe(acac)3, THF, 25 °C.
For the final phase of the lead optimization effort, a series of
carboxylic acid-containing analogues were prepared and are
summarized in Scheme 5. For the direct-linked acid analogues,
nucleophilic substitution of 35a and 35b on to the desired
chloropyrimidine intermediate 44. The ether-linked analogues
were derived from Mitsunobu reaction6c with alcohols 36a and
36b and phenols 37a or 37b to give the incipient intermediate,
which was subsequently subjected to Boc- or Cbz-deprotection
to yield piperidines 38a and 38b. The 4-phenylpiperidine
intermediate 41 was synthesized via the Suzuki coupling16 of
pinacolborane 39 with phenylbromide 40, hydrogenation of the
alkene, and Boc-deprotection. The SAR investigation also
included replacing the piperidine ring with a phenyl group. This
was accomplished via conversion of phenylchlorides 42a and
42b into pinacolborane intermediates 43a and 43b with
Pd(dba)2 and PCy3. Nucleophilic aromatic substitution of 44
with 38a, 38b, and 41, followed by saponification, completed
the syntheses of the acid-containing compounds 45a−f. In an
analogous fashion, Suzuki coupling with 43a and 43b, followed
by saponification, yielded 45g and 45h, respectively.
on to dichloropyrimidines 21a and 21b, followed by addition of
piperidine 15 to yield the final compounds. The synthesis of
compound 22c began with the addition of 20 on to 21c and
then methylation via methyl magnesiumbromide and Fe-
(acac)3.12 The intermediate was subsequently treated with 15
to give the final compound. Finally, compounds 22d and 22e
were prepared via nucleophilic aromatic substitutions of 15 on
to 21d or 21e followed by the addition of 20.
A strategy to reduce cLogP in the chemical series involved
various substitutions on the piperazine ring and incorporating
polar heterocycles as replacements for the phenyl group in the
phenethyl side chain. In Scheme 4, piperidine 15 was added on
to pyrimidine 21e to yield the putative intermediate that was
subsequently treated with Boc-piperazine, followed by depro-
tection to give 23a. Subsequently, 23a was subjected to
reductive alkylation with 2-oxetanone to yield 23b.
a
Scheme 3. Preparation of Piperazine-Containing Analogues
a
Reagents and conditions: (a) w/21a (i) DIEA, EtOH, or ACN, 0−25 °C, (ii) 15, DIEA, acetone/H2O, 145 °C; (b) w/21b (i) DIEA, EtOH, or
ACN, 0−25 °C, (ii) 15, DIEA, NMP, 100 °C; (c) w/21c (i) DIEA, EtOH, or ACN, 0−25 °C, (ii) CH3MgBr, Fe(acac)3, THF, 25 °C, (iii) 15, DIEA,
DMF, 145 °C; (d) w/21d and 21e (i) 15, DIEA, iPrOH, 0−25 °C, (ii) 20, ACN, 100 °C.
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a
Scheme 4. Preparation of Core, Pyridylethyl, and Pyrimidylethyl Analogues
a
Reagents and conditions: (a) (i) ACN, DIEA, 0−25 °C, (ii) 1-Boc-piperazine, ACN, DIEA, 165 °C, (iii) 4 M HCl; (b) 2-oxetanone, NaBH(OAc)3;
(c) NaBH4, NiCl2−6H2O; (d) Boc2O; (e) Pd(TFA)2, TrixiePhos, Zn(CN)2, Zn; (f) 4 M HCl; (g) Pd(OAc)2, RuPhos, Cs2CO3, 95 °C; (h) TFA; (i)
2-propanol, DIEA, 0−25 °C; (j) LiOH; (k) w/31b (1) 26, HATU, (2) 20, DMSO, 60 °C; (l) w/31a (1) 1-ethylpiperazine, 2-propanol, 80 °C, (2)
LiOH, (3) 29, HATU; (m) EDCI, HOBt, DIEA, DMF; (n) HATU, DIEA, DMF; (o) T3P; (p) R1-piperazine, ACN or 2-propanol, heat.
The data in Table 2 show a strong preference for the
(pyrimidin-2-yl)piperidine-3-carboxamide configuration.
Among the 2-yl analogues, monosubstitution of the pyrimidine
at the 5-position with fluorine (19a) was comparable to 13c.
Positing that the combination of the 5-fluoro and 4-methyl
substituents could be additive led to 19b, which showed
improved potency by 10-fold and 4-fold on hTGR5 and
mTGR5, respectively. By comparison, the pyrimidin-4-yl
derivatives 19d and 19e were significantly less potent than
their 2-yl congeners 13a and 13c.
We then turned our attention toward surveying substituted
pyridines as replacements to the pyrimidine. While these
compounds were generally less potent than their pyrimidine
congeners, they did provide some insight into the potential
binding mode for this series. First, the preference for the methyl
substituent to be adjacent to the nitrogen on both heterocycles
appeared to be important for gaining activity. Collectively, the
matched pairs 13a:13c, 19c:19f, and 19d:19e, yielded 6-, 4-,
and 6-fold improvements, respectively, on hTGR5 when the
methyl substituent was present adjacent to the ring nitrogen. By
contrast, when the methyl substituent was located at the 4-
position on the pyridine ring (19g), the compound was less
RESULTS AND DISCUSSION
■
The percent efficacy for all compounds in this work were
determined relative to a previously disclosed internal control
that was found to be a potent and species cross-selective full
agonist.17 At the outset hit-to-lead effort, a clear preference for
the R-enantiomer (13a) over the S-enantiomer (13b) was
demonstrated, therefore all subsequent analogues in the
nipecotamide series were made exclusively with this stereo-
chemistry (Table 1). The addition of a methyl group to the
pyrimidine ring at the 4-position (13c) yielded a ∼6-fold
improvement in hTGR5 potency. The methyl-substituted
pyrimidine was then employed as the heterocycle of choice
to probe the SAR of the phenethyl amide region. The results
showed that 4-Cl (13c) and 4-Br (13f) were preferred over 4-F
(13e). Quite strikingly, nearly complete ablation of activity was
observed by removal of the p-substituent (13d) and a loss of
approximately 5-fold was observed by replacing the 4-Cl of 13a
with a 4-CH3 (13g), suggesting perhaps that both lipophilicity
and electronics play a key role in the activity in this series.
Additional substitution patterns were also investigated, but p-
substitution was strongly preferred and no clear advantage from
substitution at other ring positions were observed.
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a
Scheme 5. Preparation of Acid-Containing Analogues
a
Reagents and conditions: (a) PPh3, DIAD, THF; (b) w/36a 4 M HCl, w/36b H2, Pd/C, EtOAc; (c) Pd(PPh3)4, NaHCO3, dioxane; (d) H2, Pd/C;
(e) 4 M HCl/dioxane; (f) Pd(dba)2, PCy3, KOAc, dioxane; (g) 35a, 35b, 38a, 38b, or 41, NMP, DIEA, 140 °C; (h) 43a or 43b, Pd2(dba)3, 5 M
K3PO4, SPhos, dioxane; (i) 3 M LiOH.
selective for mTGR5 than the (pyrimidin-2-yl)piperidine-3-
carboxamide subtype; this was particularly evident with the 4-
(trifluoromethyl)pyrimidine (19i) and 2(trifluoromethyl)-pyr-
imdine (19j) derivatives. Compounds 19i and 19j showed
selectivity for hTGR5 and mTGR5 that was similar to their
methyl counterparts 13c and 19e, respectively. While 19j was
weaker by approximately 2-fold on hTGR5 than 13c, the nearly
6-fold improvement on mTGR5 was a desirable property that
we were optimizing for as well. An important distinction for
both 19i and 19j was the replacement of the 4-chloro group
with a nitrile, R1 = CN. This modification provided a nearly
equipotent (19h vs 19b) alternative to the R1 = Cl analogues,
but with lower cLogP. Moreover, the 4-trifluoromethyl (19i)
and 2-trifluoromethyl (19j) pyrimidine substitution provided
new optimization nodes with good hTGR5 and mTGR5 cAMP
activity from which to explore further. While good potency and
reliable SAR were achievable in the series to this point,
compounds with acceptable solubility and metabolic stability
remained elusive.
Table 1. Initial SAR of Head, Tail, and Core Stereochemistry
a
cAMP EC50 (μM)
no.
R1
R2
Cl
hTGR5
mTGR5
b
5
H
0.88 23
0.35 12
>80
1.29 32
0.28 16
>80
c
13a
13b
H
Cl
Cl
Cl
H
d
H
c
13c
CH3
CH3
CH3
CH3
H
0.06 0.01
5.8 1.8
0.14 0.02
26.4 8.9
0.75 0.15
0.088 0.03
2.17 0.55
c
13d
c
13e
F
0.32 0.06
0.033 0.002
4.3 0.68
c
13f
13g
Br
CH3
c
a
EC50 values are the average of at least two assay runs performed in
triplicate SD; unless otherwise noted, all compounds are 100% full
agonists relative to an internal control (see Materials and Methods).
b
c
d
Racemic. R-Enantiomer. S-Enantiomer.
The SAR for compounds 22a−e in Table 3 clearly showed a
preference for the trifluoromethyl substituent, especially in
combination with the piperazine group. Both 22b and 22e
provided leads with low single-digit nanomolar potency on
hTGR5 and mTGR5. All of the compounds in Table 3 were
assayed in a high-throughput solubility assay at pH 6.8, and
only 22b showed poor solubility. The stronger cytochrome
P450 3A4 (Cyp3A4) inhibition of 22b and 22e compared to
the methyl counterparts (22a and 22d, respectively) became a
focus of the optimization campaign. While compound 22c also
demonstrated excellent potency compared to the CF3-
analogues and weaker Cyp3A4 inhibition, this compound had
poorer microsomal stability than 22e and was therefore
deprioritized. Additionally, all of the compounds in this series
potent by 15-fold and 35-fold on hTGR5 and mTGR5,
respectively, compared to 13c. Compound 19g was also 4-fold
and 7-fold less potent on hTGR5 and mTGR5 compared to
pyridine 19f. It seemed that retaining the methyl group
adjacent to the nitrogen was key for maintaining favorable
activity on both receptors.
When considering the mTGR5 SAR closely, it was apparent
that compounds having a heterocycle ring nitrogen on the
opposite face (N3) of the piperidine-3-carboxamide improved
mTGR5 selectivity but at the expense of hTGR5 potency. SAR
analysis of compounds 19e, 19f, and 13c confirmed that the
(pyrimidin-4-yl)piperidine-3-carboxamide subtype was more
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Table 2. N-Heteroaryl SAR
a
EC50 values are the average of at least two assay runs performed in triplicate SD; unless otherwise noted, all compounds are 100% full agonists
relative to an internal control (see Materials and Methods).
Table 3. SAR of Piperazine-Containing Analogues
a
cAMP EC50 (μM)
b,
c
c
c
no.
R
X
Y
hTGR5
mTGR5
solubility (μM)
Cyp3A4 IC50 (μM)
hERG IC50 (μM)
cLogP
22a
22b
22c
22d
22e
CH3
CF3
CH3
CH3
CF3
CH
CH
CF
N
N
0.076 0.031
0.002 0.0004
0.004 0.003
0.44 0.12
0.19 0.11
0.003 0.001
0.011 0.005
0.17 0.034
0.001 0.0006
>175
6.9
>25
2
3.1
2.1
2.7
3.1
2.8
2.7
3.1
N
N
110
7.6
>25
<1
CH
CH
>175
169
N
0.003 0.001
3.2
a
EC50 values are the average of at least two assay runs performed in triplicate SD; unless otherwise noted, all compounds are 100% full agonists
b
c
relative to an internal control (see Materials and Methods). High throughput thermodynamic solubility at pH 6.8. Data from single assay run.
showed moderate to strong hERG inhibition, which also
needed to be addressed in the next phase of the optimization.
As summarized in Table 4, compound 22e was a potent full
activator of functional cellular responses, including GLP-1
secretion from the murine GLUTag cell line (EC50 = 256 nM,
106% efficacy versus control) and inhibition of TNFα release
from human peripheral blood mononuclear cells (PBMC)
(IC50 = 92 nM, 89% efficacy versus control). In mouse, 22e
showed moderate oral bioavailability (24%), which made it an
acceptable tool compound for early stage in vivo studies and
additional profiling activities. Additional profiling showed that
22e had high intrinsic clearance in microsomes for all species
tested, especially rat. In rat, the compound exhibited poor
bioavailability and high clearance (data not shown), consistent
with the rat liver microsome (RLM) results, so the in vivo
models were limited to the mouse unless high doses of 22e
were employed.
Extensive metabolite identification was performed in micro-
somes with 22e, and the results suggested that the piperidine
core, piperazine N-methyl, and phenethyl side chains were
prone to oxidative metabolism. Unfortunately, early exploration
of the piperidine core and phenethyl side chain proved to have
particularly narrow SAR with no suitable alternatives identified
at the time. Importantly, removal of the N-methyl substituent
yielded compound 23a (Table 5), which retained hTGR5 and
mTGR5 activity and had improved intrinsic clearance (33 μL/
min/mg) in mouse liver microsomes (MLM). Unfortunately,
Cyp3A4 and hERG inhibition were not improved with this
modification. It was clear after a survey of several alkyl
substituents on the piperazine that the N-alkyl group
contributed significantly to the poor stability in the series.
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Interestingly, however, the hERG risk (>30 μM) could be
mitigated with the N-oxetanyl group (23b)18 and still retain
target potency; however, Cyp3A4 inhibition and metabolic
stability in MLMs (138 μL/min/mg) were not improved, so
additional strategies needed to be explored.
Table 4. In Vitro Activity, ADME, and PK profiles of 22e
target functional assays (μM, % efficacy)
GLP-1 secretion GLUTag
0.296 0.046 (n = 202), 116
0.090 0.008 (n = 26), 86
PBMC LPS TNFα
Microsomal Stability (μL/min/mg)
It has been reported that compounds with a basic functional
group and molecular weight >400 and cLogP > 4 are more
likely to exhibit above average Cyp3A4 inhibition and high
clearance,19 which compound 22e and related analogues
resemble. The introduction of the trifluoromethyl group placed
this series in a molecular weight (450−550) band that proved
to be challenging for multiparameter optimization, particularly
because analogues with cLogP < 4 did not significantly improve
the ADME properties. Therefore, an SAR strategy to alter the
structures and lower cLogP further was pursued in order to
identify compounds with ADME properties necessary to
progress into our preclinical animal models.
Several strategies toward reducing cLogP were considered
with the chemical series. One strategy probed replacing the side
chain phenyl ring with heterocyclic isosteres. The nitrile was
retained as a substituent because it generally provided potent
compounds while also maintaining lower cLogP than other
substituents. The pyridine and pyrimidine analogues 32a and
32c have cLogPs lower than 22e at 2.0 and 1.5, respectively,
mouse
human
rat
214
289
>500
Cyp Inhibition (μM)
3A4
2C9
2D6
<1
4.7
>10
Off-Target Activity
hERG (patch clamp)
IC50 = 6.7 μM
>10 μM
Safety Pharm panel (110 assays)
mouse pharmacokinetics
IV (5)
route (dose mg/kg)
AUC (h·nM)
CL (mL/min/kg)
Vss (L/kg)
PO (20)
2540
2667
62
1.6
Cmax (nM)
4521
0.36
1067
1.35
24
t1/2 (h)
F (%)
Table 5. SAR of Tail and Carboxamide Analogues
a
EC50 values are the average of at least two assay runs performed in triplicate SD; unless otherwise noted, all compounds are 100% full agonists
b
relative to an internal control (see Materials and Methods). Data from single assay run.
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which likely contributed to the reduced Cyp3A4 and hERG
inhibition in both cases; however, the switch to these moieties
reduced target potency on hTGR5 and mTGR5 by >20-fold.
