Design of substituted imidazolidinylpiperidinylbenzoic acids as chemokine receptor 5 antagonists: Potent inhibitors of R5 HIV-1 replication

Article abstract of DOI:10.1021/jm401101p

The redesign of the previously reported thiophene-3-yl-methyl urea series, as a result of potential cardiotoxicity, was successfully accomplished, resulting in the identification of a novel potent series of CCR5 antagonists containing the imidazolidinylpiperidinyl scaffold. The main redesign criteria were to reduce the number of rotatable bonds and to maintain an acceptable lipophilicity to mitigate hERG inhibition. The structure-activity relationship (SAR) that was developed was used to identify compounds with the best pharmacological profile to inhibit HIV-1. As a result, five advanced compounds, 6d, 6e, 6i, 6h, and 6k, were further evaluated for receptor selectivity, antiviral activity against CCR5 using (R5) HIV-1 clinical isolates, and in vitro and in vivo safety. On the basis of these results, 6d and 6h were selected for further development.

Full text of DOI:10.1021/jm401101p

Article
pubs.acs.org/jmc
Design of Substituted Imidazolidinylpiperidinylbenzoic Acids as
Chemokine Receptor 5 Antagonists: Potent Inhibitors of R5 HIV‑1
Replication
Renato Skerlj,*,† Gary Bridger,§ Yuanxi Zhou,§ Elyse Bourque,† Ernest McEachern,§ Markus Metz,†
Curtis Harwig,§ Tong-Shuang Li,§ Wen Yang,§ David Bogucki,§ Yongbao Zhu,§ Jonathan Langille,§
Duane Veale,§ Tuya Ba,§ Michael Bey,§ Ian Baird,§ Alan Kaller,§ Maria Krumpak,§ David Leitch,§
Michael Satori,§ Krystyna Vocadlo,§ Danielle Guay,§ Susan Nan,§ Helen Yee,§ Jason Crawford,§
Gang Chen,§ Trevor Wilson,§ Bryon Carpenter,§ David Gauthier,§ Ron MacFarland,§ Renee Mosi,§
Veronique Bodart,§ Rebecca Wong,§ Simon Fricker,‡ and Dominique Schols∥
†LGCR Unit, Sanofi, 153 Second Avenue, Waltham, Massachusetts 02451, United States
‡Genzyme, a Sanofi company, 49 New York Avenue, Framingham, Massachusetts 01701, United States
§AnorMED Inc., 200-20353 64th Avenue, Langley, British Columbia V2Y 1N5, Canada
∥Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, Leuven B-3000, Belgium
S
* Supporting Information
ABSTRACT: The redesign of the previously reported thiophene-3-yl-methyl urea series, as a result of potential cardiotoxicity,
was successfully accomplished, resulting in the identification of a novel potent series of CCR5 antagonists containing the
imidazolidinylpiperidinyl scaffold. The main redesign criteria were to reduce the number of rotatable bonds and to maintain an
acceptable lipophilicity to mitigate hERG inhibition. The structure−activity relationship (SAR) that was developed was used to
identify compounds with the best pharmacological profile to inhibit HIV-1. As a result, five advanced compounds, 6d, 6e, 6i, 6h,
and 6k, were further evaluated for receptor selectivity, antiviral activity against CCR5 using (R5) HIV-1 clinical isolates, and in
vitro and in vivo safety. On the basis of these results, 6d and 6h were selected for further development.
structurally diverse antagonists with improved pharmacokinetic,
pharmacodynamic, and resistance profiles.
INTRODUCTION
■
The recognition that the chemokine receptor CCR5 is required
by HIV-1 as a coreceptor for cell entry1,2 together with the
discovery that individuals lacking CCR5 because of a 32 bp
deletion in the CCR5 gene are resistant to HIV-1 infection3,4
sparked a strong research interest to identify CCR5 small-
molecule antagonists5−19 to treat HIV infection. This effort
culminated in the marketing approval of the first-in-class
chemokine receptor antagonist, maraviroc, in 2007.20 Maraviroc
represents a new class of therapeutic agents for the treatment of
patients with HIV, which becomes very crucial in light of the
viral resistance that develops over time.
Other small-molecule CCR5 inhibitors have failed in the
clinic. For example, TAK-779 failed because of poor
pharmacological and toxicological properties as well as a lack
of oral bioavailability,7 SCH-C failed because of prolongations
of the QT interval,7 vicriviroc failed because of viral rebound
issues,21 and aplaviroc failed because of liver toxicity.22 Because
of this attrition rate, there was a strong interest to develop
We have recently reported our efforts to design structurally
unique compounds using SCH-C as our starting point. In a
systematic approach, we first modified the piperidyl-amide
group and then successfully discovered several new scaffolds for
the benzoxime moiety23−25 (Figure 1). Mutation studies26
investigated how these small molecules interact with CCR5,
and, in agreement with the literature,27−35 all of the small-
molecule antagonists tested so far share a common binding site
in the transmembrane region beneath ECL 2. Because the
binding site of these allosteric inhibitors is rather hydrophobic,
we25 and others36,37 encountered liabilities with hERG
inhibition. In a recent publication,25 we reported the
identification of thiophene-3-yl-methyl ureas (1) as a novel
class of CCR5 antagonists that exhibited potent inhibition of
Received: July 19, 2013
© XXXX American Chemical Society
A
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protected diamines. Reductive amination of the primary amine
with commercially available ketones or aldehydes using
NaBH(OAc)3 or NaBH4, respectively, followed by Boc
deprotection using TFA afforded corresponding diamines 9a.
A second reductive amination with N-(t-butoxycarbonyl)-
piperidin-4-one installed the piperidine ring to give the
disubstituted secondary amine that underwent Boc depro-
tection to afford cyclic ureas 10a. Alternatively, substituted
vinyl precursors 7b (specifically, 3-vinylthiophene, 1-chloro-3-
vinylbenzene, and 1-fluoro-3-vinylbenzene) were utilized for
asymmetric aminohydroxylations39 to afford the corresponding
enantiomerically enriched amino alcohols (R)-8b (94% ee).
Utilizing methodology that we previously reported,23 com-
pound 10a was readily converted to target compounds 2a−c
and 3a−l (Tables 1 and 2). However, in the case of the single
diastereomers (Table 1) and the compounds containing the
(R)-phenyl group (Table 3), the starting materials were the
commercially available (R)- and (S)-Boc-protected amino
alcohols 8b and 8c, which were readily converted to diamines
9b and 9c. Reaction of (R)-9b and (S)-9c with (R) and (S)-tert-
butyl (3-(4-oxopiperidin-1-yl)butyl)carbamate followed by
treatment with triphosgene gave the corresponding cyclic
ureas. Boc deprotection followed by standard amino acid
coupling with 4,6-dimethylpyrimidine-5-carboxylic acid or 2-
chloro-4,6-dimethylpyridine-5-carboxylic acid afforded single
diastereomers 2d−i (Table 1). Following the conditions
described above for the synthesis of the racemic compounds,
intermediate 10b was used to synthesize target compounds
4a−c, 4e−l, and 4n−p (Table 3) as described.23−25
In several examples, the starting material was the
corresponding Boc-protected amino acid 11a and (R)-11b
(Scheme 1). Activation of the acids using iso-butylchlorofor-
mate followed by reaction with a commercially available
primary amine gave amides 12a−c. Boc deprotection followed
by borane reduction of the amide afforded diamines 13a−c,
which were converted to cyclic ureas 10a and 10b as described
above. Ultimately, compounds 3m, 4d, and 4m (Tables 2 and
3) were synthesized using this procedure.23−25
Pyridyl analogs 3n and 3o (Table 2) were prepared from the
commercially available methyl esters 14a and 14b (Scheme 1).
Installation of the oxime moiety, 15a and 15b, alpha to the
ester using sodium nitrate in the presence of acetic acid
followed by concomitant reduction of the ester and oxime
groups to the hydroxy amine and Boc protection of the amine
afforded compounds 8a, which were processed in a similar
manner as mentioned above to yield compounds 3n and
3o.23−25
Figure 1. Structures of compounds 1−6.
R5 HIV-1 (BaL) replication in PBMC (Figure 1). Although
these compounds had an acceptable hERG profile, they
unexpectedly prolonged the APD in canine Purkinje fibers,
suggesting a more complex mixed cardiac ion-channel effect. As
a consequence, the development of these compounds was
suspended.
In this Article, we describe our strategy in redesigning this
chemotype as well as the subsequent SAR of the intermediate
chemotypes (2-5) that resulted in the identification of
compounds (6) that met the criteria for the pharmacological
inhibition of HIV-1 but with no cardiac liability on the basis of
the canine Purkinje fiber and hERG assays. The SAR was based
on several in vitro assays. We utilized a gp120/CCR5 cell-
fusion assay as a primary screen to select compounds for
antiviral screening because the fusion data was found to be a
reliable predictor of antiviral activity.26 Antagonism of CCR5
was measured using the RANTES-induced Ca2+-flux inhibition
and 125I-RANTES competitive-binding assays, which were
found to correlate well with the antiviral activity evaluated in
PBMC utilizing the R5 HIV-1 clinical isolates and strains.38
The methodology used to prepare compounds 5 (Table 4) is
shown in Scheme 2 using the advanced intermediate (R)-1-
cyclohexyl-4-phenyl-3-(piperidin-4-yl)imidazolidin-2-one 10b.
