Structure-Based Design of 1-Heteroaryl-1,3-propanediamine Derivatives as a Novel Series of CC-Chemokine Receptor 5 Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.8b01077

CC-chemokine receptor 5 (CCR5) is an attractive target for preventing the entry of human immunodeficiency virus 1 (HIV-1) into human host cells. Maraviroc is the only CCR5 antagonist, and it was marketed in 2007. To overcome the shortcomings of maraviroc, structure-based drug design was performed to minimize CYP450 inhibition and to enhance anti-HIV potency and bioavailability. Thirty-four novel 1-heteroaryl-1,3-propanediamine derivatives (1-34) were synthesized, displaying CCR5-antagonist activities in the 2.3-296.4 nM range. Among these, compounds 21 and 34 were the most potent CCR5 antagonists, with excellent in vitro anti-HIV-1 activity, low cytotoxicity, and an acceptable pharmacokinetic profile. Furthermore, the X-ray crystal structures of compounds 21 and 34 bound to CCR5 were determined at 2.8 ? resolution. Compound 34 exhibited no CYP450-inhibition activity at 25 μM, which overcomes the potential drug-drug interaction of maraviroc. Compound 34 represents a promising drug candidate for HIV-infection treatment.

Full text of DOI:10.1021/acs.jmedchem.8b01077

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Article
Structure-Based Design of 1-Heteroaryl-1,3-Propanediamine Derivatives
as a Novel Series of CC-chemokine Receptor 5 (CCR5) Antagonists
Panfeng Peng, Huan Chen, Ya Zhu, Zhi-Long Wang, Jian Li, Ronghua Luo, jiang wang, Liang
Chen, Liu-Meng Yang, Hualiang Jiang, Xin Xie, Beili Wu, Yong-Tang Zheng, and Hong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01077 • Publication Date (Web): 20 Sep 2018
Downloaded from http://pubs.acs.org on September 22, 2018
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StructureꢀBased
Design
of
1ꢀHeteroarylꢀ1,3ꢀPropanediamine
Derivatives as a Novel Series of CCꢀchemokine Receptor 5 (CCR5)
Antagonists
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†,#,§,║
‡,§,║
#,§,║
†,§,║
†,#,§
Panfeng Peng
, Huan Chen , Ya Zhu , Zhilong Wang , Jian Li
,
‡
,§
†,#,§
†,#,§
‡,§
RongꢀHua Luo , Jiang Wang , Liang Chen , LiuꢀMeng Yang , Hualiang
†
,§
†,§
#,§,*
‡,§,*
†,#,§,*
Jiang , Xin Xie , Beili Wu , YongꢀTang Zheng
and Hong Liu
†
State key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China, EꢀMail: hliu@simm.ac.cn
#
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu chong zhi Road, Shanghai 201203, China
EꢀMail: beiliwu@simm.ac.cn
‡
Key Laboratory of Bioactive Peptides of Yunnan Province/Key Laboratory of Animal
Models and Human Disease Mechanisms of Chinese Academy of Sciences,
KIZꢀCUHK Joint Laboratory of Bioresources and Molecular Research in Common
Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming,
Yunnan 650223, China; EꢀMail: zhengyt@mail.kiz.ac.cn
§
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,
China
║
These authors contributed equally to this study.
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ABSTRACT:
CCꢀchemokine receptor 5 (CCR5) is an attractive target to prevent the entry of
human immunodeficiency virusꢀ1 (HIVꢀ1) into human host cells. Maraviroc is the
only one CCR5 antagonist marketed in 2007. To overcome the shortcomings of
maraviroc, structureꢀbased drug design was performed to minimize CYP450
inhibition, and to enhance antiꢀHIV potency and bioavailability. Thirtyꢀfour novel
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1ꢀheteroarylꢀ1,3ꢀpropanediamine derivatives (1ꢀ34) were synthesized, displaying
CCR5 antagonist activities in the 2.3–296.4 nM range. Among these, compounds 21
and 34 were the most potent CCR5 antagonists, with excellent in vitro antiꢀHIVꢀ1
activity and low cytotoxicity, an acceptable pharmacokinetic profile. Furthermore, the
Xꢀray crystal structures of compounds 21 and 34 bound to CCR5 were determined at
2.8 Å resolution. Compound 34 exhibited no CYP450 inhibition activity at 25 ꢁM,
which overcome the potential drugꢀdrug interaction of maraviroc. Compound 34
represents a promising drug candidate for the HIV infection treatment.
Key Words: CCꢀchemokine receptor 5, antagonist, HIVꢀ1, structureꢀbased design,
Xꢀray crystal structure
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INTRODUCTION
Despite worldwide efforts to prevent the epidemic of acquired immune
deficiency syndrome (AIDS), it remains a global threat: currently, 36.7 million
individuals live with HIV/AIDS. There are roughly 2.1 million new infections each
year, and approximately 1.1 million AIDSꢀrelated deaths in 2015. Antiretroviral
therapy (ART) delays progression to AIDS by reducing the viral load in infected
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individuals. However, there remain many problems in current HAART, including
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unwanted side effects, drugꢀdrug interactions, and viral resistance. Thus, developing
a new class of antiꢀHIVꢀ1 agents with superior efficacy and safety profiles is required
urgently. Preventing HIV entry into human host cells is an attractive way to control
3
viral infection and replication. CCR5 serves as a coꢀreceptor for HIVꢀ1 entry into
+
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CD4 Tꢀcells. The importance of CCR5 on HIVꢀ1 infection was discovered by the
observation that the naturally occurring mutation known as CCR5ꢀꢂ32 in the CCR5
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gene provides resistance to infection.
Many CCR5 allosteric antagonists have been reported as shown in Figure 1,
6
ꢀ18
however, very few have progressed into the clinic before studies were halted,
mostly because of challenging drugꢀlike properties (antiviral activity, human
etherꢀàꢀgoꢀgo related gene (hERG) activity, suitable bioavailability, and toxicity).
