Discovery of soluble epoxide hydrolase inhibitors through DNA-encoded library technology (ELT)

Article abstract of DOI:10.1016/j.bmc.2021.116216

Inhibition of soluble epoxide hydrolase (sEH) has recently emerged as a new approach to treat cardiovascular disease and respiratory disease. Inhibitors based on 1,3,5-triazine chemotype were discovered through affinity selection against two triazine-base

Full text of DOI:10.1016/j.bmc.2021.116216

Bioorg. Med. Chem. 41 (2021) 116216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of soluble epoxide hydrolase inhibitors through DNA-encoded
library technology (ELT)
,
,
,
Yun Dinga *, Svetlana Belyanskayaa 1, Jennifer L. DeLoreya 2, Jeffrey A. Messera,
G. Joseph Franklina 1, Paolo A. Centrellaa 3, Barry A. Morgana 4, Matthew A. Clarka
,
,
,
,
,
,3
Steven R. Skinnera, Jason W. Dodsonb, Peng Lib, Joseph P. Marino Jr. b, David I. Israela
4
a Encoded Library Technologies/NCE Molecular Discovery, GSK, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
b Department of Chemistry, Heart Failure Disease Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area Unit, GSK, 709 Swedeland Road, King of
Prussia, PA 19406, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inhibition of soluble epoxide hydrolase (sEH) has recently emerged as a new approach to treat cardiovascular
disease and respiratory disease. Inhibitors based on 1,3,5-triazine chemotype were discovered through affinity
selection against two triazine-based DNA-encoded libraries. The structure and activity relationship study led to
the expansion of the original 1,4-cycloalkyl series to related aniline, piperidine, quinoline, aryl-ether and
benzylic series. The 1,3-cycloalkyl chemotype led to the discovery of a clinical candidate (GSK2256294) for
COPD.
DNA-encoded library technology
Soluble epoxide hydrolase
1. Introduction
metabolism of arachidonic acid derivatives known as epoxyeicosa-
trienoic acids (EETs).12 sEH converts EETs into dihydroxyeicosatrienoic
DNA-encoded library technology (ELT) has developed over the 28
years since the concept was proposed in 1992.1 Using a split-and-pool
combinatorial synthesis strategy, ELT can provide greater coverage of
chemical space that improves the chances of finding leads that may be
considered “close to candidate”.2–4 By DNA-encoding each chemical
warhead, this technology overcomes the problem of deconvolution, the
historical challenge of other combinatorial chemistry methods. ELT has
proven to be a powerful tool for ligand identification and target vali-
dation, attracting significant attention from both academia and in-
dustry. Numerous biologically active compounds have been discovered
through affinity selection of DNA-encoded libraries against a broad
range of targets.5–11
acids (DHETs). Several publications13,14 have described the beneficial
vasodilatory, anti-inflammatory, and anti-thrombotic effects of EETs.
Taking advantage of the beneficial effects of EETs, the inhibition of sEH
can be applied to the treatment of various conditions mediated by the
sEH enzyme through the prevention of EETs degradation. In addition,
several studies have demonstrated a protective effect as a result of sEH
inhibition in models involving cigarette smoke exposure.15–17 Thus, the
inhibitors of sEH can be applied to the treatment of chronic obstructive
pulmonary disease (COPD), which accounted for 3 million deaths in
2019 and was the third leading cause of death worldwide according to
the World Health Organization’s report.18
Several classes of sEH inhibitors have been identified including
urea,19 amide,20 hydrazone derivatives,21 and others.22 Our goal was to
discover novel inhibitors of sEH through affinity selection against DNA-
encoded libraries. In this paper, we will describe the discovery of unique
Epoxide hydrolases, found in both plants and animals, are enzymes
that convert epoxides to diols by hydrolysis. In mammals, soluble
epoxide hydrolase (sEH, EPHX2) is primarily responsible for the
Abbreviations: ELT, encoded library technology; DEL, DNA-encoded library; sEH, soluble epoxide hydrolase; HP, DNA headpiece; SAR, structure activity
relationship.
* Corresponding author.
E-mail address: yun.x.ding@gsk.com (Y. Ding).
1
2
Anagenex, Inc., 1173 Hayes St. No. 2, San Francisco, CA 94117, USA.
RxVantage, 2301 Rosecrans Avenue, Suite 4195, El Segundo, CA 90245, USA.
3
X-Chem Inc., 100 Beaver Street, Waltham, MA 02453, USA.
4
HitGen Inc., No. 8 Huigu 1st East Road, Chengdu, Sichuan 610200, PR China.
https://doi.org/10.1016/j.bmc.2021.116216
Received 3 January 2021; Received in revised form 7 May 2021; Accepted 8 May 2021
Available online 13 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
triamino-triazine derivatives as potent inhibitors of sEH and the follow
up SAR study that led to the expansion of hit series. One of the hit series
led to a drug candidate, GSK2256294, for the treatment of COPD after
minimal lead optimization.17,23-24
competitively bind over some of the sEH binders from the DEL-A library.
After removing all sequences that occurred in the selection without the
target (NTC), we still observed a large number of binders with most
represented in fewer than 2 copies. This made it difficult to decide which
species to follow up with off-DNA synthesis.
2. Results and discussions
We next turned to the selection of DEL-B against sEH. We predicted
that titrating down the protein concentration would help define the
binders. The selection was performed using the same method as above
2.1. Selection with DNA-encoded libraries
with different concentrations of sEH, 1 μM, 100 nM and 10 nM, along
Two closely related libraries, A & B (Figure 1), based on the triazine
chemotype were selected against sEH. Both libraries commenced with
acylation of DNA headpiece (Supporting Information) using 192 Fmoc-
amino acids. After Fmoc-deprotection, the triazine scaffold was installed
using cyanuric chloride. Cycles 2 and 3 both employed SNAr reactions to
substitute the remained two chlorides, with 32 bifunctional acids
installed at cycle 2 and 340 amines installed at cycle 3. The libraries
diverged at the final cycle, in which amidation was performed using two
sets of building blocks. DEL A was produced using 384 amines, giving a
library containing 802,160,640 compounds and DEL B was produced
using 48 amino esters. Final hydrolysis of DEL B gave a library con-
taining 100,270,080 members. It is worth noting that as libraries
progress beyond the first cycle, it becomes difficult to quantify conver-
sions, yields and byproduct formation with each reaction. Thus nearly
all building blocks used were tested in representative validation exper-
iments using a DNA-linked substrate prior to synthesis of the libraries.
Only those building blocks which gave high conversions (>70%) were
used in the synthesis of DELs A and B.
with no target control. A visualization of the selection output in a cubic
plot (Figure 2b) showed that related structures were selected after
removing the molecules with only one copy number. More potential
ligands were observed at the higher concentration of sEH. However,
some compounds that were enriched in the higher concentrations of sEH
were also present in the 10 nM sEH sample. To prioritize the off-DNA
follow-up, we focused on the binding events from 10 nM of sEH. After
removing all sequence reads that occurred only once, only 4-(amino-
methyl) benzoic acid was selected at cycle 4, which means this building
block outwins all the other 47 aminoacids and very likely is critical for
the binding of the target. Building block preferences at other cycles are
visualized in a 3D scatter plot (Figure 2c). There were 2 highly popu-
lated planes along the cycle 3 axis, which corresponded to cis- 4-amino-
cyclohexanecarboxylic acid and its mixture of cis- and trans- isomers.
