Discovery of a novel series of GPR119 agonists: Design, synthesis, and biological evaluation of N-(Piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives

Article abstract of DOI:10.1016/j.bmc.2021.116208

We undertook an optimization effort involving propan-2-yl 4-({6-[5-(methanesulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate 1, which we had previously discovered as a novel G protein-coupled receptor 119 (GPR119) agonist. To occupy a presumed hydrophobic space between the pyrimidine and piperidine rings in interaction with GPR119, we replaced the linker oxygen with nitrogen. Subsequently, the introduction of a substituent at the bridging nitrogen atom was explored. We found that the installation of N-trifluoromethyl group 10 not only enhanced GPR119 agonist activity but also considerably improved the human ether-à-go-go-related gene (hERG) inhibition profile. These improvements were not observed for non-fluorinated substituents, such as ethyl analog 8b. The next optimization effort focused on the exploration of a new surrogate structure for the indoline ring and the isosteric replacements of the piperidine N-Boc group to improve solubility, metabolic stability, and oral bioavailability. As a result, N-{1-[3-(2-fluoropropan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}-6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)pyrimidin-4-amine (27) was identified as a potent and orally bioavailable GPR119 agonist. This compound augmented insulin secretion and effectively lowered plasma glucose excursion in a diabetic animal model after oral administration. In this study, we discuss the designs, syntheses, and biological activities of a novel series of N-(piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives as GPR119 agonists, and to determine the distinctive effect of the N-trifluoromethyl group on hERG inhibition, we also discuss the conformational preference of representative compounds.

Full text of DOI:10.1016/j.bmc.2021.116208

Bioorg. Med. Chem. 41 (2021) 116208
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a novel series of GPR119 agonists: Design, synthesis, and
biological evaluation of N-(Piperidin-4-yl)-N-(trifluoromethyl)
pyrimidin-4-amine derivatives
,
Osamu Kuboa *, Kazuaki Takamia, Masahiro Kamauraa, Koji Watanabea, Hirohisa Miyashitaa,
Shinichi Abea, Kae Matsudaa, Yoshiyuki Tsujihataa, Tomoyuki Odanib, Shinji Iwasakic,
Tomoyuki Kitazakia, Toshiki Murataa, Kenjiro Satoa
a Cardiovascular & Metabolic Drug Discovery Unit, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b Biomolecular Research Laboratories, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
c DMPK Research Laboratories, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
We undertook an optimization effort involving propan-2-yl 4-({6-[5-(methanesulfonyl)-2,3-dihydro-1H-indol-1-
yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate 1, which we had previously discovered as a novel G protein-
coupled receptor 119 (GPR119) agonist. To occupy a presumed hydrophobic space between the pyrimidine
and piperidine rings in interaction with GPR119, we replaced the linker oxygen with nitrogen. Subsequently, the
introduction of a substituent at the bridging nitrogen atom was explored. We found that the installation of N-
trifluoromethyl group 10 not only enhanced GPR119 agonist activity but also considerably improved the human
GPR119 agonist
GPCR
Type 2 diabetes mellitus
N-trifluoromethyl
hERG
Conformation
Aromatic ring count
Solubility
`
ether-a-go-go-related gene (hERG) inhibition profile. These improvements were not observed for non-fluorinated
substituents, such as ethyl analog 8b. The next optimization effort focused on the exploration of a new surrogate
structure for the indoline ring and the isosteric replacements of the piperidine N-Boc group to improve solubility,
metabolic stability, and oral bioavailability. As a result, N-{1-[3-(2-fluoropropan-2-yl)-1,2,4-oxadiazol-5-yl]
piperidin-4-yl}-6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)pyrimidin-4-amine (27) was
identified as a potent and orally bioavailable GPR119 agonist. This compound augmented insulin secretion and
effectively lowered plasma glucose excursion in a diabetic animal model after oral administration. In this study,
we discuss the designs, syntheses, and biological activities of a novel series of N-(piperidin-4-yl)-N-(tri-
fluoromethyl)pyrimidin-4-amine derivatives as GPR119 agonists, and to determine the distinctive effect of the N-
trifluoromethyl group on hERG inhibition, we also discuss the conformational preference of representative
compounds.
Fluorine
Metabolic stability
1. Introduction
drugs.4–10 To date, several compounds have been investigated under
clinical trials, with their results affirming the safety and tolerability of
Type 2 diabetes mellitus (T2DM) is a major public health problem
worldwide. Diabetes causes complications such as retinopathy, ne-
phropathy, neurological disorder when left untreated, and blindness.
Dialysis treatment may be necessary at the end of the disease. Recently,
considerable attention has been paid to incretin-related approaches,
such as dipeptidyl peptidase-4 inhibitors and glucagon-like peptide-1
mimetics as medication therapy for T2DM.1–3 G protein-coupled re-
ceptor 119 (GPR119) has also attracted considerable interest as an
incretin-related approach for the next generation of antidiabetic
GPR119 agonists in humans.
We have previously reported the lead generation and the subsequent
optimization efforts of a novel series of indoline-based GPR119 agonists.
11,12
`
To address the undesirable human ether-a-go-go-related gene
(hERG) issue of lead compound 1, structural modification was employed
for the substituent at the indoline 5-position. This successfully led to the
identification of compound 2 as a potent GPR119 agonist with favorable
pharmacokinetic profiles and reduced hERG liability (Fig. 1).
In a parallel effort, we conducted structural modification around the
* Corresponding author.
E-mail address: osamu.kubo@takeda.com (O. Kubo).
https://doi.org/10.1016/j.bmc.2021.116208
Received 16 March 2021; Received in revised form 27 April 2021; Accepted 4 May 2021
Available online 9 May 2021
0968-0896/© 2021 The Authors.
Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
central pyrimidine core of compound 1. To induce interaction with the
presumed hydrophobic space between the pyrimidine and piperidine
rings in GPR119, we introduced a substituent through a nitrogen linkage
between the two rings. As a result, the installation of an N-tri-
fluoromethyl group at the bridging nitrogen was found to be effective in
enhancing GPR119 agonist activity and reducing hERG inhibitory ac-
tivity. The replacement of the indoline ring and the optimization of the
piperidine N-substituent resulted in the identification of compound 27,
which exhibited potent GPR119 agonist activity, low risk in hERG in-
hibition, and favorable pharmacokinetic profiles (Fig. 1). In this study,
we report the design, synthesis, and biological activity of N-(tri-
fluoromethyl)pyrimidin-4-amine derivatives as a novel series of GPR119
agonists and discuss the cause of improvement in hERG inhibition by the
N-trifluoromethyl group in terms of conformational preference, basicity,
hydroxyl group. Compound 20 was furnished from 19 through the re-
action with sodium thiomethoxide, followed by meta-chloroperox-
ybenzoic acid (mCPBA) oxidation. Compound 21 was obtained by SNAr
with azetidin-3-ol, followed by sulfonamidation with methanesulfonyl
chloride (MsCl). Compound 22 was synthesized under similar conditions
used in the synthesis of 18.
Scheme 4 describes the substitute conversion on the right piperidine
nitrogen. The Boc group of compound 18 was deprotected under acidic
conditions to give compound 23. The introduction of oxadiazoles was
accomplished via N-cyanamide 24, which was prepared by the reaction
of compound 23 with BrCN. The 1,2,4-oxadiazole ring was constructed
to afford 25a, 25b, and 25c. By reacting with methyl magnesium
chloride (MeMgCl), tertiary alcohol 26 was delivered from ester 25c.
The hydroxyl group of 26 was converted to fluorine atom using Xtal-
Fluor-E® as a source of fluorine to give compound 27. In this reaction, a
small amount of dehydrated olefin by-product 28 was observed and
could not be separated from the desired compound using silica gel col-
umn chromatography. To remove this impurity, ruthenium-catalyzed
oxidation was conducted to convert the olefin of the crude mixture to
a ketone. Ketone derivative 29 could be easily separated using silica gel
column chromatography to give the pure final compound 27.
and π-electron density of compounds.
2. Chemistry
The synthetic route was used to prepare compounds 8a–e bearing
various alkyl substituents at the nitrogen atom between the pyrimidine
and piperidine rings (Scheme 1). The SNAr of 4,6-dichloropyrimidine
with 4-amino-1-Boc-piperidine and the subsequent protection of the
resulting anilinic NH with a trifluoroacetyl group yielded compound 5.
The Buchwald–Hartwig reaction of compound 5 with 5-(methylsulfonyl)
indoline, followed by the removal of the trifluoroacetyl group under
basic conditions, provided key intermediate 6. Alternatively, compound
6 could be prepared in shorter steps by changing the reaction sequence
in reverse order. Notably, the refluxing of 4,6-dichloropyrimidine and 5-
(methylsulfonyl)indoline in ethanol (EtOH) gave chloropyrimidine 7,
which was then subjected to the Buchwald–Hartwig reaction with 4-
amino-1-Boc-piperidine to attain 6. The introduction of an alkyl sub-
stituent on the nitrogen atom was accomplished by alkylation using
sodium hydride (NaH) as a base to give N-alkyl derivatives 8a–e.
The syntheses of N-trifluoromethyl derivatives 10 are outlined in
Scheme 2. According to Hiyama et al.’s method,13,14 compound 6 was
converted to dithiocarbamate 9, which was subjected to an oxidative
desulfurization–fluorination reaction using tetrabutylammonium dihy-
drogen trifluoride (TBAH2F3) and N-bromosuccinimide (NBS) to give
the desired N-trifluoromethyl derivative 10 in a moderate yield. After
the removal of the tert-butyloxycarbonyl protecting group, also known
as the Boc group, N-cyanation was followed by the construction of a
1,2,4-oxadiazole ring to deliver the final compound 13.
3. Results and discussion
The newly synthesized compounds were tested for functional
GPR119 agonism using a cAMP reporter assay in Chinese hamster ovary
(CHO) cells, stably expressing human or rat GPR119. Inhibitory activity
against the hERG channel was measured in vitro using the automated
patch clamp assay system at 10 μM concentration of compounds. In vitro
metabolic stability was assessed by monitoring the disappearance of the
parent compound after incubation with human liver microsomes, which
is expressed as CLint (μL/min/mg).
Compound design for the addition of a substituent around the central
moiety was based on the structure–activity relationship of the indoline
carbamate derivatives, which we had previously reported9 (Fig. 2). The
potency enhancement by the methyl installation on the central spacer
(3a vs. 3b) indicated a hydrophobic space around the 4-position of the
piperidine ring in binding with GPR119. Based on this observation, we
hypothesized that a hydrophobic substituent could be embedded in the
indolinylpyrimidine derivatives at the corresponding position to in-
crease GPR119 agonist activity. A flexible alignment of 3b and 1 using
the Molecular Operating Environment software15 showed that the two
molecules overlapped well; it also suggested that the methyl group of
compound 3b could be positioned around the oxygen atom between the
pyrimidine and piperidine rings of compound 1 (Fig. 2B). This prompted
us to design 6-(indolin-1-yl)-N-(piperidin-4-yl)pyrimidin-4-amine de-
rivative I, which enabled the introduction of an additional substituent at
the bridging point through N-alkylation (Fig. 2, A). To investigate the
effects of the N-substituent on GPR119 agonist activity and hERG
inhibitory activity, we planned to introduce alkyl substituents with
different sizes or fluorinated alkyl substituents at the bridging nitrogen.
Table 1 shows the structural variations of the nitrogen substituent
between the central pyrimidine and piperidine rings with their corre-
sponding GPR119 agonistic and hERG inhibitory activities. The
The synthesis of compounds with various left-hand side substitutes
was carried out (Scheme 3). The condensation of 4,6-dichloropyrimi-
dine and 1-Boc-4-aminopiperidine in the same manner described in
Scheme 1 yielded compound 14. Dithiocarbamate was introduced to
compound 14 and then trifluorinated with 1,3-dibromo-5,5-dimethylhy-
dantoin and TEA trihydrofluoride to give N-trifluoromethylated deriv-
ative 15. Compound 15 was coupled with 1-(methylsulfonyl)piperidin-
4-amine using N,N-diisopropylethylamine (DIPEA) as a base to afford
16. Methylation by MeI and NaH gave 17 in a good yield. 1-Methanesul-
fonyl piperidin-4-ol was installed on 15 using NaH to afford compound
18 in a good yield. Mesylate 19 was prepared by SNAr of 15 and cis-
cyclohexane-1,4-diol with NaH, followed by mesylation of the resulting
Fig. 1. Pyrimidine-based GPR119 agonists.
2