Reductive amination of 10b under standard conditions with a
variety of substituted benzaldehydes afforded compounds 5a−
p. The benzaldehyde precursors utilized to prepare 5a−f were
readily synthesized by Suzuki cross coupling of 4-formyl
benzene boronic acid with the appropriately substituted bromo
heterocyclic ring to afford the coupled products in excellent
yield. The precursor for 5g, 4-morpholinobenzaldehyde, was
readily prepared using Buchwald cross-coupling chemistry.
Similarly, the piperidine intermediate 1-(o-tolyl)piperidine-4-
carbaldehyde precursor to 5i was prepared by Buchwald
coupling of methyl piperidine-4-carboxylate and 1-bromo-2-
methylbenzene. The intermediate 1H-benzo[d]imidazole-6-
carbaldehyde used for 5h was simply prepared by TPAP
CHEMISTRY
■
The general methodology utilized for the synthesis of the cyclic
ureas 2−4 is shown in Scheme 1. The Strecker reaction was
used to prepare the racemic α-amino nitrile precursors from
commercially available aldehydes 7a, which were then readily
reduced to the corresponding amino acids. Borane reduction of
the acid to the alcohol followed by Boc protection of the amine
afforded the corresponding racemic Boc-protected amino
alcohols 8a. Mitsunobu reaction of alcohols 8a with N-(Z-
amino)phthalimide gave the phthaloyl carbazates, which were
cleaved with hydrazine to afford the corresponding Boc-
B
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a
Scheme 1. Synthesis of Compounds 2−4
a
Reagents: (a) NaCN, NH4Cl, NH4OH, MeOH, H2O; (b) HCl 6 N, reflux; (c) BH3·THF, reflux; (d) Boc2O, Et3N, THF; (e) NH2Boc, n-PrOH,
H2O, t-BuOCl, (DHQD)2PHAL, K2OsO2(OH)4, 0 °C, 1 h, ee = 94%; (f) N-(Z-amino)phthalimide, DEAD, Ph3P, THF; (g) hydrazine, EtOH; (h)
R2CO, NaBH(OAc)3, CH2Cl2 or RCHO, NaBH4, MeOH; (i) TFA, CH2Cl2, rt; (j) N-(t-butoxycarbonyl)piperidin-4-one, NaBH(OAc)3, CH2Cl2;
(k) triphosgene, pyridine, CH2Cl2; (l) (R)- or (S)-tert-butyl (3-(4-oxopiperidin-1-yl)butyl)carbamate, NaBH(OAc)3, CH2Cl2; (m) 4,6-
dimethylpyrimidine-5-carboxylic acid or 2-chloro-4,6-dimethylpyridine-5-carboxylic acid, EDCI, HOBT, NMM, THF/DMF; (n) iso-butyl
chloroformate, NMM, THF; (o) R2NH2; (p) BH3·THF, reflux; (q) NaNO2, AcOH, H2O; and (r) LAH, THF.
oxidation of the corresponding alcohol, which in turn was
obtained from the ester. The precursor for 5j, 4-formyl-N-
methylbenzamide, was simply prepared by treatment of 4-
carboxybenzaldehyde with methylamine hydrochloride. Finally,
the phenoxy benzaldedydes used to prepare compounds 5k−p
and 6a were synthesized as previously described.40
Compounds 5q and 5r containing the bis-piperidinyl core
were also prepared from advanced intermediate 10b (Scheme
2). Following the procedure reported by Palani,41 intermediate
10b was coupled with N-(t-butoxycarbonyl)piperidin-4-one in
the presence of titanium(IV)isopropoxide followed by the
addition of diethylaluminum cyanide. Methylation of the distal
piperidyl ring was accomplished by treating the cyano group
with an excess of methyl magnesium bromide. Boc depro-
tection followed by standard EDCI coupling with the
corresponding aromatic acids23,24 afforded compounds 5q
and 5r.
Scheme 3 summarizes the synthesis of compounds 6 (Table
5). The key step involved coupling of intermediate 10b with
the appropriately functionalized right-hand side intermediates
18a−f. Intermediates 18a−b were synthesized from the
coupling of commercially available 2-bromo-5-methylpyridine
16 with corresponding phenol 17b or thiol 17a. Intermediate
18a was used to prepare compounds 6b and 6c. For instance,
treatment of chloride 18a with Zn(CN)2 in the presence of
Pd(0) afforded the corresponding nitrile, which was ultimately
used to liberate the carboxylic acid. The toluoyl group was used
to install the electrophilic bromomethyl moiety using NBS,
which then underwent N-alkylation with key intermediate 10b.
Subsequent base hydrolysis afforded the corresponding
carboxylic acids 6b or 6c. However, intermediate acid 18b
was used to prepare compound 6i, but the carboxylic acid
group of 18b was first protected as the corresponding methyl
ester. As in the previous example, the toluoyl group was
converted to the bromomethyl moiety, which underwent N-
C
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a
Table 1. In Vitro Pharmacology of Compounds 2
a
The assays were performed in duplicate, and the values represent the mean with standard deviations of <30% of the mean. The bracketed values
represent the number of experiments.
alkylation with 10b followed by acid hydrolysis of the methyl
ester to afford carboxylic acid 6i. Compounds containing a 6-
methylpyridyl substituted linker, 6d−g and 6j−k, were
prepared using commercially available 6-bromo-2-methyl-3-
carboxaldehyde 19a. For example, coupling of 19a with 4-
hydroxybenzonitrile 17c afforded nitrile 18c, which underwent
reductive amination in the presence of NaBH(OAc)3 with
amine 10b followed by acid hydrolysis to afford carboxylic acid
6k. Compounds 6d−g and 6j were prepared in a similar
manner as described above from phenol and thiol methyl esters
17d and 17f.42 Methyl amide 6l was simply prepared from
carboxylic acid 6d via standard EDCI coupling. The same
methodology was also used to prepare ethyl-containing analog
6h starting from aldehyde 19b and ester 17e. Pyrimidine analog
6m was prepared from 4-((5-vinylpyrimidin-2-yl)thio)benzoate
21, which was obtained from the coupling of 5-bromo-2-
chloropyrimidine 19 and 4-mercaptobenzoic acid 20 followed
by the Pd(0)-catalyzed coupling of vinyltributylstannane to
install the vinyl group. Oxidative cleavage of the vinyl group
afforded the corresponding aldehyde, which was treated under
reductive amination conditions with intermediate 10b followed
by base hydrolysis to afford carboxylic acid 6m. To synthesize
methyl pyrimidine analog 6n, the intermediate 2-((4-
bromophenyl)amino)-4-methylpyrimidine-5-carbaldehyde 24
was synthesized by the Pd(0)-catalyzed amination between
ethyl 2-amino-4-methylpyrimidine-5-carboxylate 22 and dibro-
mobenzene 23 followed by conversion of the ester to the
aldehyde and the bromide to the nitrile. The coupling of
aldehyde 24 with 10b followed by nitrile hydrolysis afforded
carboxylic acid 6n.
RESULTS AND DISCUSSION
■
Rather than making incremental modifications to the existing
chemotype, our goal was more ambitious, resulting in a
complete redesign. Our main redesign criteria was to reduce the
number of rotatable bonds on the basis of the retrospective
analysis of large pharma compound collections that suggested
that compound rigidity resulted in improved pharmacokinetic
and pharmacological profiles.43 The left-hand side urea of
compound series 1 was an obvious area where the number of
rotatable bonds could be reduced by synthesizing a cyclic urea.
D
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a
Table 2. Comparative In Vitro Pharmacology of Compounds 3
CCR5 fusion
IC50 (nM)
CCR5 Ca flux
IC50 (nM)
RANTES 125I binding
IC50 (nM)
HIV-1 PBMC
IC50 (nM)
PBMC
CC50 (nM)
compound
R1
MW/cLogP
2b
3a
3b
3c
3d
3e
3f
Ph
533/3.8
547/4.3
551/3.9
567/4.5
547/4.3
612/4.7
567/4.5
551/3.9
558/3.2
591/3.6
612/4.7
561/4.8
565/4.5
534/2.3
534/2.3
548/2.8
65.7
20.7 (n = 4)
322
77
2-Me-Ph
2-F-Ph
1702
342
91
155
2-Cl-Ph
3148
1526
195
3-Me-Ph
3-Br-Ph
11.7 (n = 28)
10.4 (n = 4)
2.5 (n = 19)
62.5
6.0 (n = 27)
5.1 (n = 2)
5.8 (n = 18)
15.0 (n = 2)
32.2
31
22
347.9 (n = 3)
1777 (n = 3)
>32 699
>34 742
3-Cl-Ph
19.5
3g
3h
3i
3-F-Ph
25.9
3-CN-Ph
3-CO2Me-Ph
4-Br-Ph
56.9 (n = 2)
346.5
34.8 (n = 2)
175.8
157.6 (n = 3)
700.8
3j
3020
3k
3l
3,5-diMe-Ph
3-F,5-Me-Ph
3-Pyridyl
2-Pyridyl
79.6
4.7 (n = 2)
7.1 (n = 3)
832
36.5
18.5 (n = 2)
2938
32.0 (n = 2)
3m
3n
3o
908.8 (n = 2)
117.1
300.7 (n = 2)
149.8
6-Me-2-
Pyridyl
a
See Table 1.