Within each plane, there were several lines corresponding to selected
cycle 3 amines, some of which were structurally similar. The appearance
of lines indicated that there was little preference for the cycle 1 building
block, suggesting that cycle 1 chemistry may contribute very little to the
binding.
We first performed the selections against DEL-A using the conven-
tional affinity selection protocol as described previously.5 The library
was incubated with either 1 μM of protein alone, protein with a known
2.2. Hit confirmation
sEH inhibitor (AUDA) (Figure 2a), or with buffer only (the no-target
control). Then, streptavidin affinity matrix resin was added to capture
the protein with bound library. The unbound molecules were washed
away. The heat denaturation method was used to elute binders from the
protein and the affinity resin. The heat eluted binders were then incu-
bated with fresh aliquots of target protein and two more rounds of se-
lection were performed. After the third round of selection, we quantified
the number of recovered DEL molecules by qPCR. In comparison with
the no-target control (NTC) condition, a very high number of DEL
molecules were observed in the sEH alone selection, while the sEH with
AUDA condition provided 15-times fewer potential ligands (Supporting
Information). This result indicated that the sEH inhibitor AUDA can
To confirm the selected features from DEL-B, a representative set of
the fully elaborated library molecules were synthesized (Table 1) using
the protocol in Scheme 1. The building blocks at cycle 1 were the des-
carboxylic analogues of the cycle 1 amino acids. Biochemical assay
showed these compounds had inhibitory activity ranging from 11 nM ~
73 nM. The complete depletion of cycle 1 building block led to dramatic
loss of activity (Table 1, 4). However, activity was retained with
methylamine at cycle 1 (Table 1, 5). This confirmed the presumptive
selection result that cycle 1 was not necessary for affinity. Since cycle 1
was the position linked to the DNA, we hypothesized that it might be
exposed to the solvent and could also be used as a handle to improve the
Figure 1. Synthesis of DEL-A & DEL-B.
2

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
Figure 2. a) AUDA tool compound. b) Selection of library B against sEH. The selection output from NTC and under different concentrations of sEH. c) The cube
corresponding to 4-(aminomethyl) benzoic acid as cycle 4 is shown in detail. The copy number of the selected species is indicated by the continuous color and size of
the points (darker and bigger points indicate higher copy number). The structures shown represent the family of compounds defined by a line along the BB
axis (arrow).
Table 1
Off-DNA hit confirmation.
Cmpd
1
R1
R3
IC50
(nM)
Cmpd
4
R1
H
R3
IC50
(nM)
25
27
39
>5000
2
3
5
NHMe
24
6
38
physicochemical properties of the inhibitors.
original acid synthon at the cycle 4 position. To understand why aro-
matic amines which were included in both libraries were not selected at
the cycle 4 position, we synthesized the compound with 4-amino ben-
zeneacetic acid (Table 2a, 12) at this position. The activity dropped
>700-fold, which was consistent with the selection output.
2.2.1. SAR at cycle 4 position
From the selection data of DEL-A, we did not observe obvious fea-
tures with similar resolution as found in DEL-B. However, several cycle 4
amines were highly enriched, including (2-((trifluoromethyl)thiol)
phenyl) methanamine and (2-(trifluoromethoxyl)phenyl) methanamine.
A series of compounds with diversity at the cycle 4 position were syn-
thesized following Scheme 1C. A substitution of 4-(aminomethyl) ben-
zoic acid at cycle 4 with these two ortho-substituted benzylamines
displayed similar activity (Table 2a, 7 & 8). With a trifluoromethyl
group at the ortho position, compound 9 retained the activity. Tri-
fluoromethyl substitution at the para position led to a 2-fold loss of
activity (Table 2a, 10). With complete depletion of the substitution on
the ring, compound 11 lost about 10-fold activity compared with the
2.2.2. SAR at cycle 3 position
Considering the information inferred from the data cube and di-
versity of amines at cycle 3 being active, we further attenuated the cycle
3 structure. Substituting R3 with piperidine, pyrrolidine and benzyl-
amine yielded compounds that maintained similar excellent potency
(Table 2b, 13, 14 & 15). This showed cycle 3, similar to cycle 1, was not
critical for activity. These positions could then be modified to optimize
the physical and pharmacokinetic properties of the chemotype. To in-
crease the solubility of the compound, diamine derivatives,
3

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
Scheme 1. Synthesis of sEH off-DNA hitsa. a Reagents and conditions: (i) amine (1 equiv), EDCI (1.25 equiv), DMAP (0.2 equiv), CH2Cl2, 0 ◦C – rt; (ii) TFA (50% in
CH2Cl2), room temperature, 25 mins; (iii) amine (1 equiv), CH3CN/H2O (1/1), 1 N NaOH, pH 9 ~ 10, 0 ◦C; (iv) amine (1 equiv), CH3CN/H2O (1/1), 1 N NaOH, pH 9
~ 10, room temperature 4 hrs-overnight; (v) amine (5 ~ 10 equiv), CH3CN/H2O (1/1), 80 ◦C; (vi) 1 N NaOH (50–100% v/v), RT 3 hrs - overnight; (vii) amine (1
equiv), DIEA (2.5 equiv), CH3CN/H2O (1/1), 0 ◦C ; (viii) amine (5 equiv), CH3CN/H2O (1/1), 80 ◦C for 2 hrs.
Table 2
SAR at a) cycle 4; b) cycle 3.
trimethylethane-1,2-diamine (Table 2b, 16) and 1-methylpiperazine
(Table 2b, 17), were placed at the cycle 3 position. Both compounds
showed an increase in the biochemical activity. As the piperazine de-
rivative (Table 2b, 17) had the most potent activity (9 nM), we used that
substitution at cycle 3 position for further SAR studies and hit-to-lead
optimization.
of the aniline derivative (Table 3, 18) was approximately 4-fold lower,
while the piperidine derivative (Table 3, 19) had comparable potency to
the cyclohexyl analog (Table 3, 17). However, when azetidine was
substituted at the cycle 2 position (Table 3, 20), the activity dropped
dramatically. We further optimized the hits with diversity at the cycle 2
position (Table 3, 21-25). With different amine derivatives at cycle 2,
such as benzylamine, 1,3 cyclohexyl amine, furanylmethanamine, tet-
rahydroquinoline and phenol, all compounds had single digit nanomolar
activity.
2.2.3. SAR at cycle 2 position
From the selection data, both the cis- and mixture of cis- and trans-
stereoisomers of 4-aminocyclohexanecarboxylic acid were highly
selected cycle 2 synthons. To determine the stereochemical preference at
cycle 2, the trans-isomer of compound 5 was also prepared. Surprisingly,
the trans-isomer was found to have the same potency as the cis-isomer.
This suggested that the binding pocket for cycle 2 and cycle 4 was suf-
ficient to tolerate further modification. To avoid the stereochemistry at
the cycle 2 position, we modified the cycle 2 building block with aniline
and piperidine analogs with 1-methylpiperazine at cycle 3. The potency
2.2.4. CYP450 profile
CYP450 inhibition was also evaluated for compounds 16–19. In
general, the compounds showed a good CYP450 inhibition profile
(Table 4). Activity was in the micromolar range across all targets tested,
showing potency > 1000x lower for compounds 16, 17, and 19 and >
250x lower for compound 18 against CYP450s compared to sEH.