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
Scheme 1. Synthesis of 4,6-diaminopyrimidine de-
rivatives 6 and 8a–ea. Reagents and conditions: (a) 4-
amino-1-Boc-piperidine, triethylamine (TEA), EtOH,
80 ◦C, 84%; (b) trifluoroacetic anhydride (TFAA),
TEA, tetrahydrofuran (THF), 0 ◦C to rt, 88%; (c) 5-
(methylsulfonyl)-2,3-dihydro-1H-indole, tris(dibenzy-
lideneacetone)dipalladium(0) (Pd2(dba)3), Xantphos,
cesium carbonate (Cs2CO3), toluene, 100 ◦C; (d) so-
dium hydroxide (NaOH), H2O, THF, methanol
(MeOH), rt, 33% over two steps; (e) 5-(methyl-
sulfonyl)-2,3-dihydro-1H-indole, EtOH, reflux, 71%;
(f) 4-amino-1-Boc-piperidine, Pd2(dba)3, X-Phos, Cs2CO3, toluene, 110 ◦C, 42%; (g) RI or RBr, NaH, N, N-dimethylformamide (DMF), 0 ◦C to rt, 9–98%.
Scheme 2. Synthesis of N-trifluoromethyl derivatives
10 and 13a. Reagents and conditions: (a) (1) n-butyl
lithium (nBuLi), THF, 0 ◦C, (2) carbon disulfide (CS2),
0 ◦C to rt, (3) iodomethane (MeI), 0 ◦C to rt, 60%; (b)
TBAH2F3, NBS, toluene, ꢀ 78 ◦C to 0 ◦C, 41%; (c)
hydrochloric acid (HCl), MeOH, ethylacetate (AcOEt),
rt, 87%; (d) cyanogen bromide (BrCN), sodium bi-
carbonate (NaHCO3), THF, H2O, 0 ◦C to rt, 97%; (e)
iPrCNH(NHOH), zinc chloride (ZnCl2), p-toluene-
sulfonic acid (p-TsOH), DMF, 85 ◦C, 69%.
Scheme 3. Synthesis of N-trifluoromethyl derivatives 16, 17, 18, 20, 21, and 22a. Reagents and conditions: (a) 4-amino-1-Boc-piperidine, TEA, acetonitrile (MeCN),
85 ◦C, 75%; (b) (1) NaH, CS2, MeI, DMF, rt, 80%, (2) TEA trihydrofluoride, 1,3-dibromo-5,5-dimethylhydantoin, chlorobenzene, ꢀ 20 ◦C, 33%; (c) 1-(methylsulfonyl)
piperidin-4-amine, DIPEA, N, N-dimethylacetamide (DMA), 100 ◦C, 58%; (d) MeI, NaH, DMF, rt, 83%; (e) 1-(methylsulfonyl)piperidin-4-ol, NaH, DMA, rt, 78%; (f)
(1) cis-cyclohexane-1,4-diol, NaH, DMA, rt, (2) MsCl, 4-dimethylaminopyridine (DMAP), pyridine, rt, 60% over two steps; (g) sodium thiomethoxide, DMF, 80 ◦C,
then mCPBA, DMF, rt, 16%; (h) (1) azetidin-3-ol hydrochloride, NaH, DMA, rt, 44%; (2) MsCl, TEA, DMF, 0 ◦C to rt, 47%; (i) (1-(methylsulfonyl)azetidin-3-yl)
methanol, NaH, THF, rt, 54%.
Scheme 4. Synthesis of 1,2,4-oxadiazole derivatives 25a, 25b, and 27a. Reagents and conditions: (a) 4 M HCl in AcOEt, MeOH, AcOEt, rt, 98%; (b) BrCN, NaHCO3,
THF, H2O, rt, 88%; (c) N-hydroxyisobutyrimidamide, N-hydroxycyclopropanecarboximidamide, or ethyl 2-amino-2-(hydroxyimino)acetate, ZnCl2, p-TsOH, DMF,
80–85 ◦C, 69–81%; (d) MeMgCl, THF, 0 ◦C, 79%; (e) XtalFluor-E®, TEA trihydrofluoride, TEA, toluene, ꢀ 78 ◦C to rt; (f) ruthenium(III) chloride, sodium periodate,
MeCN, H2O, 0–80 ◦C, 72% over two steps.
3

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
Fig. 2. (A) Filling design of a presumed hydrophobic space between the pyrimidine and piperidine rings through a nitrogen linkage. (B) An alignment of compounds
3b and 1 obtained by flexible alignment using Molecular Operating Environment software (cyan: 3b, magenta: 1). The stereochemistry of the chiral center of 3b
is tentative.
To investigate the spatial tolerance around the bridging nitrogen and
Table 1
enhance agonist activity, the size of the substituent on the nitrogen atom
GPR119 agonistic and hERG inhibitory activities of 6-(indolin-1-yl)-N-(piper-
was increased by elongating the alkyl chain, such as ethyl, n-propyl, or
idin-4-yl)- pyrimidin-4-amine derivatives: effect of N-substituents at the
cyclopropylmethyl moiety (8b–d). All these compounds exhibited
bridging nitrogen.
similar potency to N-methyl analog 8a. This result suggested that, as
expected, GPR119 would have a hydrophobic space between the py-
rimidine and piperidine rings in interaction and accommodate a sub-
stituent larger than methyl. However, despite an increase in lipophilicity
of N-substituent, no further enhancement of GPR119 activity was
observed for 8b–d.
d
Compound
R
hGPR119aEC50
(nM)
hERGb%
LogD7.4
LLEe
To enhance GPR119 agonist activity, another strategy to increase the
lipophilicity of an N-substituent other than the elongation of carbon
atoms was applied. In particular, we designed an N-trifluoromethyl
analog, for which the entropic cost upon binding to GPR119 might be
reduced relative to compounds with flexible N-substituents such as
8b–d.
inhibition
at 10 μM
6
H
300
21
80 ± 3
85 ± 6
2.7
3.2
3.5
4.0
4.1
3.4
4.1
3.8
4.5
4.4
3.7
3.2
4.1
4.5
8a
8b
8c
8d
8e
10
Me
Et
14
116 ± 2
94 ± 4
nPr
18
CH2cPr
CH2CH2OCH3
CF3
51
100 ± 4
120 ± 4
13 ± 11
(16 ± 1) c
N-trifluoromethyl analog 10 gratifyingly exhibited an enhanced
agonist activity with an EC50 value of 2.5 nM and also maintained a
comparable LLE value to that of N-methyl analog 8a (LLE = 4.5 for 10
and 4.5 for 8a). The comparison between 10 and 8d with similar lip-
ophilicity also illustrated the usefulness of the trifluoromethyl group in
terms of GPR119 agonist activity.
29
2.5
a
Agonist activity against human GPR119. EC50 values are shown as mean (n
= 2).
b
Automated patch clamp assay. Inhibition percentages are represented as
We examined the effect of N-substituents on hERG inhibition. Un-
fortunately, NH derivative 6 and N-alkyl derivatives 8a–d were found to
potently block the hERG channel current with almost complete inhibi-
mean ± standard deviation (n = 4).
c
d
Data at 30 µM.
LogD value at pH 7.4.
e
tion at 10 μM, independently of the size of N-substituent. The insertion
LLE = pEC50 ꢀ LogD7.4
.
of an oxygen atom in the N-substituent to lower lipophilicity of the
compound had no clear beneficial effect on hERG inhibition profile 8e.
However, the introduction of the trifluoromethyl group had a positive
impact on hERG inhibition, and compound 10 exhibited low hERG
replacement of the oxygen atom at the bridging position of compound 1
with a nitrogen atom resulted in more than a tenfold loss of activity (6).
The drop of potency is likely associated with lower lipophilicity
(LogD7.4 = 3.1 for 1 and 2.7 for 6) or the presence of the NH proton of 6.
Compared with 6, the introduction of a methyl group at the bridging
nitrogen (8a), as we had expected, led to a substantial increase in po-
tency. The ligand–lipophilicity efficiency (LLE) value (pEC50 ꢀ LogD)16
increased with a change from 3.8 (6) to 4.5 (8a), indicating that the N-
methyl group could make some specific interaction with GPR119. Based
on the comparison of 8a with 3b (LogD7.4 = 3.4), we assumed that
increasing lipophilicity would further enhance GPR119 agonist activity.
inhibitory activity even at 30 μM.
This result might be assumed to be attributed to the lowered basicity
of the aminopyrimidine moiety by the high electronegativity of fluorine
atoms.17 To examine this assumption, we tested the pKa values of the
conjugate acids of N-methyl analog 8a and N-trifluoromethyl analog 10
using an ultraviolet (UV) spectrophotometric method. Indeed, the ba-
sicity of compound 10 was altered, reducing pKa to 0.8 (10) from 3.7
(8a). However, since the basicity of N-methyl analog 8a was not
essentially very high and oxygen-linked analog 1 with a pKa value of 1.9
4