a
Table 3. Comparative In Vitro Pharmacology of Compounds 4
CCR5 fusion
IC50 (nM)
CCR5 Ca flux
IC50 (nM)
RANTES 125I binding
IC50 (nM)
HIV-1 PBMC
IC50 (nM)
PBMC
CC50 (nM)
compound
R2
MW/cLogP
4a
4b
4c
4d
4e
4f
Et
512/3.8
526/4.4
526/4.2
540/4.7
538/4.3
552/4.8
568/3.0
582/3.7
580/6.1
574/5.2
575/3.7
589/4.2
56.6
24.7 (n = 2)
7.5 (n = 2)
5.3 (n = 2)
3.6 (n = 2)
2.3 (n = 2)
0.9 (n = 2)
6.3 (n = 2)
0.8 (n = 2)
1.8 (n = 2)
1.3 (n = 3)
2.9 (n = 2)
1.2 (n = 2)
Pr
6.4 (n = 4)
11.2 (n = 2)
3.8 (n = 4)
1.7 (n = 4)
0.9 (n = 3)
8.6 (n = 4)
1.1 (n = 2)
0.9 (n = 3)
1.2 (n = 4)
5.9 (n = 2)
1.7 (n = 3)
19.4
19.5
i-Pr
t-Bu
c-Bu
c-Pent
4-THP
351.7 (n = 3)
549.9 (n = 3)
>34 300
>33 500
4g
4h
4i
4-THPCH2
c-HexCH2
Benzyl
14.6
7.7
4j
9.0
12.3 (n = 5)
>34325
>34237
4k
4l
2-PyridylCH2
1026.7
3-(4-Me-Pyridyl)
CH2
8.0
4m
4n
4o
4p
3-Pyridyl
561/3.9
575/3.7
610/1.6
618/2.7
9.3 (n = 2)
1.0 (n = 3)
56.0 (n = 2)
1.0 (n = 4)
3.4 (n = 2)
0.8 (n = 3)
807.2
52.4
5.7
4-PyridylCH2
HO2C-4-c-Hex
HO2C-4-PhCH2
12.2 (n = 3)
14.3 (n = 4)
>32400
>32350
3.6 (n = 2)
42.2 (n = 2)
a
See Table 1.
Given that an additional stereogenic center is generated, we
sought to confirm that a phenyl substituent proximal to the
urea nitrogen would have equivalent potency to the
corresponding thiophene because the synthetic precursors to
prepare the single enantiomers of the phenyl were
commercially available. As shown in Table 1, there was a
marginal, <2-fold, loss in potency for phenyl compound 2b
compared to thiophene compound 2a based on the CCR5-
fusion and RANTES binding-inhibition assays. In addition, the
SAR for compounds 1 also tracked with cyclic ureas 2. For
example, using the optimized 2-chloro-4,6-dimethylpyridine
amide group on the right-hand side, 2c resulted in an
E
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a
Table 4. Comparative In Vitro Pharmacology of Compounds 5
a
See Table 1.
approximate 40-fold enhancement in potency compared to 2a,
Moreover, we were encouraged that 2c exhibited antiviral
IC50 of 0.9 versus 39.0 nM in the fusion-inhibition assay.
activity in the PBMC HIV-1 assay at an IC50 of 30 nM and was
F
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a
Scheme 2. Synthesis of Compounds 5
a
Reagents: (a) 1-Boc-4-piperidone, Ti(OiPr)4, CH2Cl2, rt, 48 h, then AlCNEt2, 20 h; (b) MeMgBr, THF, 0 °C to rt, 20 h; (c) TFA, CH2Cl2; (d)
ArCO2H, EDCI, HOBT, NMM, THF; and (e) RCHO, NaBH(OAc)3, CH2Cl2, AcOH, rt.
a
Scheme 3. Synthesis of Compounds 6
a
Reagents: (a) K2CO3, DMF, 125−200 °C; (b) Zn(CN)2, Zn dust, Pd2(dba)3, DPPF, DMAC, 145 °C; (c) H2SO4, MeOH, reflux, 15 h; (d) NBS,
(PhCO)2O2, CCl4, reflux, 4 h, then POH(OEt)2, DIPEA, THF; (e) 10b, CH3CN, DIPEA or 10b, NaBH(OAc)3, AcOH, CH2Cl2, rt; (f) EtOH/10N
NaOH, 60−90 °C, 5−18 h; (g) TFA, H2SO4 (4 drops); (h) MeNH2, EDCI, hOBT, NMM, THF/DMF; (i) Pd(PPh3)2Cl2, PPH3, LiCl, DMF,
CH2CHSnBu3, 100 °C, 18 h; (j) NMO, OsO4, CH2Cl2, 18h; (k) NaIO4, acetone, H2O; (l) Pd2(dba)3, 4,5-bis(diphenylphosphine)-9,9-
dimethylxanthane, t-BuOK, PhMe; (m) DIBAL-H, PhMe, −78 °C; and (n) MnO2, CH2Cl2.
noncytotoxic to PBMCs, as measured by a CC50 of >32 μM.
On the basis of the comparable potency of the phenyl and
thiophene groups, the corresponding four diastereoisomeric
phenyl substituted ureas of 2b were synthesized to rank the
diastereoisomeric pair according to potency (Table 1). It was
apparent that the preferred stereochemistry on the urea was
(R), whereas the (S) configuration of the isobutyl chain was
slightly preferred on the basis of the fusion-inhibition data. For
example, the CCR5 fusion inhibition for the (R,R) pair 2d was
27.6 nM, whereas for the (S,R) pair 2e it was 9039 nM, a 330-
fold difference in IC50. In contrast, the stereochemistry of the
isobutyl chain had a moderate impact on fusion inhibition, as
illustrated by the (R,S) pair 2f where the fusion inhibition IC50
was 14.0 nM, a 2-fold difference compared to compound 2d.
This trend was also evident using the optimum 2-chloro-4,6-
dimethyl pyridine amide on the right-hand side, which agreed
with the SAR for the thiophene-3-yl-methyl urea series 1. For
example, the (R,S) pair 2i was antivirally more potent than the
(R,R) pair 2h, with an IC50 of 18.0 versus 49.2 nM, which is
consistent with the fusion-inhibition data of 0.5 versus 1.2 nM.
G
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a
Table 5. Comparative In Vitro Pharmacology of Compounds 6
a
See Table 1.
The effect of phenyl substitution was next investigated using
pyrimidine amide 3 tested as a racemic mixture (Table 2).
Substitution at the ortho position was not tolerated. Simple
addition of a methyl group, 3a, resulted in a 26-fold reduction
in fusion inhibition compared to the unsubstituted analog 2f,
perhaps due to steric effects. Interestingly, the fluoro isostere 3b
resulted in an approximately 5-fold reduction in fusion and
calcium-flux inhibition compared to 2f, suggesting that an
electronic effect was also in play. This was confirmed by the 2-
chloro substituent 3c, which was characterized by an
approximately 50- and 70-fold reduction in inhibition for
fusion and calcium flux, respectively. In contrast, substitution at
the meta position resulted in enhanced potency comparable to
the SAR that we reported for an earlier aniline series.23 For
example, the 3-Me 3d and 3-Br 3e analogs resulted in an
approximately 6-fold enhancement in potency based on the
fusion assay, whereas the 3-Cl analog 3f resulted in a 26-fold
increase in fusion inhibition compared to 2f, with an IC50 of 2.5
versus 65.7 nM. More striking was the difference in fusion
inhibition between the 2- and 3-Cl-substituted analogs, 3c and
3f, with IC50 values of 3148 and 2.5 nM, a 1260-fold difference.
Interestingly, the fluoro and cyano analogs, 3g and 3h, had no
H
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effect on fusion or calcium-flux inhibition, whereas methyl ester
3i resulted in a 5-fold loss in potency based on fusion and an 8-
fold potency loss based on calcium-flux inhibition. A
comparable loss in potency to the 2-substituted analogs was
also observed for the 4-Br substituent 3j, where the IC50 fusion
inhibition was 3020 nM. Disubstitution of the phenyl at the 3-
and 5-positions did not provide an additive effect, as shown by
dimethyl analog 3k and fluoro methyl analog 3l. In an attempt
to reduce the lipophilicity, substitution of the phenyl group by a
pyridine group, either linked at the 2- or 3-postion, compounds
3m and 3n, resulted in potency losses based on fusion
inhibition. However, substitution of a methyl group at the 6-
position (equivalent to the meta position of the phenyl), 3o,
regained some of the fusion potency.
consequence, we sought to enhance the pharmacokinetic
properties by further reducing the number of rotatable bonds.
Replacements of the entire isobutyl linker were investigated
with the initial focus on maintaining potency (Table 4).
Synthesis of biphenyl linker 5a resulted in a substantial 360-fold
loss in potency based on fusion inhibition but only a 3−8-fold
loss in potency based on CCR5 Ca2+-flux inhibition and 125I
-RANTES binding inhibition compared to compound 2j.
Adding hydrophilicity to the molecule by including a
pyrimidine ring at the terminus 5b improved the fusion
inhibition 9-fold to an IC50 of 50.6 nM compared to 5a.
Interestingly, 2-substituted pyrimidine 5c was substantially less
potent than 5a based on fusion, Ca2+ flux, and 125I-RANTES
binding inhibition, indicating that perhaps an H-bond acceptor
at the terminal position was important for CCR5 inhibition.