Interestingly, despite the low cLogP (1.4) of the desmethyl
compound 32b, it was a highly potent Cyp3A4 inhibitor, like
the phenyl analogue 23a. Molecular docking studies with a
model of Cyp3A4 provided evidence that 22e is anchored into
the active site of the enzyme via an hydrogen bond between the
amide carbonyl and S119 and the CF3 group fits into a
hydrophobic pocket formed from F108, F213, F220, and F241
(Figure 3). The piperazine nitrogen of 22e also engages E308
in a salt bridge, which may explain why the desmethyl
piperazine analogues 23a and 32b were more potent Cyp3A4
inhibitors.
with several challenges. One caveat of carboxylic acids is that
they generally have high plasma protein binding,22 so high
plasma exposure may need to be attained in order to overcome
the potentially low free concentration of active compound.23
The first compound that was made in this series was
isonipecotic acid derivative 45a (Table 6). While not quite as
potent as 22e, it showed no Cyp inhibition and had good MLM
stability (CLint 16 μL/min/mg), consistent with the attractive
features of acids outlined above. Unfortunately, 45a had low
plasma exposure. In Caco-2 cells, 45a showed potential for
efflux with Papp (A−B) of 1.36 × 10−6 cm/s and Papp (B−A) of
12.96 × 10−6cm/s and B−A/A−B ratio of 9.53. The relatively
unhindered nature of the acid and low calculated log D (cLogD
0.9 at pH 7.4) for a high molecular weight carboxylic acid were
considered to be possible contributors to the poor permeability.
To address this, a methyl group was installed adjacent to the
carboxylic acid to yield 45b. This compound showed similar
potency to 45a while still exhibited similar Cyp inhibition and
metabolic profiles to its predecessor. Moreover, 45b showed a
nearly 10-fold improvement of plasma exposure but had nearly
identical effects in Caco-2 cells (A−B 1.02 × 10−6 cm/s and B−
A 13.51 × 10−6 cm/s) to 45a. Nevertheless, the exposure for
this compound was too low to progress into in vivo studies.
Balancing the moderately high molecular weight (>500) with
the proper level of lipophilicity (cLogD) were important design
elements for the carboxylic acid analogues. A thorough analysis
of our data set revealed that absorption and oral exposure might
be improved by increasing lipophilicity (e.g., cLogD at pH 7.4).
Therefore, the 4-position of the piperidine ring was first
explored by adding a phenyl ring via an ether or direct carbon−
carbon bond (Table 6). Compounds 45c and 45d showed good
potency and species cross-reactivity. In accordance with our
hypothesis, both compounds showed improved plasma
exposure (area under curve (AUC) 5018 h·nM and 14221 h·
nM, respectively) in the mouse using a snapshot PK study
protocol.24 More importantly, the azetidine analogue showed
high plasma exposure in the mouse, perhaps due to a lower
potential for efflux (A−B 5.73 cm/s and B−A 6.79 cm/s for
45d vs A−B 2.12 and 6.4 cm/s for 45c) and high parallel
artificial membrane permeability assay (PAMPA) bioavailability
(89%). While both of these compounds had slightly higher
intrinsic clearance in MLM (CLint 46 and 53 μL/min/mg,
respectively) than 45a and 45b, their improved plasma
exposure was likely due to improved intestinal absorption,
perhaps due to a more compact conformation. Direct
attachment of the phenyl ring bearing the acid to the piperidine
led to 45f, which was potent on both hTGR5 and mTGR5 and
had good MLM stability (CLint 14 μL/min/mg) but
unfortunately showed only moderate exposure (AUC 3742 h·
nM) relative to the other acid-containing analogues. In Caco-2
cells, 45f showed efflux potential with A−B of 2.7 cm/s and B−
A of 12.9 cm/s (B−A/A−B 4.7). Overall, because the
properties of the azetidine-2-carboxamide appeared to be
superior to the piperidine congener, subsequent analogues
employed the former as the key segment.
Figure 3. Compound 22e docked into the Cyp3A4 active site.
The next series of compounds targeted replacement of the
piperidine-3-carboxamide with progressively smaller and less
lipophilic rings (34a−34f). Generally, the 2-substituted
piperidine, pyrrolidine, and azetidine cores yielded compounds
with acceptable target potency and, in some cases, improved in
vitro ADME profiles. Stereochemical bias was observed in this
series of compounds, and in all cases the S-enantiomer was
preferred. Serendipitously, the more active S-enantiomer
derivatives 34b and 34d were about 3-fold less potent as a
Cyp3A4 inhibitors than the R-enantiomers 34c and 34e,
respectively. Azetidines 34d and 34f showed promising target
potency and solid evidence that Cyp3A4 and hERG inhibition
could be mitigated by strategically reducing lipophilicity in this
series. While this proved to be an effective strategy toward
identifying compounds with good target selectivity, this series
of molecules still suffered from poor microsomal stability and
poor in vivo plasma exposure. Therefore, to obtain compounds
with the desired balance of potency and systemic exposure, an
alternative strategy was explored.
Several functional groups were considered as replacements
for the N-alkylpiperazine group. The tactic that we chose was to
replace the basic functional group with a carboxylic acid.
Because carboxylic acids are polar and ionized at physiological
pH, they generally show better metabolic stability than basic
and neutral compounds. Moreover, they also have the added
benefits of being poor substrates for the hERG channel and
much weaker Cyp inhibitors.20 These general properties of
acids made them an attractive functional group21 to explore,
especially because the basic and neutral analogues presented us
For compounds 45e−45h, plasma AUC correlated well with
increasing cLogD. Ultimately, this effort led to the identification
of compounds 45g and 45h as potent, cross-selective agonists
having good MLM stability and excellent plasma exposure, with
45h providing a slightly more potent agonist on hTGR5;
therefore, 45h was chosen for more extensive profiling. (Table
7) Compound 45h was potent in the GLP-1 and PBMC
functional assays, showed no Cyp or hERG inhibition, and was
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Table 6. SAR of Acid-Containing Analogues
a
EC50 values are the average of at least two assay runs performed in triplicate SD; unless otherwise noted, all compounds are 100% full agonists
b
c
relative to an internal control (see Materials and Methods). CLint μL/min/mg, data from single assay run. Snapshot PK (0−5 h, pooled samples),
(20 mg/kg) AUC (h·nM).
Adult mice were deprived of food overnight and dosed orally
with either vehicle, a control (compound 22e), or 45h 30 min
prior to an oral glucose bolus (Figure 4). Both compound 22e
and 45h produced a robust GLP-1 increase, with 45h showing a
near maximal effect at 3 mg/kg. This encouraging result
prompted us to test 45h in chronic disease models, particularly
Table 7. In Vitro Activity, ADME and PK Profile of 45h
target functional assays (μM, % efficacy)
GLP-1 secretion GLUTag
PBMC LPS TNFα
0.046 0.006 (n = 10), 80
0.215 0.06 (n = 3), 99
FXR
>10 μM
All >25
IC50 >30 μM
2.5
Cyp inhibition (μM) 3A4, 2C9, 2D6
hERG (patch clamp)
log D pH 6.8
% plasma protein binding (human)
99.9
mouse pharmacokinetics
IV (5)
route dose (mg/kg)
AUC (h·nM)
CL (mL/min/kg)
Vss (L/kg)
PO (20)
53750
13925
10.4
1.31
Cmax (nM)
12002
4.14
9750
11.7
96.5
t1/2 (h)
F (%)
nearly completely bioavailable in mouse. In a safety panel
screen of 20 receptors, 45h showed only moderate inhibition of
human phosphodiesterase type 4D (hPDE4D) of 4.6 μM, while
all other receptors tested were >30 μM. This compound was
then taken forward in a chronic in vivo study.
The effects of compound 45h on the secretion of total GLP-
1 levels in wild-type C57BL/6 male mice was determined as an
in vivo screening method prior to testing in a chronic model.
Figure 4. Effect of 22e and 45h on in vivo GLP-1 levels in C57/Bl6
mice.
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Figure 5. Efficacy and pharmacokinetics of 45h in 5-week ob/ob study at 30 mg/kg qd.
an examination of the effect on glucose excursion in an oral
glucose tolerance test (OGTT).
CONCLUSION
■
In conclusion, extensive lead optimization was carried out from
an HTS hit (5), which ultimately led to the potent, species
cross-selective compound 45h. Upon leading up to the
selection of testing compound 45h in acute and chronic
disease models of obesity, we screened several of the lead
compounds mentioned in this manuscript for their ability to
produce robust GLP-1 increases in nondiseased C57 mice as
well as for their ability to decrease glucose levels in OGTT
experiments in nondiseased and diseased mice (diet-induced
obese, ob/ob, and db/db). Compound 45h met all of our
selection criteria for testing in chronic disease models in which
a modest effect on reducing peak glucose levels in ob/ob mice
was observed that was equivalent to a DPP4 inhibitor on day 1
(not statistically significant). However, the effect with 45h was
lost upon chronic dosing despite nearly equivalent compound
exposure at the beginning and end of the study and was
confirmed in a second study with the compound. Recently,
sustained TGR5 agonism has been reported to cause elevated
heart rate and blood pressure in the dog, which would likely
limit the applicability of a systemic agonist to treat metabolic
disease.10a,c,26 Moreover, concern has been raised about the
effects of TGR5 activation on gall bladder function.27 Indeed,
pilot studies in dogs with compound 22e led to changes in gall
bladder contractility and heart rate (Nguyen et al., manuscript
in preparation). Taken together, there would seem to be a
number of toxicological and therapeutic issues that limit the
Genetically obese (ob/ob) mice have elevated basal glucose
levels and possess an inflammatory cytokine profile that has
been shown to be comparable to the human disease. During a
series of pilot studies with 22e, we found that the compound
provided a similar increase of GLP-1 levels in the ob/ob model
as it did in wild types (data not shown). This result, combined
with the robust GLP-1 effect of 45h in the wild type, justified
testing the compound in ob/ob mice for 5 weeks to determine
if chronic TGR5 agonism would improve glucose disposal and
reduce inflammatory cytokine levels, the two major purported
effects of receptor activation. For this chronic study, a 30 mg/
kg dose of 45h was chosen in order to maximize the coverage
of the compound during the course of the study. Interestingly,
an OGTT on day 1 showed nearly equivalent efficacy to the
DPP4 inhibitor PKF275.25 However, after 26 days of dosing qd
with 45h, the efficacy in the OGTT was ablated (Figure 5). In
addition to a lack of robust efficacy on glycemic control, 45h
showed no significant change in insulin levels or inflammatory
cytokines in white adipose tissue (not shown). Importantly,
45h maintained plasma exposure throughout the dosing period,
showing equivalent exposure on day 1 and day 37. Therefore, it
is possible that the lack of efficacy may be due to receptor
internalization and down-regulation leading to tachyphylaxis.
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PBMC LPS Assay. Peripheral blood mononuclear cells (PBMC)
were isolated from healthy donors using Ficoll Hypaque per the
manufacturer’s recommendation (GE Healthcare). Cells were seeded
at 30000 cells/10 μL/well in a 384-well proxiplate (Perkin-Elmer) in
AIM V media (Life Technologies) supplemented with 3% FBS
(Hyclone) and 1× Pen/Strep/Glutamine. Following an overnight
incubation at 37 °C, 50 nL of compound in DMSO was added per well
by Pintool (GNF systems). Plates were incubated for 30 min at 37 °C,
followed by the addition of 2 μL/well of 6× LPS (EC80 final,
determined during LPS dose titration for each donor) and incubation
overnight at 37 °C. To detect TNFα secretion by HTRF, 2.5 μL/well
anti-TNFα cryptate, then 2.5 μL/well of anti-TNFα XL665 were
added (Cisbio) and subsequently incubated for 3 h at rt. The
emissions at 620 nM and 665 nM were read on the Envision (Perkin-
Elmer) as suggested by the manufacturer. Data was expressed as a ratio
of emission at 665/620 and subsequently normalized to a DMSO
alone control, and the percent inhibition was then determined based
on N-(3,5-dichlorophenyl)-N-(1-(2-methoxyphenyl)ethyl)-3-methyli-
sonicotinamide.17 Dose−response data points were curve fitted using
the standard logistic regression model, implemented with an in-house
developed Matlab (mathworks.com) program. The resultant IC50
values correspond to the compound concentration that inhibited
activity by 50%.
Animal Studies. All aspects of animal care, use, and welfare for all
animals used for in vivo studies were performed in compliance with
U.S. Department of Agriculture regulations and the National Institutes
of Health Guide for the Care and Use of Laboratory Animals.
Furthermore, all animals were handled in accordance with Novartis
Animal Care and Use Committee protocols and regulations. For
mouse studies, animals were obtained from the Jackson laboratories
(C57BL/6) and Harlan (ob/ob).
Mouse in Vivo GLP-1 Secretion Assay. Male C57BL/6 mice were
fasted overnight and then dosed orally with 10, 30, or 100 mg/kg of
22e. After 30 min, animals were challenged with 3 g/kg oral dextrose
(Sigma). For assessment of GLP-1 secretion, plasma was collected at 2
min postchallenge into EDTA coated tubes containing 8 μL of a DPP4
inhibitor solution (EMD Millipore) and GLP-1 levels were measured
by ELISA according to the manufacturer’s instructions (Meso Scale
Discovery). Data was analyzed using one-way ANOVA followed by
Dunnett’s post-test in Graphpad Prism. Values of p < 0.050 were
considered statistically significant. Data shown is the average SEM of
eight animals.
utility of systemic TGR5 agonists for treatment of metabolic
disease.
MATERIALS AND METHODS
■
Experimental Protocols (Biology). TGR5 Cellular cAMP Assay.
Jurkat cells overexpressing human or mouse TGR5 were seeded at
50000 cells/25 μL/well in 384-well plates (Greiner) in AIM V media
(Life Technologies) supplemented with 1 mM IBMX (Sigma) and 10
mM HEPES. Immediately after plating, 200 nL of compounds in
DMSO were transferred using the Pintool (GNF systems). Following
a 1 h at 37 °C, cryptate and D2 conjugates from the cAMP HiRange
HTRF kit (Cisbio) were added (12.5 μL each). Plates were then
incubated 1 h at rt and emissions at 620 nM and 665 nM were read on
the Envision (Perkin-Elmer) as suggested by the manufacturer. Data
was expressed as a ratio of emission at 665/620 and then normalized
to a DMSO alone control. Dose−response data points were curve
fitted using the standard logistic regression model, implemented with
an in-house developed Matlab (mathworks.com) program. The
percent efficacy was then determined based on N-(3,5-dichlorophen-
yl)-N-(1-(2-methoxyphenyl)ethyl)-3-methylisonicotinamide17 as the
internal control. The resultant EC50 values correspond to the
compound concentration that displaces 50% of the binding of the
d2-labeled cAMP.
FXR Reporter Gene Assay. 293T cells stably expressing GAL4-
luciferase-FXR receptor were maintained in Dulbecco’s Modified
Eagles Medium (Hyclone) containing 10% fetal bovine serum
(Sigma), 1% penicillin/streptomycin (Hyclone), and 200 μg/mL
hygromycin (Hyclone). Cells were detached and seeded into 384-well
white plates (Greiner Bio-one) at 4000 cells/50 μL/well in Dulbecco’s
Modified Eagles Medium (Hyclone) containing 3% charcoal/dextran
treated fetal bovine serum (Sigma) and 1% penicillin/streptomycin.
Following 24 h incubation at 37 °C, 50 nL of 1000× compound
diluted in DMSO was transferred by Pintool (GNF Systems) into the
assay plate and incubated for 24 h at 37 °C. Then 25 μL of Bright Glo
(Promega) was added per well and flash luminescence was detected on
the Viewlux (PerkinElmer Life Sciences). Dose−response data points
were curve fitted using the standard logistic regression model,
implemented with an in-house developed Matlab (mathworks.com)
program. The resultant IC50 values correspond to the compound
concentration that inhibited activity by 50%.
GLUTag GLP-1 Secretion Assay. Murine GLUTag cells were
maintained in Dulbecco’s Modified Eagles Medium (Hyclone)
containing 10% fetal bovine serum and 1% penicillin/streptomycin.