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
displayed a potent hERG inhibitory activity, it is unlikely that the
lowering of basicity by electron withdrawing property of the tri-
fluoromethyl group would be the only cause of the dramatic effect on
on further optimization based on 10. The acid-labile Boc group of 10
was replaced with 3-isopropyl-1,2,4-oxadiazole, which had been found
as a surrogate moiety of the Boc group in this series of our previous
work.11 As expected, oxadiazole derivative 13 displayed potent GPR119
agonist activity similar to 10 (Fig. 3). Our further profiling of 13 illus-
trated the low solubility of this compound. We thought that the cause of
low solubility was linked to the planarity of the molecule and hypoth-
esized that the conversion of the aromatic ring to a nonaromatic ring
would enable the escape of the planar structure and improvement in
solubility.
hERG inhibition. In effect, it can be presumed that the reduced π-elec-
tron density of the pyrimidine ring by the trifluoromethyl group is
associated with the improved hERG inhibition profile of 10, because
π
-stacking is considered
a
major determinant of hERG–drug
interaction.17
Mindful of the distinctive conformational preference of tri-
fluoromethyl aryl ethers,18 we anticipated that the introduction of the
trifluoromethyl group would affect conformational orientation around
the bridging nitrogen, leading to a different propensity for hERG inhi-
bition. To verify this hypothesis, X-ray analyses of single-crystal struc-
tures of N-methyl, ethyl, and trifluoromethyl analogs (8a, 8b, and 10)
were conducted, mainly because the conformational preference of N-
trifluoromethyl anilines has not been reported (Table 2). In general,
In our previous report, we identified that the hydrogen bond
acceptor (HBA) existing on the left-hand side of the molecule is impor-
tant for exhibiting GPR119 agonistic activity.11,12 We assumed that the
indoline ring would not be essential and could be replaced with another
motif if the HBA existed in a proper arrangement. Based on this hy-
pothesis, instead of the indoline ring, we tried to introduce various non-
aromatic rings (Fig. 3).
anilines prefer planar conformation to achieve
π-conjugation of the
anilinic nitrogen atom with the aryl system. It has been reported that
anilines are often slightly pyramidal and that dimethyl aniline, which is
the simplest N,N-disubstituted aniline, has nonsymmetrical conforma-
tion with one methyl group in plane with the ring and the other one out
of plane deviating 14◦ from the ring.19 Our X-ray examination illustrated
the same tendency for N-methyl analog 8a and N-ethyl analog 8b with
similar conformations. As shown by the distance between the nitrogen
atom and the plane formed by nitrogen’s three substituents (C4, C4′, and
C1′′), nitrogen atoms at the bridging point exhibit slight inclination to
pyramidal conformation. The carbon atoms at the 4-position of the
piperidine ring (C4′) of 8a and 8b are located in plane, whereas the other
carbon atom (C1′′) deviates 10–15◦from the pyrimidine plane. For N-
trifluoromethyl analog 10, however, both substituents on the nitrogen
The results of compounds with a non-aromatic ring system on the
left-hand side are shown in Table 3. To confirm whether the activity was
retained after each modification, the substituent on the nitrogen atom of
the right piperidine ring was fixed with the Boc group. 4-Amino piper-
idine derivative 16 exhibited an EC50 value of 43 nM, which was
attenuated nearly 20-fold relative to indoline derivative 10. N-Methyl-
ated compound 17 showed loss of activity, whereas the derivative in
which a linker atom was converted to an oxygen atom (18) showed
tenfold improved activity compared with 16, exhibiting the agonist
activity equivalent to that of indoline derivative 10. The activity of trans-
cyclohexane 20 was 70-fold weaker than that of 18. Moreover, azetidine
derivatives 21 and 22 exhibited further attenuated potency (7100 nM
and 960 nM, respectively). These results supported our hypothesis that
the indoline ring can be replaced with other saturated rings and a proper
arrangement of the HBA would be important for the expression of potent
agonistic activity.
atom are out of plane, with a larger dihedral angle of 26◦ (
τ1). There was
hardly any difference in the degree of pyramidalization observed for
these three compounds. Since the trifluoromethyl group has a similar
size to the ethyl group, which was supported by the analysis of van der
We selected 18 as the new lead compound, and the Boc group was
replaced with the 3-isopropyl 1,2,4-oxadiazole ring to give 25a as
before. Compared with 13 and consistent with our expectation, com-
pound 25a displayed potent GPR119 agonist activity and dramatically
Waals volumes and experimental assessment using
a protease
probe,20,21 the electron withdrawing nature of fluorine atoms of 10
likely reduces the degree of π-conjugation between the nitrogen atom
and the pyrimidine ring to further deviate the N-substituents from the
plane. We assumed that the improved hERG profiles of the N-tri-
fluoromethyl derivatives would be driven by this conformational pref-
improved solubility (Table 4, <0.22–29 μg/mL). This compound dis-
played good metabolic stability in rat microsomes but was rapidly
metabolized by human microsomes. The metabolic vulnerability of 25a
in human microsomes may be due to the isopropyl moiety susceptible to
metabolic oxidation. Based on this assumption, structural modification
was employed for the isopropyl group of 25a to improve its metabolic
stability (Table 4). Consequently, the cyclization of the isopropyl moiety
(25b) or the introduction of a fluorine atom at the 2-position of the
isopropyl moiety (27) remarkably improved human microsomal stabil-
ity and retained equivalent potency for 25a. This remarkable improve-
ment in microsomal stability illustrates the oxidation of the isopropyl
moiety of 25a as a major route of metabolism by human microsomes.
We selected 27 as a representative compound for further evaluation.
First, we confirmed the rat GPR119 agonist potency of compound 27,
with compound 27 displaying strong agonistic activity (EC50 = 24 nM).
We then evaluated the compound’s effects on hormone secretion in
some in vitro cell lines, because GPR119 is involved in glucose-
dependent insulin secretion and gastrointestinal hormone release (e.g.,
GLP-1). Compound 27 augmented GLP-1 secretion in mouse intestinal L
cell line (i.e., GLUTag cells) and insulin secretion in hamster pancreatic
beta cell line (i.e., HIT-T15) (Table 5). These results demonstrated the
strong activities of in vitro hormone secretion of compound 27. Next, we
investigated hERG inhibitory activity and pharmacokinetic parameters
in rats for 27 (Table 6). This compound displayed low hERG inhibitory
activity as with N-trifluoromethyl derivatives thus far. In vivo low
clearance illustrated the in vitro low clearance of 27, with the compound
displaying favorable area under the curve, prolonged mean residence
time, and good oral bioavailability (F = 57%).
erence together with the reduced
ring by the trifluoromethyl group.
π-electron density of the pyrimidine
We discovered that the N-trifluoromethyl group was a key motif of
the central moiety to attain high GPR119/hERG selectivity and focused
Table 2
Analysis of the single-crystal X-ray structures of 6-(indolin-1-yl)-N-(piperidin-4-
yl)pyrimidin- 4-amine derivatives.
Distance N-planeb (angstrom)
Compound
Dihedral angle
(degree)
τ
1
τ12
8aa
8ba
Me
Et
0.089 (0.0021)
0.077 (0.0020)
0.102 (0.0021)
0.065 (0.0020)
0.097 (0.0018)
1.8
169.3
170.6
164.7
170.4
166.9
1.3
ꢀ 1.2
ꢀ 0.9
ꢀ 26.5
10
CF3
a
Two crystallographically independent X-ray structures were observed.
Distance between the nitrogen atom at the 4-position of the pyrimidine ring
b
and the triangle formed by nitrogen’s three substituents: C4, C4′, and C1′′. Mean
The antidiabetic effects of 27 were assessed by an oral glucose
values and standard deviations are presented in parentheses.
5

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
Fig. 3. Profile of 13 and design of compounds for better solubility while retaining potency.
Table 3
Replacement of the indoline ring with various nonaromatic rings.
Compound
R
hGPR119a EC50(nM)
43
Compound
R
hGPR119a EC50(nM)
270
16
20
17
>10000
21
22
7100
960
18
3.6
a
Agonist activity against human GPR119. EC50 values are shown as mean (n = 2).
Table 4
Exploration of the substitute on 1,2,4-oxadiazole.
Table 6
hERG inhibitory activity and pharmacokinetic profiles of 27 in rats.
hERGa%
Vdssb(mL/
h/kg)
CLtotal
(mL/h/
kg)
AUCc
(ng*h/
kg)
MRTc
(h)
Fc
b
Compound
inhibitionat
(%)
10
19
μM
27
1270
373
1540
4.05
57
a
Automated patch clamp assay. Percentages of inhibition are represented as
mean ± standard deviation (n = 4).
b
hGPR119a EC50
(nM)
CLint.
(
μ
L/
Solubilityc
mL)
(μg/
b
Compound
R
Rat cassette dosing, 0.1 mg/kg, iv.
min/mg)
HLM
c
Rat cassette dosing, 1 mg/kg, po.
RLM
25a
25b
27
iPr
2.7
5.4
4.6
108
31
36
13
<1
29
15
21
tolerance test (OGTT) in N-STZ-1.5 rats (Fig. 4). Compound 27 and
vehicle were administered 1 h before the oral glucose load, and plasma
glucose and insulin concentrations were monitored over 2 h. As shown
in Fig. 4, compound 27 effectively lowered the plasma glucose levels
after oral administration, which was accompanied by dose-dependent
insulin secretion. The glucose-lowering effect of compound 27 was
significant (p < 0.025) at a dose of 0.3 mg/kg.
cPr
27
a
Agonist activity against human GPR119. EC50 values are shown as mean (n
= 2).
b
c
Human or rat hepatic microsomal clearance.
pH6.8.
4. Conclusion
Table 5
We described the optimization of an initial lead 1 to 27. The novel
potent GPR119 agonist 27 with excellent ADME-Tox profiles showed a
remarkable blood glucose-lowering effect from the dosing of 0.3 mg/kg
in the OGTT in N-STZ-1.5 rats. Through the optimization effort, we
discovered that the N-trifluoromethyl group was a key motif of the
central moiety; it not only increased agonistic activity but also yielded
high GPR119/hERG selectivity. Improvement of hERG profiles may be
Rat GPR119 agonist activity, in vitro hormone-secretion activity on HIT-T15,
and GLUTag cell line of 27.
Compound
rGPR119a EC50
(nM)
Insulin secretion in
HIT-T15b EC50 (nM)
GLP-1 secretion in
GLUTagc EC50 (nM)
27
24
4.8
3.8
a
Agonist activity against rat GPR119. EC50 values are shown as mean (n = 2).
Insulin secretion assay in HIT-T15 cell (n = 2).
b
c
ascribed to conformational preference, with reduced
π-electron density
GLP-1 secretion assay in GLUTag cell (n = 2).
of the pyrimidine ring linked to the trifluoromethyl group. A second key
6

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
Fig. 4. OGTT results using 27 in N-STZ-1.5 rats. Glucose-lowering and insulinotropic effects of compound 27 during OGTT by a single dose in N-STZ-1.5 rats. (A)
AUC0–120min of plasma glucose levels and (C) plasma insulin levels. Values are mean ± standard deviation (n = 6): (#) p ≤ 0.025 vs. control by one-tailed Wil-
liams’ test.
challenge in this program was the weak solubility of the new lead 13. We
focused on the number of aromatic rings and succeeded in improving
solubility by replacing the indoline ring with a piperidin-4-yl-oxy moi-
ety. Subsequent modification to the potential metabolic site of 25a,
which was the alkyl moiety on the 1,2,4-oxadiazole ring, led to the
identification of 27 having a fluorine atom introduced at the branching
site of the isopropyl group. More follow-up findings will be reported in
the near future.
tert-butyl 4-aminopiperidine-1-carboxylate (10.0 g, 49.9 mmol), and
TEA (10.5 mL, 75.3 mmol) in EtOH was stirred at 80 ◦C for 16 h. After
the mixture was concentrated under reduced pressure, the residue was
diluted with AcOEt, washed with water and brine, and dried over
magnesium sulfate (MgSO4). The solvent was removed by evaporation
to give tert-butyl 4-[(6-chloropyrimidin-4-yl)amino]piperidine-1-
carboxylate as a solid (13.2 g, 84%). 1H NMR (300 MHz, CDCl3) δ
1.30–1.55 (11H, m), 1.93–2.11 (2H, m), 2.81–3.06 (2H, m), 3.86 (1H,
bs), 3.98–4.23 (2H, m), 5.11 (1H, bs), 6.34 (1H, s), 8.34 (1H, s).
Step B. To a mixture of tert-butyl 4-[(6-chloropyrimidin-4-yl)amino]
piperidine-1-carboxylate (8.00 g, 25.6 mmol) and TEA (5.44 mL, 39.0
5. Experimental section
◦
5.1. Chemistry
mmol) in THF was added TFAA (6.45 g, 30.7 mmol) at 0 C, and the
mixture was stirred at room temperature for 16 h. The reaction was
quenched by addition of water and extracted with AcOEt. The organic
layer was washed with brine and dried over MgSO4. After removal of the
solvent, the residue was purified by silica gel column chromatography
(hexane/AcOEt = 95/5 to 60/40) to give the title compound as an oil (5,
9.22 g, 88%). 1H NMR (300 MHz, CDCl3) δ 1.43 (9H, s), 1.48–1.71 (2H,
m), 1.87 (2H, d, J = 12.1 Hz), 2.78 (2H, t, J = 12.6 Hz), 4.06–4.36 (2H,
m), 4.45–4.67 (1H, m), 7.27 (1H, s), 9.01 (1H, s).
NMR spectra were recorded on Bruker AVANCE III or Bruker
AVANCE 300 spectrometer (1H at 300 MHz and 19F at 282 MHz).
Chemical shifts for 1H NMR are given in parts per million (ppm)
downfield from tetramethysilane (δ) as the internal standard in
deuterated solvent and coupling constants (J) are in Hertz (Hz). Data are
reported as follows: chemical shift, integration, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of
doublet, td = triplet of doublets, tt = triplet of triplets, dtd = doublet of
triplet of doublets, ddd = doublet of doublet of doublets, bs = broad
singlet), and coupling constants. All solvents and reagents were obtained
from commercial suppliers and used without further purification. Thin-
layer chromatography (TLC) was performed on Merck silica gel plates
60F254. Column chromatography was performed on silica gel 60
(0.063–0.200 or 0.040–0.063 mm, E. Merck), basic silica gel (Chroma-
torex NH, 100–200 mesh, Fuji Silysia Chemical Ltd.) or Purif-Pack (Si or
NH, Moritex Corporation). LC–MS analysis was performed on a Waters,
Agilent, or Shimadzu Liquid Chromatography–Mass Spectrometer Sys-
tem, operating in APCI (+or ꢀ ) or ESI (+or ꢀ ) ionization mode. Analytes
were eluted using a linear gradient of 0.05% TFA containing water/
acetonitrile or 5 mM ammonium acetate containing water/acetonitrile
mobile phase. Determination of chemical purity by HPLC (detection at
220 nm) was conducted using a Shimadzu Liquid Chromatography
System with 0.05% TFA containing water/acetonitrile mobile phase.
Elemental analyses were performed by Takeda Analytical Research
Laboratories, Ltd. Yields are not optimized.
5.1.2. 1-(6-Chloropyrimidin-4-yl)-5-(methylsulfonyl)-2,3-dihydro-1H-
indole (7)
A mixture of 4,6-dichloropyrimidine (4.10 g, 27.5 mmol), 5-(meth-
ylsulfonyl)-2,3-dihydro-1H-indole (5.00 g, 25.0 mmol), and EtOH (160
mL) was refluxed for 4 h. After the mixture was concentrated under
reduced pressure, saturated aqueous NaHCO3 solution was added to the
residue. The precipitated solid was collected by filtration, washed with
water, and dried under reduced pressure to give the title compound as a
colorless solid (5.50 g, 71%). MS (ESI/APCI) m/z 310 [M+H]+. 1H NMR
(300 MHz, DMSO‑d6) δ 3.17 (3H, s), 3.24–3.37 (2H, m), 4.16 (2H, t, J =
8.7 Hz), 7.09 (1H, s), 7.73–7.85 (2H, m), 8.57 (1H, d, J = 9.4 Hz), 8.68
(1H, s).
5.1.3. tert-Butyl 4-({6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]
pyrimidin-4-yl}amino)piperidine-1-carboxylate (6)
Method A (from compound 5). A mixture of compound 5 (9.00 g,
22.0 mmol), 5-(methylsulfonyl)-2,3-dihydro-1H-indole (3.00 g, 15.2
mmol), Pd2(dba)3 (641 mg, 0.700 mmol), Xantphos (810 mg, 1.40
mmol), and Cs2CO3 (8.10 g, 24.9 mmol) in toluene (120 mL) was stirred
at 100 ◦C under Argon (Ar) atmosphere for 16 h. The reaction mixture
was partitioned between AcOEt and water. The organic layer was
5.1.1. tert-Butyl 4-[(6-chloropyrimidin-4-yl)(trifluoroacetyl)amino]piperi-
dine-1-carboxylate (5)
Step A. A mixture of 4,6-dichloropyrimidine (8.93 g, 59.9 mmol),
7