Thus, only one CCR5 antagonist (maraviroc in 2007) has been approved for the
1
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treatment of HIV patient. By systematical SAR optimizations, it was found that the
cytochrome P450 (CYP450) inhibition and hERG inhibition were associated with the
tropane moiety, triazole moiety, and amide moiety. Reducing the lipophilicity and
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maintaining antiviral activity is very important for the CCR5 antagonist. Maraviroc
was discovered and development by the optimization of binding potency against
CCR5, antiretroviral activity, absorption, pharmacokinetics, and selectivity for the
hERG channel. However, there are some factors that limit its wide prescription, such
as CYP450 inhibition (CYP3A4ꢀM IC50 = 3.1 ꢁM), drugꢀdrug interactions (the dose
should be adjusted when administrated with CYP3A4 inhibitors or CYP3A4 inducers),
and viral resistance. Therefore, with the rapid rise in the number of patients with HIV
worldwide, discovering novel therapeutics to treat HIV with better efficacy and fewer
side effects has been a research focus in academia and industry.
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Figure 1. Representative structures of known CCR5 antagonists.
RESULT AND DISCUSSION
Design of 1ꢀheteroarylꢀ1,3ꢀ propanediamine scaffold.
Maraviroc is associated with some side effects in clinical use, such as
hepatotoxicity, skin hypersensitivity reaction, and cardiovascular events, which result
from its shortcomings, such as potential CYP450 enzyme inhibition, drugꢀdrug
interactions, viral resistance, and high administration dose. Moreover, the
physicochemical properties and pharmacokinetic profile of maraviroc need to be
further optimization. In rat pharmacokinetic study, after a single oral dose of 10
mg/kg maraviroc, the maximum plasma concentration (Cmax), the area under the
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plasma concentration–time curve (AUC), and bioavailability (F) were 5.5 ng/mL,
12.4 ng·h/mL, and 6.0%, respectively. Metabolic studies indicated there were four
primary metabolic sites/pathways for maraviroc (Mꢀ1–Mꢀ4): hydroxylation at the
paraꢀposition of the phenyl ring (Mꢀ1), oxidation in the difluorocyclohexyl ring
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(Mꢀ2), oxidation of the triazole moiety (Mꢀ3) and Nꢀdealkylation adjacent to the
tropane ring (Mꢀ4). Accordingly, we aimed to block these metabolism sites to
improve the metabolic stability, and thus increase the oral bioavailability of
maraviroc.
Figure 2. Metabolic pathways of maraviroc in rat, dog, and human.
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Recently, Wu et al. reported the structure of the CCR5ꢀmaraviroc complex (Figure
20
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a). This work reveals that maraviroc binds to a deep pocket formed by residues of
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the transmembrane helices and this binding site does not overlap with the major
recognition site for chemokines, suggesting that maraviroc stabilizes CCR5 in an
inactive state to inhibit the receptor’s function via an allosteric mode of action. The
interactions between maraviroc and CCR5 involve four distinct regions (Figure 3b).
(1) P1: The fluorines in the cyclohexane ring form two hydrogen bonds with Thrꢀ195
and Thrꢀ259, the carboxamide nitrogen forms a hydrogen bond with Tyrꢀ251, and the
cyclohexane group makes hydrophobic contacts with a CCR5 subpocket. (2) P2: The
phenyl group reaches deep into a pocket, and forms hydrophobic interactions with
five aromatic residues (Tyrꢀ108, Pheꢀ109, Pheꢀ112, Trpꢀ248, and Tyrꢀ251), especially
the πꢀinteraction with the conserved aromatic residue Trpꢀ248, preventing the residue
from moving to an active state conformation. (3) The linker: tropane moiety. The
protonated nitrogen is engaged in a saltꢀbridge interaction with Gluꢀ283, and the
length of the carbon chain between the aboveꢀmentioned two nitrogens was reported
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to be critical to maintain those main forces. (4) P4: The triazole group forms a
hydrogen bond with Tyrꢀ37 via a water molecule. Based on the crystal structure of the
maraviroc with CCR5, the amide moiety (“head” part) and triazole moiety (“tail” part)
are very important for the CCR5 binding activity. In addition, analyzing the metabolic
pathways of the maraviroc, the phenyl group, amide moiety and triazole moiety are
the major metabolic sites. In order to improve the CCR5 binding affinity and
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increased the metabolic stability, a novel series of 1ꢀheteroarylꢀ1,3ꢀpropanediamine
derivatives (1ꢀ34) were designed (Figure 4).
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Figure 3. (a): CCR5 (PDB ID: 4MBS, light blue)/maraviroc (magenta carbons)
complex structure. Maraviroc is shown in magenta stick representation. The area in
black frame is known as the recognition site for chemokine. (b): CCR5 (gray) binding
pocket for maraviroc (magenta carbons). Some key interactions are shown as blue
(hydrogen bond) and red (salt bridge) dashed lines. Black frames highlight four
distinct positions of maraviroc (P1ꢀP4).
Figure 4. Design of 1ꢀheteroarylꢀ1,3ꢀpropanediamine scaffold.
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Chemistry.
The general synthetic route to compounds 1ꢀ34 is shown in Scheme 1. Condensation
following protection of ketone 35 provided intermediate oxime 37. The intermediate
38 was obtained by the sodium metal reduction of 37, and acetylation of 38 provided
intermediate amides 39aꢀc. The triazoles 40aꢀe were synthesized in three steps:
activation of 39aꢀc to their corresponding imidoyl chlorides, followed by trapping
with hydrazides, and acid catalyzed cyclization to the triazoles. Deprotection of
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compounds 40aꢀe provided key intermediate compounds 41aꢀe. The chiral βꢀamino
acids 45aꢀl were prepared in three steps from aldehydes 42aꢀl reacting with Ellman’s
chiral auxiliary. Oxidation following reduction of 45aꢀl provided intermediates 47aꢀl,
which further reacted with 41aꢀe producing compounds 48aꢀr. Deprotection of
compounds 48aꢀr, and introduction of a range of acyl groups provided the final
compounds 1ꢀ34.