On day one of the assay, cells from a confluent flask were detached
using 0.25% trypsin (Life Technologies) and seeded in 96-well poly-D-
lysine plates (BD Biosciences) at 42000 cells/150 μL/well in
maintenance medium. After incubation at 37 °C overnight, medium
was changed to phenol red free DMEM with low glucose (Sigma) and
then incubated for another 24 h at 37 °C. GLUTag cells were then
washed and incubated in glucose free EBSS buffer with 0.1% BSA
(Sigma) at 37 °C to starve, followed by two additional EBSS buffer
washes. Following the last wash, EBSS buffer containing 15 mM
glucose, DPP4 inhibitor PKF275 (600 nM final concentration28), and
agonist compounds was added to the cells and incubated for another 2
h at 37 °C to allow for GLP-1 secretion. Then 6 μL of supernatant was
transferred to HEK293 cells stably expressing a GLP-1 luciferase
reporter construct, seeding the morning of GLUTag cell treatment at
12000 cells/40 μL/well in a 384-well plate (Greiner). After overnight
incubation, 15 μL of 25% Bright Glo (Promega) was added to the
reporter cells, incubated at rt for 2 min, and luminescence was then
read on Envision per the manufacturer’s instructions (Perkin-Elmer).
Data were normalized to a DMSO alone control, and the percent
efficacy was then determined based on N-(3,5-dichlorophenyl)-N-(1-
(2-methoxyphenyl)ethyl)-3-methylisonicotinamide.17 Dose−response
data points were curve fitted using the standard logistic regression
model, implemented with an in-house developed Matlab (math-
works.com) program. The resultant EC50 values correspond to the
compound concentration that induced half-maximal activity.
4-Week ob/ob Study Oral Glucose Tolerance Test (OGTT). Male
ob/ob mice (Harlan) were dosed once-daily (SID) for 4 weeks with
vehicle, PKF275 30 mg/kg, or 45h 30 mg/kg as the N-methyl-D-
glucamine salt. On day 1 and after 4 weeks, mice were overnight fasted
then orally dosed with vehicle or compound. After 30 min, all mice
were orally challenged with 1 g/kg dextrose (Sigma) and blood
glucose measured via tail tip by hand-held glucometer (Embrace).
Blood glucose was measured predose, preglucose dose, and 20, 40, 60,
and 120 min postglucose challenge. AUC was calculated and data
analyzed using one-way ANOVA followed by Dunnett’s post-test in
Graphpad Prism. Values of p < 0.050 were considered statistically
significant. Data shown is the average SEM of eight animals.
Experimental Protocols (Metabolite Identification). In vitro
incubations in liver microsomes from mouse, rat, dog, monkey, and
human were used. Stock solutions of trifluoromethyl(pyrimidin-2-
yl)azetidine-2-carboxamide analogues (TGR5 agonists) at 10 mmol/L
were prepared in DMSO. For the characterization of in vitro
metabolites, compounds were incubated at 37 °C for 60 min with
liver microsomes from mouse, rat, dog, monkey, and human in
individual incubations. All incubations were conducted on the same
date using the same batch of compound material. A 4 μL stock
solution (0.5 mM in ACN/water) of TGR5 agonists were added to an
enzyme reaction mixture (50 μL liver microsomes, containing 1 mg
protein/mL with phosphate buffer) and preincubated for 3 min at 37
°C. After preincubation, the final reaction was started by addition of 46
μL of the NADPH-regenerating system or containing NADPH
cofactors. After 60 min, the reaction was stopped with 200 μL of
ice-cold ACN. The reaction mixture was stored at −20 °C. The 20 μM
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concentration is used for incubation because the detection of
metabolites is very challenging at 1 μM initial substrate levels.
Prior to use, the mixture was centrifuged (10000g, 5 min) and 200
μL of supernatant was used without prior evaporation and transferred
to a clean 96-well plate. From these samples, 5 μL aliquots were used
for analysis.
Samples were then analyzed by HPLC-MS/MS. High resolution
chromatography was performed using the Acquity UPLC and a 1.0
mm × 50 mm C18 RP column (1.8 μm particles). The mobile phases
consisted of 0.1% formic acid in water for buffer A and 0.1% formic
acid in ACN for buffer B. A standard binary gradient was used for
reverse phase chromatography.
irradiated by microwave at 150 °C for 30 min. Upon completion of the
reaction, the mixture was cooled to rt and diluted with water (2 mL),
precipitate was collected by filtration, and the title compound was
obtained as a white solid (50 mg, 29%). 1H NMR (400 MHz, CDCl3)
δ 8.23 (d, J = 4.7, 2H), 7.22−7.16 (m, 2H), 7.02 (d, J = 8.4, 2H), 6.44
(t, J = 4.7, 1H), 6.28 (s, 1H), 4.07 (dd, J = 3.7, 13.8, 1H), 3.88 (s, 1H),
3.76 (dd, J = 7.4, 13.8, 1H), 3.62−3.33 (m, 4H), 2.71 (dd, J = 6.8, 12.5,
2H), 2.40−2.27 (m, 1H), 2.16−2.00 (m, 1H), 1.81 (s, 1H). HPLC-MS
C18H21ClN4O (M + H+): 345.1, 6.91 min. The S-enantiomer 13b was
synthesized in an identical manner starting from (S)-1-Boc-nipecotic
acid and has spectral data that matches 13a.
(R)-N-(4-Chlorophenethyl)-1-(4-methylpyrimidin-2-yl)piperidine-
3-carboxamide (13c). To a microwave reaction tube was added (R)-
N-(4-chlorophenethyl)piperidine-3-carboxamide (120 mg, 0.33
mmol), 2-propanol (1.2 mL), DIEA (122 μL, 0.7 mmol), and 2-
chloro-4-methylpyrimidine (10b). The vessel was sealed and heated to
150 °C for 30 min. The solvent removed en vacuo, and the residue
was purified by flash chromatography (12 g ISCO, 0−10% methanol/
DCM) to yield a pale-yellow oil, which was then repurified by
preparative HPLC to give 5 mg (4%) of the title compound. 1H NMR
(400 MHz, CDCl3) δ 8.03 (d, J = 5.0, 1H), 7.13−7.10 (m, 2H), 6.94
(dd, J = 2.3, 8.8, 2H), 6.56 (s, 1H), 6.27 (d, J = 5.0, 1H), 3.88 (d, J =
5.0, 2H), 3.71−3.64 (m, 2H), 3.50 (td, J = 6.6, 13.2, 1H), 3.38−3.25
(m, 1H), 2.74−2.59 (m, 2H), 2.37−2.27 (m, 1H), 2.22 (s, 3H), 2.08
(td, J = 6.4, 13.1, 1H), 1.79−1.70 (m, 1H), 1.51−1.45 (m, 2H).
HPLC-MS C19H23ClN4O (M + H+): 359.1, 6.72 min.
(R)-2,5-Dioxopyrrolidin-1-yl 1-(4-Methylpyrimidin-2-yl)-
piperidine-3-carboxylate (11b). In a 35 mL microwave reaction
vessel was combined (R)-nipecotic acid (1.07 g, 8.2 mmol), 2-
propanol (10 mL), DIEA (1.43 mL, 8.2 mmol), and 2-chloro-4-
methylpyrimidine (1.06 g, 8.2 mmol). The vessel was sealed and
heated to 150 °C for 30 min. The solvent was removed en vacuo, and
the residue was then dissolved in water (5 mL), acidified with 0.1 M
HCl, and extracted with DCM (3 × 10 mL), dried over sodium sulfate,
filtered, and concentrated to give 1.27 g (70%) of a pale-yellow oil that
was carried forward without further purification. The crude
intermediate was dissolved in DCM (30 mL) and treated with
EDCI (1.21 g, 6.3 mmol). After 5 min, N-hydroxysuccinimide (791
mg, 6.8 mmol) was added and the mixture was allowed to stir for 18 h.
The reaction mixture was then quenched with water (30 mL), and the
layers were partitioned. The aqueous layer was washed once with
DCM (30 mL). and the combined organics were washed with 10%
citric acid (aq) (20 mL) and water (20 mL), dried over sodium sulfate,
filtered, and concentrated to yield 1.21 g (66%) of the title compound
that was used without purification. The same procedure was employed
to prepare 11a.
General Method A for Compounds 13d−g. (R)-1-(4-Methylpyr-
imidin-2-yl)-N-phenethylpiperidine-3-carboxamide (13d). A stock
solution of 11b was prepared with a concentration of 0.3 M in ACN.
Phenethylamine (50 μL, 0.4 mmol) was dispensed into the reaction
vial and was subsequently dosed with 2 mL of ACN and 350 μL of the
stock solution of 11b. The reaction mixture was kept stirring until 11b
was consumed, and then the reaction mixture was quenched with
water (2 mL), extracted with ethyl acetate (2 × 20 mL), and
concentrated to dryness. The residue was purified via preparative
HPLC to give (R)-1-(4-methylpyrimidin-2-yl)-N-phenethylpiperidine-
3-carboxamide (33 mg, 96%). 1H NMR (400 MHz, CDCl3) δ 8.03 (d,
J = 5.2, 1H), 7.23−7.15 (m, 2H), 7.15−7.09 (m, 1H), 7.08−7.03 (m,
2H), 6.48−6.36 (br, 1H), 6.26 (d, J = 5.2, 1H), 4.10−4.04 (m, 1H),
3.95−3.85 (m, 1H), 3.72−3.64 (m, 1H), 3.53−3.35 (m, 3H), 2.75−
2.62 (m, 2H), 2.34−2.26 (m, 1H), 2.23 (s, 3H), 2.08−1.95 (m, 1H),
1.84−1.72 (m, 1H), 1.58−1.38 (m, 2H). HPLC-MS C19H24N4O (M +
H+): 325.2, 5.56 min.
Computational Modeling of Cyp3A4. Flexible docking was
performed using Glide 5.8 (Schrodinger, Inc., Portland, OR, 2012).
The CYP3A4 coordinates were taken from the Protein Data Bank
(2J0D.pdb). The grid box was centered on the cocrystallized ligand
erythromycin and extended 10 Å from the center, with the outer box
extending an additional 20 Å. The ligand was docked using the
standard precision (SP) algorithm and scored using GlideScore.
Experimental Protocols (Chemistry). Unless otherwise noted,
materials were obtained from commercial suppliers and were used
without purification. Removal of solvent under reduced pressure (i.e.,
en vacuo) refers to distillation using Buchi rotary evaporator attached
to a vacuum pump (∼3 mmHg). Products obtained as solids or high
boiling oils were dried under vacuum (∼1 mmHg). Purification of
compounds by HPLC was achieved using a Waters 2487 series with
Ultra 120 5 μm C18Q column with a linear gradient from 10% solvent
A (ACN with 0.035% TFA) in solvent B (water with 0.05% TFA) to
1
90% A in 7.5 min, followed by 2.5 min elution with 90% A. H NMR
spectra were recorded on Bruker XWIN-NMR (400 or 600 MHz).
Proton resonances are reported in parts per million (ppm) downfield
from tetramethylsilane (TMS). 1H NMR data are reported as
multiplicity (s singlet, d doublet, t triplet, q quartet, quint quinted,
sept septed, dd doublet of doublets, dt doublet of triplet, br s broad
singlet), number of protons, and coupling constant in hertz (Hz). For
spectra obtained in CDCl3, DMSO-d6, and CD3OD, the residual
protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the
reference.
Analytical thin-layer chromatography (TLC) was performed on
commercial silica plates (Merck 60-F 254, 0.25 mm thickness);
compounds were visualized by UV light (254 nm). Flash
chromatography was performed using ISCO CombiFlash cartridges.
The purity and quantitative analysis were determined with a Waters
ZQ 2000 LC/MS system, which employed an Acquity UPLC system
with PAL autosampler, Waters 3100 single quadrupole mass
spectrometer, Waters Acquity 2.1 mm × 50 mm C18 column (1.8
μM), flow rate set at 0.4 mL/min, gradient 5−95%, 0.5−15 min
eluting with mobile phase A (water +0.05% formic acid) and mobile
phase B (ACN + 0.035% formic acid). Unless otherwise stated, all
compounds reported in this manuscript were tested at >95% HPLC
purity by internal quality control protocols.
Unless otherwise stated, all purifications performed via preparative
HPLC used the general conditions: C18 (19 mm × 50 mm), 10−90%
ACN/water, 0.05% TFA, 100 mL/min.
(R)-N-(4-Chlorophenethyl)-1-(pyrimidin-2-yl)piperidine-3-carbox-
amide (13a). (R)-1-Boc-nipecotic acid (0.115 g, 0.5 mmol) and
HATU (209 mg, 0.55 mmol) were dissolved in ACN (2 mL) and
treated with DIEA (0.16 mL, 1.0 mmol, 2 equiv) and 4-
chlorophenethylamine (0.077 mL, 0.55 mmol), and the reaction was
stirred at ambient temperature overnight. The reaction was
concentrated and partitioned between ether and water. The organic
was concentrated, and purification of the residue by flash
chromatography (0−10% methanol/DCM) gave the intermediate.
The material was then dissolved in DCM (2 mL) and treated with
excess TFA (1 mL). The reaction was complete after 1 h and then
concentrated to dryness and carried forward crude into the next step.
In a 5 mL microwave reaction vessel was combined (R)-N-(4-
chlorophenethyl)piperidine-3-carboxamide 2,2,2-trifluoroacetate (130
mg, 0.5 mmol), DMA (2 mL), DIEA (0.25 mL, 1.5 mmol), and 2-
chloropyrimidine (63 mg, 0.55 mmol). The vessel was sealed and
(R)-N-(4-Fluorophenethyl)-1-(4-methylpyrimidin-2-yl)piperidine-
3-carboxamide (13e). 13e was prepared by general method A in 74%
yield. 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 4.9, 1H), 7.07−6.97
(m, 2H), 6.96−6.84 (m, 2H), 6.56 (s, 1H), 6.31 (d, J = 5.0, 1H), 3.96
(dd, J = 3.8, 13.9, 1H), 3.88 (dd, J = 6.6, 13.9, 1H), 3.78−3.65 (m,
2H), 3.53 (td, J = 6.8, 13.2, 1H), 3.37 (dt, J = 6.9, 12.6, 1H), 2.70 (tq, J
= 6.9, 13.9, 2H), 2.41−2.34 (m, 1H), 2.27 (s, 3H), 2.11 (td, J = 7.4,
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12.5, 1H), 1.83−1.74 (m, 1H). HPLC-MS C19H23FN4O (M + H+):
343.1, 5.86 min.
(R)-N-(4-Chlorophenethyl)-1-(5-fluoro-4-methylpyrimidin-2-yl)-
piperidine-3-carboxamide (19b). (R)-1-(4-Amino-5-fluoropyrimidin-
2-yl)-N-(4-chlorophenethyl)piperidine-3-carboxamide (prepared by
general method B) (50 mg, 0.13 mmol) and CuCl2 (21 mg, 0.16
mmol) were added into 1 mL of ACN and cooled to 0 °C. After tert-
butyl nitrite (19 μL, 0.16 mmol) was added, the reaction mixture was
warmed to rt and left overnight. The mixture was concentrated, and
the residue was purified by preparative HPLC (10−90% ACN/water,
0.05% TFA, 100 mL/min) to give (R)-1-(4-chloro-5-fluoropyrimidin-
2-yl)-N-(4-chlorophenethyl)piperidine-3-carboxamide. (R)-1-(4-
Chloro-5-fluoropyrimidin-2-yl)-N-(4-chlorophenethyl)piperidine-3-
carboxamide (50 mg, 0.13 mmol), methylzinc chloride (95 μL, 2.0 M
solution in THF, 0.19 mmol), and Pd(PPh3)2Cl2 (4.4 mg, 0.006
mmol) were added into 1 mL of THF. After flushing with argon, the
mixture was stirred at rt overnight. The reaction was quenched with
aqueous NH4Cl and purified by preparative HPLC (10−90% ACN/
water, 0.05% TFA, 100 mL/min) to give (R)-N-(4-chlorophenethyl)-
1-(5-fluoro-4-methylpyrimidin-2-yl)piperidine-3-carboxamide (9 mg,
(R)-N-(4-Bromophenethyl)-1-(4-methylpyrimidin-2-yl)piperidine-
3-carboxamide (13f). 13f was prepared by general method A in 62%
1
isolated yield. H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 5.0, 1H),
7.26 (d, J = 8.3, 2H), 6.89 (d, J = 8.3, 2H), 6.60 (s, 1H), 6.27 (d, J =
5.0, 1H), 3.87 (d, J = 5.1, 2H), 3.67 (s, 2H), 3.49 (dt, J = 6.6, 13.1,
1H), 3.31 (td, J = 6.8, 12.6, 1H), 2.63 (dq, J = 7.0, 13.9, 2H), 2.37−
2.27 (m, 1H), 2.22 (s, 3H), 2.07 (dd, J = 6.6, 13.0, 1H), 1.73 (dd, J =
5.4, 12.7, 1H), 1.48 (dd, J = 5.9, 11.5, 2H). HPLC-MS C19H23BrN4O
(M + H+): 402.1, 1.91 min (4 min method).