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
washed with brine and dried over MgSO4. The solvent was removed by
evaporation to give an oil. The oil was dissolved in THF/MeOH (1:1 v/v,
200 mL), and then 1 M aqueous NaOH solution (15 mL) was added. The
resulting mixture was stirred at room temperature for 2 h. After removal
of THF and MeOH, the residue was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and concentrated under
reduced pressure to give a crude material, which was purified by silica
gel column chromatography (hexane/AcOEt = 50/50 to 10/90) to give
the title compound as a white solid (2.40 g, 33% over 2 steps). MS (ESI/
APCI) m/z 474 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.14–1.38 (2H,
m), 1.41 (9H, s), 1.78–1.95 (2H, m), 2.80–3.03 (2H, m), 3.12 (3H, s),
3.25 (2H, t, J = 8.7 Hz), 3.79–4.13 (5H, m), 5.78 (1H, s), 7.14 (1H, d, J
= 7.5 Hz), 7.59–7.75 (2H, m), 8.25 (1H, s), 8.47 (1H, d, J = 9.0 Hz).
Anal. Calcd for C23H31N5O4S: C, 58.33; H, 6.60; N, 14.79. Found: C,
58.33; H, 6.71; N, 14.74.
5.1.7. tert-Butyl 4-[(cyclopropylmethyl){6-[5-(methylsulfonyl)-2,3-
dihydro-1H-indol-1-yl]pyrimidin-4-yl}amino]piperidine-1-carboxylate (8d)
Compound 8d was prepared from compound 6 and (bromomethyl)
cyclopropane in a manner similar to that described for compound 8b.
White solid. Yield 52%. MS (ESI/APCI) m/z 528 [M+H]+. 1H NMR (300
MHz, CDCl3) δ 0.25–0.39 (2H, m), 0.57–0.70 (2H, m), 0.91–1.07 (1H,
m), 1.48 (9H, s), 1.55–1.85 (4H, m), 2.78–2.93 (2H, m), 3.03 (3H, s),
3.21 (2H, d, J = 5.7 Hz), 3.28 (2H, t, J = 8.7 Hz), 4.10 (2H, t, J = 8.9 Hz),
4.16–4.40 (2H, m), 4.66–4.90 (1H, m), 5.79 (1H, s), 7.68 (1H, s), 7.75
(1H, dd, J = 8.5, 2.1 Hz), 8.40 (1H, s), 8.51 (1H, d, J = 8.7 Hz). Anal.
Calcd for C27H37N5O4S: C, 61.46; H, 7.07; N, 13.27. Found: C, 61.16; H,
7.25; N, 13.37.
5.1.8. tert-Butyl 4-[(2-methoxyethyl){6-[5-(methylsulfonyl)-2,3-dihydro-
1H-indol-1-yl]pyrimidin-4-yl}amino]piperidine-1-carboxylate (8e)
Compound 8e was prepared from compound 6 and 2-bromoethyl
methyl ether in a manner similar to that described for compound 8b.
White solid. Yield 9%. MS (ESI/APCI) m/z 532 [M+H]+. 1H NMR (300
MHz, CDCl3) δ 1.48 (9H, s), 1.53–1.84 (4H, m), 2.75–2.94 (2H, m), 3.03
(3H, s), 3.28 (2H, t, J = 8.7 Hz), 3.38 (3H, s), 3.51 (4H, s), 4.00–4.38
(4H, m), 4.65–4.87 (1H, m), 5.83 (1H, s), 7.64–7.80 (2H, m), 8.40 (1H,
s), 8.49 (1H, d, J = 8.7 Hz). Anal. Calcd for C26H37N5O5S: C, 58.74; H,
7.01; N, 13.17. Found: C, 59.13; H, 7.11; N, 12.77.
Method B (from compound 7). A mixture of compound 7 (11.0 g,
35.6 mmol), tert-butyl 4-aminopiperidine-1-carboxylate (10.0 g, 49.9
mmol), Pd2(dba)3 (1.83 g, 2.00 mmol), XPhos (1.91 g, 4.01 mmol), and
Cs2CO3 (23.4 g, 71.8 mmol) in toluene (150 mL) was stirred at 110 ◦C
under nitrogen (N2) atmosphere for 16 h. The reaction mixture was
partitioned between AcOEt and water. The organic layer was washed
with brine and dried over MgSO4. After removal of the solvent, the
residue was purified by silica gel column chromatography (hexane/
AcOEt = 70/30 to 20/80) to give the title compound as a white solid
(7.10 g, 42%).
5.1.9. tert-Butyl 4-([(methylsulfanyl)carbonothioyl]{6-[5-
(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}amino)
piperidine-1-carboxylate (9)
5.1.4. tert-Butyl 4-(methyl{6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-
yl]pyrimidin-4-yl}amino)piperidine-1-carboxylate (8a)
To a mixture of compound 6 (8.32 g, 17.6 mmol) in THF (500 mL)
was added dropwise nBuLi (1.6 M hexane solution, 13.2 mL, 21.1 mmol)
at 0 ◦C under N2 atmosphere, and the mixture was stirred at 0 ◦C for 1 h.
Carbon disulfide (1.59 mL, 26.4 mmol) was added to the mixture at 0 ◦C.
The mixture was allowed to warm to room temperature followed by
Compound 8a was prepared from compound 6 and iodomethane in a
manner similar to that described for compound 8b. White solid. Yield
1
88%. MS (ESI/APCI) m/z 488 [M+H]+. H NMR (300 MHz, CDCl3) δ
1.48 (9H, s), 1.57–1.77 (4H, m), 2.78–2.94 (5H, m), 3.02 (3H, s), 3.27
(2H, t, J = 8.7 Hz), 3.98–4.17 (2H, m), 4.17–4.39 (2H, m), 4.81–5.04
(1H, m), 5.59 (1H, s), 7.68 (1H, s), 7.75 (1H, dd, J = 8.7, 1.9 Hz), 8.39
(1H, s), 8.53 (1H, d, J = 8.7 Hz). Anal. Calcd for C24H33N5O4S: C, 59.12;
H, 6.82; N, 14.36. Found: C, 58.95; H, 6.90; N, 14.63.
◦
stirring for 7 h. After cooling to 0 C, MeI (3.28 mL, 52.7 mmol) was
added, and the resulting mixture was stirred at room temperature for 63
h. The reaction was quenched by addition of water and extracted with
AcOEt. The organic layer was washed with brine and dried over sodium
sulfate (Na2SO4). After removal of the solvent, the residue was purified
by silica gel column chromatography (hexane/AcOEt = 60/40 to 0/100)
to give the title compound as a pale yellow amorphous solid (5.91 g,
60%). 1H NMR (300 MHz, CDCl3) δ 1.41 (9H, s), 1.49 (2H, dd, J = 12.5,
4.5 Hz), 2.10 (2H, d, J = 12.1 Hz), 2.60 (3H, s), 2.82 (2H, t, J = 13.0 Hz),
3.07 (3H, s), 3.38 (2H, t, J = 8.5 Hz), 4.07–4.29 (4H, m), 5.54 (1H, tt, J
= 11.9, 3.6 Hz), 6.57 (1H, d, J = 0.8 Hz), 7.79 (1H, d, J = 1.5 Hz), 7.86
(1H, dd, J = 8.5, 2.1 Hz), 8.65 (1H, d, J = 8.7 Hz), 8.90 (1H, d, J = 1.1
Hz).
5.1.5. tert-Butyl 4-(ethyl{6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-
yl]pyrimidin-4-yl}amino)piperidine-1-carboxylate (8b)
To a mixture of compound 6 (1.30 g, 2.75 mmol) and iodoethane
(0.328 mL, 4.10 mmol) in DMF (20 mL) was added sodium hydride (60%
oil dispersion, 164 mg, 4.1 mmol) at 0 ◦C, and the mixture was stirred at
room temperature for 16 h. The reaction was quenched by addition of
water and extracted with AcOEt. The organic layer was washed with
brine and dried over MgSO4. After removal of the solvent, the residue
was purified by silica gel column chromatography (hexane/AcOEt = 90/
10 to 50/50) to give the title compound as a white solid (903 mg, 65%).
MS (ESI/APCI) m/z 502 [M+H]+. 1H NMR (300 MHz, CDCl3) δ 1.22 (3H,
t, J = 7.0 Hz), 1.48 (9H, s), 1.56–1.84 (4H, m), 2.76–2.95 (2H, m), 3.03
(3H, s), 3.19–3.48 (4H, m), 4.09 (2H, t, J = 8.7 Hz), 4.22 (2H, bs), 4.87
(1H, bs), 5.61 (1H, s), 7.68 (1H, s), 7.75 (1H, dd, J = 8.7, 1.9 Hz), 8.39
(1H, s), 8.50 (1H, d, J = 8.7 Hz). Anal. Calcd for C25H35N5O4S: C, 59.86;
H, 7.03; N, 13.96. Found: C, 59.57; H, 6.98; N, 13.78.
5.1.10. tert-Butyl 4-[{6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]
pyrimidin-4-yl}(trifluoromethyl)amino]piperidine-1-carboxylate (10)
Condition A. To a mixture of compound 9 (170 mg, 0.302 mmol) and
TBAH2F3 (455 mg, 1.51 mmol) in toluene (20 mL) was added NBS (215
mg, 1.21 mmol) at room temperature. The mixture was stirred at room
temperature for 6 h. The reaction mixture was diluted with AcOEt and
quenched by addition of aqueous NaOH solution. The organic layer was
separated, washed with water and brine, and dried over Na2SO4. After
removal of the solvent, the residue was purified by silica gel column
chromatography (hexane/AcOEt = 70/30 to 0/100) to give a solid. The
obtained solid was suspended in diisopropylether (IPE) and the precip-
itated solid was collected to give the title compound as a white solid (34
mg, 21%). MS (ESI/APCI) m/z 542 [M+H]+. 1H NMR (300 MHz, CDCl3)
δ 1.48 (9H, s), 1.80–1.93 (2H, m), 1.99–2.14 (2H, m), 2.79 (2H, t, J =
12.1 Hz), 3.04 (3H, s), 3.27–3.39 (2H, m), 4.06–4.33 (4H, m), 4.57–4.75
(1H, m), 6.26 (1H, d, J = 1.5 Hz), 7.73 (1H, d, J = 1.5 Hz), 7.80 (1H, dd,
J = 8.7, 1.9 Hz), 8.56 (1H, d, J = 8.7 Hz), 8.62 (1H, s). 19F NMR (282
MHz, DMSO‑d6) δ ꢀ 51.16 (s). Anal. Calcd for C24H30F3N5O4S: C, 53.22;
H, 5.58; N, 12.93. Found: C, 53.22; H, 5.47; N, 12.93.
5.1.6. tert-Butyl 4-[{6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]
pyrimidin-4-yl}(propyl)amino]piperidine-1-carboxylate (8c)
Compound 8c was prepared from compound 6 and 1-iodopropane in
a manner similar to that described for compound 8b. White solid. Yield
1
66%. MS (ESI/APCI) m/z 516 [M+H]+. H NMR (300 MHz, CDCl3) δ
0.89–1.06 (3H, m), 1.49 (9H, s), 1.59–1.82 (6H, m), 2.76–2.94 (2H, m),
3.03 (3H, s), 3.11–3.23 (2H, m), 3.28 (2H, t, J = 8.7 Hz), 4.08 (2H, t, J =
8.7 Hz), 4.15–4.38 (2H, m), 4.85 (1H, bs), 5.58 (1H, s), 7.69 (1H, s), 7.75
(1H, dd, J = 8.7, 1.9 Hz), 8.39 (1H, s), 8.48 (1H, d, J = 8.7 Hz). Anal.
Calcd for C26H37N5O4S: C, 60.56; H, 7.23; N, 13.58. Found: C, 60.52; H,
7.21; N, 13.50.
Condition B. To a mixture of compound 9 (564 mg, 1.00 mmol) and
8