4

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
Table 3
SAR at cycle 2.
4. Experimental section
Table 4
P450 inhibition summary.
4.1. Chemical synthesis
Cmpd
IC50 (µM)
All reagents and solvents were of commercial quality and used
without further purification unless indicated otherwise.
1A2
2C9
2C19
2D6
3A4 (DEF)
3A4 (7BQ)
16
17
18
19
>33
>33
>33
>33
32
32
>33
>33
21
33
>33
23
22
22
>33
>33
>33
>33
NMR spectra were recorded on a Varian Mercury 500. Chemical
shifts are expressed in parts per million (ppm, δ units). Coupling con-
stants (J) are in units of hertz (Hz). Splitting patterns describe apparent
multiplicities and are designated as s (singlet), d (doublet), t (triplet), q
(quartet), dd (double doublet), dt (double triplet), m (multiplet), br
(broad). All mass spectra were performed under electrospray ionization
(ESI) method. All the final compounds for biological assay were purified
on Gilson system with a Phenomenex Luna 5 um C8(2), 100 mm × 21
mm 100A column with MeCN/H2O (+0.1% TFA) solvents. High-
resolution mass measurements were acquired on an Orbitrap Exactive
Plus mass spectrometer (Thermo-Fisher) equipped with a heated elec-
trospray source (HESI) and operated at 70 k resolution. HPLC utilized a
Kinetex XB C18 column (Phenomenex, 1.7u, 2.1x50 mm at 30C), eluted
with a 0.1% formic acid-acetonitrile gradient.
12
19
>33
>33
>33
>33
3. Conclusion
In summary, employing DNA-encoded library technology, a series of
potent sEH inhibitors were developed. Guided by selection output,
extensive SAR studies and hit-to-lead optimization yielded a series of
leads with diversity at cycle 2 and flexibility at both cycle 1 and cycle 3
positions. These positions were later utilized to improve oral bioavail-
ability and physiochemical properties. Since few triazines have been
developed as drugs,25–26 and based on the unknown properties of this
template, attempts to replace the triazine core with pyrimidine, pyri-
dine, or phenyl were undertaken.27 However, the triazine proved to be
the optimal core from a potency and CYP450 standpoint. Among those
triazine cores, 1,3-aminocycleohexanecarboxylate core 22 led to a drug
candidate GSK2256294 as a novel anti-inflammatory for the treatment
of COPD.23 As a potent, selective, and orally available sEH inhibitor,
GSK2256294 was well-tolerated with no serous adverse events attrib-
utable to the drug. These data support further investigation in patients
with endothelial dysfunction or abnormal tissue repair.
4.1.1. Synthesis of DNA-encoded libraries
The synthesis of library A has been reported previously.5 Library B
was synthesized similarly as library A from cycle 1–4. After pooling and
precipitation with EtOH, the cycle 4 library material was further treated
with 200 equivalents of NaOH to hydrolyze the ester prior to the final
purification with reverse-phase HPLC.
4.1.2. Synthesis of compounds off-DNA.
General method A.
A
solution of 4-(Boc-amino)cyclo-
hexanecarboxylic acid (2.0 g, 8.22 mmol, 1 equivalent), methyl 4-
(aminomethyl)benzoate hydrochloride (1.89 g, 9.37 mmol, 1.14
5

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
equivalents) and N,N-dimethylamino pyridine (DMAP, 200.8 mg, 1.644
mmol, 0.2 equivalent) in methylene chloride (50 mL) was cooled with
stirring in an ice bath. DIEA (1.79 mL, 10.275 mmol, 1.25 equivalents)
was added, followed by addition of 1-ethyl-3-[3-(dimethylamino)pro-
pyl]carbodiimide hydrochloride (EDCI, 1.97 g, 10.275 mmol, 1.25
equivalents). The reaction mixture was stirred at 0 ◦C for 2 h and at room
temperature for 3 h. The solution was diluted with methylene chloride
(50 mL), which was further washed with saturated sodium bicarbonate,
water, salt and dried over MgSO4. The solvent was removed in vacuo to
give the crude compound which was further purified by silica chroma-
tography (60%-70% EtOAc in Hexane) to give acylation product (2.97 g,
92.5%). This compound (2.97 g) was treated with 50% TFA in
dichloromethane (80 mL) at room temperature for 25 min. The reaction
mixture was condensed to give a light-yellow oil which were the desired
compound I as the TFA salt. MS: calcd for C16H22N2O3 + H+ 291.16,
found 291.11.
(Luna 5
μ
C8(2), 100x21mm, 20–65% CH3CN/H2O, 0.1% TFA, 20
min). HRMS [M + H]+ calcd for C35H41N7O3S + H+ 640.30644, found
640.30497.
4-((cis-4-((4-((2-phenylpropyl)amino)-6-(((R)-2-(thiophen-2-yl)
ethyl)amino)-1,3,5-triazin-2-yl)amino)cyclohexane-1-carboxamido)
methyl)benzoic acid (3). By use of (R)-(+)-beta-methylphenethylamine
as R1, compound I as R2, and 2-thiopheneethylamine as R3, the title
compound was prepared by following general method A. The product
was purified with RP-HPLC (Luna 5
μ C8(2), 100x21mm, 25–80%
CH3CN/H2O, 0.1% TFA, 20 min). HRMS [M + H]+ calcd for
C33H39N7O3S + H+ 614.29079, found 614.28956.
4-((cis-4-((4-(((R)-2-phenylpropyl)amino)-1,3,5-triazin-2-yl)amino)
cyclohexane-1-carboxamido)methyl)benzoic acid (4). 2,4-Dichlorotria-
zine (96.6 mg, 0.612 mmol, 1 eq) was mixed with CH3CN/H2O (1/1,
6 mL) in an ice bath. Compound I as the TFA salt (272 mg, 0.673 mmol,
1.1 equivalents) was added. The pH of the reaction mixture was adjusted
to about 9 by adding dropwise 1 N NaOH. After the addition, the reac-
tion mixture determined to be complete based on LC-MS monitoring.
Without work-up, the reaction mixture (1 mL, ~ 0.1 mmol) was added
(R)-(+)-beta-methylphenethylamine (67.7 mg, 0.5 mmol, 5 equiva-
lents). The reaction was heated at 80 ◦C for 2 hrs. The reaction mixture
was concentrated in vacuo and the residue was purified with RP-HPLC
To a mixture of cyanuric chloride (1.22 mmol, 1 equivalent) in
◦
CH3CN/H2O (1/1, 2 mL) was cooled to 0 C. A primary or secondary
amine (1 equivalent) was added. The reaction mixture was adjusted to a
pH of about 9–10 using 1 N NaOH. LC-MS was used to monitor the re-
action. Without work-up and purification, the second amine (1 equiva-
lent) was added to the reaction . The pH of the reaction solution was
adjusted to 9 ~ 10 with 1 N NaOH and the reaction mixture was stirred
at room temperature for 4 h. Then the third amine (10 equivalents) was
added to the reaction mixture. The reaction was heated at 80 ◦C for 4 ~ 6
hrs monitored with LC-MS. Cooled to room temperature, the reaction
solution was added 1 N NaOH (50% ~ 100% v/v) and stirred at room
temperature for 3 h to overnight monitored with LC-MS to complete the
hydrolysis of ester. The reaction mixture was concentrated in vacuo and
the residue was purified with preparative HPLC to afford the final
compound (V).