(R)-N-(4-Methylphenethyl)-1-(pyrimidin-2-yl)piperidine-3-carbox-
amide (13g). 13g prepared by general method A in 43% isolated yield.
1H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 4.7, 2H), 7.00 (d, J = 8.0,
2H), 6.95 (d, J = 8.1, 2H), 6.37 (t, J = 4.7, 1H), 6.05 (s, 1H), 4.22−
4.14 (m, 1H), 4.01−3.97 (m, 1H), 3.57 (dd, J = 8.2, 13.7, 1H), 3.43−
3.33 (m, 3H), 2.68−2.63 (m, 2H), 2.29−2.21 (m, 4H), 1.99−1.96 (m,
1H), 1.81−1.76 (m, 1H), 1.55−1.52 (m, 1H), 1.45−1.42 (m, 1H).
HPLC-MS C19H24N4O (M + H+): 325.2, 6.75 min.
1
19%). H NMR (400 MHz, CD3OD) δ 7.27−7.23 (m, 3H), 7.20−
(R)-N-(4-Cyanophenethyl)piperidine-3-carboxamide (15). (R)-1-
Boc-nipecotic acid (57 g, 249 mmol), EDCI (52.5 g, 274 mmol), and
HOBT (37.0 g, 274 mmol) were dissolved in DMF (600 mL) and
stirred for 60 min at ambient temperature. DIEA (95 mL, 548 mmol)
and 4-(2-aminoethyl)benzonitrile hydrochloride (14) (50 g, 274
mmol) were sequentially added, and the reaction was stirred at
ambient temperature overnight. The reaction was concentrated and
partitioned between ethyl acetate (200 mL) and water (200 mL). The
aqueous layer was extracted twice with ethyl acetate (100 mL), and the
combined organic extracts were washed consecutively with 100 mL of
10% citric acid (2×), bicarbonate (2×), water (2×), and brine (1×).
The extracts were dried over magnesium sulfate, filtered, and
concentrated to give crude (R)-tert-butyl 3-((4-cyanophenethyl)-
carbamoyl)piperidine-1-carboxylate (89 g, 99%). 1H NMR (400
MHz, CDCl3) δ 7.60 (d, J = 8.3, 2H), 7.31 (d, J = 8.3, 2H), 3.75
(s, 1H), 3.72−3.59 (m, 1H), 3.58−3.44 (m, 2H), 3.30−3.18 (m, 1H),
3.15−2.96 (m, 1H), 2.88 (t, J = 7.2, 2H), 2.24 (s, 1H), 1.96−1.82 (m,
1H), 1.78 (s, 1H), 1.68 (s, 2H), 1.55 (s, 1H), 1.44 (s, 9H). HPLC-MS
calculated C20H27N3O3 (M + H+-tBu): 302.2, 1.38 min (2.5 min
method). (R)-tert-Butyl 3-((4-cyanophenethyl)-carbamoyl)piperidine-
1-carboxylate was dissolved in ethyl acetate (475 mL) and was cooled
to 0 °C, then 4 M HCl in dioxane (190 mL, 760 mmol) was added
slowly and the mixture was stirred to completion. At 1 h the solution
began to precipitate and at 90 min a viscous oil fell out of solution
which impeded stirring. Then 100 mL of DCM was added and the
mixture was allowed to stand overnight then evaporated to dryness to
give a white foam. The salt was converted into the free base by
dissolving in water and neutralizing with potassium carbonate (s) until
pH ∼10, then extracted with DCM (2 × 300 mL), followed by
chloroform/2-propanol (2 × 150 mL). The organic extracts were dried
over sodium sulfate, filtered through a thin pad of Celite, and
concentrated to dryness to yield 17g. The pH was then adjusted to pH
12 and extracted with 3/1 chloroform/2-propanol (2 × 250 mL),
combined with the initial extract, dried over sodium sulfate, filtered
through a thin pad of Celite, and concentrated to dryness to give 15 as
7.17 (m, 2H), 6.55 (dd, J = 2.6, 9.1, 1H), 4.10−4.02 (m, 1H), 3.94−
3.90 (m, 1H), 3.47 (dt, J = 7.0, 13.8, 1H), 3.39 (dd, J = 6.8, 13.7, 1H),
3.01 (dd, J = 10.0, 12.9, 1H), 2.92−2.83 (m, 1H), 2.79 (t, J = 7.1, 2H),
2.39 (ddd, J = 3.9, 8.9, 14.1, 1H), 2.31 (d, J = 3.0, 2H), 1.89−1.80 (m,
1H), 1.79−1.66 (m, 2H), 1.64−1.49 (m, 2H).
(R)-N-(4-Chlorophenethyl)-1-(pyridin-2-yl)piperidine-3-carboxa-
1
mide (19c). 19c was prepared by general method B in 38% yield. H
NMR (400 MHz, CD3OD) δ 7.98−7.92 (m, 1H), 7.47−7.41 (m, 1H),
7.21−7.14 (m, 2H), 7.12−7.06 (m, 2H) 6.68 (d, J = 8.8, 1H), 6.57−
6.51 (m, 1H), 4.09−4.02 (m, 1H), 3.98−3.91 (m, 1H), 3.31 (td, J =
7.2, 2.8, 2H), 2.93 (dd, J = 13.2, 10.4, 1H), 2.86−2.77 (m, 1H), 2.68
(t, J = 7.2, 2H), 2.34−2.24 (m, 1H), 1.81−1.73 (m, 1H), 1.70−1.57
(m, 2H), 1.51−1.38 (m, 1H). HPLC-MS C19H22ClN3O (M + H+):
344.0, 0.69 min (2 min).
(R)-N-(4-Chlorophenethyl)-1-(pyrimidin-4-yl)piperidine-3-carbox-
amide (19d). 19d was prepared by general method B in 41% yield. 1H
NMR (400 MHz, CD3OD) δ 8.34 (s, 1H), 8.00 (s, 1H), 7.22−7.16
(m, 2H), 7.13−7.07 (m, 2H), 6.66 (d, J = 6.3, 1H), 4.34 (s, 1H), 4.18
(d, J = 11.6, 1H), 3.31 (d, J = 7.1, 2H), 3.06−2.86 (m, 2H), 2.71−2.67
(m, 2H), 2.23 (tt, J = 4.0, 10.8, 1H), 1.86−1.76 (m, 1H), 1.76−1.63
(m, 2H), 1.47−1.35 (m, 1H). HPLC-MS C18H21ClN4O (M + H+):
345.1, 4.56 min.
(R)-N-(4-Chlorophenethyl)-1-(2-methylpyrimidin-4-yl)piperidine-
3-carboxamide (19e). 19e was prepared by general method B in 63%
yield. 1H NMR (400 MHz, CD3OD) δ 7.89 (d, J = 6.5, 1H), 7.17 (dd,
J = 4.3, 6.3, 2H), 7.09 (d, J = 8.4, 2H), 6.47 (d, J = 6.6, 1H), 4.32 (s,
1H), 4.15 (s, 1H), 3.39−3.25 (m, 2H), 3.00 (dd, J = 10.6, 13.3, 1H),
2.97 (s, 1H), 2.77−2.65 (m, 2H), 2.34−2.27 (m, 3H), 2.23 (tt, J = 4.0,
10.7, 1H), 1.85−1.77 (m, 1H), 1.76−1.62 (m, 2H), 1.38 (ddd, J = 8.3,
14.7, 16.5, 1H). HPLC-MS C19H23ClN4O (M + H+): 359.1, 4.79 min.
(R)-N-(4-Chlorophenethyl)-1-(6-methylpyridin-2-yl)piperidine-3-
carboxamide (19f). 19f was prepared by general method B in 41%
1
yield. H NMR (400 MHz, CD3OD) δ 7.30 (dd, J = 7.3, 8.4, 1H),
7.17−7.11 (m, 2H), 7.08−7.02 (m, 2H), 6.40 (t, J = 8.4, 2H), 4.06−
3.98 (m, 1H), 3.86 (dt, J = 3.9, 12.8, 1H), 3.36 (dt, J = 7.0, 14.0, 1H),
3.33−3.23 (m, 1H), 2.98 (dd, J = 9.9, 13.0, 1H), 2.85−2.74 (m, 1H),
2.72−2.62 (m, 2H), 2.34−2.21 (m, 4H), 1.80−1.38 (m, 4H). HPLC-
MS C20H24ClN3O (M + H+): 358.1, 5.19 min.
1
a white to off-white solid (54 g, 84%, 2 steps). H NMR (400 MHz,
CD3OD) δ 7.66 (d, J = 8.2, 2H), 7.41 (d, J = 8.2, 2H), 2.88 (t, J = 7.1,
4H), 2.73−2.47 (m, 2H), 2.29 (m, 1H), 1.68 (m, 5H).
(R)-N-(4-Chlorophenethyl)-1-(5-fluoropyrimidin-2-yl)piperidine-
3-carboxamide (19a): General Method B. To a 5 mL reaction vial
with a screw cap was added 15 (100 mg, 0.37 mmol), 2-propanol (2
mL), DIEA (122 μL, 0.7 mmol, 2 equiv), and 2-chloro-5-
fluoropyrimidine (54 μL, 0.44 mmol). The vessel was sealed and
heated to 100 °C for 2 h. The solvent was removed en vacuo and the
residue purified by flash chromatography (24 g ISCO, 0−10%
methanol/DCM) to yield the title compound (93 mg, 69%) as a pale-
(R)-N-(4-Chlorophenethyl)-1-(4-methylpyridin-2-yl)piperidine-3-
carboxamide (19g). 19g was prepared by general method B in 8%
1
yield. H NMR (400 MHz, CD3OD) δ 7.78 (d, J = 5.5, 1H), 7.20−
7.14 (m, 2H), 7.10 (d, J = 8.4, 2H), 6.64 (s, 1H), 6.48 (d, J = 5.4, 1H),
3.99−3.88 (m, 2H), 3.32 (t, J = 6.6, 2H), 3.07−3.01 (m,, 1H), 2.93−
2.91 (m, 1H), 2.69 (t, J = 7.1, 2H), 2.38−2.27 (m, 1H), 2.21 (s, 3H),
1.78 (s, 1H), 1.74−1.57 (m, 2H), 1.48 (s, 1H). HPLC-MS
C20H24ClN3O (M + H+): 358.2, 5.24 min.
1
yellow oil. H NMR (400 MHz, CDCl3) δ 8.17 (s, 2H), 7.25 (d, J =
(R)-N-(4-Cyanophenethyl)-1-(5-fluoro-4-methylpyrimidin-2-yl)-
piperidine-3-carboxamide (19h). A solution of 2,4-dichloro-5-
fluoropyrimidine (1g, 6 mmol) and Fe(acac)3 (317 mg, 0.69 mmol)
in dry THF (15 mL) was cooled to −78 °C and was subsequently
treated with methyl magnesiumbromide (3.4 mL, 3 M in diethyl ether)
8.3, 2H), 7.09 (d, J = 8.3, 2H), 6.06 (s, 1H), 4.21−4.12 (m, 1H), 3.99
(s, 1H), 3.67−3.39 (m, 4H), 2.81−2.77 (m, 2H), 2.40−2.29 (m, 1H),
2.11−1.98 (m, 1H), 1.88−1.85 (m, 1H), 1.68−1.64 (m,, 1H), 1.56−
1.53 (m, 1H). HPLC-MS C18H20ClFN4O (M + H+): 363.1, 8.46 min.
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via dropwise addition. After 1 h, the reaction was quenched with
saturated ammonium chloride (2 mL) and extracted with diethyl ether.
dried over magnesium sulfate, and filtered. Purification by flash
chromatography (24 g cartridge, 0−30% ethyl acetate/hexanes) gave
596 mg (68%) of 2-chloro-5-fluoro-4-methylpyrimidine as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 0.9, 1H), 2.54 (d, J =
2.5, 3H). Caution should be taken when removing solvent en vacuo, as
the product is volatile. The title compound was then prepared by
general method B, starting from (R)-N-(4-cyanophenethyl)piperidine-
1.55−1.41 (m, 1H). HPLC-MS C25H30F3N7O (M + H+): 502.2, 5.66
min.
(R)-N-(4-Cyanophenethyl)-1-(5-fluoro-4-methyl-6-(4-methylpi-
perazin-1-yl)pyrimidin-2-yl)piperidine-3-carboxamide (22c). To a
solution of 2,4,6-trichloro-5-fluoropyrimidine (21c) (149 mg, 0.74
mmol) in ethanol (2 mL) was added DIEA (131 μL, 0.75 mmol) and
20 (81 μL, 0.73 mmol). The mixture was heated to 80 °C until
complete by LC-MS, then cooled to rt, diluted with water (2 mL),
extracted with DCM (5 mL), dried over magnesium sulfate, filtered,
and concentrated to give 2,4-dichloro-5-fluoro-6-(4-methylpiperazin-1-
yl)pyrimidine as an off-white solid (152 mg, 79%) that was carried
forward without further purification. 2,4-Dichloro-5-fluoro-6-(4-
methylpiperazin-1-yl)pyrimidine (100 mg, 0.37 mmol) and ferric
acetylacetonate (20 mg, 0.056 mmol) were dissolved in THF (2 mL)
and N-methylpyrroldinone (0.1 mL). The solution was cooled to 0 °C
and treated with methylmagnesium bromide (3 M in diethyl ether, 211
μL, 0.63 mmol). The reaction was allowed to warm to rt over 1 h and
was complete by LC-MS. The reaction was quenched with water,
extracted with ethyl acetate, dried over magnesium sulfate, filtered, and
concentrated then purified by flash chromatography (12 g ISCO, 0−
7% gradient methanol/DCM) to give 2-chloro-5-fluoro-4-methyl-6-(4-
methylpiperazin-1-yl)pyrimidine as a colorless oil that crystallized on
standing (74 mg, 80%). The title compound was then prepared as
described in general method B by combining 2-chloro-5-fluoro-4-
methyl-6-(4-methylpiperazin-1-yl)pyrimidine with 15 to give a 6%
isolated yield after preparative HPLC purification. 1H NMR (400
MHz, CDCl3) δ 7.40 (d, J = 8.2, 2H), 7.08 (d, J = 8.2, 2H), 6.99 (s,
1H), 4.06 (dd, J = 4.9, 14.0, 1H), 3.83−3.78 (m, 1H), 3.72−3.55 (m,
5H), 3.45 (dd, J = 3.4, 14.1, 1H), 3.33−3.17 (m, 2H), 2.84−2.64 (m,
2H), 2.47−2.33 (m, 5H), 2.26 (s, 3H), 2.15−2.10 (m, 4H), 1.69−1.62
(m, 1H), 1.47−1.45 (m, 1H), 1.37−1.28 (m, 1H). HPLC-MS
C25H32FN7O (M + H+): 466.3, 1.1 min (3 min method).
1
3-carboxamide and the pyrimidine prepared above in 36% yield. H
NMR (400 MHz, CDCl3) δ 7.99 (d, J = 1.6, 1H), 7.53 (d, J = 8.3, 2H),
7.22 (d, J = 8.2, 2H), 6.35 (s, 1H), 3.98 (dd, J = 3.6, 13.8, 1H), 3.86−
3.71 (m, 2H), 3.67−3.51 (m, 2H), 3.44 (dd, J = 6.2, 13.1, 1H), 2.85
(dq, J = 7.0, 17.9, 2H), 2.42−2.34 (m, 1H), 2.33 (d, J = 2.5, 3H), 2.07
(d, J = 8.2, 1H), 1.81 (s, 1H), 1.55 (d, J = 5.6, 2H). HPLC-MS
C20H22FN5O (M + H+): 367.2, 2.07 min (4 min method).