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
TBAH2F3 (1.51 g, 5.00 mmol) in toluene (10 mL) was added NBS (712
mg, 4.00 mmol) at ꢀ 78 ◦C. After stirring at ꢀ 78 ◦C for 30 min, the
mixture was allowed to warm to at 0 ◦C followed by stirring at 0 ◦C for 7
h. The reaction mixture was diluted with AcOEt and quenched by
addition of aqueous NaOH solution. The organic layer was separated,
washed with water and brine, and dried over Na2SO4. After removal of
the solvent, the residue was purified by silica gel column chromatog-
raphy ((hexane/AcOEt = 70/30 = 0/100) to give the title compound as
a white solid (220 mg, 41%).
mL, 248 mmol) in MeCN (250 mL) was stirred at 85 ◦C for 10 h. After the
reaction mixture was concentrated in vacuo, the residue was diluted
with AcOEt and THF, washed with 0.2 M aqueous NaOH solution and
brine, and dried over Na2SO4. The solvent was removed by evaporation
to give a pale yellow solid. The solid was suspended in AcOEt and the
precipitated solid was collected to give the title compound as a white
solid (29.0 g, 74.7%). MS (ESI/APCI) m/z 313 [M+H]+. 1H NMR (300
MHz, DMSO‑d6) δ 1.16–1.47 (11H, m), 1.74–1.92 (2H, m), 2.78–3.01
(2H, m), 3.77–4.12 (3H, m), 6.48 (1H, bs), 7.71 (1H, d, J = 7.6 Hz), 8.27
(1H, s).
5.1.11. 6-[5-(Methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]-N-(piperidin-4-
yl)-N-(trifluoromethyl)pyrimidin-4-amine (11)
5.1.15. tert-Butyl 4-[(6-chloropyrimidin-4-yl)(trifluoromethyl)amino]
piperidine-1-carboxylate (15)
A mixture of compound 10 (220 mg, 0.405 mmol), 4 M HCl in AcOEt
(2 mL), AcOEt (10 mL), and MeOH (10 mL) was stirred at room tem-
perature for 20 h. After the reaction mixture was concentrated under
reduced pressure, the residue was diluted with AcOEt and water, and
then basified with aqueous NaOH solution. The organic layer was
separated and dried over Na2SO4. The solvent was removed by evapo-
ration and dried to give the title compound as a white solid (156 mg,
87%). MS (ESI/APCI) m/z 442 [M+H]+. 1H NMR (300 MHz, CDCl3) δ
1.83–1.96 (2H, m), 1.98–2.16 (2H, m), 2.72 (3H, td, J = 12.6, 2.8 Hz),
3.04 (3H, s), 3.19 (2H, dd, J = 10.6, 2.6 Hz), 3.32 (2H, t, J = 8.5 Hz),
4.12 (2H, t, J = 8.7 Hz), 4.48–4.66 (1H, m), 6.27 (1H, d, J = 1.1 Hz),
7.73 (1H, d, J = 1.1 Hz), 7.80 (1H, dd, J = 8.7, 2.3 Hz), 8.56 (1H, d, J =
8.7 Hz), 8.63 (1H, d, J = 0.8 Hz).
Step A. To a solution of 14 (30.0 g, 95.9 mmol) in DMF(dry) (450 mL)
was added sodium hydride (60% oil dispersion, 3.84 g, 95.9 mmol) at
room temperature. After the mixture was stirred at room temperature
for 1 h, carbon disulfide (9.81 mL, 163 mmol) was added dropwise
followed by stirring at room temperature for further 2 h. MeI (10.2 mL,
163 mmol) was added and the resulting mixture was stirred at room
temperature for 1 h. The reaction was quenched with water. The mixture
was extracted with AcOEt, washed with water (3 times) and brine, and
dried over Na2SO4. After removal of the solvent, the residue was purified
by column chromatography (hexane/AcOEt = 90/10 to 80/20) to give
tert-butyl 4-((6-chloropyrimidin-4-yl)(methylthiocarbonothioyl)amino)
piperidine-1-carboxylate as a yellow oil (30.8 g, 80%). MS (ESI/APCI)
m/z 403 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.22–1.43 (11H, m),
1.94–2.06 (2H, m), 2.56 (3H, s), 2.69–2.93 (2H, m), 3.91–4.07 (2H, m),
5.30 (1H, tt, J = 12.0, 3.5 Hz), 8.07 (1H, s), 9.19 (1H, s).
5.1.12. 4-[{6-[5-(Methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-
yl}(trifluoromethyl)amino]piperidine-1-carbonitrile (12)
To a mixture of compound 11 (330 mg, 0.748 mmol), NaHCO3 (190
mg, 2.26 mmol), THF (15 mL), and water (5 mL) was added cyanogen
bromide (105 mg, 0.991 mmol) at 0 ◦C, and the mixture was stirred at
room temperature for 16 h. The reaction mixture was diluted with
saturated aqueous NaHCO3 solution and extracted with a mixed solvent
of AcOEt and THF. The organic layer was separated, washed with brine,
and dried over MgSO4. The solvent was removed by evaporation and
dried to give the title compound as a white solid (335 mg, 96%). MS
Step B. To a solution of tert-butyl 4-((6-chloropyrimidin-4-yl)
(methylthiocarbonothioyl)amino)piperidine-1-carboxylate (1.80 g,
4.47 mmol) in PhCl (36 mL) were added triethylamine trihydrofluoride
(3.60 g, 22.3 mmol) and 1,3-dibromo-5,5-dimethylimidazolidine-2,4-
dione (3.96 g, 13.85 mmol)) at ꢀ 20 ◦C. The mixture was stirred at
ꢀ 20 ◦C under N2 for 20 min. The reaction was quenched with saturated
aqueous NaHCO3 solution at the same temperature. The mixture was
extracted with AcOEt, successively washed saturated aqueous NaHCO3
solution, aqueous Na2S2O3 solution and brine, and dried over Na2SO4.
After removal of the solvent, the residue was purified by column chro-
matography (NH, hexane/AcOEt = 88/12) to give the title compound as
(ESI/APCI) m/z 467 [M+H]+. 1H NMR (300 MHz, DMSO‑d6)
δ
1.84–2.12 (4H, m), 3.14–3.30 (7H, m), 3.40–3.54 (2H, m), 4.12–4.21
(2H, m), 4.26–4.42 (1H, m), 6.38 (1H, s), 7.73–7.82 (2H, m), 8.56 (1H,
d, J = 9.0 Hz), 8.70 (1H, s).
1
a white solid (560 mg, 32.9%). MS (ESI/APCI) m/z 381 [M+H]+. H
NMR (300 MHz, DMSO‑d6) δ 1.40 (9H, s), 1.75–1.94 (4H, m), 2.74–2.94
5.1.13. 6-[5-(Methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]-N-{1-[3-
(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}-N-(trifluoromethyl)
pyrimidin-4-amine (13)
(2H, m), 3.95–4.09 (2H, m), 4.42–4.56 (1H, m), 7.31 (1H, s), 8.81 (1H,
s).
To a mixture of compound 12 (500 mg, 1.07 mmol) and N-hydroxy-
2-methylpropanimidamide (131 mg, 1.29 mmol) in anhydrous DMF (20
mL) were added ZnCl (1.0 M Et2O solution, 0.640 mL, 0.640 mmol) and
p-TsOH monohydrate (122 mg, 0.641 mmol) at room temperature, and
the mixture was stirred at 85 ◦C for 10 h. The reaction mixture was
diluted with AcOEt successively washed with 0.03 M hydrochloric acid,
0.03 M aqueous NaOH solution, and brine, and dried over Na2SO4. After
removal of the solvent, the residue was purified by silica gel column
chromatography (hexane/AcOEt = 50/50 to 20/80) to give the title
compound as a white powder (410 mg, 69%). MS (ESI/APCI) m/z 552
[M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.18 (6H, d, J = 6.8 Hz),
1.94–2.07 (4H, m), 2.74–2.89 (1H, m), 3.16 (3H, s), 3.19–3.39 (4H, m),
4.01–4.11 (2H, m), 4.17 (2H, t, J = 8.5 Hz), 4.36–4.52 (1H, m), 6.40
(1H, s), 7.73–7.80 (2H, m), 8.51–8.59 (1H, m), 8.69 (1H, s). 19F NMR
5.1.16. tert-Butyl 4-[(6-{[1-(methanesulfonyl)piperidin-4-yl]amino}pyri-
midin-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate (16)
A mixture of 15 (300 mg, 0.790 mmol), 1-(methylsulfonyl)piperidin-
4-amine (211 mg, 1.18 mmol) and DIPEA (0.274 mL, 1.58 mmol) in
DMA (6 mL) was stirred at 100 ◦C for 15 h. The mixture was stirred at
room temperature for 15 h. The mixture was extracted with AcOEt,
washed water (3 times) and brine, and dried over Na2SO4. After removal
of the solvent, the residue was purified by column chromatography
(hexane/AcOEt = 50/50 to 20/80) and crystallization from diethyl
ether (Et2O) to give the title compound as a white solid (240 mg, 58.3%).
MS (ESI/APCI) m/z 523 [M+H]+.1H NMR (300 MHz, DMSO‑d6) δ
1.33–1.54 (11H, m), 1.67–1.85 (4H, m), 1.90–2.03 (2H, m), 2.67–2.97
(7H, m), 3.44–3.55 (2H, m), 3.85–4.08 (3H, m), 4.17–4.31 (1H, m), 6.15
(1H, bs), 7.50 (1H, d, J = 7.6 Hz), 8.29 (1H, s). Anal. Calcd for
(282 MHz, DMSO‑d6)
24H28F3N7O3S⋅0.1AcOEt: C, 52.30; H, 5.18; N, 17.50. Found: C, 52.49;
H, 5.28; N, 17.11.
δ
ꢀ
51.34 (s). Anal. Calcd for
C21H33N6O4SF3:C,48.26;H,6.36;N,16.08;S,6.14;F,10.91.Found:C,
C
48.12;H,6.41;N,15.89.
5.1.17. tert-Butyl 4-[(6-{[1-(methanesulfonyl)piperidin-4-yl](methyl)
amino}pyrimidin-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate
(17)
5.1.14. tert-Butyl 4-(6-chloropyrimidin-4-ylamino)piperidine-1-
carboxylate (14)
A mixture of 4,6-dichloropyrimidine (4, 18.5 g, 124 mmol), tert-butyl
4-aminopiperidine-1-carboxylate (24.8 g, 124 mmol), and TEA (34.5
To a mixture of 16 (140 mg, 0.270 mmol) in DMF(dry) (6 mL) was
added NaH (12.9 mg, 0.320 mmol) at room temperature. After the
9