Several compounds containing H-bond acceptor moieties were
synthesized, such as pyridine 5d−e, tetrazole 5f, morpholine
5g, and benzimidazole 5h. However, none of these compounds
resulted in an improvement in fusion or Ca2+-flux inhibition
compared to 5b. The incorporation of a phenyl piperidine, 5i,
resulted in a complete loss in activity. However, we were
encouraged by the addition of a secondary amide, 5j. For
instance, the activity was restored to that of 5b where the fusion
IC50 was 62.7 versus 50.6 nM, and the Ca2+-flux inhibition IC50
was 15.8 versus 17.3 nM. In addition, replacement of the linker
with a phenoxy phenyl group, 5k, also resulted in encouraging
activity. On the basis of our modeling/mutagenesis studies,44
we determined that extension of the chain containing a
hydrophilic group would pick up additional interactions in the
extracellular region. As a consequence, phenoxy amide 5l was
synthesized, and the inhibition profile showed marginal
enhancements in the CCR5 Ca2+-flux inhibition and 125I-
RANTES binding-inhibition assays, but more importantly the
anti-HIV-1 activity was 3.2 nM. Primary amide 5n and tertiary
amide 5m were also synthesized. These compounds had
comparable fusion inhibition compared to 5l but were
approximately 2-fold less potent based on 125I-RANTES
binding inhibition, which resulted in an approximately 10-fold
loss in anti-HIV-1 potency. In an effort to reduce the
lipophilicity and to mitigate potential hERG liabilities, the m-
and p-carboxylic acids, 5p and 5o, were synthesized. p-
Substituted acid 5o was 7-fold more potent based on fusion
inhibition than the m-carboxylic acid, 5p, and the anti-HIV-1
activity was 34.3 nM.
Given that the imidazolidinyl moiety is a novel chemotype in
the context of CCR5 inhibitors as well as modeling studies that
suggested that attachment of the (4,6-dimethylpyrimidin-5-
yl)(piperidin-1-yl)methanone moiety of vicriviroc would be
tolerated, we synthesized compound 5q, which resulted in a
fusion inhibition of 1.7 nM and anti-HIV-1 activity of 107.1
nM. On the basis of this encouraging result, we replaced the
pyrimidine amide with our optimized 2-chloro-4,6-dimethyl
amide, resulting in a very potent compound, 5r; >250-fold
enhancement in anti-HIV-1 activity compared to 5q (IC50 of
<0.4 nM).45
Having demonstrated that suitable replacements for the
isobutyl linker could be achieved without compromising
antiviral potency, our goal shifted to reducing lipophilicity to
mitigate off-target effects (Table 5). We demonstrated that
incorporation of a tetrahydropyran group on the left-hand side
rather than a cyclohexyl group lowered the cLogP by almost 2.5
orders of magnitude, 4g (Table 3, cLogP 3.0) versus 2j (Table
1, cLogP 5.4). In addition, we showed that the potency could
Having determined that the optimum stereochemistry of the
cyclic urea phenyl was (R), we kept this stereochemistry fixed
to determine the optimum substitution at the urea nitrogen
atom using the racemic isobutyl chain (the (S) isomer gave a
moderate 2-fold enhancement in potency) attached to the
optimal 2-chloro-4,6-dimethyl pyridine amide group, com-
pounds 4 (Table 3). Although the cyclohexyl analog of
compound 4 was not prepared, it can be inferred on the basis of
the SAR from Table 1 that the activity would be comparable to
the (R,R) isomer 2j (e.g., comparison of 2a to 2e), which had a
fusion inhibition IC50 of 1.2 nM. The effect of varying the size
of the alkyl group from ethyl to cyclopentyl, 4a−f, was
investigated, and it appears that bulkier groups provide a
potency comparable to the cyclohexyl analog. For example, the
fusion inhibition for the t-Bu, c-Bu, and c-pent analogs 4d−f
were 3.8, 1.7, and 0.9 nM, which are comparable to the
estimated IC50 of the cyclohexyl analog (1.2 nM). The addition
of an oxygen atom, a potential hydrogen-bond acceptor, by the
incorporation of a tetrahydropyran (THP) moiety, 4g, resulted
in an approximately 8-fold loss in fusion inhibition compared to
the cyclohexyl analog. Interestingly, the potency was restored
by adding a methylene linker, 4h, between the THP group and
the nitrogen urea. A similar level of fusion inhibition was
observed for c-hexCH2 4i and benzyl analogs 4j, which
correlated well with the anti-HIV-1 activity. For example, the
IC50 value in the HIV-1 PBMC assay for compound 4j was 12.3
nM. The incorporation of more hydrophilic pyridine groups,
4k−n, was also investigated as a means of reducing the
lipophilicity. It was found that the 4-pyridylmethylene analog
4n had a comparable fusion inhibition to the cyclohexyl analog
and, more importantly, had acceptable anti-HIV-1 potency of
12.2 nM. Previously, we have shown in the thiophene-3-yl-
methyl urea series (1) that the addition of a carboxylic acid
tethered to an appropriate sized linker, to pick up an ionic
interaction with lysines located in the extracellular loop region
of the receptor, mitigated hERG liability.44 On the basis of the
mutagenesis/molecular-modeling approach that we developed,
we hypothesized that attaching the same carboxylic acid linker
to cyclic urea 4 should generate potent compounds. This was
indeed confirmed by the synthesis of compound 4p, which had
a fusion inhibition IC50 of 1.0 nM and an anti-HIV-1 activity of
14.3 nM. Similar to what had previously been reported,44 the
length of the linker chain is important, as highlighted by
compound 4o where the fusion inhibition was approximately
50-fold lower compared to 4p.
Although the antiviral potency of compounds 4n and 4p met
the criteria for the pharmacological inhibition of R5 HIV-1
replication, the oral bioavailability based on rat and dog
pharmacokinetics was poor (data not shown). As a
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Table 6. Selected In Vitro Properties for Compounds 6d, 6e, 6h, 6i, and 6k
microsomal stability
(%)
HIV-1 PBMC
IC50 (nM)
HIV-1 PBMC IC50
a
HIV-1 PBMC
IC90 (nM)
HIV-1 PBMC IC90
a
Pe value
IC50 values in CYP
b
compound
AGP fold change
AGP fold change
(cm/sec × 10−6
)
assays (μM)
rat dog human
6d
6e
6h
6i
24.1 (n = 8)
0.6 (n = 2)
34.7 (n = 6)
4.0 (n = 5)
7.9 (n = 3)
5.1
0.3
1.4
1.3
0.3
222.0 (n = 8)
3.9 (n = 5)
0.8
0.7
0.7
1.0
0.3
7.8
>40
>100
>73
>60
>26
16
38
36
49
71
6
8
nd
13
35
6
18
4
279.4 (n = 8)
43.2 (n = 5)
12.2
11.7
12.0
9
6k
112.5 (n = 3)
22
44
a
b
Ratio of HIV-1 PBMC IC90 or IC50 with and without AGP. The lower limit in any of the CYP assays is shown: CYP450 1A2, 2B6, 2C8, 2C9,
2C19, 2D6, and 3A4.
Table 7. Chemokine Receptor Selectivity Profiles for Compounds 6d, 6e, 6h, 6i, and 6k
a
a
b
a
a
a
compound CCR1 inhibition (%)
CCR2b inhibition (%)
CCR4 IC50 (nM)
CCR7 inhibition (%)
CXCR3 inhibition (%)
CXCR4 inhibition (%)
6d
6e
6h
6i
91.4 (n = 2)
84.9 (n = 2)
103.4 (n = 2)
73.8 (n = 2)
81.9 (n = 2)
89.2 (n = 2)
87.2 (n = 2)
88.7 (n = 2)
95.6 (n = 2)
97.3 (n = 2)
566.2
83.8 (n = 2)
80.1 (n = 2)
93.6 (n = 2)
90.1 (n = 2)
75.5 (n = 2)
89.5 (n = 2)
91.0% (n = 2)
99.7 (n = 2)
66.7 (n = 2)
67.8 (n = 2)
103.1 (n = 2)
90.4 (n = 2)
93.8 (n = 2)
88.6 (n = 2)
84.7 (n = 2)
283.4 (n = 2)
a
73.4% (n = 2)
3723 (n = 1)
a
6k
22.5% (n = 2)
a
b
Percent inhibition of receptor-mediated calcium flux with 5 μM compound. Inhibition results for receptor-mediated calcium flux are expressed as
IC50 (nM) if not otherwise specified.
be enhanced by substitution of the phenyl ring with a 3-Cl or 3-
Me substituent on the basis of the SAR from Table 2. Aside
from antiviral potency, the properties that were used to
prioritize these analogs was the effect of AGP on fusion
inhibition (AGP fusion shift) because HIV-infected patients
exhibit elevated levels of AGP that can reduce the level of free
drug, particularly of a basic compound, human plasma protein
binding, and hERG inhibition based on the percent inhibition
and/or IC50.
fold more potent with an IC50 of 0.6 nM. However, it was
surprising to observe that the anti-HIV-1 activity of the 6-F
derivative, 6g, was lower at 91.8 nM. The addition of an ethyl
group, 6h, resulted in a comparable property profile compared
to 6d. Replacing the ether linkage with a thioether, 6i, also
provided an enhancement in anti-HIV-1 activity compared to
the corresponding ether analog 6d, with an IC50 of 4.0 versus
24.1 nM. Moreover, the other critical parameters remained
unchanged. The addition of a 6-methylpyridine moiety, 6j, to
the linker provided a 40-fold enhancement in antiviral activity
similar to what was observed in the ether examples.