(R)-N-(4-Cyanophenethyl)-1-(2-(trifluoromethyl)pyrimidin-4-yl)-
piperidine-3-carboxamide (19i). 19i was prepared by general method
1
B in 80% yield. H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 4.8, 1H),
7.55 (d, J = 8.2, 2H), 7.24 (d, J = 8.2, 2H), 6.76 (d, J = 4.8, 1H), 6.23
(s, 1H), 4.13 (s, 1H), 3.99 (s, 1H), 3.79 (d, J = 5.9, 1H), 3.74−3.57
(m, 2H), 3.52−3.34 (m, 1H), 2.92−2.82 (m, 2H), 2.45−2.35 (m, 1H),
2.14 (s, 1H), 1.87 (s, 1H), 1.60 (q, J = 4.6, 2H). HPLC-MS
C20H20F3N5O (M + H+): 404.2, 8.53 min.
(R)-N-(4-Cyanophenethyl)-1-(4-(trifluoromethyl)pyrimidin-2-yl)-
piperidine-3-carboxamide (19j). 19j was prepared by general method
1
B in 50% yield. H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 6.3, 1H),
7.56 (d, J = 8.2, 2H), 7.26 (d, J = 8.1, 2H), 6.58 (d, J = 6.3, 1H), 6.35−
6.13 (m, 1H), 4.02 (d, J = 10.4, 1H), 3.96−3.75 (m, 2H), 3.71−3.58
(m, 1H), 3.49 (s, 1H), 3.34 (s, 1H), 2.98−2.80 (m, 2H), 2.41 (s, 1H),
2.22−2.08 (m, 1H), 1.89 (s, 1H), 1.62 (s, 2H). HPLC-MS
C20H20F3N5O (M + H+): 404.2, 7.20 min.
(R)-N-(4-Cyanophenethyl)-1-(4-methyl-6-(4-methylpiperazin-1-
yl)pyrimidin-2-yl)piperidine-3-carboxamide (22a). A solution of 2,4-
dichloro-6-methylpyrimidine (326 mg, 2 mmol) in ethanol (4 mL)
was treated with DIEA (0.35 mL, 2 mmol), and the mixture was
cooled to −10 °C, when a solution of 1-methylpiperazine (0.22 mL, 2
mmol) in ethanol (1 mL) was added dropwise. The cooling bath was
removed, and the mixture was slowly warmed to rt. After 2 h, the
reaction was complete and the solvent was removed en vacuo and was
purified by flash chromatography (12 g, 0−10% methanol/DCM) to
give 320 mg of 2-chloro-4-methyl-6-(4-methylpiperazin-1-yl)-
pyrimidine. 2-Chloro-4-methyl-6-(4-methylpiperazin-1-yl)pyr-imidine
(35 mg, 0.15 mmol) was dissolved in DMA (1 mL) and treated with
DIEA (26 μL, 0.15 mmol) and 15 (46 mg, 0.18 mmol). The mixture
was heated to 150 °C for 2 h, cooled to rt, and subsequently purified
by preparative HPLC to give the title compound as a white solid in
(R)-N-(4-Cyanophenethyl)-1-(2-methyl-6-(4-methylpiperazin-1-
yl)pyrimidin-4-yl)piperidine-3-carboxamide (22d). A solution of 15-
HCl salt (123 mg, 0.41 mmol) in 2-propanol (1.5 mL) and DIEA
(0.18 mL, 1 mmol) was cooled to 0 °C. To this was added 4,6-
dichloro-2-methylpyrimidine 21d (68 mg, 0.41 mmol). After warming
to rt over 30 min, 15 was added and the mixture was heated to 160 °C
in a microwave reaction vessel for 15 min. The crude was purified by
preparative HPLC to yield the title compound (16 mg, 9%). 1H NMR
(400 MHz, CDCl3) δ 7.57 (s, 1H), 7.46 (d, J = 8.2, 2H), 7.15 (d, J =
8.2, 2H), 5.33 (s, 1H), 4.32 (d, J = 14.1, 1H), 3.72−3.50 (m, 5H), 3.37
(ddd, J = 4.9, 13.0, 17.1, 3H), 3.20 (t, J = 9.3, 1H), 2.87 (dt, J = 6.8,
13.7, 1H), 2.77 (dt, J = 6.9, 14.0, 1H), 2.46 (dt, J = 4.6, 8.5, 5H), 2.34
(s, 3H), 2.28 (s, 3H), 2.21 (s, 1H), 1.78−1.66 (m, 1H), 1.52 (s, 1H),
1.38 (s, 1H). HPLC-MS C25H33N7O (M + H+): 448.2, 2.58 min.
(R)-N-(4-Cyanophenethyl)-1-(2-methyl-6-(4-methylpiperazin-1-
yl)pyrimidin-4-yl)piperidine-3-carboxamide (22e). A solution of 15-
HCl salt (360 mg, 1.22 mmol) in 2-propanol (4 mL) and DIEA (0.64
mL, 3.7 mmol) was cooled to 0 °C. To this was added 4,6-dichloro-2-
trifluoromethylpyrimidine (265 mg, 1.22 mmol). After warming to rt
over 15 min, 20 (0.67 mL, 6.1 mmol) was added and the mixture was
heated to 160 °C in a microwave reaction vessel for 15 min. The
solvent was removed en vacuo, and the crude was suspended in water.
The precipitate was collected by filtration and purified by flash
chromatography (40 g, 0−7% methanol/DCM, 0.1% TEA) to give the
1
41% yield. H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.44 (d, J =
8.2, 2H), 7.12 (d, J = 8.2, 2H), 5.73 (s, 1H), 4.45−4.43 (m, 1H),
4.20−4.18 (m, 1H), 3.78 (dd, J = 6.6, 13.4, 1H), 3.59 (s, 4H), 3.42−
3.41 (m, 1H), 3.29−3.18 (m, 2H), 2.83 (dd, J = 6.9, 13.8, 1H), 2.74
(dd, J = 6.8, 13.6, 1H), 2.50−2.47 (m, 5H), 2.36−2.30 (m, 4H), 2.17
(s, 3H), 1.78−1.70 (m, 1H), 1.57−1.50 (m, 1H), 1.29−1.24 (m, 1H).
HPLC-MS C25H33N7O (M + H+): 448.2, 2.62 min.
(R)-N-(4-Cyanophenethyl)-1-(4-(4-methylpiperazin-1-yl)-6-
(trifluoromethyl)pyrimidin-2-yl)piperidine-3-carboxamide (22b). 20
(0.079 mL, 0.71 mmol) was dissolved in N-methylpyrrolidinone (2.5
mL) and cooled to 0 °C. The mixture was then treated with DIEA
(0.17 mL, 0.95 mmol) and 2,4-dichloro-6-(trifluoromethyl)pyrimidine
(21b) (114 mg, 0.52 mmol). The mixture was warmed to rt over 30
min. To this was then added 15-HCl salt (140 mg, 0.48 mmol), and
the mixture was heated to 100 °C for 17 h. The reaction was cooled,
diluted with water, extracted with ethyl acetate, and the organic layer
was washed with water, dried over magnesium sulfate, and
concentrated to dryness. It was purified by ISCO (24 g, 0−10%
methanol/DCM) to yield the title compound as a white solid (51 mg,
21%). 1H NMR (400 MHz, CD3OD) δ 7.63 (d, J = 8.1, 2H), 7.38 (d, J
= 8.1, 2H), 6.33 (s, 1H), 4.47 (s, 2H), 3.69 (s, 4H), 3.59−3.48 (m,
1H), 3.41 (d, J = 6.8, 1H), 3.21−3.11 (m, 1H), 3.05 (s, 1H), 2.96−
2.84 (m, 2H), 2.52 (t, J = 5.0, 4H), 2.35 (s, 4H), 1.75 (d, J = 6.8, 4H),
1
title compound (381 mg, 62%) as a white solid. H NMR (CDCl3) δ
7.45 (d, J = 8.2, 2H), 7.17 (d, J = 8.2, 2H), 7.1 (br s, 1H), 5.5 (s, 1H),
4.27 (dd, J = 14.2, 4.6, 1H), 3.70−3.59 (m, 5H), 3.48−3.26 (m, 4H),
2.91−2.84 (m, 1H), 2.8.1−2.74 (m, 1H), 2.49 (t, J = 5.1, 4H), 2.46−
2.42 (m, 1H), 2.34 (s, 3H), 2.30−2.22 (m, 1H), 1.78−1.70 (m, 1H),
1.62−1.53 (m, 1H), 1.50−1.40 (m, 1H). 13C NMR (CDCl3) δ 176.2,
164.9, 164.5, 146.6, 133.3, 131.1, 119.9, 111.2, 83.1, 55.5, 47.7, 46.2,
45.9, 44.8, 43.8, 44.2, 36.5, 29.3, 25.1. HPLC-MS C25H30F3N7O (M +
H+): 502.3, 5.70 min. Melting point 169−171 °C.
(R)-1-(6-Chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)piperidine-3-carboxamide. A 3-neck 3 L round-
bottom flask was fitted with an overhead stirrer and an addition funnel.
Compound 15 (54 g, 210 mmol) was dissolved in a mixture of ACN
(900 mL) and DIEA (105 mL) at rt. The mixture was then cooled to 0
3276
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Article
°C. A solution of 21e (50 g, 231 mmol) in ACN (diluted to volume up
to 100 mL) was added via an addition funnel over 30 min. During the
addition, the mixture thickened considerably, and an additional 200
mL of ACN was added (total 1100 mL). Upon complete addition, the
mixture was allowed to stir for 4 h, stirring was stopped, and the
material was allowed to settle and precipitate for 1 h and was then
100 °C for 3 h. After being cooled down to rt, the reaction was diluted
into ethyl acetate and washed with brine. The organic phase was
concentrated, and the crude product was purified by flash
chromatography, eluting with 40% ethyl acetate in hexane to yield
tert-butyl (2-(6-chloropyridin-3-yl)ethyl)carbamate (25) (0.26 g,
55%). 1H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 1.2, 1H),
7.98 (d, J = 7.6, 1H), 7.89 (dd, J = 8.0, 2.4, 1H), 6.95 (t, J = 5.6, 1H),
3.24−3.19 (m, 2H), 2.82 (t, J = 6.8, 2H), 1.32 (s, 9H). C13H17N3O2
(M + H+) 248.2. To the solution of tert-butyl (2-(6-cyanopyridin-3-
yl)ethyl)carbamate (25) (0.25 g, 1 mmol) in DCM (4 mL) was added
4 M HCl in dioxane (0.5 mL, 2 mmol). The reaction was stirred at
ambient temperature for 16 h. The solvent was removed en vacuo to
give the title compound 26. 1H NMR (400 MHz, DMSO-d6) δ 8.71−
8.68 (m, 1H), 8.13 (s, 3H8.03−7.99 (m, 2H), 3.17−3.08 (m, 2H),
3.06−3.00 (m, 2H). HPLC-MS C8H9N3 (M + H+) 148.2.
5-(2-Aminoethyl)pyrimidine-2-carbonitrile (29). Potassium (2-
((tert-butoxycarbonyl)-amino)ethyl) trifluoroborate (92 mg, 0.5
mmol), 5-bromopyrimidine-2-carbonitrile (125 mg, 0.5 mmol),
Pd(OAc)2 (11 mg, 0.05 mmol), 2-dicyclohexylphosphino-2′,6′-
diisopropoxybiphenyl (RuPhos) (47 mg, 0.1 mmol), and Cs2CO3
(487 mg, 1.5 mmol) were mixed in a mixture solvent of toluene (2.5
mL) and water (0.25 mL). The reaction was complete after being
heated at 90 °C for 10 h. After being cooled down to rt, the reaction
mixture was diluted with ethyl acetate and washed with saturated
sodium bicarbonate and brine. The organic phase was dried over
sodium sulfate, and solvent was removed by rotary evaporation. The
residue was purified by flash chromatography, eluting with 50% ethyl
acetate/hexanes to give tert-butyl (2-(5-cyanopyrimidin-2-yl)ethyl)-
carbamate as an off-white solid (11 mg, 9%). Next, a solution of tert-
butyl (2-(5-cyanopyrimidin-2-yl)ethyl)carbamate (11 mg, 0.044
mmol) in DCM (0.5 mL) was treated with TFA (0.05 mL). The
reaction was stirred at rt for 1 h. The solvent and excess TFA were
removed by rotary evaporation, and the crude product was used
directly in the next step without further purification.
1
collected by filtration as a white solid (85.8 g, 93%). H NMR (400
MHz, DMSO) δ 8.03 (s, 1H), 7.76 (d, J = J =7.9, 2H), 7.42 (d, J = 8.0,
2H), 7.25 (s, 1H), 4.67−4.41 (m, 1H), 4.14−3.89 (m, 1H), 3.33−3.30
(m, 3H obscured by solvent), 3.06 (t, J = 11.2, 1H), 2.81 (t, J = 6.9,
2H), 2.39−2.21 (m, 1H), 1.85−1.68 (m, 3H), 1.43−1.40 (m, 1H).
HPLC-MS C20H19ClF3N5O (M + H+): 438.2, 1.27 min (3 min
method).
(R)-N-(4-Cyanophenethyl)-1-(6-(piperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl)piperidine-3-carboxamide (23a). A
solution of (R)-1-(6-chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)piperidine-3-carboxamide (150 mg, 0.34 mmol) in 2-
propanol (3 mL) was treated with DIEA (0.06 mL, 0.34 mmol) and 1-
Boc-piperazine (316 mg, 1.7 mmol). The mixture was heated 165 °C
for 25 min. The solvent was removed and then purified by flash
chromatography (24 g ISCO cartridge, 0−8% methanol/DCM). The
isolated product was then dissolved in DCM (1 mL) and treated with
4 M HCl in dioxane (1 mL, 4 mmol). After 1 h, the solvent was
removed en vacuo, yielding the title compound as the HCl salt (135
1
mg, 76%). H NMR (400 MHz, CD3OD) δ 7.54−7.49 (m, 2H), 7.27
(d, J = 8.2, 2H), 5.74 (s, 1H), 4.20 (d, J = 13.3, 1H), 4.06−3.98 (m,
1H), 3.57−3.48 (m, 4H), 3.41 (dt, J = 6.8, 13.6, 1H), 3.32 (dd, J = 6.8,
13.6, 1H), 3.05 (dd, J = 10.0, 13.4, 1H), 2.96−2.85 (m, 1H), 2.83−
2.74 (m, 6H), 2.31−2.19 (m, 1H), 1.81−1.56 (m, 3H), 1.43−1.39 (m,
1H). HPLC-MS C24H28F3N7O (M + H+): 488.2, 5.70 min.
(R)-N-(4-Cyanophenethyl)-1-(6-(4-(oxetan-3-yl)piperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl)piperidine-3-carboxamide (23b).
Compound 23a (135 mg, 0.25 mmol) was dissolved in THF (1
mL) and treated with acetic acid (0.043 mL, 0.75 mmol) and 3-
oxetanone (0.03 mL, 0.5 mmol). The mixture was heated to 55 °C for
1 h under an atmosphere of nitrogen. To this was then added sodium
triacetoxyborohydride (106 mg, 0.5 mmol). Heating was continued at
65 °C for 3 h. The mixture was then cooled to rt and quenched with
10% citric acid (1 mL). Water (1 mL) was added, and the aqueous
layer was extracted with ethyl acetate (3 × 3 mL). The combined
organics were dried over magnesium sulfate, filtered, and concentrated.
Purified by flash chromatography (12 g ISCO cartridge, 0−8%
methanol/DCM, 0.1% TEA). The product was further purified by
preparative HPLC to give the title compound (35 mg, 25%). 1H NMR
(400 MHz, CDCl3) δ 7.42 (d, J = 8.2, 2H), 7.14 (d, J = 8.2, 2H), 7.02
(s, 1H), 5.43 (s, 1H), 4.68 (t, J = 6.6, 2H), 4.62 (t, J = 6.2, 2H), 4.29−
4.25 (m, 1H), 3.73−3.58 (m, 5H), 3.57−3.47 (m, 1H), 3.41 (dd, J =
3.4, 14.4, 1H), 3.35−3.29 (m, 3H), 2.88 (dt, J = 7.1, 14.2, 1H), 2.82−
2.72 (m, 1H), 2.47−2.37 (m, 5H), 2.32−2.21 (m, 1H), 1.72 (ddd, J =
4.2, 9.4, 17.9, 1H), 1.56 (d, J = 4.1, 1H), 1.51−1.37 (m, 1H). HPLC-
MS C27H32F3N7O2 (M + H+): 544.3, 5.87 min.