(Luna 5μ C8(2), 100x21mm, 20–65% CH3CN/H2O, 0.1% TFA, 17 min)
to give the desired ester product (2.2 mg). MS [M + H]+ calcd for
C28H34N6O3 + H+ 503.27, found 503.39.
Methyl ester of compound 4 (2.2 mg) in MeOH (0.4 mL) was treated
with 1 N NaOH (0.2 mL). The reaction mixture was stirred at room
temperature for 2 to 3 hrs. The solution was neutralized with 1 N HCl
and the crude product was purified with RP-HPLC (Luna 5
μ C8(2),
100x21mm, 10–65% CH3CN/H2O, 0.1% TFA, 20 min). HRMS [M + H]+
calcd for C27H32N6O3 + H+ 489.26087, found 489.25929.
4-((cis-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-
General method B. Following the general method A, 3 amines, one
of which was free aminoacid, were added to cyanuric chloride subse-
quently. Then the acid was acylated with amine using EDCI/DMAP as
described in general method A.
triazin-2-yl)amino)cyclohexane-1-carboxamido)methyl)benzoic
acid
(5). By use of (R)-(+)-beta-methylphenethylamine as R1, compound I as
R2, and methylamine as R3, the title compound was prepared by
following general method A. The product was purified with RP-HPLC
General method C. 2,4-Dichlorotriazine (96.6 mg, 0.612 mmol, 1
eq) was mixed with CH3CN/H2O (1/1, 6 mL) in an ice bath. Compound I
as the TFA salt (272 mg, 0.673 mmol, 1.1 equivalents) was added. The
pH of the reaction mixture was adjusted to about 9 by adding dropwise
1 N NaOH. After the addition, the reaction was determined to be com-
plete based on LC-MS monitoring. Without work-up, the crude product
was added second amine (5 equivalents). The reaction mixture was
(Luna 5
μ C8(2), 100x21mm, 25–65% CH3CN/H2O, 0.1% TFA, 20
min). HRMS [M + H]+ calcd for C28H35N7O3 + H+ 518.28741, found
1
◦
518.28618. H NMR (500 MHz, DMSO‑d6, 80 C) δ ppm 1.26 (d, J =
7.02 Hz, 3H), 1.58–1.74 (m, 4H), 1.75–1.92 (m, 4H), 2.34–2.43 (m, 1H),
2.86 (br s, 3H), 3.06–3.15 (m, 1H), 3.40–3.50 (m, 1H), 3.51–3.62 (m,
1H), 4.02 (br s, 1H), 4.37 (d, J = 5.80 Hz, 2H), 7.22 (t, J = 6.80 Hz, 1H),
7.27 (d, J = 7.02 Hz, 1H), 7.32 (t, J = 7.63 Hz, 2H), 7.37 (d, J = 8.24 Hz,
2H), 7.90 (d, J = 8.24 Hz, H), 8.12 (br t, J = 5.65 Hz, 1H).
◦
heated at 80 C for 2–3 h. The reaction mixture was concentrated in
vacuo and the residue was purified with preparative HPLC. To a solution
of above purified compound (0.044 mmol, 1 equivalent) in MeOH (0.4
mL) was added 1 N NaOH (0.2 mL). The reaction mixture was stirred at
room temperature for 2 to 3 h. The solution was neutralized with 1 N
HCl and the crude product was purified with RP-HPLC.
4-((cis-4-((4-(isobutylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-
triazin-2-yl)amino)cyclohexane-1-carboxamido)methyl)benzoic
acid
(6). By use of (R)-(+)-beta-methylphenethylamine as R1, compound I as
R2, and ibutylamine as R3, the title compound was prepared by following
4-((cis-4-((4-(phenethylamino)-6-(((R)-2-phenylpropyl)amino)-
1,3,5-triazin-2-yl)amino)cyclohexane-1-carboxamido)methyl)benzoic
acid (1). By use of (R)-(+)-beta-methylphenethylamine as R1, compound
I as R2, and phenethylamine as R3, the title compound was prepared by
following general method A. The product was purified with RP-HPLC
general method A. The product was purified with RP-HPLC (Luna 5μ C8
(2), 100x21mm, 30–85% CH3CN/H2O, 0.1% TFA, 20 min). MS: calcd for
C31H41N7O3 + H+ 560.33, found 560.41.
cis-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-tri-
azin-2-yl)amino)-N-(2-((trifluoromethyl)thio)benzyl)cyclohexane-1-
carboxamide (7). By use of (R)-(+)-beta-methylphenethylamine as first
amine for chloro-substitution, cis-4-amino-cyclohexanecarboxylic acid
as second amine for chloro-substitution, methylamine as third amine for
substitution, and 2-(trifluoromethyl)thio-benzenemethanamine for
acylation, the title compound was prepared by following general
method B. HRMS [M + H]+ calcd for C28H34F3N7OS + H+ 574.25704,
found 574.25602. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 1.26 (d, J
= 7.02 Hz, 3H), 1.59–1.72 (m, 4H), 1.75–1.89 (m, 4H), 2.35–2.42 (m,
1H), 2.85 (br s, 3H), 3.39–3.61 (m, 2H), 4.02 (br. s, 1H), 4.55 (d, J =
5.80 Hz, 2H), 7.22 (t, J = 7.02 Hz, 1H), 7.27 (d, J = 7.32 Hz, 2H), 7.32 (t,
J = 7.32 Hz, 2H), 7.43 (td, J = 7.63, 1.53 Hz, 1H), 7.49 (dd, J = 7.93,
0.92 Hz, 1H), 7.59 (td, J = 7.48, 1.22 Hz, 1H), 7.71 (d, J = 7.93 Hz, 1H),
8.06 (br t, J = 5.19 Hz, 1H).
(Luna 5
μ C8(2), 100x21mm, 25–80% CH3CN/H2O, 0.1% TFA, 20
min). HRMS [M + H]+ calcd for C35H41N7O3 + H+ 608.33436, found
608.33277. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ 1.26 (d, J = 7.02 Hz,
3H), 1.58–1.73 (m, 4H), 1.73–1.92 (m, 4H), 2.33–2.43 (m, 1H), 2.88 (t,
J = 7.32 Hz, 2H), 3.10 (tq, J = 7.02 Hz, 1H), 3.42–3.49 (m, 1H),
3.52–3.60 (m, 3H), 4.01 (br. s, 1H), 4.37 (d, J = 5.80 Hz, 2H), 7.19–7.34
(m, 10H), 7.35–7.41 (m, 2H), 7.87–7.94 (m, 2H), 8.12 (br. t, J = 5.65
Hz, 1H).