a
Scheme 1. Synthetic routes to thedesired compounds 1ꢀ34
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a
Reagents and conditions: (a) BnBr, K
2
CO
3
, MeCN; (b) NH
2
OH·HCl, K
, R
, EtOH; (g)
2
CO
3
, EtOH;
o
(c) Na,
n
ꢀC
5
H
11OH, 100 C; (d) R
3
COCl, Na
2
CO , DCM; (e) (1) PCl
3
5
4
CONHNH ,
2
o
DCM; (2) AcOH, 1,4ꢀdioxane, 80 C; (f) 10% Pd/C, HCOONH
4
(R
)ꢀ(+)ꢀ2ꢀMethylꢀ2ꢀpropanesulfinamide, Ti(EtO) , THF; (h) LDA, CH COOMe,
4
3
(iꢀPrO)
3
TiCl, THF; (i)(1) 2
N
HCl, MeOH; (2) (Boc)
2
3 4
O, NEt , MeOH; (j) NaBH , THF,
3
reflux; (k) DessꢀMartin periodinane, DCM; (l) 41aꢀe, NaBH(OAc) , AcOH, DCE; (m)
(1) 2
N
HCl, MeOH; (2) R
2
COOH, EDCI, HOBt, NEt
3
, DCM.
StructureꢀActivity
Derivatives.
Relationships
of
1ꢀheteroarylꢀ1,3ꢀpropanediamine
Initially, all 1ꢀheteroarylꢀ1,3ꢀpropanediamine derivatives (1ꢀ34) were evaluated
their inhibitory effects on the CCR5 binding activities in the HEK293 cells with
maraviroc as the positive control. As shown in Table 1, most of the compounds
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generally showed potent inhibitory activity against CCR5. Firstly, different substituted
heterocyclic groups were investigated at the R
group showed excellent activity toward CCR5 (IC50 = 9.1 nM). Replacement of the
ꢀthienyl group of compound 1 to 4ꢀthiazolyl (compound 2) and 3ꢀpyridinyl
compound 3) resulted in the decreased inhibitory activities. When the 2ꢀthienyl group
1
. Compound 1 containing the 2ꢀthienyl
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(
was replaced by 2ꢀfluoroꢀ5ꢀpyridinyl, compound 4 showed significantly decreased the
potency (IC50 = 251.6 nM). However, introducing the bulky 3ꢀbenzothienyl group,
compound 5 maintained the potent antagonism of CCR5, with an IC50 value of 9.7 nM,
indicating that large group could be tolerated at R . Then, introduction of different
1
substituted group at the 2ꢀthienyl group, compounds 6ꢀ11 bound to CCR5 with IC50
values of 2.8, 6.8, 15.8, 12.0, 8.1, and 6.8 nM, respectively. Compounds 6, 7, and 10
were slightly more potent than that of maraviroc.
Second, we turned the structure modification to the “head” carboxamide group,
the carboxamide N1 forms a hydrogen bond with Tyrꢀ251, and cyclohexane group
makes hydrophobic contacts with the CCR5 subpocket. To probe the
structure−activity relationships at this site, compounds (12ꢀ18) were designed and
synthesized with different carboxamide groups, and their binding affinities to CCR5
were determined. Replacement the 4,4ꢀdifluorocyclohexyl group with a phenyl group,
4
ꢀfluorobenzyl group, 4ꢀacetylpiperidinyl group, 4ꢀmethylpiperidinyl group,
4ꢀtetrahydroꢀ2 ꢀpyranyl group, cyclopentyl group or isopropyl group afforded
compounds 12ꢀ18. Among them, compound 17 was the most active CCR5 antagonist
IC50 = 2.6 nM), which was about 3ꢀfold more potent than that of maraviroc (IC50
.0 nM).
Then, replacing the 4,4ꢀdifluorocyclohexyl group with a 4ꢀfluorocyclohexyl
group, cyclohexyl group, cyclopentyl group, 1ꢀtrifluoromethylꢀ1,1ꢀdimethyl group,
H
(
=
8
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3ꢀoxocyclopentyl group, 5ꢀoxopyrrolidine group, 1ꢀmethylꢀ5ꢀoxopyrrolidine group,
3,3ꢀdifluorocyclopentyl group or 3,3ꢀdifluorocyclobutyl group yielded compounds
19ꢀ27. The 2ꢀthienyl substituted derivatives (19ꢀ27) presented more potent inhibitory
effects against CCR5 than the 5ꢀmethoxythienyl derivatives (12ꢀ18). Among them, six
compounds (19, 20, 21, 23, 26, and 27) exhibited significant inhibitory potency
against CCR5, which was approximately more than 2ꢀfold higher than that of
maraviroc (IC50 = 8.0 nM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We further investigated the effect of the “tail” triazole group. Finally, to block
the metabolic sites, replacing isopropyl with cyclopropyl or ethyl afforded compounds
28ꢀ30, Compounds 28ꢀ30 displayed potent CCR5 inhibitory activity, with IC50 values
of 2.6, 2.9, and 3.0 nM, respectively. These results indicated that the cyclopropyl
moiety could improve the activity. Replacement the methyl group with a
trifluoromethyl group yielded compounds 31, 32, and 33. These compounds proved to
1
be potent CCR5 antagonists with IC50 values below 5.0 nM. Replacing R phenyl
group to the 3ꢀthienyl group afforded compound 34 (IC50 = 3.0 nM), which
exhibited2ꢀfold more potent than maraviroc (IC50 = 8.0 nM) for antagonizing CCR5.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. CCR5 binding activity of 1ꢀheteroarylꢀ1,3ꢀpropanediamine derivatives
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
CCR5 antagonist activity
cLog P
Compd.