Substitution of the carboxylic acid for an amide, 6k and 6l,
provided an approximately 3-fold increase in anti-HIV-1
potency. However, comparison of 6k to 6d showed that the
hERG inhibition was higher, with an IC50 of 18 versus >30 μM
for 6d, and compared to 6l the hERG inhibition was 22.7
versus 4.9% for 6d. In addition, generally, the amide analogs, 6k
and 6l, were more highly metabolized on the basis of their in
vitro metabolic stability in human and rat liver microsomes, and
this was correlated with low oral bioavailability in rat (data not
shown). Importantly, although 6k and 6l had very comparable
profiles, 6k was chosen for further evaluation because plasma
protein binding was lower compared to 6l (89.4 vs 94.5%), as
shown in Table 5. To increase polarity further, a series of
pyrimidine analogs40 were synthesized, represented by 6m and
6n, as shown in Table 5. The increased polarity of the
pyrimidine group compensated for the additional 3-chloro
group, 6m, and methyl group, 6n. For example, the cLogP of
1.5 of compound 6m remained virtually unchanged compared
to the parent compound 6i. The fusion inhibition, anti-HIV-1
activity, and human plasma protein binding did not change
significantly. The replacement of the thioether linkage by an
aniline group improved the human plasma protein binding to
89%. Although this modification was tolerated in the fusion
inhibition assay, the anti-HIV-1 activity dropped more than 10-
fold.
As predicted, the cLogP of compound 6a containing a
tetrahydropyran group was lower when compared to cyclohexyl
analog 5o, 2.4 versus 4.8. Although the fusion inhibition of 6a
was comparable to 5o, the anti-HIV potency was approximately
5-fold lower. However, the other properties were acceptable;
for example, the hERG IC50 was >40 μM, the fold AGP fusion
shift was 1.0, and the human plasma protein binding was 90.2%.
To reduce the lipophilicity further, the phenyl linker was
replaced with a pyridine ring, 6b, reducing the cLogP from 2.4
to 0.9. This resulted in a 2-fold increase in fusion inhibition, a
5-fold increase in anti-HIV-1 potency (IC50 of 39.7 nM), and in
comparison to 6a the other properties remained essentially
unchanged. In an attempt to increase the potency further, 3-Cl
substituent 6c was synthesized, and although the fusion
inhibition increased 5-fold the anti-HIV-1 activity remained
unchanged at an IC50 of 36.5 nM. On the basis of the SAR
earlier developed for the isobutyl linker,23 we were curious to
determine if the addition of a methyl group to the pyridine ring
would enhance the potency. A comparison of des-methyl
compound 6b with the 6-methylpyridine compound 6d
provided approximately a 5-fold increase in fusion inhibition
but a comparable anti-HIV-1 activity, 39.7 versus 24.1 nM,
respectively. In addition, the hERG inhibition was 4.9%,
corresponding to an IC50 of >30 μM. Interestingly, when the
6-methylpyridine moiety was combined with the 3-Cl-, 6e, 3-
Me-, 6f, or 6-F-, 6g, substituted phenyl groups, the fusion
inhibition remained unchanged at approximately 1 nM, but the
anti-HIV-1 activity was significantly enhanced for 6e and 6f.
For example, in comparison to 6c, the 3-Cl analog 6e was 60-
In Table 6, the ADME in vitro properties of five lead
compounds, 6d, 6e, 6h, 6i, and 6k, are shown in addition to the
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a
Table 8. Dog Purkinje Fiber Data and hERG Data for Compounds 6d, 6e, 6i, and 6k
Δ%
ΔmV
b
compound
BCL (s)
APD60
APD90
RMP
APA
Vmax (Δ%)
hERG IC50 (μM)
6d
2
−2.9 2.5
−4.0 2.8
−4.0 2.6
−10.5 7.2
−8.2 7.7
−4.9 6.0
−6.1* 1.7
−4.6* 1.5
−4.2* 2.0
1.2 3.2
−2.8 2.1
−3.8 2.4
−3.5 2.1
−7.8 5.3
−6.4 5.8
−3.4 4.2
−7.0* 0.5
−6.2* 0.8
−6.2* 1.3
1.0 2.1
0.1 1.4
0.4 1.0
1.2 1.2
0.7 0.4
0.1 0.2
−0.1 0.4
−2.2 0.8
0.4 1.7
0.7 1.9
−1.6 1.3
−0.5 3.3
−0.5 2.8
2.6 2.7
11.0 9.7
8.4 6.3
>30
1
2.4 2.1
0.5
2
0.8 2.1
11.8 5.1
−10.5 14.7
−11.2 11.2
5.4 16.2
10.2 4.5
4.5 2.3
6e
6i
−4.4 4.0
−3.4 3.4
−1.8 3.6
4.1 1.9
9.9
1
0.5
2
>30
18.0
1
−0.9 3.3
−1.5 2.3
−3.4 4.7
7.8 14.3
7.9 14.0
0.5
2
2.2 2.7
6k
−0.8 8.3
−15.6 12.9
−16.7 9.9
1
−1.7 5.4
−6.0 6.2
1.8 1.7
0.5
−0.3 1.1
a
Action-potential parameters are reported as means SDs, percent change (Δ%), increase (+), or decrease (−) in test-compound-exposed Purkinje
fibers over those measured in time-matched vehicle control. APD60 and APD90 are the action-potential durations at 60 and 90% repolarization,
respectively; RMP is the resting membrane potential; APA is the action-potential amplitude; Vmax is the maximum velocity of phase 0 depolarization;
b
and BCL is the basic cycle length. The compounds were tested at 10 μM. * P < 0.05.
a
Table 9. Anti-HIV-1 Activity Profile of Compounds 6d, 6e, 6h, 6i, and 6k and Maraviroc
6d
6e
6h
6i
6k
maraviroc
clade
IC50
IC90
IC50
IC90
IC50
IC90
200
IC50
78.0
IC90
IC50
61.9
IC90
IC50
IC90
UG273 clade A
US2 clade B
8.0
93.8
3.8
27.9
0.5
86.2
12.7
80.1
21.5
89.8
81.9
29.2
6.1
68.0
1.4
522.6
4.7
546.1
0.8
7.1
22.7
2.6
0.23
14.7
9.4
5.7
0.6
<0.1
>1000
4.0
0.3
ETH2220 clade C
ID12 clade E
BZ163 clade G
I-2478B clade O
SF162 clade B
Ada clade B
77.8
30.0
78.9
8.2
2.8
173.9
13.6
6.5
>1000
40
>1000
4.0
>1000
56.9
463.0
0.1
>1000
56.9
463.0
0.1
50.4
14.6
14.8
6.4
101.2
57.5
71.8
16
7.0
15.0
2.2
33.2
31.2
10.6
2.0
105.3
1.2
16.2
<0.1
nd
16.2
<0.1
nd
0.4
17.6
6.0
83.4
26.5
16.1
19.5
84.6
nd
nd
nd
nd
18.6
<0.3
1.4
105.3
33.6
8.3
1.4
5.6
0.6
15.6
nd
<0.3
nd
12.9
nd
CI#15 clade B
CI#19 clade B
BaL clade B
2.5
35.8
5.4
82.1
16
nd
nd
nd
0.7
nd
nd
nd
nd
nd
nd
1.3
11.9
89.1
9.8
23.4
78.2
nd
nd
nd
nd
nd
nd
13.9
a
All IC50 and IC90 values are reported in nM and were evaluated in PBMCs.
antiviral activity expressed both in IC50 and IC90. Importantly
and similar to what was observed with fusion inhibition, the
anti-HIV-1 activity is not significantly affected by AGP (HIV-1
PBMC IC50 or IC90 fold change), indicating that the probability
of AGP reducing the in vivo efficacy of these compounds is low.
Even though several of these compounds are zwitterionic, they
had good cell permeability, as measured by the PAMPA assay,
and more importantly they had good exposure when dosed
orally in dog (vide infra). The propensity for drug−drug
interactions was very low on the basis of cytochrome P450 IC50
inhibition ranging from >26 to >100 μM for seven primary
drug-metabolizing isozymes. The microsomal stability varied
across species, but our optimization efforts focused on human,
and it was evident that stability in general was the best in
human followed by dog. Although the in vitro microsomal
stability in rat was poor, this observation did not correlate well
with the observed in vivo stability (vide infra).
Compounds 6d, 6e, 6h, 6i, and 6k were tested against a
panel of chemokine receptors for selectivity. The data in Table
7 shows that these compounds demonstrated little or no
inhibition of the closely related chemokine receptors CCR1,
CCR2b, CCR7, CXCR3, and CXCR4, with IC50 values of at
least >5 μM (the maximum concentration tested) compared to
the IC50 values against CCR5, which are in the range of 0.6−1.4
nM. Limited inhibition of CCR4 was observed for 6d, 6e, 6i,
and 6k, with the lowest IC50 value of 283 nM for 6e, which is
approximately a 470-fold selectivity for CCR5. The IC50 for 6h
was >5 μM. The potential pharmacological or toxicological
effect of this limited inhibition of CCR4 is unknown, although
it can be predicted that this will not be of toxicological
significance because CCR4 knockout mice develop normally
and are viable.46−48
The four compounds, 6d, 6e, 6i, and 6k, were evaluated in
the canine Purkinje fiber assay to understand the risk for drug-
induced arrhythmias further (Table 8). There was an
insignificant prolongation of the action-potential duration
(APD) when all of the compounds were tested at 10 μM
using the three standard stimulation frequencies, which is
consistent with a lack of effect on the hERG potassium channel.