4-((cis-4-((4-((2-(benzylthio)ethyl)amino)-6-(phenethylamino)-
1,3,5-triazin-2-yl)amino)cyclohexane-1-carboxamido)methyl)benzoic
acid (2). By use of S-benzylcysteamine hydrochloride as R1, compound I
as R2, and phenethylamine as R3, the title compound was prepared by
following general method A. The product was purified with RP-HPLC
6

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
cis-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-tri-
substitution, pyrrolidine as third amine for substitution, and 2-trifluoro-
methyl benzylamine for acylation, the title compound was prepared by
following general method B. HRMS [M + H]+ calcd for C23H30F3N7O
+ H+ 478.25367, found 478.25266. 1H NMR (500 MHz, DMSO‑d6,
80 ◦C) δ ppm 1.68 (m, 4H), 1.83 (m, 4H), 1.94 (m, 4H), 2.41 (m, 1H),
2.86 (br. s, 3H), 3.53 (m, 4H), 4.09 (br. s, 1H), 4.49 (d, J = 5.80 Hz, 2H),
7.48 (t, J = 7.93 Hz, 1H), 7.52 (d, J = 8.24 Hz, 1H), 7.65 (t, J = 7.63 Hz,
1H), 7.70 (d, J = 7.93 Hz, 1H), 8.06–8.15 (m, 1H).
azin-2-yl)amino)-N-(2-(trifluoromethoxy)benzyl)cyclohexane-1-car-
boxamide (8). By use of (R)-(+)-beta-methylphenethylamine as first
amine for chloro-substitution, cis-4-amino-cyclohexanecarboxylic acid
as second amine for chloro-substitution, methylamine as third amine for
substitution, and 2-(trifluoromethyl)-benzenemethanamine for acyla-
tion, the title compound was prepared by following general method B.
HRMS [M + H]+ calcd for C28H34F3N7O2 + H+ 558.27988, found
558.27882. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 1.21–1.31 (m,
3H), 1.57–1.72 (m, 4H), 1.72–1.90 (m, 4H), 2.36–2.41 (m, 1H), 2.85 (br.
s, 3H), 3.38–3.60 (m, 2H), 3.96–4.09 (m, 1H), 4.37 (d, J = 5.80 Hz, 2H),
7.22 (m, 1H), 7.27 (m, 2H), 7.30–7.36 (m, 3H), 7.36–7.44 (m, 3H),
8.00–8.07 (m, 1H). 1H NMR (500 MHz, DMSO‑d6 & D2O) δ ppm 1.19 (d,
J = 7.02 Hz, 3H), 1.57–1.78 (m, 8H), 2.33 (m, 1H), 2.78 (br. s, 3H), 3.01
(m, 1H), 3.38 (m, 1H), 3.49 (m, 1H), 4.31 (br. s, 2H), 7.16 (m, 1H), 7.19
(m, 2H), 7.26 (m, 3H), 7.30–7.34 (m, 3H).
cis-4-((4-(benzylamino)-6-(methylamino)-1,3,5-triazin-2-yl)amino)-
N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carboxamide (15). By use
of cis-4-amino-cyclohexanecarboxylic acid as first amine for chloro-
substitution, methylamine as second amine for chloro-substitution,
benzylamine as third amine for substitution, and 2-trifluoromethyl
benzylamine for acylation, the title compound was prepared by
following general method B. HRMS [M + H]+ calcd for C26H30F3N7O
+ H+ 514.25367, found 514.25275.
cis-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-tri-
azin-2-yl)amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carbox-
amide (9). By use of (R)-(+)-beta-methylphenethylamine as first amine
for chloro-substitution, cis-4-amino-cyclohexanecarboxylic acid as sec-
ond amine for chloro-substitution, methylamine as third amine for
substitution, and 2-trifluoromethyl benzylamine for acylation, the title
compound was prepared by following general method B. HRMS [M +
H]+ calcd for C28H34F3N7O + H+ 542.28497, found 542.28375.
cis-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-1,3,5-tri-
azin-2-yl)amino)-N-(4-(trifluoromethyl)benzyl)cyclohexane-1-carbox-
amide (10). By use of (R)-(+)-beta-methylphenethylamine as first amine
for chloro-substitution, cis-4-amino-cyclohexanecarboxylic acid as sec-
ond amine for chloro-substitution, methylamine as third amine for
substitution, and 4-trifluoromethyl benzylamine for acylation, the title
compound was prepared by following general method B. HRMS [M +
cis-4-((4-((2-(dimethylamino)ethyl)amino)-6-(methylamino)-1,3,5-
triazin-2-yl)amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-car-
boxamide (16). By use of cis-4-amino-cyclohexanecarboxylic acid as first
amine for chloro-substitution, methylamine as second amine for chloro-
substitution, N,N,N’-trimethylethylenediamine as third amine for sub-
stitution, and 2-trifluoromethyl benzylamine for acylation, the title
compound was prepared by following general method B. HRMS [M +
1
H]+ calcd for C24H35F3N8O + H+ 509.29587, found 509.29506. H
NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 1.61–1.73 (m, 4H), 1.77–1.91
(m, 4H), 2.37–2.47 (m, 1H), 2.86 (s, 3H), 2.89 (s, 6H), 3.12 (s, 3H), 3.35
(t, J = 6.41 Hz, 2H), 3.89–3.96 (m, 2H), 4.06 (br. s, 1H), 4.49 (br. d, J =
5.49 Hz, 2H), 7.47 (t, J = 7.63 Hz, 1H), 7.52 (d, J = 7.63 Hz, 1H), 7.65 (t,
J = 7.32 Hz, 1H), 7.70 (d, J = 7.93 Hz, 1H), 8.09 (br. t, J = 5.04 Hz, 1H).
cis-4-((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yl)amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carboxamide
(17). By use of cis-4-amino-cyclohexanecarboxylic acid as first amine for
chloro-substitution, methylamine as second amine for chloro-
substitution, N-methylpiperazine as third amine for substitution, and
2-trifluoromethyl benzylamine for acylation, the title compound was
prepared by following general method B. HRMS [M + H]+ calcd for
C24H33F3N8O + H+ 507.28022, found 507.27936. 1H NMR (500 MHz,
DMSO‑d6, 80 ◦C) δ ppm 1.66 (m, 4H), 1.84 (m, 4H), 2.41 (m, 1H),
2.80–2.91 (m, 6H), 3.27 (br, s, 4H), 3.70–4.29 (m, 5H), 4.49 (br. d, J =
5.19 Hz, 2H), 7.47 (t, J = 7.63 Hz, 1H), 7.52 (br. d, J = 7.63 Hz, 1H),
7.65 (t, J = 7.32 Hz, 1H), 7.70 (br. d, J = 7.90 Hz, 1H), 8.08 (br s, 1H).
4-((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)
amino)-N-(2-(trifluoromethyl)benzyl)benzamide (18). By use of
methylamine as first amine for chloro-substitution, 4-amino benzoic
acid as second amine for chloro-substitution, N-methylpiperazine as
third amine for substitution, and 2-trifluoromethyl benzylamine for
acylation, the title compound was prepared by following general
method B. HRMS [M + H]+ calcd for C24H27F3N8O + H+ 501.23327,
found 501.23255. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 2.87 (m,
6H), 3.15–3.42 (m, 4H), 4.70 (d, J = 5.49 Hz, 2H), 4.55–4.8 (m, 4H),
7.48 (t, J = 7.70 Hz, 1H), 7.58 (d, J = 7.63 Hz, 1H), 7.64 (t, J = 7.48 Hz,
1H), 7.72 (d, J = 7.63 Hz, 1H), 7.78–7.93 (m, 4H), 8.65 (br. t, J = 5.65
Hz, 1H).