R
1
R
2
R
3
R
4
a
IC (nM)
5
0
1
2
thiophenꢀ2ꢀyl
thiazolꢀ4ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
methyl
methyl
isopropyl
isopropyl
9.1±3.0
3.37
2.07
4
27.1±6.8
3
4
5
6
7
pyridinꢀ3ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
methyl
methyl
methyl
methyl
methyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
82.6±20.8
251.6±57.0
9.7±1.6
1.77
1.99
4.75
3.87
3.82
6ꢀfluoropyridinꢀ3ꢀyl
benzo[b]thiophenꢀ3ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
4ꢀmethylthiophenꢀ2ꢀyl
2.8±1.4
6.8±3.2
1
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5ꢀfluorothiophenꢀ2ꢀyl
5ꢀchlorothiophenꢀ2ꢀy
4ꢀmethylthiophenꢀ3ꢀyl
5ꢀmethoxythiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
15.8±3.4
12.0±1.2
8.1±0.9
3.55
4,74
3.87
3.56
4.20
4.33
1.11
1.71
2.08
3,92
3.38
3.43
3,98
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
phenyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
10
11
6.8±1.4
12
51.7±7.2
33.0±9.2
87.9±21.6
296.4±47.6
24.5±4.6
2.6±0.1
13
14
15
16
17
18
4ꢀfluorobenzyl
1ꢀacetylpiperidinꢀ4ꢀyl
1ꢀmethylpiperidinꢀ4ꢀyl
tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl
cyclopentyl
iꢀPr
16.2±3.1
3.0±0.4
19
4ꢀfluorocyclohexyl
cyclohexyl
20
thiophenꢀ2ꢀyl
2.3±0.3
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
2
1
2
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ3ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ3ꢀyl
5ꢀmethylthiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ3ꢀyl
cyclopentyl
methyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
cyclopropyl
cyclopropyl
ethyl
3.1±0.2
5.4±2.0
2.9±0.4
20.6±3.7
5.2±2.0
2.3±0.2
2.6±0.2
2.6±0.2
2.9±0.1
2.8±0.1
4.0±1.1
4.7±0.8
2.9±0.1
3.42
3.36
1.87
1.19
2.40
2.81
2.25
2.88
2.88
2,97
3.97
4.37
2.88
2
2,2,2ꢀtrifluoroꢀ1,1ꢀdimethylethyl
3ꢀoxocyclopentyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
23
24
(S)ꢀ5ꢀoxopyrrolidinꢀ2ꢀyl
25
1ꢀmethylꢀ5ꢀoxopyrrolidinꢀ3ꢀyl
3,3ꢀdifluorocyclopentyl
26
27
28
29
30
31
3,3ꢀdifluoroꢀcyclobutane ꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
trifluoromethyl isopropyl
trifluoromethyl isopropyl
trifluoromethyl cyclopropyl
32
33
1
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3
4
thiophenꢀ3ꢀyl
4,4ꢀdifluoroꢀcyclohexanꢀ1ꢀyl
methyl
isopropyl
3.0±0.3
8.0±2.7
3.37
3.26
MVC
IC50 values are exhibited by means ± standard deviations, n≥3.
ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AntiꢀHIVꢀ1 Activity, and the Cellular Cytotoxicity of Compounds of
1ꢀheteroarylꢀ1,3ꢀpropanediamine Derivatives.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on the CCR5 binding results, some high binding affinity CCR5
antagonists were selected for further screening in vitro properties. The data from an
evaluation in an antiviral assay using TZMꢀbl cells (a frequently used HIVꢀ1 reporter
cell line) from labꢀadapted HIVꢀ1SF162 are shown in Table 2. All compounds showed
potent antiꢀHIVꢀ1 activity: the IC50 values of compounds 1, 6, 20, 21, and 34 were 3.6,
11.5, 11.1, 1.3, and 1.5 nM, respectively. The TI (therapeutic index) values of
4
4
4
4
compounds 1, 6, 20, 21, and 34 were more than 5.5×10 , 1.7×10 , 1.8×10 , 1.5×10 ,
4
and 1.3×10 respectively. Compounds 1, 21, and 34 possessed 2.3ꢀ, 6.6ꢀ, and 4.5ꢀfold
enhanced antiviral activity, respectively, compared to that of maraviroc. It is worth
noting the similarity in cLog
which displayed excellent in vitro antiꢀHIVꢀ1SF162 activity with high therapeutic index
TI) values.
P (3.37, 3.87, 3.98, 3.42 and 3.37 for 1, 6, 20, 21, and 34),
(
Table
2.
AntiꢀHIVꢀ1
Activity,
Cellular
Cytotoxicity
of
1ꢀheteroarylꢀ1,3ꢀpropanediamine Derivatives
TZMꢀbl
TZMꢀbl
c
Compd.
TI
a
b
IC50 (nM)
CC50 (ꢁM)
4
1
3
5
>200
3
.6±0.3
520.3±184.5
10.5±299.5
>5.5×10
>100
96.1
1
>65.8
118.6
8
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2
3
4
5
6
7
8
9
4
6
8
>200
>200
>200
>200
>200
>200
1
1.5±0.5
>1.7
×10
519.5±127.5
11.1±1.8
>385.0
4
4
4
4
2
0
1
4
>1.8
>1.5
>1.3
>2.9
×
×
×
×
10
10
10
10
2
3
1
1
6
.3±0.1
.5±0.1
.7±0.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MVC
a
b
c
IC s are exhibited by means ± standard deviations, n≥3.
5
0
CC50s are exhibited by means ± standard deviations, n≥3.
TI, therapy index, the rate of CC50/EC50
.
Further Assessment of antiꢀHIVꢀ1 Activity, and the Cellular Cytotoxicity of
compounds 1, 6, 20, 21, and 34.
To establish their broad activity spectrum, compounds 1, 6, 20, 21, and 34
wereselected for further testing as these offered a better balance of in vitro properties.
The antiviral activity against a range of CCR5ꢀtropic HIVꢀ1 isolates (HIVꢀ1SF162
,
HIVꢀ1BaꢀL, and HIVꢀ1KM018) in peripheral blood mononuclear cells (PBMC),
HOSꢀCD4ꢀCCR5 (human osteosarcoma (HOS) cells expressing CDꢀ4 and CCR5) and
PMꢀ1cells (Tꢀlymphoid cells) are shown in Table 3. Compounds 1, 6, 20, 21, and 34
were active at low nanomolar concentrations against HIVꢀ1 with low cytotoxicity.