5-(2-Aminoethyl)picolinonitrile Hydrochloride (26). 2-(6-Chloro-
3-pyridinyl)acetonitrile (24) (0.68 g, 4.46 mmol), di-tert-butyl
dicarbonate (1.95 g, 8.92 mmol), and nickel chloridehexahydrate
(1.06 g, 4.46 mmol) were added to methanol (30 mL) at rt. Sodium
borohydride (0.50 g, 13.37 mmol) was added portionwise to the
reaction mixture. The reaction mixture was allowed to stir at rt for 3 h.
After filtration on a pad of Celite and concentration en vacuo, 50 mL
of ethyl acetate was added to the crude material, which was washed
twice with 50 mL of water. The organic phase was dried over sodium
sulfate, filtered, and concentrated en vacuo. The crude product was
purified by flash chromatography (ethyl acetate/hexane = 30/70) to
give 0.56 g (49%) of tert-butyl-2-(6-chloro-3-pyridinyl) ethylcarbamate
HPLC-MS C12H17ClN2O2, (M + H+) 257.1). A reaction vessel was
then charged with tert-butyl-2-(6-chloro-3-pyridinyl) ethylcarbamate
(0.5 g, 1.9 mmol), palladium trifluoroacetate (64 mg, 0.19 mmol),
racemic-2-di-t-butylphosphino-1,1′-binaphthyl (155 mg, 0.39 mmol),
Zn(CN)2 (181 mg, 1.6 mmol), and zinc dust (25 mg, 0.39 mmol).
The vessel was evacuated and backfilled with nitrogen. DMA (5 mL)
was added via syringe. The reaction was stirred at rt for 5 min and then
(R)-N-(2-(6-Cyanopyridin-3-yl)ethyl)-1-(6-(4-methylpiperazin-1-
yl)-2-(trifluoromethyl)pyrimidin-4-yl)piperidine-3-carboxamide
(32a). To a solution of 21e (4.92 g, 22.8 mmol) in 2-propanol (100
mL) at 0 °C were added (R)-ethyl piperidine-3-carboxylate (3.6 g, 22.9
mmol) and TEA (4.6 g, 45.6 mmol). The reaction was warmed to rt
and stirred for 1 h. The reaction mixture was diluted with ethyl acetate,
washed with saturated sodium bicarbonate and brine, and dried over
sodium sulfate. The organic phase was passed through a pad of silica
gel to give (R)-ethyl 1-(6-chloro-2-(trifluoromethyl)-pyrimidin-4-
yl)piperidine-3-carboxylate (31a) as an oil (5.6 g, 73%). Then, to a
100 mL round-bottom flask containing 31a (5.52 g, 16.4 mmol) and
lithium hydroxide monohydrate (0.76 g, 18 mmol) were added THF
(30 mL), methanol (10 mL), and water (10 mL). The reaction was
stirred at 60 °C for 1 h. After cooling to 0 °C, the reaction was
neutralized with 1N HCl (19.6 mL). The solid was collected by
filtration to give (R)-1-(6-chloro-2-(trifluoromethyl)pyrimidin-4-yl)-
piperidine-3-carboxylic acid (31b) as a white solid (4.8 g, 95%). To a
solution of 31b (0.88 g, 2.8 mmol) in DMF (10 mL) were added
HATU (1.12 g, 2.9 mmol), DIEA (1.1 g, 8.5 mmol), and 26 (0.42 g,
2.8 mmol). The reaction was stirred at rt for 2 h. The reaction mixture
was diluted with ethyl acetate, washed with saturated sodium
bicarbonate and brine, and dried over sodium sulfate. The organic
phase was dried, and the residue was purified by flash chromatography
to give (R)-1-(6-chloro-2-(trifluoromethyl)-pyrimidin-4-yl)-N-(2-(6-
cyanopyridin-3-yl)ethyl)piperidine-3-carboxamide as an off-white solid
(1.1 g, 89%). Then, to a solution of (R)-1-(6-chloro-2-
(trifluoromethyl)pyrimidin-4-yl)-N-(2-(6-cyanopyridin-3-yl)ethyl)-
piperidine-3-carboxamide (44 mg, 0.1 mmol) in DMSO (0.3 mL) was
added 20 (20 mg, 0.2 mmol), and the mixture was heated to 60 °C for
16 h. The reaction was cooled to rt and purified by HPLC to give the
1
title compound (40 mg, 80%). H NMR (400 MHz, DMSO-d6) δ
8.64−8.59 (m, 1H), 8.02 (t, J = 5.7, 1H), 7.97 (dd, J = 0.7, 7.9, 1H),
7.89 (dd, J = 2.1, 8.0, 1H), 6.12 (s, 1H), 4.32 (s, 2H), 3.75 (s, 3H),
3.50−3.25 (m, 6H), 2.95−2.77 (m, 5H), 2.66 (d, J = 15.2, 3H), 2.21
(tt, J = 3.7, 11.2, 1H), 1.73 (ddd, J = 7.6, 10.0, 14.6, 2H), 1.55 (qd, J =
3277
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Journal of Medicinal Chemistry
Article
3.5, 12.4, 1H), 1.34 (qd, J = 4.0, 12.5, 1H). HPLC-MS C24H29F3N8O
(M + H+): 503.2, 5.01 min.
22e. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.72 (d, J = 8.4,
2H), 7.38 (d, J = 8.4, 2H), 5.92 (s, 1H), 4.52−4.44 (m, 3H), 3.53−
3.50 (m, 3H), 3.30−3.03 (m, 7H), 2.84 (s, 3H), 2.77 (b, 2H), 2.09−
2.08 (b, 1H), 1.91−1.76 (m, 3H). HPLC-MS C24H28F3N7O (M +
H+): 488.2, 5.35 min.
(R)-N-(2-(6-Cyanopyridin-3-yl)ethyl)-1-(6-(piperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl)piperidine-3-carboxamide (32b).
Compound 32b was synthesized in a manner similar to compound
32a from tert-butyl piperazine-1-carboxylate (20 mg, 0.1 mmol) and
(R)-1-(6-chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(2-(6-cyanopyr-
idin-3-yl)ethyl)piperidine-3-carboxamide (40 mg, 0.09 mmol). Follow-
ing removal of the DIEA by evaporation, a 1:1 solution of DCM:TFA
was added and mixture was stirred for 1 h, purified by preparative
HPLC, and converted into the free base by filtration through Si-
(S)-1-(6-Chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)azetidine-2-carboxamide. (S)-1-(tert-
Butoxycarbonyl)azetidine-2-carboxylic acid (2.01 g, 10 mmol) and 5-
(2-aminoethyl)benzontrile (2.0 g, 11 mmol) were dissolved in ethyl
acetate (20 mL), pyridine (2.7 mL, 34 mmol), and DIEA (1.9 mL, 11
mmol). The mixture was cooled to 0 °C when 1-propanephosphonic
anhydride (T3P, 50% w/w in ethyl acetate, 12.7 mL, 20 mmol) was
added slowly over 3 min. After 1 h, 10% citric acid solution (30 mL)
was added, followed by ethyl acetate (20 mL). The layers were
partitioned and the aqueous layer was extracted once with ethyl acetate
(20 mL), and the combined organics were washed with 10% citric acid
(2 × 20 mL) and then brine (20 mL). Dried over magnesium sulfate,
filtered and concentrated. Finally, 2.8 g (85%) of (S)-tert-butyl 2-((4-
cyanophenethyl)carbamoyl)azetidine-1-carboxylate was obtained as a
1
carbonate column to yield the title compound (19.7 mg, 44%). H
NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 1.2, 1H), 8.02 (t, J = 5.6,
1H), 7.98 (d, J = 8.0, 1H), 7.90 (dd, J = 8.0, 2.0, 1H), 6.10 (s, 1H),
4.32 (m, 2H), 3.82 (t, J = 5.2, 4H), 3.38 (m, 2H), 3.18 (m, 5H), 2.94−
2.83 (m, 4H), 2.23−2.18 (m, 1H), 1.80−1.70 (m, 2H), 1.57−1.53 (m,
1H), 1.36−1.32 (m, 1H). HPLC-MS C23H27F3N8O (M + H+): 489.2,
4.93 min.
(R)-N-(2-(2-Cyanopyrimidin-5-yl)ethyl)-1-(6-(4-ethylpiperazin-1-
yl)-2-(trifluoromethyl)pyrimidin-4-yl)piperidine-3-carboxamide
(32c). To a solution of 31a (3.85 g, 11.4 mmol) in 2-propanol (50
mL) was added 1-ethylpiperazine (2.6g, 22.8 mmol). The reaciton was
heated to 80 °C for 16 h. The solvent was removed en vacuo, and the
crude product was purified by flash chromatography to give (R)-ethyl
1-(6-(4-ethylpiperazin-1-yl)-2-(trifluoro-methyl)pyrimidin-4-yl)-
piperidine-3-carboxylate as an oil (3.9 g, 82%). Then, to a 100 mL
round-bottom flask containing (R)-ethyl 1-(6-(4-ethylpiperazin-1-yl)-
2-(trifluoromethyl)-pyrimidin-4-yl)piperidine-3-carboxylate (3.9 g, 9.4
mmol) were added THF (30 mL), methanol (10 mL), and water (10
mL) followed by lithium hydroxide monohydrate (0.5 g, 11.9 mmol).
The reaction was heated to 60 °C for 16 h. The reaction was cooled to
0 °C and neutralized with 1N HCl (12 mL). The solid was collected
by filtration to give (R)-1-(6-(4-ethylpiperazin-1-yl)-2-(trifluorometh-
yl)-pyrimidin-4-yl)piperidine-3-carboxylic acid as a white solid (3.3 g,
91%). To the solution of (R)-1-(6-(4-ethylpiperazin-1-yl)-2-(trifluor-
omethyl)-pyrimidin-4-yl)piperidine-3-carboxylic acid (15 mg, 0.04
mmol) in DMF (0.3 mL) was added DIEA (12.5 mg, 1 mmol),
HATU (15 mg, 0.04 mmol), and 29 (6 mg, 0.04 mmol). The reaction
was stirred at rt for 2 h. The reaction was then diluted with DMSO
(0.5 mL) and purified by preparative HPLC to give the title
compound (10 mg, 48%). 1H NMR (400 MHz, DMSO-d6) δ 8.83 (s,
2H), 7.94 (t, J = 5.6, 1H), 5.92 (s, 1H), 4.27−4.17 (m, 2H), 3.51 (m,
4H), 3.35 (m, 2H), 2.82−2.69 (m, 4H), 2.34−2.26 (m, 6H), 2.17−
2.10 (m, 1H), 1.70−1.60 (m, 2H), 1.48−1.44 (m, 1H), 1.28−1.23 (m,
1H), 0.98−0.94 (t, J = 6.8, 3H). HPLC-MS C24H30F3N9O (M + H+):
518.2, 5.20 min.
1
white solid that was carried forward without further purification. H
NMR (400 MHz, CDCl3) δ 7.58 (d, J = 8.2, 2H), 7.32 (d, J = 8.3, 2H),
4.60 (s, 1H), 3.87 (d, J = 8.3, 1H), 3.72 (dd, J = 8.1, 14.3, 1H), 3.55
(dd, J = 6.8, 13.2, 2H), 2.90 (dd, J = 6.9, 13.9, 2H), 2.40 (s, 2H), 1.42
(s, 9H). The crude material from two batches of the previous step
were combined and were dissolved in DCM (50 mL) and treated with
4 M HCl in dioxane (14 mL) and stirred for 20 h. Concentrated to
dryness and then re-evaporated 3× from methanol to yield (S)-N-(4-
cyanophenethyl)azetidine-2-carboxamide hydrochloride (4.7 g, 88%).
1H NMR (400 MHz, CD3OD) δ 7.71−7.64 (m, 2H), 7.45 (d, J = 8.2,
2H), 4.93 (d, J = 8.9, 1H), 4.11 (dd, J = 9.5, 18.3, 1H), 3.91 (dd, J =
10.0, 16.3, 1H), 3.55 (tdd, J = 6.5, 12.8, 18.9, 2H), 2.95 (td, J = 3.7,
7.0, 2H), 2.83−2.70 (m, 1H), 2.40 (ddd, J = 7.9, 12.1, 17.6, 1H). (S)-
N-(4-Cyanophenethyl)azetidine-2-carboxamide hydrochloride (4.7 g,
17 mmol) was suspended in ACN (60 mL) and treated with DIEA
(8.9 mL, 51 mmol). The mixture was cooled to 0 °C when 21e (4.1 g,
18.7 mmol) was added. The mixture was then allowed to stir to rt
overnight. Approximately 75% of the solvent was removed by
evaporation and then diluted with ethyl acetate (70 mL), washed
with 10% citric acid (2 × 30 mL), and brine (30 mL), dried over
magnesium sulfate, filtered, and concentrated. The crude was
suspended in ethyl acetate and filtered to give a white solid (2.4 g).
The filtrate was then charged with excess Celite and concentrated to
dryness for dryloading for flash chromatography (80g ISCO, 30−100%
ethyl acetate/hexanes) to yield an additional 1.3 g. The mixed fractions
were repurified by the same method to obtain an additional 0.69 g for
a combined yield of 4.39 g (63%) of (S)-1-(6-chloro-2-
(trifluoromethyl)pyrimidin-4-yl)-N-(4-cyanophenethyl)azetidine-2-
carboxamide as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
8.35−8.16 (m, 2H), 7.74 (s, 2H), 7.42 (s, 2H), 4.87−4.70 (m, 1H),
4.10−4.03 (m, 2H), 2.92−2.75 (m, 2H), 2.67−2.58 (m, 1H), 2.21−
2.08 (m, 1H) (peaks at 3.3 and 2.5 ppm partially obs by solvent).
HPLC-MS C18H15ClF3N5O (M + H+): 410.2, 3.19 min (4.5 min
method).
(2S)-N-[2-(4-Cyanophenyl)ethyl]-1-[6-(4-methylpiperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl]piperidine-2-carboxamide (34a).
34a was synthesized in a manner similar to compound 22e in 70%
1
isolated yield. H NMR (400 MHz, CD3OD) δ 7.49 (d, J = 8.4, 2H),
7.26 (d, J = 8.4, 2H), 5.8 (s, 1H), 5.15 (s, 1H), 3.90 (d, J = 13.2, 1H),
3.6 (m, 4H), 3.41−3.33 (m, 2H), 3.02−2.95 (m, 1H), 2.78 (t, J = 6.8,
2H), 2.47 (t, J = 5.2, 4H), 2.28 (s, 3H), 2.05 (m, 1H), 1.63−1.51 (m,
3H), 1.44−1.29 (m, 2H). HPLC-MS C25H30F3N7O (M + H+): 502.2,
6.12 min.
(2S)-N-[2-(4-Cyanophenyl)ethyl]-1-[6-(4-methylpiperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl]azetidine-2-carboxamide (34d).
Compound 34d was synthesized in a manner similar to compound
(2S)-N-[2-(4-Cyanophenyl)ethyl]-1-[6-(4-methylpiperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl]pyrrolidine-2-carboxamide. (34b).
Compound 34b was synthesized in a manner similar to compound
1
22e in 55% yield. H NMR (400 MHz, CD3OD) δ 7.50 (d, J = 8.4,
2H), 7.28 (d, J = 8.4, 2H), 5.27 (s, 1H), 4.82 (m, 1H), 3.97 (m, 1H),
3.84 (m, 1H), 3.69−3.45 (m, 6H), 2.95−2.85 (m, 3H), 2.52 (m, 5H),
2.37 (s, 3H). HPLC-MS C23H26F3N7O (M + H+): 474.2, 5.16 min.
(2R)-N-[2-(4-Cyanophenyl)ethyl]-1-[6-(4-methylpiperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl]azetidine-2-carboxamide (34e).
Compound 34e was synthesized in a manner similar to compound
1
22e on 1 mmol scale in 50% isolated yield. H NMR (400 MHz,
DMSO-d6) δ 7.98 (s, 1H), 7.72 (d, J = 8.4, 2H), 7.38 (d, J = 8.4, 2H),
5.91 (s, 1H), 4.52−4.45 (m, 3H), 3.53−3.50 (m, 3H), 3.30−3.03 (m,
7H), 2.84 (s, 3H), 2.77 (b, 2H), 2.09−2.08 (b, 1H), 1.91−1.75 (m,
3H). HPLC-MS C24H28F3N7O (M + H+): 488.2, 5.37 min.