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
mixture was stirred at room temperature for 30 min, MeI (0.0250 mL,
0.400 mmol) was added. The resulting mixture was stirred at room
temperature for 1 h. The mixture was quenched with water, extracted
with AcOEt, washed with water (3 times) and brine, and dried over
Na2SO4. After removal of the solvent, the residue was purified by col-
umn chromatography (Hexane/AcOEt = 60/40 to 30/70) to give the
title compound as a pale yellow solid (120 mg, 83%). MS (ESI/APCI) m/
z 537 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.38 (9H, s), 1.60–1.92
(8H, m), 2.69–2.97 (10H, m), 3.58–3.73 (2H, m), 3.91–4.06 (2H, m),
4.10–4.23 (1H, m), 4.53–4.83 (1H, m), 6.15 (1H, s), 8.37 (1H, s). Anal.
to give the title compound as a white powder (70.0 mg, 16.0%). MS
(ESI/APCI) m/z 523 [M+H]+. 1H NMR (300 MHz, DMSO‑d6)
δ
1.31–1.69 (13H, m), 1.72–1.90 (4H, m), 2.09–2.29 (4H, m), 2.70–2.90
(2H, m), 2.94 (3H, s), 3.05–3.21 (1H, m), 3.92–4.09 (2H, m), 4.28–4.45
(1H, m), 4.96–5.11 (1H, m), 6.41 (1H, s), 8.60 (1H, s). Anal. Calcd for
C
22H33N4O5SF3:C,50.56;H,6.36;N,10.72;S,6.14;F,10.91.Found:C,50.60;
H,6.41;N,10.50.
5.1.21. tert-Butyl 4-[(6-{[1-(methanesulfonyl)azetidin-3-yl]oxy}pyrimi-
din-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate (21)
Calcd
for
C22H35N6O4SF3:C,49.24;H,6.57;N,15.66;S,5.98;F,10.62.
MsCl (0.0980 mL, 1.26 mmol) was added to a solution of 15 (0.48 g,
1.15 mmol) and TEA (0.176 mL, 1.26 mmol) in DMF(dry) (10 mL) at
0 ◦C. The mixture was stirred at room temperature under N2 overnight.
The mixture was poured into water and extracted with AcOEt. The
organic layer was separated, washed with water and brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt = 50/50 to 0/100) to give the
title compound as a beige solid (0.268 g, 47.0%). MS (ESI/APCI) m/z
496 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.39 (9H, s), 1.69–1.86
(4H, m), 2.80 (2H, bs), 3.29 (3H, s), 3.99 (2H, d, J = 11.7 Hz), 4.14 (2H,
dd, J = 10.4, 3.6 Hz), 4.19–4.32 (1H, m), 4.44 (2H, dd, J = 10.2, 6.4 Hz),
5.32–5.56 (1H, m), 5.95 (1H, s), 8.37 (1H, s). Anal. Calcd for
Found:C,49.10;H,6.63;N,15.46.
5.1.18. tert-Butyl 4-[(6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}pyrimi-
din-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate (18)
To a solution of 1-(methylsulfonyl)piperidin-4-ol (21.2 g, 118 mmol)
in DMA (450 mL) was added sodium hydride (4.73 g, 118 mmol) at room
temperature, and the mixture was stirred at room temperature for 1.5 h.
15 (30.0 g, 78.8 mmol) was added to the mixture at room temperature in
one portion. The resulting mixture was stirred at room temperature for
5 h. The reaction was quenched with water. The mixture was extracted
with AcOEt, successively washed with water (3 times) and brine, and
dried over Na2SO4. After removal of the solvent, the residue was dis-
solved in AcOEt (150 mL) at 80 ◦C. IPE (300 mL) was added to the
mixture at 80 ◦C and then the resulting mixture was allowed to cool to
room temperature. After 15 h, the precipitated solid was collected and
washed with IPE to give the title compound as a white powder (32.1 g,
78%). MS (ESI/APCI) m/z 524 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ
1.40 (9H, s), 1.69–1.89 (6H, m), 1.99–2.13 (2H, m), 2.71–2.88 (2H, m),
2.90 (3H, s), 3.06–3.19 (2H, m), 3.28–3.43 (2H, m), 3.94–4.07 (2H, m),
4.32–4.47 (1H, m), 5.19–5.30 (1H, m), 6.45 (1H, s), 8.61 (1H, d, J = 0.8
Hz). Anal. Calcd for C21H32N5O5SF3:C,48.17;H,6.16;N,13.38;S,6.12;
F,10.89.Found:C,48.26;H,6.21;N,13.25.
C
19H28N5O5SF3:C,46.05;H,5.70;N,14.13;S,6.47;F,11.50.Found:C,46.28;
H,5.90;N,13.87.
5.1.22. tert-Butyl 4-[(6-{[1-(methanesulfonyl)azetidin-3-yl]methoxy}pyr-
imidin-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate (22)
Sodium hydride (13.0 mg, 0.330 mmol) was added to a solution of
(1-(methylsulfonyl)azetidin-3-yl)methanol (44.8 mg, 0.27 mmol) in
THF(dry) (3 mL) at room temperature. The mixture was stirred at the
same temperature under N2 for 1 h. 15 (103 mg, 0.27 mmol) was added
to the mixture. The mixture was stirred at room temperature for 3 h. The
mixture was quenched with ice at room temperature and extracted with
AcOEt. The organic layer was separated, washed with water and brine,
dried over MgSO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/AcOEt = 50/50 to 0/100)
to give the title compound as a white solid (74.8 mg, 54.1%). MS (ESI/
APCI) m/z 454 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.40 (9H, s),
1.71–1.91 (4H, m), 2.68–2.90 (2H, m), 2.93–3.08 (4H, m), 3.72–3.80
(2H, m), 3.89–4.08 (4H, m), 4.29–4.44 (1H, m), 4.49 (2H, d, J = 6.4 Hz),
6.48 (1H, s), 8.62 (1H, s). Anal. Calcd for C20H30N5O5SF3:C,47.14;
H,5.93;N,13.74;S,6.29;F,11.19.Found:C,47.29;H,6.11;N,13.43.
5.1.19. tert-Butyl 4-{[6-({(cis)-4-[(methanesulfonyl)oxy]cyclohexyl}oxy)
pyrimidin-4-yl](trifluoromethyl)amino}piperidine-1-carboxylate (19)
To a solution of tert-butyl 4-((6-((trans)-4-hydroxycyclohexyloxy)
pyrimidin-4-yl)(trifluoromethyl)amino)piperidine-1-carboxylate (1.28
g, crude, as 2.63 mmol) in pyridine (15 mL) was added methanesulfonyl
chloride (0.447 mL, 5.78 mmol) at room temperature. The mixture was
stirred at room temperature for 2 h. After the reaction mixture was
concentrated in vacuo, the residue was diluted with AcOEt, successively
washed with water, 10% aqueous KHSO4 solution, water and brine, and
dried over Na2SO4. After removal of the solvent, the residue was purified
by column chromatography (hexane/AcOEt = 70/30 to 50/50) to give
the title compound as a colorless amorphous solid(0.850 g, 60.1%). MS
(ESI/APCI) m/z 539 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.40 (9H,
s), 1.71–1.97 (12H, m), 2.71–2.92 (2H, m), 3.19 (3H, s), 3.93–4.09 (2H,
m), 4.31–4.47 (1H, m), 4.73–4.85 (1H, m), 5.13–5.23 (1H, m), 6.45 (1H,
s), 8.59 (1H, s).
5.1.23. 6-{[1-(Methanesulfonyl)piperidin-4-yl]oxy}-N-(piperidin-4-yl)-N-
(trifluoromethyl)pyrimidin-4-amine dihydrochloride (23)
To a mixture of 18 (20.4 g, 38.96 mmol), AcOEt (115 mL) and MeOH
(70 mL) was added 4 M HCl in AcOEt (117 mL, 468 mmol) at room
temperature, and the mixture was stirred at room temperature for 3 h.
IPE (250 mL) was added to the mixture. The precipitated solid was
collected and washed with IPE to the title compound as a white powder
1
(18.9 g, 98%). MS (ESI/APCI) m/z 424 [M+H]+. H NMR (300 MHz,
5.1.20. tert-Butyl 4-[(6-{[(trans)-4-(methanesulfonyl)cyclohexyl]oxy}
pyrimidin-4-yl)(trifluoromethyl)amino]piperidine-1-carboxylate (20)
A mixture of 19 (450 mg, 0.840 mmol) and sodium thiomethoxide
(88.0 mg, 1.25 mmol) in DMF(dry) (10 mL) was stirred at 80 ◦C for 2 h.
The mixture was diluted with AcOEt, washed with water (3 times) and
brine, and dried over Na2SO4. The solvent was removed by evaporation
to give tert-butyl 4-((6-((trans)-4-(methylthio)cyclohexyloxy)pyrimidin-
4-yl)(trifluoromethyl)amino)piperidine-1-carboxylate as a crude mate-
rial (pale yellow oil).
DMSO‑d6) δ 1.70–1.86 (2H, m), 1.95–2.12 (4H, m), 2.14–2.31 (2H, m),
2.91 (3H, s), 2.94–3.19 (4H, m), 3.25–3.45 (4H, m), 4.37–4.54 (1H, m),
5.18–5.31 (1H, m), 6.55 (1H, s), 8.62 (1H, s), 8.75–8.95 (1H, m),
9.10–9.26 (1H, m), 1H was not observed.
5.1.24. 4-[(6-{[1-(Methanesulfonyl)piperidin-4-yl]oxy}pyrimidin-4-yl)
(trifluoromethyl)amino]piperidine-1-carbonitrile (24)
Cyanogen bromide (16.3 g, 154 mmol) was added to a solution of 23
(63.5 g, 128 mmol) and NaHCO3 (53.7 g, 640 mmol) in THF (200 mL)
and water (200 mL) at room temperature. The mixture was stirred at
room temperature for 1 h. The mixture was poured into water and
extracted with AcOEt and THF. The organic layer was separated, washed
with water and brine, dried over Na2SO4 and concentrated in vacuo. The
residue was suspended in IPE and the mixture was stirred for 1 h at room
temperature. The precipitate was filtered to obtain the title compound as
The oil was dissolved in DMF(dry) (10 mL), and mCPBA(70%) (618
mg, 2.51 mmol) was added at room temperature. The resulting mixture
was stirred at room temperature for 2 h. The reaction mixture was
diluted with AcOEt, successively washed with water (3 times) and brine,
and dried over Na2SO4. After removal of the solvent, the residue was
purified by column chromatography (hexane/AcOEt = 60/40 to 40/60)
10