Especially, compound 34 showed good activity against HIVꢀ1SF162 in PBMCs (IC50
=
5
4
.3 nM, CC50 = 674.8 ꢁM, TI = 1.6×10 ), HIVꢀ1
in HOSꢀCD4ꢀCCR5 (IC50 = 1.2
BaꢀL
5
nM, CC50 = 337.5 ꢁM, TI = 2.8×10 ), HIVBaꢀL in PMꢀ1 (IC50 = 2.8 nM, CC50 = 735.9
5
ꢁ
M, TI = 2.6×10 ) and HIVꢀ1KM018 in PBMC (IC50 = 2.3 nM, CC50 = 674.8 ꢁM, TI =
5
2
.9×10 ).
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 3. AntiꢀHIVꢀ1 Activity, Cellular Cytotoxicity of Compounds 1, 6, 20, 21, and 34
HIVꢀ1SF162
HIVꢀ1BaꢀL
HOS
HIVꢀ1KM018
PBMC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
PBMC
HOS
CC50
PBMC
CC50
PMꢀ1
Compd.
CC50
a
a
b
a
b
a
b
b
a
b
IC50
IC50
IC50
IC50
CC50
CC50
IC50
CC50
b
c
c
c
c
a
c
c
TI
TI
TI
TI
IC50 (nM)
TI
TI
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
2
6
5
.9±1
268.0
±9.7
9.2×1
13.1±0.
8
369.7±
11.0
2.8×1
268.0±9.
7
9.4×1
369.7±
11.0
617.5±
49.6
9.9×1
268.0±
9.7
5
4
1
6
28.5±8.8
240±4.3
3.6±2.4
11.4±1.7
12.5±2.4
1.6±0.3
1.2±0.4
1.9±0.7
1.0×10
6.2±0.8
14.9±7.9
8.2±2.9
3.4±0.7
2.8±0.3
1.0±0.1
4.8±1.1
5.6×10
4
4
3
4
.
3
0
0
0
0
.7±1
.6
>2.9× 18.8±1.
>1.1×
>833.
3
>1.8×1
>1.3× 126.2±23
>1.6×1
>200
>200
>200
>200
>200
>200
4
4
4
3
3
10
4
43.7±7.
4
10
0
10
.0
504.5±58
.5
0
24.2
266.7
342.9±
12.7
7.8×1 463.9±99
266.7±12
342.9±
12.7
360.7±
15.9
4.4×1
266.7±
12.7
4
2
2
3
0
1
4
508.8
575.1
5.9×1
2.7×10
3.3×10
2.8×10
528.8
3
4
±
1
±
69.0 ±12.7
0
.3
.7
755.0±37
.4
0
23.4
755.0
6.1×1
21.0±2.
8
525.42
±8.94
337.5±
24.3
2.5×1 127.4±24
525.4±
8.9
758.4±
6.5
2.2×1
90.0±33.
0
755.0±
37.4
5
5
3
2.0×10
3
4
3
5
30.6 ±37.4
0
0
.9
0
0
4.3±1
674.8
±6.9
1.6×1
6.9×1
674.8±6.
9
2.2×1
337.5±
24.3
735.9±
4.4
2.6×1
674.8±
6.9
5
4.9±4.3
4.7±0.4
3.0±1.8
2.3±0.1
2.0±0.5
2.9×10
5
4
4
5
.
6
0
0
0
0
1
.0±0
>2.0×
>4.3×
>1.6×
>1.1×1
>2.0×
>1.0×1
MVC
>200
>200
12.3±2.9
>200
>200
>200
>200
5
4
4
5
5
5
.
5
10 .0
10
10
0
10
0
a
b
EC50s are exhibited by means ± standard deviations, n≥3.
CC50s are exhibited by means ± standard deviations, n≥3.
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c
TI, therapy index, the rate of CC50/EC50
.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
9
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4
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4
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5
5
5
5
5
5
5
5
6
Determination of Coꢀcrystal Structure of CCR5 Bound to Compounds 21 and
34.
To understand the structural basis for the high binding affinity of this series of
compound to CCR5, we determined the crystal structure of CCR5 in complex with
0
1
2
3
4
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8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
21 and 34 at 2.8 Å resolution respectively (Figure 5). In the CCR5ꢀ21 structure
(Figure 5a), the binding model indicates that compound 21 interact with CCR5 in a
similar way to the maraviroc: the thiophene moiety reaches deep into the binding
pocket, forming hydrophobic interactions with five aromatic residues: Tyrꢀ108,
Pheꢀ109, Pheꢀ112, Trpꢀ248, and Tyrꢀ251, which stabilize CCR5 in an inactive
conformation. The protonated nitrogen of the tropane moiety is engaged in a
saltꢀbridge interaction with Gluꢀ283. The triazole group forms a hydrogen bond with
Tyrꢀ371 via a water molecule. The carboxamide nitrogen forms a hydrogen bond
with Tyrꢀ251, and the cyclopentane group makes hydrophobic contacts with CCR5.
In the CCR5ꢀ34 complex structure (Figure 5b), the metaꢀposition sulphur on
thiophene is buried deeper into the binding pocket than the sulphur at the
orthoꢀposition in 34. In addition to the polar interactions between the ligand and the
residues Gluꢀ283, Tyrꢀ37, and Tyrꢀ251 in the receptor, another two hydrogen bonds
are formed by one of the fluorines in the cyclohexane ring of compound 34 with
Thrꢀ195 and Thrꢀ259 of CCR5.
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2
2
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0
1
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0
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. (a) Compound 21 (PDB ID: 6AKX), (b) Compound 34 (PDB ID: 6AKY),
and receptor residues involved in ligand binding are shown in stick representation.
Some key interactions are shown as blue dashed lines.
Pharmacokinetic (PK) Profiles of the Selected CCR5 Antagonists.