(2R)-N-[2-(4-Cyanophenyl)ethyl]-1-[6-(4-methylpiperazin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl]pyrrolidine-2-carboxamide. Com-
pound 34c was obtained via chiral HPLC (16 mL/min 80/20
hexane/2-propanol, 21 mm × 250 mm Lux-Celluclose-2 colmun, 35
min elution run time) of N-[2-(4-cyanophenyl)ethyl]-1-[6-(4-
methylpiperazin-1-yl)-2-(trifluoro-methyl)pyrimidin-4-yl]pyrrolidine-
2-carboxamide that was synthesized in a manner similar to compound
1
22e in 34% yield. H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.43
(m, 2H), 7.21 (m, 2H), 5.22 (s, 1H), 4.75 (m, 1H), 3.89 (m, 1H), 3.75
(m, 1H), 3.50 (m, 3H), 2.85 (m, 3H), 2.58 (m, 1H), 2.43 (m, 5H),
2.30 (m, 3H), 1.05 (m, 2H). HPLC-MS C23H26F3N7O (M + H+):
474.2, 5.16 min.
(2S)-N-[2-(6-Cyanopyridin-3-yl)ethyl]-1-[6-(4-ethylpiperazin-1-
yl)-2-(trifluoromethyl)pyrimidin-4-yl]azetidine-2-carboxamide (34f).
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Compound 34f was synthesized in a manner similar to compound 32c
in 63% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.15 (t,
J = 5.6, 1H), 7.85−7.79 (m, 2H), 5.52 (s, 1H), 4.53 (m, 1H), 3.91−
3.80 (m, 2H), 3.47 (b, 4H), 3.37−3.30 (m, 2H), 2.80 (t, J = 6.4, 2H),
2.47 (m, 1H), 2.34−2.27 (m, 6H), 2.10−2.01 (m, 1H), 0.97−0.93 (t, J
= 7.2, 3H). HPLC-MS C23H27F3N8O (M + H+): 489.2, 4.50 min.
Methyl 4-(N-Boc-piperidin-4-yloxy) benzoate (38a). A mixture of
N-boc-4-hydroxypiperidine (201 mg, 1 mmol), methyl 4-hydrox-
ybenzoate (215 mg, 1 mmol), and triphenylphosphine (514 mg, 2
mmol) in THF (10 mL) was cooled to 0 °C and then treated with a
solution of DEAD (358 mg, 2 mmol) in THF (1 mL) via dropwise
addition. The reaction temperature was slowly raised to rt and stirred
for 2 h. The solvent was removed en vacuo, and the crude product was
purified by flash chromatography to give tert-butyl 4-(4-(methoxy-
carbonyl)phenoxy)piperidine-1-carboxylate (214 mg, 63%), which was
subsequently treated with 4 M HCl solution in dioxane (2 mL). After
stirring 2 h at rt, the solvent was removed by vacuum and the product
was lyophilized overnight to yield the hydrochloride salt. (150 mg,
99%). 1H NMR (400 MHz, CD3OD) δ 7.98 (d, J = 8.9, 2H), 7.08 (d, J
= 8.9, 2H), 4.85 (tt, J = 6.5, 3.5, 1H), 3.87 (s, 3H), 3.41 (td, J = 9.0,
4.5, 2H), 3.34−3.13 (m, 3H), 2.24 (m, 2H), 2.05 (m, 2H). 13C NMR
(101 MHz, MeOD) δ 168.18, 162.13, 132.77, 124.23, 116.46, 69.81,
52.45, 41.88, 28.30.
Methyl 2-(4-(Piperidin-4-yloxy)phenyl)acetate (38b). A mixture of
benzyl 4-hydroxypiperidine-1-carboxylate (1.18 g, 5 mmol), methyl 2-
(4-hydroxyphenyl)acetate (0.83 g, 5 mmol), and triphenylphosphine
(1.57 g, 6 mmol) in THF (10 mL) was cooled to 0 °C and then
treated with a solution of diisopropyl azodicarboxylate (DIAD) (0.75
mL, 6 mmol, 1.2 equiv 95%) in THF (1 mL) via dropwise addition.
The reaction temperature was slowly raised to rt and stirred for 2 h.
The solvent was removed by rotary evaporator, and the crude product
was purified by flash chromatography to give benzyl 4-(4-(2-methoxy-
2-oxoethyl)phenoxy)piperidine-1-carboxylate (0.924 g, 47%). 1H
NMR (400 MHz, CDCl3) δ 7.39 (d, J = 4.5, 4H), 7.34 (ddt, J =
8.4, 5.7, 2.9, 1H), 7.21 (d, J = 8.6, 2H), 6.88 (d, J = 8.7, 2H), 5.16 (s,
2H), 4.49 (tt, J = 6.8, 3.4, 1H), 3.83−3.71 (m, 2H), 3.70 (d, J = 1.4,
3H), 3.58 (s, 2H), 3.48 (ddd, J = 13.3, 7.2, 4.0, 2H), 2.02−1.85 (m,
2H), 1.81 (m 2H). The obtained intermediate (244 mg) was dissolved
in ethyl acetate (8 mL) andPd/C (10%, 24 mg) was added under a
stream of nitrogen. The reaction vessel was backfilled with hydrogen
gas and was stirred under hydrogen atmosphere for 4 h. The catalyst
was removed by filtration and was washed by methanol (3 × 10 mL),
and then the solvent was removed en vacuo. After drying overnight,
the title compound was obtained as a light-yellow solid (156 mg,
98%). HPLC-MS: 100% (ELSD), (M + H+): 250.
(m, 2H), 2.89 (s, 2H), 2.61 (m, 1H), 2.34 (m, 1H), 2.01 (m, 2H), 1.68
(m, 5H), 1.28 (m, 1H). HPLC-MS C26H29F3N6O3 (M + H+) 531.2,
8.25 min.
1-{6-[(3R)-3-{[2-(4-Cyanophenyl)ethyl]carbamoyl}piperidin-1-yl]-
2-(trifluoromethyl)-pyrimidin-4-yl}-4-methylpiperidine-4-carboxylic
Acid (45b). was prepared in a manner similar to 45a from 35b in 29%
1
yield. H NMR (400 MHz, CD3CN) δ 7.64 (m, 2H), 7.36 (m, 2H),
6.64 (m, 1H), 5.77 (m, 1H), 4.02 (m, 3H), 3.44 (m, 2H), 3.21 (m,
3H), 2.99 (m, 1H), 2.86 (m, 2H), 2.26 (m, 2H), 2.07 (m, 2H), 1.99
(m, 1H), 1.74 (m, 4H), 1.45 (m, 2H), 1.24 (s, 3H). HPLC-MS
C27H31F3N6O3 (M + H+) 545.0, 8.84 min.
2-{4-[(1-{6-[(3R)-3-{[2-(4-Cyanophenyl)ethyl]carbamoyl}-
piperidin-1-yl]-2-(trifluoromethyl)pyrimidin-4-yl}piperidin-4-yl)oxy]-
phenyl}acetic Acid (45c). 45c was prepared in a manner similar to 45a
1
from 38b in 66% yield. H NMR (400 MHz, CD3OD) δ 7.51 (d, J =
8.4, 2H), 7.27 (d, J = 8.0, 2H), 7.13 (d, J = 8.8, 2H), 6.84 (d, J = 8.8,
2H) 5.73 (s, 1H), 4.55−4.49 (m, 1H), 4.16 (dd, J = 2.8, 13.2, 1H),
3.98−3.80 (m, 3H), 3.54−3.44 (m, 5H), 3.38−3.29 (m, 1H), 3.17 (dd,
J = 9.6, 13.6, 1H), 2.95 (dt, J = 2.8, 13.2, 1H), 2.88−2.74 (m, 2H),
2.34−2.26 (m, 1H), 1.99−1.90 (m, 3H), 1.85−1.59 (m, 5H), 1.54−
1.42 (m, 1H). HPLC-MS C33H35F3N6O4 (M + H+) 637.3, 9.67 min.
(S)-2-(4-((1-(6-(2-((4-Cyanophenethyl)carbamoyl)azetidin-1-yl)-
2-(trifluoromethyl)pyrimidin-4-yl)piperidin-4-yl)oxy)phenyl)acetic
Acid (45d). 45d was prepared in a manner similar to 45a from 38b
and (S)-1-(6-chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)azetidine-2-carboxamide in 59% yield. 1H NMR
(400 MHz, CD3OD) δ 8.51 (s, 1H), 7.54 (d, J = 8.3, 2H), 7.36 (d,
J = 8.3, 2H), 7.21 (d, J = 8.6, 2H), 6.98−6.89 (m, 2H), 5.59 (s, 1H),
4.75−4.61 (m, 2H), 4.09−3.86 (m, 4H), 3.67−3.55 (m, 3H), 3.50−
3.39 (m, 1H), 2.89 (t, J = 6.9, 2H), 2.61−2.40 (m, 2H), 2.12−1.96 (m,
4H), 1.85−1.69 (m, 2H). HPLC-MS C31H31F3N6O4 (M + H+) 608.2,
1.56 min (2 min method).
4-[(1-{6-[(2S)-2-{[2-(4-Cyanophenyl)ethyl]carbamoyl}azetidin-1-
yl]-2-(trifluoromethyl)pyrimidin-4-yl}piperidin-4-yl)oxy]benzoic Acid
(45e). To a mixture of 38a and (S)-1-(6-chloro-2-(trifluoromethyl)-
pyrimidin-4-yl)-N-(4-cyanophenethyl)azetidine-2-carboxamide (75
mg, 0.3 mmol) dissolved in NMP (6 mL) was added excess DIEA
(0.2 mL). The reaction was stirred at 120 °C overnight in a seal tube.
The reaction was quenched by a saturated aqueous solution of sodium
bicarbonate and then was extracted by ethyl acetate (3 × 15 mL). The
combined organic phase was washed by water (2 × 10 mL), saturated
sodium bicarbonate (10 mL), and brine (10 mL). After drying over
sodium sulfate, the solvent was removed and the crude product was
dissolved in a mixture of THF/methanol/water (2 mL, 3/1/1). Excess
lithium hydroxide (200 mg) was then added. The reaction was stirred
at rt for 2 h and was quenched by saturated solution of sodium
bisulfate (10 mL). The solution was extracted with a mixture of (2-
propanol/chloroform: 1/3, 3 × 20 mL). The combined organic phase
was washed with water (20 mL) and brine (20 mL) and was dried over
sodium sulfate, and the solvent was removed en vacuo. The crude
product was then purified by flash chromatography (24 g ISCO, 0−
1-{6-[(3R)-3-{[2-(4-Cyanophenyl)ethyl]carbamoyl}piperidin-1-yl]-
2-(trifluoromethyl)-pyrimidin-4-yl}piperidine-4-carboxylic Acid
(45a). (R)-1-(6-Chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)piperidine-3-carboxamide (200 mg, 0.457 mmol),
ethyl piperidine-4-carboxylate (141 mg, 0.914 mmol), and diisopro-
pylethyl amine (159 uL, 0.914 mmol) were dissolved in 2-propanol
(50 mL) in a sealed tube. The reaction mixture was then heated at 130
°C for 20 h. The solvents were removed en vacuo, and the residue was
purified by flash chromatography (40 g ISCO, 0−10% DCM/
1
10% methanol/DCM) to give the title compound (86 mg, 73%). H
NMR (400 MHz, CD3OD) δ 8.51(m, 1H), 8.08 (d, J = 7.8, 2H), 7.46
(m, J = 7.8, 2H), 7.26 (d, J = 7.8, 2H), 6.98 (d, J = 7.8, 2H), 5.32 (s,
1H). 4.82 (m, 1H), 4.74 (m, 1H), 4.12(m, 1H), 4.02−3.77 (m, 4H);
3.77−3.55 (m, 2H), 3.49 (m, 1H), 3.25−2.64 (m, 3H), 2.48 (m, 1H),
2.34−2.00 (m, 3H), 1.95 (m, 1H). HPLC-MS C30H30F4N6O4 (M +
H+) 595.2, 9.08 min.
4-(1-{6-[(2S)-2-{[2-(4-Cyanophenyl)ethyl]carbamoyl}azetidin-1-
yl]-2-(trifluoromethyl)-pyrimidin-4-yl}piperidin-4-yl)-2-methoxyben-
zoic Acid (45f). A mixture of methyl 4-bromo-2-methoxybenzoate
(490 mg, 2.0 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (927 mg, 3.0
mmol), sodium bicarbonate (504 mg, 6.0 mmol), and Pd(PPh3)4
(231 mg, 0.2 mmol) in dioxane (5.0 mL) and water (2.0 mL) in a
sealed tube was heated 130 °C for 30 min. After cooling to rt, the
reaction mixture was diluted with water (20 mL) and extracted with
ethyl acetate (3 × 40 mL), the combined extracts were then dried over
MgSO4, filtered, and the residue purified by flash chromatography
(10−30% ethyl acetate/hexanes) to give tert-butyl 4-(3-methoxy-4-
(methoxycarbonyl)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate
1
methanol) to yield the intermediate ester (212 mg, 85%). H NMR
(400 MHz, CD3OD) δ 7.63 (d, J = 8.1, 2H), 7.38 (d, J = 8.1, 2H), 5.85
(s, 1H), 4.35 (m, 3H), 4.15 (m, 3H), 3.42 (m, 3H), 3.17 (m, 1H), 3.03
(m, 3H), 2.89 (m, 2H), 2.64 (m, 1H), 2.34 (m, 1H), 1.71 (m, 7H),
1.26 (t, J = 7.1, 3H). HPLC-MS C28H33F3N6O3 (M + H+) 559.2, 3.297
min (4.5 min method). (R)-Ethyl 1-(6-(3-((4-cyanophenethyl)-
carbamoyl)piperidin-1-yl)-2-(trifluoromethyl)pyrimidin-4-yl)-
piperidine-4-carboxylate (81.2 mg, 0.145 mmol) was dissolved in THF
(4 mL) and ethanol (0.5 mL), then treated with lithium hydroxide
(3M, 0.15 mL, 0.435 mmol). The reaction was stirred at rt overnight.
Upon completion, the reaction was quenched with 6 N HCl to pH 3,
and the residue was taken up in ethyl acetate (3×), and the combined
organic layers were dried over magnesium sulfate, filtered, and
1
concentrated to yield the title compound (115 mg, 48%). H NMR
(400 MHz, CD3OD) δ 7.63 (d, J = 8.2, 2H), 7.39 (d, J = 8.2, 2H), 5.85
(s, 1H), 4.33 (m, 3H), 4.11 (m, 1H), 3.50 (m, 3H), 3.17 (m, 1H), 3.03
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(713 mg, 99%). 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.0, 1H),
7.00 (dd, J = 1.6, 8.0, 1H), 6.96 (d, J = 1.6, 1H), 6.15 (m, 1H), 4.12
(m, 2H), 3.95 (s, 3H), 3.90 (s, 3H), 3.67 (t, J = 5.2, 2H), 2.55 (m,
2H), 1.51 (s, 9H). HPLC-MS C19H26NO5 (M + H+) 348.18, 2.19 min
(3.5 min method). tert-Butyl 4-(3-methoxy-4-(methoxycarbonyl)-
phenyl)-5,6-dihydropyridine-1(2H)-carboxylate (705 mg, 2.03
mmol) was dissolved in methanol (20 mL), and 10% Pd/C (200
mg) was added. The mixture was then stirred overnight under H2
atmosphere. The catalyst was removed by filtration, and the solvent
was removed en vacuo to afford the product (623 mg, 84%, two steps).
HPLC-MS C19H28NNaO5 (M + Na+) 372.18, 2.21 min (3.5 min). A
solution of tert-butyl 4-(3-methoxy-4-(methoxy-carbonyl)phenyl)-
piperidine-1-carboxylate (250 mg, 0.715 mmol) in dioxane was treated
with 4N HCl solution in dioxane (6.0 mL) overnight at rt; solvent was
removed en vacuo to afford 41. HPLC-MS C14H20NO3 (M + H+)
250.1, 1.11 min (3.5 min method).