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
a pale yellow solid(50.2 g, 88%). MS (ESI/APCI) m/z 449 [M+H]+. 1H
NMR (300 MHz, DMSO‑d6) δ 1.68–1.94 (4H, m), 1.96–2.19 (4H, m),
2.90 (3H, s), 3.03–3.26 (4H, m), 3.32–3.51 (4H, m), 4.34 (1H, t, J =
12.1 Hz), 5.25 (1H, tt, J = 7.8, 3.7 Hz), 6.48 (1H, s), 8.63 (1H, d, J = 0.8
Hz).
6.50 (1H, s), 8.62 (1H, s).
5.1.28. 2-(5-{4-[(6-{[1-(Methanesulfonyl)piperidin-4-yl]oxy}pyrimidin-
4-yl)(trifluoromethyl)amino]piperidin-1-yl}-1,2,4-oxadiazol-3-yl)propan-
2-ol (26)
MeMgCl (45.2 mL, 135 mmol) was added to a solution of 25c (25.5 g,
45.2 mmol) in THF(dry) (250 mL) at 0 ◦C. The mixture was stirred at
room temperature overnight. The mixture was quenched with saturated
aqueous ammonium chloride solution at 0 ◦C and extracted with AcOEt.
The organic layer was separated, washed with water and brine, dried
over Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (AcOEt) to give the title compound as
a white solid (19.7 g, 79%). MS (ESI/APCI) m/z 550 [M+H]+. 1H NMR
(300 MHz, DMSO‑d6) δ 1.40 (6H, s), 1.79 (2H, dtd, J = 12.4, 8.3, 3.8 Hz),
1.91–2.13 (6H, m), 2.90 (3H, s), 3.07–3.18 (2H, m), 3.25 (2H, t, J =
10.7 Hz), 3.32–3.44 (2H, m), 4.00–4.13 (2H, m), 4.37–4.56 (1H, m),
5.17–5.34 (2H, m), 6.49 (1H, s), 8.63 (1H, s).
5.1.25. 6-{[1-(Methanesulfonyl)piperidin-4-yl]oxy}-N-{1-[3-(propan-2-
yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}-N-(trifluoromethyl)pyrimidin-4-
amine (25a)
To a mixture of 24 (125 mg, 0.280 mmol) and N-hydroxyisobutyr-
imidamide (34.2 mg, 0.330 mmol) in DMF(dry) (10 mL) were added
ZnCl2 (1 M Et2O solution, 0.167 mL, 0.167 mmol) and p-TsOH mono-
hydrate (31.8 mg, 0.167 mmol) at room temperature, and the mixture
was stirred at 85 ◦C for 6 h. The mixture was diluted with AcOEt, suc-
cessively washed 0.03 M HCl solution., water, 0.03 M aqueous NaOH
solution and brine, and dried over Na2SO4. After removal of the solvent,
the residue was purified by column chromatography (hexane/AcOEt =
55/45 to 30/70) and crystallized from Et2O-hexane to give the title
compound as a white solid(103 mg, 69.3%). MS (ESI/APCI) m/z 534
[M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.18 (6H, d, J = 7.2 Hz),
1.71–1.85 (2H, m), 1.90–2.13 (6H, m), 2.75–2.87 (1H, m), 2.90 (3H, s),
3.07–3.43 (6H, m), 3.98–4.10 (2H, m), 4.38–4.53 (1H, m), 5.19–5.30
(1H, m), 6.49 (1H, s), 8.62 (1H, s). Anal. Calcd for C21H30N7O4SF3:
C,47.27;H,5.67;N,18.38;S,6.01;F,10.68.Found:C,47.14;H,5.69;N,18.31.
5.1.29. N-{1-[3-(2-fluoropropan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-
yl}-6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)
pyrimidin-4-amine (27)
Xtalfluor-E® (12.3 g, 53.7 mmol) was added to a solution of 26 (19.7
g, 35.8 mmol), TEA (4.99 mL, 35.8 mmol) and TEA trihydrogen fluoride
(11.6 mL, 71.6 mmol) in toluene (200 mL) at ꢀ 78 ◦C. The mixture was
stirred overnight (-78 ◦C to room temperature). The mixture was
quenched with saturated aqueous NaHCO3 solution at 0 ◦C and extrac-
ted with AcOEt. The organic layer was separated, washed with water
and brine, dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography (NH, hexane/AcOEt = 100/
0 to 50/50) to obtain the crude title compound as a white solid (18.8 g,
95%). The crude was dissolved in MeCN (100 mL) and water (100 mL).
Sodium periodate (3.46 g, 16.2 mmol) and ruthenium(III) chloride
5.1.26. N-[1-(3-Cyclopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl]-6-{[1-
(methanesulfonyl)piperidin-4-yl]oxy}-N-(trifluoromethyl)pyrimidin-4-
amine (25b)
To
a mixture of 24 (1.00 g, 2.23 mmol) and N-hydrox-
ycyclopropanecarboximidamide (0.268 g, 2.68 mmol) in DMF(dry) (20
mL) were added ZnCl2 (1 M Et2O solution, 1.34 mL, 1.34 mmol) and p-
TsOH monohydrate (0.254 g, 1.34 mmol) at room temperature, and the
mixture was stirred at 85 ◦C for 10 h. The mixture was diluted with
AcOEt, successively washed 0.03 M HCl solution., water, 0.03 M
aqueous NaOH solution and brine, and dried over Na2SO4. After removal
of the solvent, the residue was purified by column chromatography
(hexane/AcOEt = 60/40 to 20/80) and crystallized from Et2O-hexane to
give the title compound as a white solid (870 mg, 73.4%). MS (ESI/
APCI) m/z 532 [M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 0.75–0.98 (4H,
m), 1.71–2.12 (9H, m), 2.90 (3H, s), 3.07–3.43 (6H, m), 3.93–4.07 (2H,
m), 4.38–4.51 (1H, m), 5.19–5.31 (1H, m), 6.48 (1H, s), 8.62 (1H, d, J =
0.8 Hz). Anal. Calcd for C21H28N7O4SF3:C,47.45;H,5.31;N,18.45;S,6.03;
F,10.72.Found:C,47.55;H,5.48;N,18.23.
◦
(0.390 g, 1.73 mmol) were added at 0 C. The mixture was stirred at
80 ◦C overnight. The mixture was diluted with water at room temper-
ature and extracted with AcOEt. The organic layer was separated,
washed with water and brine, dried over Na2SO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(NH, AcOEt) to give the title compound as a white solid, which was
crystallized from Et2O/hexane (12.9 g, 72.2%). MS (ESI/APCI) m/z 552
[M+H]+. 1H NMR (300 MHz, DMSO‑d6) δ 1.61 (6H, s), 1.79 (2H, dtd, J
= 12.6, 8.4, 4.0 Hz), 1.91–2.14 (6H, m), 2.90 (3H, s), 3.13 (2H, ddd, J =
12.0, 8.4, 3.4 Hz), 3.22–3.44 (4H, m), 4.07 (2H, d, J = 13.3 Hz),
4.40–4.55 (1H, m), 5.25 (1H, tt, J = 7.8, 3.6 Hz), 6.50 (1H, s), 8.63 (1H,
s). Anal. Calcd for
C21H29N7O4SF4:C,45.73;H,5.30;N,17.78;S,5.81;
5.1.27. Ethyl 5-{4-[(6-{[1-(methanesulfonyl)piperidin-4-yl]oxy}
pyrimidin-4-yl)(trifluoromethyl)amino]piperidin-1-yl}-1,2,4-oxadiazole-3-
carboxylate (25c)
F,13.78.Found:C,45.77;H,5.38;N,17.64;S,5.81;F,13.60.
5.2. Estimation of LogD at pH 7.4
ZnCl2 (1 M Et2O solution, 111 mL, 111 mmol) was added to a solu-
tion of 24 (25 g, 55.8 mmol) and ethyl 2-amino-2-(hydroxyimino)ace-
tate (14.7 g, 111 mmol) in DMF(dry) (250 mL) at room temperature.
The mixture was warmed to 80 ◦C with stirring. p-TsOH monohydrate
(15.9 g, 83.6 mmol) was added at the same temperature and the mixture
was stirred for 4 h. The reaction was quenched with saturated aqueous
NaHCO3 solution (300 mL) then water (200 mL) was added. The mixture
was stirred at room temperature for 1 h. The emerged solid was filtered
and the filtrate was suspended in IPE. The suspension was stirred for 1 h.
The water layer was extracted with AcOEt. The organic layer was
washed with water and brine, dried over MgSO4, filtered and the pre-
cipitate was added. The suspension was passed to silica gel pad. The
fraction was concentrated. The residue was suspended in IPE and the
mixture was stirred for 4 h and then filtered to obtain the title compound
LogD7.4, which is a partion coefficient between 1-octanol and
aqueous buffer pH 7.4, of the compounds was measured on the chro-
matographic procedure whose condition was developed based on a
published method.22, 23
5.3. Solubility determination
Small volumes of the compound DMSO solutions were added to the
aqueous buffer solution (pH 6.8). After incubation, precipitates were
separated by filtration. The solubility was determined by HPLC analysis
of each filtrate.
5.4. In vitro GPR119 agonist activity
1
as a white solid (25.5 g, 81%). MS (ESI/APCI) m/z 564 [M+H]+. H
NMR (300 MHz, DMSO‑d6) δ 1.29 (3H, t, J = 7.2 Hz), 1.79 (2H, dtd, J =
12.6, 8.5, 3.8 Hz), 1.92–2.16 (6H, m), 2.90 (3H, s), 3.13 (2H, ddd, J =
12.0, 8.4, 3.4 Hz), 3.25–3.46 (4H, m), 4.10 (2H, d, J = 13.3 Hz), 4.34
(2H, q, J = 7.1 Hz), 4.41–4.58 (1H, m), 5.25 (1H, tt, J = 7.6, 3.5 Hz),
GPR119 agonist activities were evaluated in the reporter gene assay
using CHO cells stably co-expressing cyclic AMP response element
(CRE)–luciferase reporter gene (Promega) and GPR119. Cells were
seeded at 10,000 cells/well in Minimum essential medium (MEM)
α
11