It was reported that maraviroc has poor bioavailability following oral administration
in rats. To gain an initial understanding of the pharmacokinetic parameters of our
series, we selected compounds 1, 6, 20, 21, and 34, which had potent antiviral
activities, as representatives for further investigation following intravenous (i.v.) and
oral (p.o.) dosing in rats. The results (Table 4) showed that all compounds had better
bioavailability compared with maraviroc. Compound 6 showed a high maximal
concentration (Cmax = 580.2 ng/mL), a much longer halfꢀlife (t1/2 = 32.8 h) and a much
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
better oral bioavailability (40.1% vs 6.0%). Compound 20 showed a high maximal
concentration (Cmax = 631.3 ng/mL), a low halfꢀlife (t1/2 = 1.16 h) and an acceptable
oral bioavailability (F = 14.8%). Compounds 21 and 34 showed high maximal
concentration (Cmax = 436.3 ng/mL and 306.4 ng/mL), appropriate halfꢀlife (t1/2 = 1.65
h and 1.6 h) and much better oral bioavailability (33.4%, 14.5%, vs 6.0%). These
results suggested that this series of compounds could serve as possible lead
compounds for the development of CCR5 antagonists.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Pharmacokinetic Parameters of Compounds 1, 6, 20, 21, and 34 in Rats
dose
mg/kg)
20
C
max
T
max
t
1/2
AUC0−
∞
MRT
(h)
Cl
compd.
1
admin.
F (%)
(
(ng/mL)
266.2
6841
(h)
(h)
(ng·h/mL)
1108
(L/h/kg)
p.o.
i.v.
4
2.1
3.3
ꢀ
1.42
ꢀ
7.8
ꢀ
10
0.25
2
0.77
32.8
1.60
1.16
0.31
1.65
1.31
1.60
1.02
7053
0.7
6
p.o.
i.v.
20
580.2
1692.5
631.3
4316.7
436.3
2221.7
306.4
3778
1870.7
2330.2
1512.3
5114.9
1671.8
2504.6
1259
4.8
40.1
ꢀ
10
0.25
0.25
0.25
2
2.3
4.3
ꢀ
20
21
34
p.o.
i.v.
20
2.1
14.8
ꢀ
10
0.8
1.96
ꢀ
p.o.
i.v.
20
3.02
0.91
2.6
33.4
ꢀ
10
0.25
2
3.99
ꢀ
p.o.
i.v.
20
14.5
ꢀ
10
0.25
2
4162
0.8
1.0
a
MVC
p.o.
10
5.5
12.4
6.0
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9
i.v.
1
0.9
74
ꢀ
a
Ref 23.
Compounds 21 and 34 were selected for further investigation following intravenous
(i.v.) and oral (p.o.) dosing in dogs since it had the good results of antiꢀHIV activity
and pharmacokinetic parameters in rats. The results (Table 5) showed that
compounds 21 and 34 had lower clearance (2.8, 0.3, vs 21 L/h/kg), and compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1 displayed equivalent oral bioavailability (38.1% vs 40.0%) compared with
maraviroc, suggesting that it would have acceptable pharmacokinetic properties in
humans.
Table 5. Pharmacokinetic Properties of Compounds 21 and 34 in Dogs
dose
C
max
T
max
T
1/2
AUC0−∞
(ng·h/mL)
2157
MRT
(h)
2.4
0.7
2.1
1.6
ꢀ
Cl
compd.
admin.
F
po (%)
(
mg/kg)
(ng/mL)
(h)
(h)
2.5
0.9
3.9
1.7
ꢀ
(L/h/kg)
2
1
4
p.o.
i.v.
15
3
1155
0.8
ꢀ
2.8
ꢀ
38.1
ꢀ
ꢀ
4613
ꢀ
ꢀ
0.8
ꢀ
1100
3
p.o.
i.v.
15
3
5520
10.0
10889
583
0.3
ꢀ
a
MVC
p.o.
2
256
ꢀ
0.75
ꢀ
40.0
ꢀ
i.v.
0.5
2.3
ꢀ
ꢀ
21
a
Ref 23.
Metabolic pathways of compound 34 in different species liver cells.
High levels of unchanged drug in the liver cells of all species (>99% human, >98%
monkey, >97% dog, >97% rat, and >97% mouse). The major metabolic pathways
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
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5
5
5
5
5
5
5
5
6
were defluorination (M1) and oxidation (M2ꢀM8) of the difluorocyclohexyl moiety,
oxidation hydrogenation of the left moiety (M9), and oxidation of the triazole
moiety (M10ꢀM12) (Figure 6). According to the metabolic pathways of compound
34 in different species liver cell, the unchanged drug level is much higher than
0
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2
3
4
5
6
7
8
9
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3
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3
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7
8
9
0
maraviroc, which improved the metabolic stability and increased bioavailability.
F
N
O
S
NH
N
N
N
M1
human: <0.1%
monkey: <0.1%
dog: <0.1%
rat: <0.1%
mouse: <0.1%
F
F
F
F
F F
mono-oxidation
N
O
S
NH
N
N
N
N
O
NH
O
S
NH
N
N
mono-oxidation
N
N
N
N
N
S
M10-M12
34
M2-M8
human: <0.1%
monkey: <0.1%
dog: <0.1%
rat: <3%
human: >99%
monkey: >98%
dog: >97%
human: <1%
monkey: <2%
dog: <3%
rat: >97%
rat: <0.1%
mouse: <1%
mouse: >99%
mouse: <0.1%
F
F
H2O
N
O
NH
N
N
N
S
M9
dog: <0.1%
Figure 6. Metabolic pathways of compound 34 in human, monkey, dog, rat, and
mouse with percentage of liver cell.
hERG and liver metabolic enzymes P450 tests of compounds 1, 6, 20, 21, and 34.
Blockade of the hERG channel is a significant hurdle encountered in drug discovery.
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Compounds 1, 6, 20, 21, and 34 were selected for hERG testing (Table 6). The result
showed that the IC50 values of compounds 1, 6, 20, 21, and 34 on hERG were 3.50,
3.38, 2.06, 1.52 and more than 40 ꢁM, respectively. Compound 34 showed low
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hERG potassium ion channel inhibition.