(trifluoromethyl)pyrimidin-4-yl)-[1,1′-biphenyl]-4-carboxylate (10.5
g, 17.9 mmol) was suspended in THF (50 mL) and methanol (50
mL) and treated with lithium hydroxide (3M, 13 mL, 38 mmol) and
heated to at 50 °C for 1 h and then at 70 °C for 30 min. The reaction
mixture was diluted with DCM, acidified with 10% citric acid, and
poured into a separatory funnel. The aqueous layer was extracted with
DCM (2 × 30 mL), and the combined organics were washed with
water, then dried over magnesium sulfate, filtered, and concentrated to
1
yield the title compound (9.47g, 93%). H NMR (400 MHz, CDCl3
CD3OD) δ 8.03 (t, J = 8.7, 4H), 7.62 (dd, J = 8.4, 20.3, 4H), 7.37 (d, J
= 8.1, 2H), 7.16 (d, J = 8.2, 2H), 6.65 (s, 1H), 4.82 (s, 1H), 4.13−3.96
(m, 2H), 3.49−3.39 (m, 2H), 2.80−2.60 (m, 3H), 2.51 (br s, 1H).
HPLC-MS C31H24F3N5O3 (M + H+) 572.3, 9.13 min.
4-(4-{6-[(2S)-2-{[2-(4-Cyanophenyl)ethyl]carbamoyl}azetidin-1-
yl]-2-(trifluoromethyl)pyrimidin-4-yl}phenyl)cyclohexane-1-carbox-
ylic Acid (45h). To a 350 mL pressure vessel was added
bis(dibenzylideneacetone)palladium(0) (605 mg, 1.05 mmol), tricy-
clohexylphosphine (708 mg, 2.52 mmol), and dioxane (25 mL). The
mixture was stirred under Ar (g) for 30 min and was then charged with
bis(pinacolato)diboron (12.85 g, 50.6 mmol), potassium acetate (6.77
g, 69 mmol), and trans-methyl 4-(4-chlorophenyl)-cyclohexanecarbox-
ylate (11.6 g, 46 mmol) in 70 mL of dioxane. The vessel was then
evacuated and backfilled with Ar (g) twice. The vessel was capped and
heated to 120 °C for 1 h and then 100 °C overnight. It was cooled to
rt, diluted with DCM (40 mL), and filtered on Celite to remove
palladium. Water was added to the filtrate, and layers were partitioned.
The aqueous layer was extracted with DCM, then dried, filtered, and
concentrated by 75%. Then 10% ethyl acetate/hexanes was added
(∼200 mL) and reduced volume by 50% en vacuo and the product
began to precipitate. The precipitate was collected by filtration to yield
a brownish-gray solid. The crude material was dissolved in DCM and
loaded on to a bed of silica gel and washed successively with 20% ethyl
acetate/hexanes, 30% ethyl acetate/hexanes, and then 50% ethyl
acetate/hexanes to give a white solid. A second crop was also collected
from the original mother liquor. The mother liquor was then
evaporated to dryness, and the material was subjected to flash
chromatography (330 g ISCO, 5−30% ethyl acetate/hexanes). Mixed
fractions were pooled, evaporated, and repurified under the same
conditions. Clean fractions were combined with the original precipitate
to yield 43b (10.5 g, 67%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J =
8.0, 2H), 7.21 (d, J = 8.0, 2H), 3.69 (s, 3H), 2.53 (m, 1H), 2.36 (m,
1H), 2.10 (m, 2H), 1.97 (m, 2H), 1.61- 1.42 (m, 4H), 1.33 (s, 12H).
Dicyclohexyl(2′,6′-dimethoxy-[1,1′-biphenyl]-2-yl)phosphine (487
mg, 1.2 mmol) and tris(dibenzylideneacetone)dipalladium(0) (162
mg, 0.18 mmol) were combined in a 350 mL pressure vessel. To this
was added dry dioxane (20 mL), and the mixture was stirred for 20
min. In a separate flask were dissolved (S)-1-(6-chloro-2-
(trifluoromethyl)pyrimidin-4-yl)-N-(4-cyanophenethyl)azetidine-2-
carboxamide (7.2 g, 17.7 mmol) and 43b (7.32 g, 21.3 mmol) in
dioxane (80 mL). The starting materials were then added to the
preformed catalyst, and the flask was rinsed (3 × 10 mL) with dry
dioxane. Potassium phoshate tribasic (5 M, 30 mL) was then added,
and the vessel was evacuated and backfilled with Ar (g) and heated to
90 °C for 3 h. The mixture was allowed to cool and stand overnight
and diluted with ethyl acetate (100 mL) and brine (40 mL). The
aqueous layer was extracted with ethyl acetate, and the combined
organics were washed with brine (2×) and then dried over magnesium
sulfate, filtered, concentrated en vacuo, and purified by flash
chromatography (330 g ISCO, 20−100% ethyl acetate/hexanes) to
give trans-methyl 4-(4-(6-((S)-2-((4-cyanophenethyl)carbamoyl)-
azetidin-1-yl)-2-(trifluoromethyl)-pyrimidin-4-yl)phenyl)-
A mixture of 41 (178 mg, 0.72 mmol), (S)-1-(6-chloro-2-
(trifluoromethyl)pyrimidin-4-yl)-N-(4-cyanophenethyl)-azetidine-2-
carboxamide (195 mg, 0.477 mmol), and DIEA (0.65 mL, 4.77 mmol)
in NMP (6.0 mL) was heated to 140 °C for 2 h. The reaction mixture
was then cooled to 35 °C and treated with lithium hydroxide (2 M, 2.4
mL) for 6 h. The reaction mixture was brought pH 3.0 with 2N HCl.
This material was then purified by reverse phase HPLC to yield the
1
title compound (150 mg, 34%). H NMR (400 MHz, CDCl3) δ 8.56
(s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.42 (m, 2H), 7.19 (m, 2H), 6.92
(dd, J = 1.6, 8.4 Hz, 1H), 6.85 (d, J = 1.2 Hz,1H), 5.35 (s, 1H), 4.92 (t,
J = 8.0 Hz, 1H), 4.54 (m, 2H), 4.01 (s, 3H), 3.90 (m,2H), 3.58 (m,
1H), 3.47 (m, 1H), 3.05 (m, 2H), 2.87 (m, 3H), 2.67 (m, 1H),
2.54(m, 1H), 1.97 (m, 2H), 1.72 (m, 2H). HPLC-MS C31H32F3N6O4
(M + H+) 609.2, 9.13 min.
4-(4-{6-[(2S)-2-{[2-(4-Cyanophenyl)ethyl]carbamoyl}azetidin-1-
yl]-2-(trifluoromethyl)-pyrimidin-4-yl}phenyl)benzoic Acid (45g). To
a 100 mL pressure vessel was added [1,1′-bis(diphenylphosphino)-
ferrocene]dichloropalladium(II) (1.63 g, 1.56 mmol, 10 mol %),
potassium acetate (5.6 g, 57.5 mmol), methyl 4′-bromo-[1,1′-
biphenyl]-4-carboxylate (4.5 g, 15.6 mmol), and bis(pinacolato)-
diboron (4.8 g, 18.6 mmol). The vessel was evaculated and backfilled
with argon then charged with dioxane (45 mL). The vessel was then
purged with argon, sealed, and heated to 90 °C for 18 h. Upon
completion, the mixture was cooled to rt, diluted with DCM (40 mL),
and filtered on Celite. Water was added to the filtrate, and the layers
were partitioned. The aqueous layer was extracted with DCM (2 × 30
mL) then dried over sodium sulfate, filtered, and concentrated. The
crude material was then subjected to flash chromatography (330 g
ISCO, 5−30% ethyl acetate/hexanes) to yield methyl 4′-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1′-biphenyl]-4-carboxylate
(41a) (3.2 g, 62%).
(S)-1-(6-Chloro-2-(trifluoromethyl)pyrimidin-4-yl)-N-(4-
cyanophenethyl)azetidine-2-carboxamide (3.1 g, 7.5 mmol), 43a (3.2
g, 9.43 mmol), dicyclohexyl(2′,6′-dimethoxy-[1,1′-biphenyl]-2-yl)-
phosphine (207 mg, 0.51 mmol), and tris(dibenzylideneacetone)-
dipalladium(0) (69 mg, 0.075 mmol) were combined in a 100 mL
pressure vessel. The vessel was evacuated and backfilled with argon
twice. To this mixture was added dry dioxane (30 mL) and aqueous
potassium phosphate (5 M, 8 mL, 30 mmol). The vessel was
evacuated and backfilled once more, then heated to 95 °C for 3 h. The
crude reaction mixture was filtered on Celite while the product
remained in solution. Upon filtering the hot solution, the product
precipitated. The Celite was washed with ethyl acetate (40 mL), and
the Celite cake was suspended in ethyl acetate and heated to reflux and
refiltered. The mixture was allowed to stand overnight and the product
precipitated and was collected in four crops of white solid to yield (S)-
methyl 4′-(6-(2-((4-cyanophenethyl)carbamoyl)azetidin-1-yl)-2-
(trifluoromethyl)pyrimidin-4-yl)-[1,1′-biphenyl]-4-carboxylate (4.06
1
cyclohexanecarboxylate. H NMR (400 MHz, CDCl3) δ 8.31−8.05
(m, 1H), 8.01 (d, J = 8.3, 2H), 7.49 (d, J = 8.2, 2H), 7.36 (d, J = 8.3,
2H), 7.27 (d, J = 9.4, 3H), 6.66 (s, 1H), 4.95 (s, 1H), 4.19−4.09 (m,
1H), 4.05 (s, 1H), 3.72 (s, 3H), 3.60 (dd, J = 6.4, 12.4, 2H), 3.07−2.97
(m, 1H), 2.91 (dd, J = 7.1, 9.5, 2H), 2.61 (d, J = 11.7, 2H), 2.41 (t, J =
11.9, 1H), 2.15 (d, J = 10.8, 2H), 2.02 (d, J = 11.1, 2H), 1.82−1.47 (m,
6H), 1.30−1.25 (m, 1H).
1
g, 93%). H NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 8.4, 12.6,
4H), 7.77 (dd, J = 8.4, 20.3, 4H), 7.51 (d, J = 8.2, 2H), 6.74 (s, 1H),
5.04−4.92 (m, 1H), 4.23−4.14 (m, 1H), 4.13−4.03 (m, 1H), 3.98 (s,
3H), 3.63 (s, 2H), 2.93 (s, 3H), 2.72−2.52 (m, 2H). HPLC-MS
C32H26F3N5O3 (M + H+) 586.2, 2.36 min (3.5 min method). (S)-
Methyl 4′-(6-(2-((4-cyanophenethyl)carbamoyl)azetidin-1-yl)-2-
trans-Methyl 4-(4-(6-((S)-2-((4-Cyanophenethyl)carbamoyl)-
azetidin-1-yl)-2-(trifluoro-methyl)pyrimidin-4-yl)phenyl)-
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cyclohexanecarboxylate. trans-Methyl 4-(4-(6-((S)-2-((4-
cyanophenethyl)carbamoyl)azetidin-1-yl)-2-(trifluoro-methyl)-
pyrimidin-4-yl)phenyl)cyclohexanecarboxylate (12.6 g, 21.3 mmol)
was dissolved in methanol (50 mL) and THF (50 mL) and was then
charged with lithium hydroxide (3 M, 14 mL, 43 mmol). The mixture
was heated to 40 °C for 1 h. Upon cooling to rt, the mixture was
treated with 50 mL of 1 M HCl. The mixture was diluted with ethyl
acetate, and the resulting white precipitate was collected by filtration.
The filtrate was then extracted with ethyl acetate, washed with brine,
dried, filtered, and concentrated to a white solid. The original
precipitate and the solid collected from the extracted filtrate were
stirred in ACN for 20 min, and the solid was collected by filtration.
The resulting filtrate was then concentrated to dryness and subjected
to flash chromatography on 220 g of ISCO (0−100% DCM/ACN) to
toward interleukin-12 hypo-producing dendritic cells via a TGR5-
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1
give 45h (11.4 g, 92%). H NMR (400 MHz, CD3OD) δ 8.09−7.99
(m, 2H), 7.60−7.49 (m, 2H), 7.40 (d, J = 8.2, 3H), 6.99−6.81 (m,
2H), 4.88−4.82 (m, 1H), 4.28−4.09 (m, 3H), 3.60−3.50 (m, 2H),
2.92 (m, 2H), 2.80−2.58 (m, 3H), 2.48−2.36 (m, 2H), 2.19−2.09 (m,
2H), 2.03−1.95 (m, 2H), 1.61 (s, 4H). 13C NMR (101 MHz, DMSO)
δ 175.58, 168.73, 162.18, 160.48, 149.02, 144.31, 132.35, 130.86,
128.64, 126.07, 125.84, 117.76, 107.80, 98.15, 61.44, 58.60, 53.75,
41.64, 40.81, 38.95, 33.83, 31.42, 19.59. HPLC-MS C31H30F3N5O3 (M
+ H+) 578.2, 9.15 min. Melting point 249−252 °C.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 858-332-4433. Fax: 858-812-1648. E-mail: dphillips@
gnf.org.
(9) Duan, H.; Ning, M.; Chen, X.; Zou, Q.; Zhang, L.; Feng, Y.;
Zhang, L.; Leng, Y.; Shen, J. Design, synthesis, and antidiabetic activity
of 4-phenoxynicotinamide and 4-phenoxypyrimidine-5-carboxamide
derivatives as potent and orally efficacious TGR5 agonists. J. Med.
Chem. 2012, 55, 10475−10489.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
(10) (a) Piotrowski, D. W.; Futatsugi, K.; Warmus, J. S.; Orr, S. T.
M.; Freeman-Cook, K. D.; Londregan, A. T.; Wei, L.; Jennings, S. M.;
Herr, M.; Coffey, S. B.; Jiao, W.; Storer, G.; Hepworth, D.; Wang, J.;
Lavergne, S. Y.; Chin, J. E.; Hadcock, J. R.; Brenner, M. B.; Wolford, A.
C.; Janssen, A. M.; Roush, N. S.; Buxton, J.; Hinchey, T.; Kalgutkar, A.
S.; Sharma, R.; Flynn, D. A. Identification of Tetrahydropyrido[4,3-
d]pyrimidine Amides as a New Class of Orally Bioavailable TGR5
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Piotrowski, D. W.; Futatsugi, K.; Warmus, J. S.; Boehm, M.; Carpino,
P. A.; Chin, J. E.; Janssen, A. M.; Roush, N. S.; Buxton, J.; Hinchey, T.
Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as
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Med. Chem. Lett. 2013, 2, 1407−1411. (c) Futatsugi, K.; Bahnck, K. B.;
Brenner, M. B.; Buxton, J.; Chin, J. E.; Coffey, S. B.; Dubins, J.; Flynn,
D.; Gautreau, D.; Guzman-Perez, A.; Hadcock, J. R.; Hepworth, D.;
Herr, M.; Hinchey, T.; Janssen, A. M.; Jennings, S. M.; Jiao, W.;
Lavergne, S. Y.; Li, B.; Li, M.; Munchhof, M. J.; Orr, S. T. M.;
Piotrowski, D. W.; Roush, N. S.; Sammons, M.; Stevens, B. D.; Storer,
G.; Wang, J.; Warmus, J. S.; Wei, L.; Wolford, A. C. Optimization of
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MedChemComm 2013, 4, 205.
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IBMX, 3-isobutyl-1-methylxanthine; HEPES, 4-(2-hydroxyeth-
yl)-1-piperazineethanesulfonic acid; HTRF, homogeneous
time-resolved fluorescence; FXR, farnesoid-X-recptor;
DMEM, Dulbecco’s Modified Eagle Medium; DPP4, dipepti-
dylpeptidase-4; HEK293, human embryonic kidney-293 cells;
GLP-1, glucagon-like peptide-1; FBS, fetal bovine serum;
DIEA, diisopropylethylamine; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; HOBT, hydroxybenzotriazole-hydrate; ACN,
acetonitrile; TEA, triethylamine; TrixiePhos, racemic-2-di-t-
butylphosphino-1,1′-binaphthyl; RuPhos, 2-dicyclohexylphos-
phino-2′,6′-diisopropoxybiphenyl; Sphos, 2-dicyclohexylphos-
phino-2′,6′-dimethoxy-1,1′-biphenyl
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