1
H]+ calcd for C28H34F3N7O + H+ 542.28497, found 542.28412. H
◦
NMR (500 MHz, DMSO‑d6, 80 C) δ ppm 1.26 (d, J = 7.02 Hz, 3H),
1.58–1.73 (m, 4H), 1.74–1.89 (m, 4H), 2.34–2.41 (m, 1H), 2.85 (s, 3H),
3.38–3.49 (m, 1H), 3.49–3.60 (m, 1H), 3.97–4.08 (m, 1H), 4.38 (d, J =
5.80 Hz, 2H), 7.22 (tt, J = 7.02, 1.53 Hz, 1H), 7.27 (d, J = 7.02 Hz, 2H),
7.32 (t, J = 7.63 Hz, 2H), 7.48 (d, J = 7.93 Hz, 2H), 7.67 (d, J = 8.24 Hz,
2H), 8.11–8.18 (m, 1H).
cis-N-benzyl-4-((4-(methylamino)-6-(((R)-2-phenylpropyl)amino)-
1,3,5-triazin-2-yl)amino)cyclohexane-1-carboxamide (11). By use of
(R)-(+)-beta-methylphenethylamine as first amine for chloro-
substitution, cis-4-amino-cyclohexanecarboxylic acid as second amine
for chloro-substitution, methylamine as third amine for substitution,
and benzylamine for acylation, the title compound was prepared by
following general method B. HRMS [M + H]+ calcd for C27H35N7O +
H+ 474.29759, found 474.29698. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ
ppm 1.26 (d, J = 7.02 Hz, 3H), 1.58–1.71 (m, 4H), 1.81 (br. d, J = 5.49
Hz, 4H), 2.32–2.39 (m, 1H), 2.85 (br s, 3H), 3.41–3.62 (m, 2H), 4.02 (m,
1H), 4.31 (d, J = 6.10 Hz, 2H), 7.19–7.35 (m, 10H), 7.96–8.05 (m, 1H).
cis-4-((4-(methylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)
amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carboxamide (13).
By use of cis-4-amino-cyclohexanecarboxylic acid as first amine for
chloro-substitution, methylamine as second amine for chloro-
substitution, piperidine as third amine for substitution, and 2-trifluoro-
methyl benzylamine for acylation, the title compound was prepared by
following general method B. HRMS [M + H]+ calcd for C24H32F3N7O
+ H+ 492.26932, found 492.26826. 1H NMR (500 MHz, DMSO‑d6,
80 ◦C) δ ppm 1.47–1.55 (m, 4H), 1.59–1.66 (m, 6H), 1.75–1.91 (m, 4H),
2.33–2.42 (m, 1H), 2.79 (d, J = 4.27 Hz, 3H), 3.65–3.74 (m, 4H), 4.00
(br. s, 1H), 4.49 (d, J = 5.80 Hz, 2H), 7.47 (t, J = 7.63 Hz, 1H), 7.51 (d, J
= 7.63 Hz, 1H), 7.65 (t, J = 7.32 Hz, 1H), 7.70 (d, J = 7.93 Hz, 1H),
7.99–8.08 (m, 1H).
1-(4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)-
N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (19). By use of
methylamine as first amine for chloro-substitution, methyl piperidine-4-
carboxylate as second amine for chloro-substitution, N-methylpiper-
azine as third amine for substitution, and 2-trifluoromethyl benzylamine
for acylation, the title compound was prepared by following general
method B. HRMS [M + H]+ calcd for C23H31F3N8O + H+ 493.26457,
found 493.2637. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 1.55 (qd, J
= 12.10, 4.27 Hz, 2H), 1.81 (br. dd, J = 12.97, 2.90 Hz, 2H), 2.56 (tt, J
= 11.60, 3.66 Hz, 1H), 2.81 (s, 3H), 2.85 (s, 3H), 2.92 (td, J = 12.82,
2.44 Hz, 2H), 3.10–3.42 (m, 4H), 3.75 – 4.20 (br, 4H), 4.48 (d, J = 5.80
Hz, 2H), 4.61 (br. d, J = 13.12 Hz, 2H), 7.47 (t, J = 7.63 Hz, 1H), 7.51 (d,
J = 7.63 Hz, 1H), 7.65 (t, J = 7.93 Hz, 1H), 7.70 (d, J = 7.93 Hz, 1H),
cis-4-((4-(methylamino)-6-(pyrrolidin-1-yl)-1,3,5-triazin-2-yl)
amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carboxamide (14).
By use of cis-4-amino-cyclohexanecarboxylic acid as first amine for
chloro-substitution, methylamine as second amine for chloro-
7

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
8.16 (br. t, J = 5.49 Hz, 1H).
7.65 (t, J = 7.63 Hz, 1H), 7.69 (d, J = 7.93 Hz, 1H), 8.19 (br s, 1H).
5-methyl-4-(((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-
triazin-2-yl)amino)methyl)-N-(2-(trifluoromethyl)benzyl)furan-2-car-
boxamide (23). By use of methylamine as first amine for chloro-
substitution, 4-aminomethyl-5-methyl-furan-2-carboxylic acid as sec-
ond amine for chloro-substitution, N-methylpiperazine as third amine
for substitution, and 2-trifluoromethyl benzylamine for acylation, the
title compound was prepared by following general method B. HRMS:
calcd for C24H29F3N8O2 + H+ 519.24383, found 519.24344. 1H NMR
(500 MHz, DMSO‑d6, 80 ◦C) δ ppm 2.37 (s, 3H), 2.81 (s, 3H), 2.83 (s,
3H), 2.95–3.50 (m, 4H), 3.90–4.20 (m, 4H), 4.26–4.30 (m, 2H), 4.62 (d,
J = 5.80 Hz, 2H), 7.06 (s, 1H), 7.47 (t, J = 7.93 Hz, 1H), 7.51 (d, J =
7.93 Hz, 1H), 7.63 (t, J = 7.93 Hz, 1H), 7.71 (d, J = 7.93 Hz, 1H), 8.48
(br. t, J = 5.80 Hz, 1H)
1-(4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)-
N-(2-(trifluoromethyl)benzyl)azetidine-3-carboxamide (20). By use of
methylamine as first amine for chloro-substitution, azetidine-3-carbox-
ylic acid as second amine for chloro-substitution, N-methylpiperazine as
third amine for substitution, and 2-trifluoromethyl benzylamine for
acylation, the title compound was prepared by following general
method B. HRMS [M + H]+ calcd for C21H27F3N8O + H+ 465.23327,
found 465.23241.
4-(((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)
amino)methyl)-N-(2-(trifluoromethyl)benzyl)benzamide (21). By use of
methylamine as first amine for chloro-substitution, 4-aminomethyl
benzoic acid as second amine for chloro-substitution, N-methylpiper-
azine as third amine for substitution, and 2-trifluoromethyl benzylamine
for acylation, the title compound was prepared by following general
method B. HRMS: calcd for C25H29F3N8O + H+ 515.24892, found
1-(4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)-
N-(2-(trifluoromethyl)benzyl)-1,2,3,4-tetrahydroquinoline-6-carbox-
amide (24). By use of methylamine as first amine for chloro-substitution,
1,2,3,4-tetrahydroquinoline-6-carboxylic acid as second amine for
chloro-substitution, N-methylpiperazine as third amine for substitution,
and 2-trifluoromethyl benzylamine for acylation, the title compound
was prepared by following general method B. HRMS: calcd for
C27H31F3N8O + H+ 541.26457, found 541.26408. 1H NMR (500 MHz,
DMSO‑d6, 80 ◦C) δ ppm 1.95 (quin, J = 6.33 Hz, 2H), 2.79–2.90 (m, 8H),
3.11–3.46 (m, 4H), 4.00 (br. t, J = 5.95 Hz, 2H), 4.10 – 4.45 (br. s, 4H),
4.70 (d, J = 5.49 Hz, 2H), 7.48 (t, J = 7.63 Hz, 1H), 7.58 (d, J = 7.63 Hz,
1H), 7.65 (t, J = 7.93 Hz, 1H), 7.69 (dd, J = 8.85, 2.14 Hz, 1H),
7.71–7.76 (m, 2H), 7.96 (br. d, J = 8.24 Hz, 1H), 8.68 (t, J = 5.80 Hz,
1H).