In addition, compounds 1 and 34 were found to show no significant inhibition of any
of the major P450 isoforms tested (1A2, 2C9, 2C19, 2D6, 3A4ꢀM, and 3A4ꢀT)(IC50
>
2
5 ꢁM). The inhibitory activities of liver metabolic enzyme CYP3A4ꢀM of
compounds 1, 6, 20, 21, and 34 are better than maraviroc (> 25, 12.3, > 25, 10.9, >
5, vs 3.1 ꢁM). Compounds 20 and 21 displayed higher inhibition activity against
CYP2D6 than compounds 1, 6, 34, and maraviroc.
2
Table 6. hERG and Live Metabolic Enzymes P450 Testing of Compounds of 1, 6,
20, 21 and 34
CYP450 IC50 (ꢁM)
hERG
Compd.
IC50 (ꢁM)
CYP1A2
>25
CYP2C9
>25
CYP2C19
CYP2D6
>25
CYP3A4ꢀM CYP3A4ꢀT
3.50±0.97
1
6
>25
>25
>25
>25
>25
>25
>25
10.9
12.3
>25
>25
3.1
>25
23.4
21.7
>25
>25
14.9
3
2
1
.38±0.61
.06±0.76
.52±0.32
>25
>25
>25
20
21
34
>25
>25
15.1
>25
>25
16.8
>
40
>25
>25
>25
a
MVC
> 1
>25
14.4
>25
a
Ref 24.
SingleꢀDose Toxicity of Compound 34
Singleꢀdose toxicity studies on 34 were performed in GLP laboratories. No rat died
after receiving 2000 mg/kg 34 by oral gavage in the 14ꢀday observation period. After
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
oral administration of 34 to beagle dogs, the maximal tolerance dose (MTD) was
higher than 1000 mg/kg. The above data indicate that 34 has very low acute toxicity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
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2
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4
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7
8
9
0
CONCLUSIONS
In this study, we reported the structureꢀbased design, synthesis, and evaluation of a
new class of CCR5 antagonists. To overcome the shortcomings of maraviroc and
based on the coꢀcrystal structure of CCR5 in complex with maraviroc, a new series of
compounds with a 1ꢀheteroarylꢀ1,3ꢀpropanediamine scaffold. The most potent
compound 34 bound to CCR5 with an IC50 values of 3.0 nM, and showed excellent in
vitro antiꢀHIVꢀ1 activity. The therapeutic index (TI) against antiꢀHIVꢀ1SF162 infection
5
5
of TZMꢀbl cells, PBMC cells, and HOSꢀCD4ꢀCCR5 cells was 1.3×10 , 1.6×10 , and
4
6
.9×10 , respectively. The oral bioavailability of compound 34 in rats and dogs are
14.6% and 10.0%, respectively, suggesting that it would have acceptable
pharmacokinetic properties in humans. Furthermore, the Xꢀray crystal structure of
CCR5 bound to compounds 21 and 34 shows that it forms strong interactions with
CCR5. Compound 34 displayed no inhibition with hERG at 40
μM and CYP450 at
25 ꢁM. In summary, compound 34 holds great promise as a more potent and a safer
HIV agent that might have the following advantages over maraviroc, the CCR5
inhibition activity of compound 34 is better than maraviroc, and no CYP3A4
inhibitory activity at 25 ꢁM. Further preclinical trials on compound 34 are currently
being conducted in our laboratories.
EXPERIMENTAL SECTION
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Chemistry. Materials and General Methods.
The reagents (chemicals) were purchased and used without further purification.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker
AMXꢀ400 and AMXꢀ300 NMR (IS as TMS). Chemical shifts were reported in parts
per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and
broad (br). Lowꢀ and highꢀresolution mass spectra (LRMS and HRMS) were given
with electric, electrospray, and matrixꢀassisted laser desorption ionization (EI, ESI,
and MALDI) produced by a Finnigan MATꢀ95, LCQꢀDECA spectrometer and
IonSpec 4.7 T. HPLC analysis of all final biological testing compounds was carried
out on an Agilent 1260 Series HPLC with an Agilent ExtendꢀC18 column (150×4.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mm, 5 µm) with three solvent systems (methanol/water, methanol/bu
ff
er (5 mM
OAc, 0.1%
95%
NH OAc, 0.1% HCOOH in water) or acetonitrile/bu er (5 mM NH
4
ff
4
HCOOH in water)). Purity was determined by reversedꢀphase HPLC and was
for all biological tested compounds.
≥
4
8
,4ꢀdifluoroꢀ
Nꢀ((S)ꢀ3ꢀ((1R,3R,5S)ꢀ3ꢀ(3ꢀisopropylꢀ5ꢀmethylꢀ4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)ꢀ
ꢀazabicyclo[3.2.1]octanꢀ8ꢀyl)ꢀ1ꢀ(thiophenꢀ2ꢀyl)propyl)cyclohexaneꢀ1ꢀcarboxam
ide (1).
To a solution of 48b (47.2 mg, 0.1 mmol) in dichloromethane (5 mL) was added
triethylamine (28 ꢁL, 0.2 mmol), HOBt (14.8 mg, 0.11 mmol), EDCI (21.1 mg, 0.11
mmol), and 4,4ꢀdifluorocyclohexanecarboxylic acid (18 mg, 0.11 mmol). The
mixture was stirred at room temperature for 12 h and quenched with 10 ml of water.