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
containing 10% fetal bovine serum, 100 U/mL penicillin, 100
μ
g/mL
colorless, block; triclinic, space group P-1, a = 7.09110(10) Å, b =
streptomycin, and 500
μ
g/mL Geneticin in 384-well white opaque
12.6309(2) Å, c = 14.7884(3) Å,
α
= 99.5130(10)◦, β = 90.0550(10)◦, γ
◦
plates, and cultured at 37 C under 5% CO2 with saturated humidity
overnight. The cells were washed once with assay buffer (MEM , 20
mmol/L HEPES, 0.1% bovine serum albumin, 100 U/mL penicillin, 100
g/mL streptomycin), and incubated with various concentrations of test
= 94.3820(10)◦,V = 1302.39(4) Å3, Z = 2, Dx = 1.381 g/cm3, T = 298
α
K,
1.075.
All measurements were made on a Rigaku R-AXIS RAPID diffrac-
tometer using graphite monochromated Cu-K radiation. The structure
μ = 1.647 mm-1, λ = 1.54184 Å, R1 = 0.0544, wR2 = 0.1503, S =
μ
compounds diluted in assay buffer for 2 h. After removal of culture su-
pernatant, cAMP-induced luciferase activities were measured with
Steady-Glo reagent (Promega) and EnVision Multilabel Plate Reader
(PerkinElmer). Agonist activities of test compounds on GPR119 were
expressed as [(A ꢀ B)/(C ꢀ B)] × 100 (luciferase activities (A) in test
compounds-treated cells, (B) in vehicle-treated cells, and (C) in cells
α
was solved by direct methods with SHELXT-2018/21) and was refined
using full-matrix least-squares on F2 with SHELXL-2018/3.2) All non-H
atoms were refined with anisotropic displacement parameters.24, 25
CCDC 2,063,007 for compound 8a, CCDC 2,063,008 for compound
8b, and CCDC 2,063,009 for compound 10 the supplementary crystal-
lographic data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via http://www.ccdc.
cam.ac.uk/structures.
treated with 10 μM N-[4-(methylsulfonyl)phenyl]-5-nitro-6-{4-[4-(tri-
fluoromethoxy)phenoxy]piperidin-1-yl}pyrimidin-4-amine29).
values were obtained with XLfit software (ID Business Solutions).
EC50
5.8. In vitro GLP-1 secretion assay
5.5. hERG inhibition assay
GLUTag cells, the murine L cell line, were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, high glucose) containing 10% heat-
hERG/CHO cells stably expressing hERG channel were purchased
from Millipore (UK) Ltd. (cat. # CYL3038). Cells were cultured at 32 ◦C,
5% CO2 in Ham’s F-12 medium supplemented with 10% fetal bovine
inactivated FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin.
GLUTag cells were seeded at density of 1 × 104 cells/well in a 96 well
poly-L-lysine coated plate. The following day, the medium was replaced
with DMEM (low glucose) containing 10% heat-inactivated FBS, 100 IU/
serum, 500 μg/mL Geneticin (Invitrogen). The hERG inhibition assay
was performed on the IonWorks Quattro (Molecular Devices) system in
population patch clamp (PPC) mode. The extracellular solution was
phosphate-buffered salines (PBS) with calcium and magnesium. The
intracellular solution contained 140 mM KCl, 2 mM MgCl2, 1 mM EGTA
mL penicillin and 100 μg/mL streptomycin, and the cells were incubated
overnight before experiments. After washing with Hank’s balanced salt
solution, Krebs-Ringer-bicarbonate HEPES buffer containing 0.2% fatty
acid free BSA, 10 mmol/L glucose, and compounds was added, and the
and 20 mM HEPES, pH 7.3 with KOH. After perforation using 100 μg/mL
amphotericin B, hERG current was measured under the potential-clamp
protocol (Holding potential ꢀ 80 mV, the first voltage 40 mV: 2 sec, the
second voltage ꢀ 50 mV: 2 sec). The peaktail current before addition of
the compounds was measured as the pre hERG current. Test compounds
were incubated on the cells for a period of 5 min. The peaktail current
after addition of the compounds was measured as the post hERG current.
%hERG inhibition was calculated (n = 4) to the following.
◦
cells were incubated for 2 h at 37 C. After incubation, supernatants
from each well were collected, and secreted active GLP-1 concentration
was measured using active GLP-1 ELISA kit (Millipore, EGLP-35 K) ac-
cording to the manufacture’s instruction.
5.9. In vitro insulin secretion assay
%hERG inhibition = 100 ꢀ (post hERG current/pre hERG current) × 100
HIT-T15 cells, the hamster pancreatic beta cell line, were cultured in
Ham’s F12 containing 10% heat-inactivated FBS, 100 IU/mL penicillin,
5.6. In vitro metabolic clearance in human and rat hepatic microsomes
100 μg/mL streptomycin, and 2 mM L-Glutamine. HIT-T15 cells were
seeded at density of 5 × 104 cells/well in a 96 well plate. The following
day, the medium was replaced with Krebs-Ringer-bicarbonate HEPES
(KRBH) buffer (116 mM NaCl, 4.7 mM KCl, 1.17 mM KH2PO4, 1.17 mM
MgSO4・7H2O, 25 mM NaHCO3, 2.52 mM CaCl2・2H2O, 24 mM
HEPES, 0.2% BSA) and the cells were pre-incubated 2 h before experi-
ments. After pre-incubation, KRBH buffer containing 0.2% fatty acid
free BSA, 10 mmol/L glucose, and compounds was added, and the cells
Human and mouse liver microsomes were purchased from Xenotech,
LLC (Lenexa, KS). An incubation mixture consisted of microsomal pro-
tein in 50 mM KH2PO4–K2HPO4 phosphate buffer (pH 7.4) and 1 μM test
compound. The concentration of microsomal protein was 0.2 mg/mL.
An NADPH-generating system containing 5 mM MgCl2, 5 mM glucose-6-
phosphate, 0.5 mM β-NADP+, and 1.5 units/mL glucose-6-phosphate
dehydrogenase was added to the incubation mixture to initiate the
enzyme reaction. The reaction was terminated 15 and 30 min after the
initiation of the reaction by mixing the reaction mixture with acetoni-
trile, followed by centrifugation. The supernatant was subjected to LC/
MS/MS analysis. The metabolic velocity was calculated as the slope of
the concentration–time plot.
◦
were incubated for 2 h at 37 C. After incubation, supernatants from
each well were collected, and secreted insulin concentration was
measured using AlphaLISA insulin kit (Perkin Elmer) according to the
manufacture’s instruction.
5.10. Pharmacokinetic analysis in rat cassette dosing
Test compounds were administered intravenously (0.1 mg/kg) or
orally (1 mg/kg, solvent: 0.5% methylcellulose aqueous solution) by
cassette dosing to non-fasted mice. After administration, blood samples
were collected and centrifuged to obtain the plasma fraction. The
plasma samples were deproteinized followed by centrifugation. The
compound concentrations in the supernatant were measured by LC/MS/
MS.
5.7. Single-crystal X-ray structure analysis
Crystal data for compound 8a: C24H33N5O4S MW = 487.62; crystal
size, 0.37 × 0.19 × 0.14 mm; colorless, block; monoclinic, space group
P21/c, a = 11.0270(3) Å, b = 22.6674(5) Å, c = 20.4331(6) Å,
α = γ =
90◦, β = 100.9880(17)◦, V = 5013.7(2) Å3, Z = 8, Dx = 1.292 g/cm3, T
= 298 K,
μ = 1.473 mm-1, λ = 1.54184 Å, R1 = 0.0554, wR2 = 0.1500,
S = 1.045. Crystal data for compound 8b: C25H35N5O4S MW = 501.64;
crystal size, 0.25 × 0.15 × 0.14 mm; colorless, block; triclinic, space
5.11. Oral glucose tolerance test
group P-1, a = 10.5215(2) Å, b = 14.3969(3) Å, c = 17.9065(3) Å,
α =
91.9556(7)◦, β = 96.7813(7)◦, γ = 109.6530(7)◦, V = 2528.63(8) Å3, Z
The care and use of the animals and the experimental protocols used
in this research were approved by the Experimental Animal Care and
Use Committee of Takeda Pharmaceutical Company Limited. Male N-
STZ1.5 rats were obtained from Takeda Rabics, Ltd. (Hikari, Japan).
= 4, Dx = 1.318 g/cm3, T = 100 K,
μ = 1.475 mm-1, λ = 1.54184 Å, R1
= 0.0611, wR2 = 0.1729, S = 1.069. Crystal data for compound 10:
C24H30F3N5O4S MW = 541.59; crystal size, 0.61 × 0.37 × 0.25 mm;
12

O. Kubo et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116208
6
7
8
9
Shah U, Kowalski TJ. GPR119 agonists for the potential treatment of type 2 diabetes
and related metabolic disorders. Vitam Horm. 2010;84:415–448. https://doi.org/
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They were fed a commercial diet CE-2 (Clea Japan Co.) and tap water ad
libitum. Male N-STZ1.5 rats (25–30 weeks of age) were fasted overnight
and orally given vehicle (0.5% methylcellulose) or compounds. Sixty
minutes later, all animals were received an oral glucose load (1.5 g/kg).
Blood samples were collected from tail vein before drug administration
(pre), and just before glucose load (time 0), and 10, 30, 60, and 120 min
after glucose load. Plasma glucose and plasma insulin levels were
measured by Autoanalyzer 7080 (Hitachi, Japan) and radioimmuno-
assay (Millipore, USA), respectively. Differences between two groups
were analyzed by one-tailed Williams test.
Shah U. GPR119 agonists: a promising new approach for the treatment of type 2
diabetes and related metabolic disorders. Curr Opin Drug Discov Dev. 2009;12:
519–532.
Jones RM, Leonard JN, Buzard DJ, Lehmann J. GPR119 agonists for the treatment of
type 2 diabetes. Expert Opin Ther Pat. 2009;19:1339–1359. https://doi.org/10.1517/
13543770903153878.
Fyfe MC, McCormack JG, Overton HA, Procter MJ, Reynet C. GPR119 agonists as
potential new oral agents for the treatment of type 2 diabetes and obesity. Expert
Opin Drug Discov. 2008;3:403–413. https://doi.org/10.1517/17460441.3.4.403.
10 Overton HA, Fyfe MC, Reynet C. GPR119, a novel G protein-coupled receptor target
for the treatment of type 2 diabetes and obesity. Br J Pharmacol. 2008;153:S76–S81.
https://doi.org/10.1038/sj.bjp.0707529.
Declaration of Competing Interest
11 Sato K, Sugimoto H, Rikimaru K, et al. Discovery of a novel series of indoline
carbamate and indolinylpyrimidine derivatives as potent GPR119 agonists. Bioorg
Med Chem. 2014;22:1649–1666. https://doi.org/10.1016/j.bmc.2014.01.028.
12 Kamaura M, Kubo O, et al. Discovery of a novel series of indolinylpyrimidine-based
GPR119 agonists: elimination of ether-a-go-go-related gene liability using a
hydrogen bond acceptor-focused approach. Bioorg Med Chem. 2021;34, 116034.
https://doi.org/10.1016/j.bmc.2021.116034.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13 Kuroboshi M, Hiyama T. A facile synthesis of trifluoromethylamines by oxidative
desulfurization-fluorination of dithiocarbamates. Tetrahedron Lett. 1992;33:
4177–4178. https://doi.org/10.1016/S0040-4039(00)74682-X.
14 Kiyoshi K, Katsuya M, Manabu K, Tamejiro H. A Facile Synthesis of
Trifluoromethylamines by oxidative desulfurization–fluorination of
dithiocarbamates. Bull Chem Soc Jpn. 1998;71:1973–1991. https://doi.org/10.1246/
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Acknowledgments
We thank Tatsuru Tomokuni, Kana Furuyabu, and Chihiro Kawate
for conducting hERG inhibition assay. We also thank Mitsuyoshi Nish-
itani for X-ray crystallographic analysis, the members in charge of
determination of LogD value, and the members of the Takeda Analytical
Research Laboratories, Ltd. for elemental analyses. Finally, we
acknowledge Dr. Tsuyoshi Maekawa for supervision of the research,
careful reading of the manuscript, and valuable suggestions.
15 MOE. (Molecular Operating Environment) Software. Version: MOE; vol 2012.10. QC,
Canada: Chemical Computing Group, M. MOE (Molecular Operating Environment)
software. Version: MOE. Montreal, QC, Canada: Chemical Computing Group;
2012.10.
16 Leeson PD, Springthorpe B. The influence of drug-like concepts on decision-making
in medicinal chemistry. Nat Rev Drug Discov. 2007;6:881–890. https://doi.org/
10.1038/nrd2445.
Funding
17 Jamieson C, Moir EM, Rankovic Z, Wishart G. Medicinal chemistry of hERG
optimizations: highlights and hang-ups. J Med Chem. 2006;49:5029–5046. https://
doi.org/10.1021/jm060379l.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
¨
18 Bohm HJ, Banner D, Bendels S, et al. Fluorine in medicinal chemistry. ChemBioChem.
2004;5:637–643. https://doi.org/10.1002/cbic.200301023.
19 Brameld KA, Kuhn B, Reuter DC, Stahl M. Small molecule conformational
preferences derived from crystal structure data. A medicinal chemistry focused
analysis. J Chem Inf Model. 2008;48:1–24. https://doi.org/10.1021/ci7002494.
20 Müller K, Faeh C, Diederich F. Fluorine in pharmaceuticals: looking beyond intuition.
Science. 2007;317:1881–1886. https://doi.org/10.1126/science.1131943.
21 Jagodzinska M, Huguenot F, Candiani G, Zanda M. Assessing the bioisosterism of the
trifluoromethyl group with a protease probe. ChemMedChem. 2009;4(1):49–51.
https://doi.org/10.1002/cmdc.200800321.
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13