1
◦
515.24837. H NMR (500 MHz, DMSO‑d6, 80 C) δ ppm 2.80 (s, 3H),
2.83 (s, 3H), 3.10 – 3.30 (m, 8H), 4.54 (br. d, J = 4.27 Hz, 2H), 4.69 (d, J
= 5.49 Hz, 2H), 7.43 (d, J = 7.93 Hz, 2H), 7.48 (t, J = 7.63 Hz, 1H), 7.56
(d, J = 7.93 Hz, 1H), 7.64 (t, J = 7.32 Hz, 1H), 7.73 (d, J = 7.93 Hz, 1H),
7.87 (d, J = 8.24 Hz, 2H), 8.76 (br. t, J = 5.65 Hz, 1H).
3-((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)
amino)-N-(2-(trifluoromethyl)benzyl)cyclohexane-1-carboxamide (22).
A mixture of cyanuric chloride (3.86 g, 20.9 mmol, 1equivalent) in
CH3CN/H2O (1/1, 200 mL) was cooled to 0 ◦C. Methylamine solution in
water (40% w, 1.83 mL, 20.9 mmol, 1 equivalent) was added, followed
by adding 1 N NaOH (20.9 mL, 20.9 mmol, 1 equivalent). The reaction
mixture was stirred at cold for 15 min and LC-MS showed the reaction
has completed. At cold, N-methylpiperazine (2.3 mL, 20.9 mmol, 1
equivalent) was added, followed by adding 1 N NaOH (4.2 mL, 0.2
equivalents). The solution turned to clear. The solvent was evaporated to
give the crude product which was further purified with silica gel chro-
matography (eluant 70% B, solvent B is 10% MeOH in DCM with 1%
Et3N) to give 4-Chloro-N-methyl-6-(4-methylpiperazin-1-yl)-1,3,5-tri-
azin-2-amine (1.8 g, 36% in 2 steps). 1H NMR (CDCl3, 400 MHz, mixture
of rotamers): δ 2.33 (s, 3H), 2.44 (br, 4H), 2.95&2.96 (s, 3H), 3.86 (br,
4H), 5.1–5.7 (br, 1H).
3-((4-(methylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)
oxy)-N-(2-(trifluoromethyl)benzyl)benzamide (25). To a solution of 3-
hydroxybenzoic acid (1.0 g, 7.24 mmol, 1 equivalent) in 75 mL dime-
thylformamide was added O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetra-
methyluronium hexafluorophosphate (HATU, 2.75 g, 7.24 mmol, 1
equivalent) and diisopropylethylamine (2.52 mL, 14.48 mmol, 2
equivalents). Immediately, (2-(trifluoromethyl)phenyl)methenamine
was added (1.27 g, 7.24 mmol, 1 equivalent). The reaction mixture was
stirred at room temperature for 1 h. The reaction solution was
condensed and the residue was purified with silica chromatography
To a solution of 3-(Boc-amino)cyclohexanecarboxylic acid (500 mg,
2.055 mmol, 1 equivalent) in dimethylformamide (10 mL) was added O-
(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluoropho
sphate (HATU, 781 mg, 2.055 mmol, 1 equivalent) and diisopropyle-
thylamine (0.716 mL, 4.11 mmol, 2 equivalents). 2-trifluoromethyl
benzylamine (360 mg, 2.055 mmol, 1 equivalent) was added and the
reaction mixture was stirred at room temperature overnight. Additional
diisopropylethylamine (0.358 mL, 2.055 mmol, 1 equivalent) was added
and the reaction was stirred at room temperature for 2 h. The solvent
was evaporated to give the crude product which was further purified
with silica gel chromatography (eluant 40% Ethyl Acetate in Hexanes).
The organic layer was dried over MgSO4, filtered and concentrated in
vacuo. The resulting compound was re-purified with RP-HPLC. The
purified compound was treated with 50% TFA in DCM (20 mL) at room
temperature for 2 h, and then the solvents were removed in vacuo. The
desired product was carried onto the next step without further purifi-
cation. MS: calcd for C15H19F3N2O + H+ 301.16, found 301.06.
The solution of 4-chloro-N-methyl-6-(4-methylpiperazin-1-yl)-1,3,5-
triazin-2-amine(30.0 mg, 0.124 mmol, 1 equivalent) and 3-amino-N-(2-
(trifluoromethyl)benzyl)cyclohexanecarboxamide (103 mg, 0.248
mmol, 2 equivalents) in CH3CN/H2O (1/1, 2 mL) was adjusted to pH
9–10 with 1 N NaOH. The resulting solution was heated at 80 ◦C for 16 h.
The solvent was removed under vacuo to give the crude which was
purified with RP-HPLC to give the desired compound (0.94 mg, 1.5%).
HRMS: calcd for C24H33F3N8O + H+ 507.28022, found 507.27994. 1H
NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 1.20–1.54 (m, 4H), 1.83 (m,
2H), 1.95 (m, 1H), 2.07 (br. d, J = 12.21 Hz, 1H), 2.46 (m, 1H), 2.77 (br.
s, 3H), 2.91 (s, 3H), 3.05 – 3.65 (br, 8H), 3.82–3.90 (m, 1H), 4.47 (br. d,
J = 5.49 Hz, 2H), 7.47 (t, J = 7.32 Hz, 1H), 7.51 (d, J = 7.93 Hz, 1H),
(eluant 60% EtOAc), followed with RP-HPLC (Luna
5μ C8(2),
100x21mm, 20–60% CH3CN/H2O, 0.1% TFA, 20 min) to yield acylation
product 3-hydroxy-N-(2-(trifluoromethyl)benzyl)benzamide (1.04 g,
49%). MS: calcd for C15H12F3NO2 + H+ 296.09, found 295.94.
By use of methylamine for the first chloro-substitution, 3-hydroxy-N-
(2-(trifluoromethyl)benzyl)benzamide for the second chloro-
substitution, and N-methylpiperazine for the last chloro-substitution,
the title compound was prepared by following general method A.
HRMS [M + H]+ calcd for C24H26F3N7O2 + H+ 502.21728, found
502.21672. 1H NMR (500 MHz, DMSO‑d6, 80 ◦C) δ ppm 2.71–2.88 (m,
6H), 4.70 (br. d, J = 5.80 Hz, 2H), 7.21–7.30 (m, 1H), 7.35 (m, 1H),
7.46–7.55 (m, 2H), 7.58 (d, J = 7.93 Hz, 1H), 7.65 (t, J = 7.93 Hz, 1H),
7.73 (br. d, J = 7.63 Hz, 1H), 7.80 (d, J = 7.93 Hz, 1H), 8.87 (m, 1H).