For example, the four compounds inhibited hERG at IC50
values of 9.9 to >30 μM, and the percent change in ADP60 at a
basic cycle length (BCL) of 2 s was insignificant in the range of
3−10%. There was also no occurrence of early after
depolarizations (EADs), and because the prolongation of
APD60 and APD90 was similar at every stimulation cycle, there
was no evidence of triangulation of the action potential. The
lack of an effect on the resting membrane potential (RMP) or
the rate of depolarization (Vmax) also suggests that sodium
channels are not inhibited at the concentrations tested. Hence,
K
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a
Table 10. Pharmacokinetics of Compounds 6d, 6e, 6h, 6i, and 6k in Dog
compound
Cmax (μM)
AUC0→inf (h μM)
CL (mL/min/kg)
V (L/kg)
t1/2 (h)
F (%)
6d
6e
6h
6i
14.4
9.31
38.6
0.91
11.3
37.96
18.14
128.55
0.75
0.30
0.52
0.11
2.05
0.22
1.23
5.18
0.49
7.91
1.16
2.83
6.97
3.00
2.67
3.61
91.4
75.5
117.2
12.3
85.5
6k
47.45
a
Clearance (CL), volume of distribution (V), and half-life (t1/2) were calculated following a 5 μmol/kg iv dose. Cmax, AUC, and oral bioavailability
(F) were calculated following solution doses of 12.5 μmol/kg.
on the basis of these results, the risk for drug-induced
arrhythmias is low.
CONCLUSIONS
■
The successful redesign of an earlier reported series, as a result
of potential cardiotoxicity, was accomplished by focusing on
reducing the number of rotatable bonds as well as maintaining
an acceptable lipophilicity to mitigate hERG inhibition. As a
result, a potent series of CCR5 antagonists containing the novel
imidazolidinylpiperidinyl scaffold were identified that demon-
strate significantly reduced cardiac liability based on hERG and
canine Purkinje fiber experiments. Five compounds from this
series, 6d, 6e, 6i, 6h, and 6k, were further evaluated for receptor
selectivity, antiviral activity against CCR5 using (R5) HIV-1
clinical isolates, and in vitro and in vivo safety. All five
compounds showed comparable or superior potency compared
to mariviroc in a HIV-1 clade analysis study and were well
tolerated in 7-day repeat toxicology studies in rat. However, on
the basis of differences in chemokine receptor selectivity,
metabolic stability, and pharmacokinetics, 6d and 6h were
identified as candidates for further development.
A limited clade analysis, against six of the most common
clades, was also undertaken. Included in this analysis were six
strains of clade B, dominant in Europe, the Americas, and parts
of Asia. The IC50 and IC90 of the five lead compounds were
measured using maraviroc as a comparator (Table 9).
Comparison of the five compounds against maraviroc using
clade B (US2) showed that all compounds were at least
comparable to or superior to maraviroc regarding their antiviral
potency. This observation also held up against the other five
clades. Interestingly, the relative potency of the five compounds
reflected what was observed in the internal screening using
PBMCs. Clearly, the excellent potency of these compounds
across a diverse set of clades was an important criteria that was
fulfilled.
Pharmacokinetic parameters were important in choosing
compounds for further evaluation. However, because we
observed a poor in vivo/in vitro correlation regarding their
stability in rat, we conducted only oral rat exposure (data not
shown) and used this data as a prescreen for a more complete
pharmacokinetic analysis in dogs. Table 10 shows the
pharmacokinetic data for compounds 6d, 6e, 6h, 6i, and 6k
in dog, all of which had acceptable exposure in rat. With the
exception of compound 6i, the compounds exhibited low
clearance and reasonable exposure, resulting in good oral
bioavailability. The volume of distribution was low for
compounds 6d, 6h, and 6k, and all of the compounds exhibited
reasonable half-lives.
The in vivo safety of the five lead compounds was evaluated
in a 7-day repeat-dose toxicology study in rat at three dose
levels (low, mid, and high). We were pleased to observe that all
of the compounds were well tolerated across all doses, resulting
in a NOAEL equivalent to the high dose (150−800 mg/kg). In
addition, a comparison of the exposures at days 1 and 7 showed
no evidence of accumulation.
Because the five lead compounds containing the novel
chemotype, imidazolidinylpiperidinylbenzoic acid, were well
tolerated in the rat safety study and ex vivo safety cardiotoxicity
studies (Purkinje Fiber assay and hERG), other criteria were
considered to prioritize the compounds for further develop-
ment. An important factor taken into consideration was the off-
target risk associated with inhibiting other receptors. For
example, compounds 6e and 6k showed cross reactivity against
CCR4 at 284 nM and 22% inhibition, respectively (Table 7). In
addition, on the basis of the lower human microsomal stability
of 6e and 6k, these compounds were not considered for further
evaluation. Of the three remaining compounds, because 6i
exhibited poor dog PK and the next safety study was a 7-day
repeat-dose toxicology study in dog, compounds 6d and 6h
were prioritized for further development.
EXPERIMENTAL SECTION
■
General Information. Unless otherwise noted, most of the
reagents and solvents were purchased at the highest commercial
quality and used without further purification. All reactions involving
air- and/or moisture-sensitive reagents were performed under a
nitrogen or argon atmosphere. Silica gel chromatography was
performed using glass columns packed with silica gel 60 (40−63
μM, VWR International). H and 13C NMR spectra were recorded at
1
300 and 75 MHz, respectively, on a Bruker Avance 300 spectrometer,
with shifts referenced to the residual proton shift of the internal
deuterated solvent. Electrospray mass spectra were recorded on a
Bruker-HP Esquire-LC ion-trap mass spectrometer. Microanalyses for
C, H, N, and halogen were performed by Atlantic Microlab, Inc.
(Norcross, GA) and were within 0.4% of theoretical values. Purity was
determined by reversed-phase HPLC and was ≥95% for all
compounds tested.
General Procedure A. To a stirred solution of the amine (1 equiv)
in CH2Cl2 (concentration ∼0.2M) at room temperature were added
the carbonyl compound (1 to 2 equivs), glacial AcOH (0−2 equivs),
and NaBH(OAc)3 (1.5−3.0 equivs), and the resultant solution was
stirred at room temperature. In a standard workup, the reaction
mixture was poured into either saturated aqueous NaHCO3 or 1 N
NaOH. The phases were separated, and the aqueous phase was
extracted with CH2Cl2. The combined organic extracts were dried
(Na2SO4 or MgSO4), filtered, and concentrated under reduced
pressure. The crude material was purified by flash column
chromatography on silica gel or by recrystallization.
General Procedure B. The BOC-protected amine was dissolved in
CH2Cl2 (∼4 mL/mmol), and trifluoroacetic acid (TFA) (∼2 mL/
mmol) was added. The mixture was stirred at room temperature for
0.5−5 h. In a standard workup, the mixture was neutralized with
saturated aqueous NaHCO3 or 1 N NaOH, and the aqueous phase
was extracted with CH2Cl2. The combined extracts were dried
(Na2SO4 or MgSO4), filtered, and concentrated under reduced
pressure. The crude material was used in the next reaction as is or
was purified by flash column chromatography on silica gel.
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General Procedure C. The ester (1.0 equiv) was dissolved in a 1:1
MeOH/2 N NaOH solution. The reaction was stirred at 50 °C for 5−
18 h. The mixture was concentrated under reduced pressure, and water
was added. The pH of the solution was adjusted to ∼4 to 5 with a 6 N
HCl solution. The aqueous solution was then extracted with CH2Cl2
or a mixture of CH2Cl2/MeOH (9:1). The combined organic extracts
were dried (Na2SO4 or MgSO4), filtered, and concentrated under
reduced pressure. The crude material was purified by flash column
chromatography on silica gel or by recrystallization.
General Procedure D. The secondary amine (1.1 equivs) was
dissolved in CH3CN (concentration ∼0.1 M). DIPEA (1.5 equivs)
followed by a halide reagent (1.0 equiv) were added. The reaction was
heated at 50−75 °C for 18 h. The mixture was concentrated under
reduced pressure, and CH2Cl2 and saturated aqueous NaHCO3 were
added. The aqueous layer was extracted with CH2Cl2, and the
combined organic extracts were dried (Na2SO4 or MgSO4), filtered,
and concentrated under reduced pressure. The crude material was
purified by flash column chromatography or by radial chromatography
on silica gel.