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The mixture was extracted with dichloromethane. The combined organic layers were
dried over anhydrous Na
2
SO
4
and evaporated in vacuum. The residue was purified
1
by silica gel column (DCM/MeOH, 10/1) to afford compound 1 (29.1 mg, 56%)
。
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (400 MHz, CDCl ) δ 7.31 (dd, J = 5.0, 2.9 Hz, 1H), 7.12 (ddd, J = 3.0, 1.4, 0.8
3
Hz, 1H), 7.01 (dd, J = 5.0, 1.3 Hz, 1H), 6.47 (d, J = 8.4 Hz, 1H), 5.25 (dd, J = 14.5,
7
.2 Hz, 1H), 4.36 – 4.23 (m, 1H), 3.39 (dd, J = 13.7, 4.7 Hz, 2H), 2.98 (dt, J = 13.8,
.9 Hz, 1H), 2.50 (s, 3H), 2.46 (t, J = 6.9 Hz, 2H), 2.23 – 1.98 (m, 12H), 1.94 – 1.59
6
1
3
(
m, 11H), 1.37 (dd, J = 6.9, 1.7 Hz, 6H). C NMR (125 MHz, CDCl
3
) δ 159.16,
50.63, 142.93, 126.55, 126.13, 121.06, 77.28, 77.03, 76.78, 58.99, 58.40, 47.98,
7.64, 47.26, 42.95, 35.55, 35.37, 34.46, 32.82, 26.72, 25.92, 21.65, 13.14. ESIꢀMS
1
4
+
+
40 2 5
m/z: 520.3 [M + H] . HRMS (ESI) cacld for C27H F N OS ([M+H] ): 506.2843;
found: 520.2855.
4,4ꢀdifluoroꢀ
N
ꢀ((S)ꢀ3ꢀ((1
R
,3R,5
S
)ꢀ3ꢀ(3ꢀisopropylꢀ5ꢀmethylꢀ4
H
ꢀ1,2,4ꢀtriazolꢀ4ꢀyl)ꢀ
8
ꢀazabicyclo[3.2.1]octanꢀ8ꢀyl)ꢀ1ꢀ(thiazolꢀ4ꢀyl)propyl)cyclohexaneꢀ1ꢀcarboxamid
e (2).
1
Compound 2 was prepared in a similar manner as described for compound 1. H
NMR (400 MHz, CDCl
3
) δ 8.72 (d, J = 1.7 Hz, 1H), 7.15 (dd, J = 9.6, 5.1 Hz, 2H),
.29 (q, J = 7.3 Hz, 1H), 4.29 – 4.13 (m, 1H), 3.66 (s, 2H), 3.33 (d, J = 13.0 Hz, 2H),
.93 (dt, J = 13.5, 6.8 Hz, 1H), 2.44 (s, 3H), 2.33 (d, J = 6.1 Hz, 2H), 2.19 (dd, J =
5
2
24.1, 12.3 Hz, 3H), 2.13 – 1.91 (m, 7H), 1.91 – 1.67 (m, 6H), 1.56 (d, J = 8.5 Hz,
13
6
H), 1.29 (d, J = 6.8 Hz, 7H). C NMR (150 MHz, CDCl
3
) δ 173.74, 159.17, 157.09,
153.51, 150.72, 122.71, 114.66, 59.04, 58.38, 48.10, 47.87, 47.39, 42.60, 35.81,
28
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
3
4.23, 32.81, 26.52, 26.42, 26.01, 21.60, 19.52, 13.05. ESIꢀMS m/z: 521.2 [M + H] .
+
HRMS (ESI) cacld for C26
,4ꢀdifluoroꢀ ꢀ(( )ꢀ3ꢀ((1
H
39
F
2
N
6
OS ([M+H] ): 521.2796; found: 521.3119.
4
N
S
R
,3R,5
S
)ꢀ3ꢀ(3ꢀisopropylꢀ5ꢀmethylꢀ4
H
ꢀ1,2,4ꢀtriazolꢀ4ꢀyl)ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8ꢀazabicyclo[3.2.1]octanꢀ8ꢀyl)ꢀ1ꢀ(pyridinꢀ3ꢀyl)propyl)cyclohexaneꢀ1ꢀcarboxamid
e (3).
1
Compound 3 was prepared in a similar manner as described for compound 1. H
NMR (600 MHz, CDCl
3
) δ 8.55 (d, J = 1.9 Hz, 1H), 8.49 (dd, J = 4.7, 1.3 Hz, 1H),
7
4
2
.61 (d, J = 7.9 Hz, 1H), 7.28 – 7.24 (m, 2H), 7.19 (s, 1H), 5.14 (q, J = 7.1 Hz, 1H),
.30 (ddd, J = 13.5, 11.5, 6.2 Hz, 1H), 3.43 (d, J = 15.8 Hz, 2H), 3.02 – 2.93 (m, 1H),
.51 (d, J = 10.2 Hz, 5H), 2.26 (dt, J = 22.0, 11.8 Hz, 4H), 2.18 – 1.97 (m, 6H), 1.95
13
– 1.60 (m, 10H), 1.36 (dd, J = 6.8, 2.6 Hz, 6H), 0.89 (dt, J = 14.4, 7.2 Hz, 1H). C
NMR (150 MHz, CDCl ) δ 173.87, 159.19, 150.59, 148.75, 148.17, 137.64, 134.28,
123.65, 68.18, 59.21, 58.25, 50.16, 47.74, 47.11, 42.72, 38.72, 35.97, 35.22, 34.09,
3
3
5
5
4
8
3.13, 32.79, 30.36, 26.66, 25.94, 23.74, 23.00, 21.66, 13.21, 10.98. ESIꢀMS m/z:
+
+
15.2 [M + H] . HRMS (ESI) cacld for C28
H
41
2
F N
6
O ([M+H] ): 515.32323; found:
15.3265.
,4ꢀdifluoroꢀ
Nꢀ((S)ꢀ3ꢀ((1R,3R,5S)ꢀ3ꢀ(3ꢀisopropylꢀ5ꢀmethylꢀ4Hꢀ1,2,4ꢀtriazolꢀ4ꢀyl)ꢀ
ꢀazabicyclo[3.2.1]octanꢀ8ꢀyl)ꢀ1ꢀ(4ꢀfluoropyridinꢀ3ꢀyl)propyl)cyclohexaneꢀ1ꢀcar
boxamide (4).
Compound 4 was prepared in a similar manner as described for compound 1. H
NMR (500 MHz, CDCl ) δ 8.13 – 8.03 (m, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.71 (s,
1H), 7.19 – 7.12 (m, 1H), 5.25 (dd, J = 14.0, 8.1 Hz, 1H), 4.37 – 4.25 (m, 1H), 3.53
1
3
29
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