4.2. Affinity selection.
The selection of sEH specific binders was done using conventional
“in-solution” selection protocol as reported.5 Different concentrations of
purified dual-tagged sEH protein were incubated with ELT libraries in
1X Selection Buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl
and 1 mg/ml of salmon sperm DNA (Invitrogen, AM9680) for 1 h. The
protein with bound ELT molecules was captured on streptavidin affinity
matrix resin and washed several times with selection buffer. Bound li-
brary members were eluted from the protein by denaturation. The eluted
molecules were subjected to a second and third rounds of selection with
fresh protein at each round. The selections, described herein, were
performed against 0.01 µM, 0.1 µM, 1 µM hsEH protein and no protein
control (selection buffer only) in the presence and absence of 1 µM
8

Y. Ding et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116216
7 Ding Y, O’Keefe H, DeLorey JL, et al. Discovery of potent and selective inhibitors for
ADAMTS-4 through DNA-encoded library technology. ACS Med Chem Lett. 2015;6:
888–893.
AUDA. The outputs from the third selection round were PCR amplified,
sequenced, translated into chemical structures and analyzed using
TIBCO Spotfire software as described previously.5
8 Disch JS, Evindar G, Chiu CH, et al. Discovery of Thieno[3,2-d]pyrimidine-6-
carboxamides as Potent Inhibitors of SIRT1, SIRT2, and SIRT3. J Med Chem. 2013;56:
3666–3679.
4.3. Assay against human recombinant sEH
9 Wu Z, Graybill TL, Zeng X, et al. Cell-based selection expands the utility of DNA-
encoded small-molecule library technology to cell surface drug targets: identification
of novel antagonists of the NK3 tachykinin receptor. ACS Comb Sci. 2015;17(12):
722–773.
The biological activity of the compounds was measured using the
nonfluorescent EnzChek epoxide hydrolase substrate (Molecular Probes,
Catalog # E33956). Assay was performed in the buffer containing 50
mM HEPES (pH 8). The compounds were re-suspended in 100% DMSO
and then serially diluted in the assay buffer (final concentration of
DMSO 0.8%). 10 nM hsEH protein was pre-incubated with compounds
for 5 min at room temperature, followed with addition of diluted Enz-
Chek substrate (final concentration 10 µM). The reaction was run at
room temperature, with fluorescence being measured every 30 s for 30
min at an excitation of 350 nm and an emission of 455 nm.
10 Seigal BA, Connors WH, Fraley A, et al. The discovery of macrocyclic XIAP
antagonists from a DNA-programmed chemistry library, and their optimization to
give lead compounds with in vivo antitumor actvity. J Med Chem. 2015;58:
2855–2861.
11 Wellaway CR, Amans D, Bamborough P, et al. Discovery of a Bromodomain and
Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of
Encoded Library Technology and Fragment Screening. J Med Chem. 2020;63(2):
714–746.
12 Harris TR, Hammock BD. Soluble epoxide hydrolase: Gene structure, expression and
deletion. Gene. 2013;526(2):61–74.
13 Spector AA, Fang X, Snyder GD, Weintraub NL. Epoxyeicosatrienoic acids (EETs):
metabolism and biochemical function Prog. Lipid Res. 2004;43:55–90.
14 Imig JD. Cardovascular therapeutic aspects of soluble epoxide hydrolase inhibitors
Cardiovasc. Drug Rev. 2006;24:169–188.
The activity of the compounds was calculated as the % of inhibition
using the following equation: 100*(1-(slope of enzyme + inhibitor/
slope of enzyme alone) and expressed as IC50 values (the concentration
of inhibitor that blocks enzyme activity to 50%).
15 Smith KR, Pinkerton KE, Watanabe T, Pedersen TL, Ma SJ, Hammock BD.
Attenuation of tobacco smike-induced lung inflammation by treatment with a soluble
epoxide hydrolase inhibitor. PNAS. 2005;102:2186–2191.
16 Wang L, Yang J, Guo L, et al. Use of a soluble epoxide hydrolase inhibitor in smoke-
induced chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2012;46:
614–622.
Declaration of Competing Interest
17 Podolin PL, Bolognese BJ, Foley JF, et al. In vitro and in vivo characterization of a
novel soluble epoxide hydrolase inhibitor. Prostaglandins Other Lipid Mediat. 2013;
104–105:25–31.
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.
18 The 10 leading causes of death in the world, 2000 and 2019.
19 Kim I-H, Morisseau C, Watanabe T, Hammock BD. Design, synthesis, and biological
activity of 1,3-disubstituted urea as potent inhibitors of the soluble epoxide
hydrolase of increased water solubility. J Med Chem. 2004;47:2110–2122.
20 Eldrup AB, Soleymanzadeh F, Taylor SJ, et al. Structure-based optimization of
arylamides as inhibitors of soluble epoxide hydrolase. J Med Chem. 2009;52(19):
5880–5895.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116216.
21 Cardozo, M. P.; Ingraham, R. H. Methods of Using Acyl Hydrazones as sEH Inhibitors
WO2006/121684, PCT/US2006/011673.
22 Ota, T.; Takahashi, H.; Kakinuma, H.; Busujima, T. Preparation of benzimidazole-5-
carboxamide derivatives as soluble epoxide hydrolase inhibitors.
WO2007043653A1, 2007.
References
23 Lazaar AL, Yang L, Boardley RL, et al. Pharmacokinetics, pharmacodynamics and
adverse event profile of GSK2256294, a novel soluble epoxide hydrolase inhibitor. Br
J Clin Pharmacol. 2016;81(5):971–979.
1 Brenner S, Lerner RA. Encoded Combinatorial chemistry. PNAS. 1992;89:5381–5383.
2 Kleiner RE, Dumelin CE, Liu DR. Small-Molecule Discovery from DNA-Encoded
Chemical Libraries. Chem Soc Rev. 2011;40:5707–5717.
24 Belyanskaya SL, Ding Y, Callahan JF, Lazaar AL, Israel DI. Discovering Drugs with
DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of
Soluble Epoxide Hydrolase. ChemBioChem. 2017;18(9):837–842.
25 Allain H, Bentue-Ferrer D. Clinical efficacy of almitrine-raubasine. Eur Neurol. 1998;
39(suppl. 1):39–44.
3 Mannocci L, Leimbacher M, Wichert M, Scheuermann J, Neri D. 20 Years of DNA-
Encoded Chemical Libraries. Chem Commun. 2011;47:12747–12753.
4 Halpin DR, Harbury PB. DNA Display II. Genetic Manipulation of Combinatorial
Chemistry Libraries for Small-molecule Evolution. PLoS Biol. 2004;2:1022–1030.
5 Clark MA, Acharya RA, Arico-Muendel CC, et al. Design, Synthesis and Selection of
DNA-Encoded Small-Molecule Libraries. Nat. Chem Bio. 2009;5:647–654.
6 Harris PA, Berger SB, Jeong JU, et al. Discovery of a First-in-Class Receptor
Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the
Treatment of Inflammatory Diseases. J Med Chem. 2017;60(4):1247–1261.
26 Kim E, Enasidenib S. First global approval. Drug. 2017;77:1705–1711.
27 Thalji RK, McAtee JJ, et al. Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-
carboxamides as inhibitors of soluble epoxide hydrolase. Bioorg Med Chem Lett. 2013;
23:3584–3588.
9