(R)-4-((6-Methyl-5-((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-
pyran-4-yl)imidazolidin-1-yl)piperidin-1-yl)methyl)pyridin-2-
yl)oxy)benzoic Acid (6d). A mixture of 6-bromo-2-methylpyridine-
3-carboxaldehyde (4.00 g, 20.0 mmol), 4-hydroxybenzoic acid methyl
ester (3.80 g, 25.0 mmol), and K2CO3 (1.73 g, 12.5 mmol) in DMF
(30 mL) was heated at 130 °C for 2 h. The mixture was cooled to
room temperature, and DMF was removed. Aqueous workup and
purification by flash chromatography on silica gel (CH3OH/CH2Cl2,
1:50 in v/v) afforded methyl 4-((5-formyl-6-methylpyridin-2-yl)oxy)-
benzoate as a white solid (3.20 g, 59%). 1H NMR (300 MHz, CDCl3)
δ 2.72 (s, 3H), 3.93 (s, 3H), 6.86 (d, 1 H, J= 8.4 Hz), 7.20−7.25 (m,
2H), 8.08−8.15 (m, 3H), 10.25 (s, 1H). Following general procedure
A, (R)-1-cyclohexyl-4-phenyl-3-(piperidin-4-yl)imidazolidin-2-one 10b
(R1Ph, R2 = 4-THP) and the above aldehyde provided (R)-methyl
4-((6-methyl-5-((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-pyran-4-yl)-
imidazolidin-1-yl)piperidin-1-yl)methyl)pyridin-2-yl)oxy)benzoate,
which was treated under general procedure C to afford 6d as a white
solid (164 mg, 51% over two steps). 1H NMR (300 MHz, CD3OD) δ
1.32 (dq, 1H, J= 12.0, 3.6 Hz), 1.48 (br d, 1H, J= 12.0 Hz), 1.60−1.80
(m, 5H), 1.95−2.20 (m, 3H), 2.39 (s, 3H), 2.78 (d, 1H, J = 11.4 Hz),
2.94 (d, 1H, J = 11.l Hz), 3.13 (m, 1H), 3.40−3.60 (m, 5H), 3.77 (t,
1H, J = 9.0 Hz), 3.93 (m, 3H), 4.73 (m, 1H), 6.69 (d, 1H, J= 8.1 Hz),
7.07 (d, 2H, J= 8.7 Hz), 7.30−7.40 (m, 5H), 7.64 (d, 1H, J = 8.4 Hz),
8.01 (d, 2H, J= 8.7 Hz). 13C NMR (75 MHz, CD3OD) δ 20.71, 28.44,
29.82 (2C), 30.14, 48.57, 49.44, 52.08, 52.85, 53.05, 56.74, 57.98,
67.18, 67.27, 109.02, 119.53 (2C), 125.88, 127.02 (2C), 128.46,
129.03 (2C), 130.67, 131.50 (2C), 142.62, 142.91, 157.37, 158.09,
160.87, 162.01, 171.02. ES−MS m/z 571 [M + H]+. Anal. Calcd. for
C33H38N4O5·0.7CH2Cl2·0.3NH3: C, 63.38; H, 6.45; N, 9.87. Found:
C, 63.20; H, 6.60; N, 9.85.
m/z 605 [M + H]+. Anal. Calcd. for C33H37N4ClO5·0.6CH2C12: C,
61.51; H, 5.87; N, 8.54. Found: C, 61.56; H, 5.82; N, 8.44.
(R)-4-((6-Ethyl-5-((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-
pyran-4-yl)imidazolidin-1-yl)piperidin-1-yl)methyl)pyridin-2-
yl)oxy)benzoic Acid (6h). Following general procedure A, (R)-4-
phenyl-3-(piperidin-4-yl)-1-(tetrahydro-2H-pyran-4-yl)imidazolidin-2-
one 10b (R1Ph, R2 = 4-THP) and t-butyl 4-((6-ethyl-5-
formylpyridin-2-yl)oxy)benzoate provided (R)-t-butyl 4-((6-ethyl-5-
((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-pyran-4-yl)imidazolidin-1-yl)-
piperidin-1-yl)methyl)pyridin-2-yl)oxy)benzoate, which was treated
under general procedure B to afford 6h as a white powder (71 mg,
59% over two steps). 1H NMR (300 MHz, CDCl3) δ 1.11 (t, 3H, J =
7.5 Hz), 1.25−1.42 (m, 2H), 1.66 (m, 5H), 2.14−2.28 (m, 3H), 2.88
(br, s, 1H), 3.08 (m, 2H), 3.48−3.81 (m, 5H), 4.01 (m, 3H), 4.59 (t,
1H, J = 7.5 Hz), 6.62 (d, 1H, J = 9.0 Hz), 7.05−7.26 (m, 5H), 7.14 (d,
2H, J = 9.0 Hz), 7.65 (br s, 1H), 8.02 (br s, 2H). 13C NMR (75 MHz,
CDCl3) δ 13.74, 28.01, 30.20, 30.52, 48.74, 49.14, 51.52, 52.96, 56.33,
57.64, 67.53, 67.62, 109.12, 120.24, 122.03, 127.09, 128.39, 128.72,
129.30, 131.97, 142.66, 142.88, 158.56, 160.33, 161.79, 162.07, 170.32.
ES−MS m/z 585 [M + H]+. Anal. Calcd. for C33H40N4O5·0.5CH2Cl2:
C, 66.07; H, 6.59; N, 8.93. Found: C, 66.02; H, 6.65; N, 8.91.
(R)-4-((5-((4-(2-Oxo-5-phenyl-3-(tetrahydro-2H-pyran-4-yl)-
imidazolidin-1-yl)piperidin-1-yl)methyl)pyridin-2-yl)thio)-
benzoic Acid (6i). 2-Bromo-5-methyl-pyridine (2.23 g, 13.0 mmol),
4-mercaptobenzoic acid (333 mg, 2.16 mmol), and K2CO3 (597 mg,
4.32 mmol) were heated at 200 °C for 2 h. The mixture was
partitioned between H2O (70 mL) and diethyl ether (20 mL). The
aqueous phase was extracted with diethyl ether (20 mL) and then
acidified to pH 3 with 10% HCl (aq). The aqueous phase was
extracted with 10% MeOH/CH2Cl2 (4 × 20 mL), and the combined
organic layers were dried (MgSO4) and concentrated to give a yellow
solid (412 mg). A solution of the yellow solid from above (412 mg)
and H2SO4 (0.11 mL) in MeOH (16 mL) was heated to reflux for 15 h
and then concentrated. The residue was dissolved in CH2Cl2 (15 mL),
washed with H2O (5 mL) and saturated NaHCO3 (aq) (10 mL), dried
(MgSO4), and concentrated. Purification by chromatography on silica
gel (0−5% EtOAc/CH2Cl2) gave methyl 4-((5-methylpyridin-2-
yl)thio)benzoate as colorless crystals (280 mg, 50% over two steps),
which was added to a mixture of NBS (231 mg, 1.30 mmol) and
benzoyl peroxide (39 mg, 0.16 mmol) in CCl4 (2.7 mL), heated to
reflux for 4 h, and filtered and concentrated. Purification by
chromatography on silica gel (1% EtOAc/CH2Cl2) gave methyl 4-
((5-(bromomethyl)pyridin-2-yl)thio)benzoate as colorless crystals
1
(161 mg, 44%). H NMR (300 MHz, CDCl3) δ 3.94 (s, 3H), 4.43
(s, 2H), 7.05 (d, 1H, J= 8.4 Hz), 7.56 (dd, 1H, J = 8.4, 2.4 Hz), 7.61
(dd, 2H, J= 6.6, 1.8 Hz), 8.06 (dd, 2H, J= 6.8, 1.7 Hz), 8.45 (d, 1H, J=
2.1 Hz). Following general procedure D, (R)-1-cyclohexyl-4-phenyl-3-
(piperidin-4-yl)imidazolidin-2-one 10b (R1Ph, R2 = 4-THP) (80
mg, 0.24 mmol) and methyl 4-((5-(bromomethyl)pyridin-2-yl)thio)-
benzoate (75 mg) in CH3CN (4 mL) afforded (R)-methyl 4-((5-((4-
(2-oxo-5-phenyl-3-(tetrahydro-2H-pyran-4-yl)imidazolidin-1-yl)-
piperidin-1-yl)methyl)pyridin-2-yl)thio)benzoate as a white foam (105
mg, 81%). Following general procedure C, the ester prepared above
(105 mg. 0.18 mmol) afforded 6i as a white precipitate (64 mg, 62%).
1H NMR (300 MHz, CDCl3) δ 1.48 (br s, 1H), 1.64−1.74 (m, 4H),
1.86−1.90 (m, 1H), 2.53 (br s, 2H), 2.72 (br s, 1H), 3.12−3.21 (m,
2H), 3.43−3.52 (m, 4H), 3.70 (t, 1H, J = 9.3 Hz), 3.83−4.20 (m, 6H),
4.61−4.65 (m, 1H), 6.98 (d, 1H, J = 6.0 Hz), 7.19−7.31 (m, 6H), 7.60
(d, 2H, J = 8.1 Hz), 8.01 (d, 2H, J = 8.1 Hz), 8.30 (s, 1H). 13C NMR
(75 MHz, CDCl3) δ 25.7, 27.3, 29.8, 30.1, 48.3, 48.9, 49.3, 51.5, 53.5,
55.7, 67.1, 67.2, 122.0, 126.7, 128.6, 129.2, 131.0, 132.1, 134.4, 135.4,
140.0, 141.8, 151.1, 159.7, 162.5, 168.5. ES−MS m/z 573 [M + H]+.
Anal. Calcd. for C32H36N4O4S·1.6CH2Cl2: C, 56.95; H, 5.58; N, 7.91.
Found: C, 56.91; H, 5.90; N, 8.00.
(R)-4-((5-((4-(5-(3-Chlorophenyl)-2-oxo-3-(tetrahydro-2H-
pyran-4-yl)imidazolidin-1-yl)piperidin-1-yl)methyl)-6-methyl-
pyridin-2-yl)oxy)benzoic Acid (6e). Following general procedure
A, (R)-4-(3-chlorophenyl)-3-(piperidin-4-yl)-1-(tetrahydro-2H-pyran-
4-yl)imidazolidin-2-one 10b (R1Ph-3-Cl, R2 = 4-THP) and methyl
4-((5-formyl-6-methylpyridin-2-yl)oxy)benzoate provided (R)-methyl
4-((5-((4-(5-(3-chlorophenyl)-2-oxo-3-(tetrahydro-2H-pyran-4-yl)-
imidazolidin-1-yl)piperidin-1-yl)methyl)-6-methylpyridin-2-yl)oxy)-
benzoate, which was treated under general procedure C to afford 6e as
1
a white solid (368 mg, 88% over two steps). H NMR (300 MHz,
CD3OD) δ 1.42 (dq, 1H, J = 12.6, 3.9 Hz), 1.50−1.80 (m, 6H), 2.05
(dq, 1H, J = 12.6, 3.6 Hz), 2.30 (m, 2H), 2.41 (s, 3H), 2.93 (d, 1H, J =
11.7 Hz), 3.05 (d, 1H, J = 11.7 Hz), 3.11 (m, 1H), 3.47 (t, 2H, J= 11.7
Hz), 3.55 (m, 1H), 3.63 (s, 2H), 3.78 (t, 1H, J = 9.3 Hz), 3.94 (m,3H),
4.56 (m, 1H), 6.75 (d, 1H, J = 8.1 Hz), 7.10 (d, 2H, J = 8.4 Hz), 7.30−
7.42 (m, 4H), 7.69 (d, 1H, J= 8.4 Hz), 8.03 (d, 2H, J= 8.7 Hz). 13C
NMR (75 MHz, CD3OD) δ 22.48, 28.01, 30.27, 30.31, 30.44, 48.64,
49.19, 51.49, 53.07 (2C), 55.68, 58.16, 67.59 (2C), 109.31, 120.14
(2C), 125.27, 126.94, 128.03, 128.97, 130.65, 132.10 (2C), 135.22,
142.94, 144.99, 157.38, 158.77, 160.24, 161.99, 169.83, 174.11. ES−S
(R)-4-((6-Methyl-5-((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-
pyran-4-yl)imidazolidin-1-yl)piperidin-1-yl)methyl)pyridin-2-
yl)oxy)benzamide (6k). A mixture of 6-bromo-2-methylpyridine-3-
carboxaldehyde (1.00 g, 5.00 mmol), 4-hydroxybenzonitrile (0.596 g.
5.00 mmol), and K2CO3 (0.414 g, 3.00 mmol) in DMF (10 mL) was
heated at 130 °C for 1 h. The mixture was cooled to room
M
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temperature, and DMF was removed. Aqueous workup and
purification by flash chromatography on silica gel (EtOAc/hexanes,
2:3 in v/v) afforded 4-(5-formyl-6-methylpyridin-2-yloxy)benzonitrile
wild-type CCR5.26 CCR5 membranes were diluted in assay buffer to a
concentration of 0.16−0.28 ug/μL in a final volume of 50 μL. The 125I-
RANTES (PerkinElmer Life Sciences, 2200 Ci/mmol) was diluted to a
final concentration of 50 pM. The membrane was incubated with 25
μL of 125I-RANTES and 50 μL of buffer or compound over a
concentration range of 1 × 10−5 to 6.4 × 10−10 M for 45 min at room
temperature in Millipore Multiscreen plates with GF-B membranes
(Millipore) blocked with 0.15% poly(ethyleneimine). The plates were
washed three times with ice-cold 50 mM HEPES, 0.5 M NaCl, pH 7.4,
dried, and counted on a Wallac 1450 Microbeta Jet liquid scintillation
counter. Ligand binding dose/response curves were analyzed, and IC50
values were calculated using Prism 3.0 (GraphPad).
CCR5 Fusion Assay. A cell−cell fusion assay was used to mimic
the first stage of the HIV infection process. The principle of the assay
is the fusion of one cell line expressing the HIV-1 JRFL viral envelope
protein and the Tat transcription factor (CHO-Tat10) with a second
cell line (HeLa-CD4-LTR-β-gal) expressing CD4 and CCR5 and the
LacZ gene under the control of the HIV-1 LTR promoter.
For compound screening, the P4-CCR5 cell line, which expresses
CCR5, CD4, and a reporter gene, β-galactosidase, was used. The cells
were washed by centrifugation and resuspended in fusion-assay
medium (DMEM/RPMI 1640 1:1 supplemented with 10% FBS) and
the concentration was adjusted to 0.4 × 106 cells/ml. Similarly,
confluent CHO-Tat cells were washed with PBS, lifted with TEN
buffer, and resuspended to a concentration of 0.4 × 106 cells/ml in
assay medium. Stock solutions of all compounds were prepared in
DMSO and subsequently diluted into the fusion-assay medium. Equal
volumes of CCR5-expressing cells and CHO-Tat cells were then
mixed just prior to plating, and 50 μL of the cell mixture was added to
the wells of a 96-well plate. Fifty microliters of diluted compound was
then added to the cells to give a final concentration range of either 1 ×
10−5 to 6.4 × 10−10 M or 1 × 10−6 to 6.4 × 10−12 M. The plates were
incubated for 20 h at 37 °C, 5% CO2, and then assayed for β-
galactosidase activity using the Gal-Screen homogeneous chemilumi-
nescent reporter gene assay (Applied Biosystems). The plates were
read on a Victor 2 plate reader. IC50 values were calculated using
nonlinear regression curve fitting (ExcelFit 3.0).
HIV-1 Infection Assay. Human PBMCs were isolated from a fresh
buffy coat (Blood Transfusion Center Leuven), cultured in RPMI
1640 medium supplemented with 10% FBS and 1% glutamine, and
stimulated for 3 days with 2 μg/mL of phytohemagglutinin (PHA).
Next, 500 000 cells/well in 200 μL of medium were infected with R5
HIV-1 strains and isolates (500 pg of p-24 Ag virus/500 μL) and
treated with 2.5 ng/mL of IL-2 (R&D Systems) and serial dilutions of
the test compound (compounds were only given one time at the
beginning of the assay) to give a final volume of 500 μL. The cells
were treated with further IL-2 after 5 days. After 10 days of culture, the
viral replication was assayed by measuring viral p-24 in the culture
supernatant by P-24 Ag ELISA (PerkinElmer). Cell viability was
assayed using the MTS assay. The IC50 for HIV-1 inhibition
(concentration of compound causing 50% inhibition of viral
replication) and the CC50 (the concentration causing 50% cell
cytotoxicity) were calculated.
1
as a white solid (0.497 g, 41%). H NMR (300 MHz, CDCl3) δ 2.72
(s, 3H), 6.92 (d, 1 H, J= 8.4 Hz), 7.28−7.31 (m, 2H), 7.69−7.73 (m,
2H), 8.17 (d, 1H, J = 8.4 Hz), 10.26 (s, l H). Following general
procedure A, (R)-1-cyclohexyl-4-phenyl-3-(piperidin-4-yl)-
imidazolidin-2-one 10b (R1Ph, R2 = 4-THP) (63 mg, 0.19 mmol)
and the above aldehyde (55 mg, 0.23 mmol) afforded (R)-4-((6-
methyl-5-((4-(2-oxo-5-phenyl-3-(tetrahydro-2H-pyran-4-yl)imidazol-
idin-1-yl)piperidin-1-yl)methyl)pyridin-2-yl)oxy)benzonitrile as a
white solid (56 mg, 53%). To a solution of the above nitrile (53
mg, 96 μmol) in TFA (2 mL) was added concd H2SO4 (4 drops). The
reaction was heated at 95 °C for 16 h and then concentrated to
dryness under reduced pressure. The crude residue was then purified
by column chromatography on silica gel (25:1:0.1, CH2Cl2/MeOH/
1
NH4OH) to afford 6k as a white solid (49 mg, 89%). H NMR (300
MHz, CDCl3) δ 1.22 (dq, 1H, J = 12.0, 3.6 Hz), 1.42 (d, 1H, J = 12.0
Hz), 1.67 (m, 5H), 1.92 (m, 2H), 2.04 (m, 1H), 2.38 (s, 3H), 2.64 (d,
1H, J = 10.5 Hz), 2.83 (d, 1H, J = 9.3 Hz), 3.07 (m, 1H), 3.32 (s, 2H),
3.46 (m, 2H), 3.61 (m, 1H), 3.63 (t, 1H, J = 9.0 Hz), 4.02 (m, 3H),
4.59 (m, 1H), 6.63 (d, 1H, J = 8.1 Hz), 7.14 (d, 2H, J = 8.7 Hz), 7.34
(s, 5H), 7.50 (d, 1H, J = 8.1 Hz), 7.82 (d, 2H, J = 8.1 Hz). ES−MS m/
z 570 [M + H]+.
Cell Lines. The HEK293F human embryonic kidney cell line was
obtained from the ATCC. The CHO-Tat-10 cell line constitutively
expressing HIV-1 JRFLenv and HIV-1 Tat was obtained under license
from the Gladstone Institute. The HeLa-CD4-LTR-β-gal and P4-
CCR5 cell lines were obtained from the NIH AIDS Research and
Reference Reagent Program. HEK293F cells were transfected to
express CCR1, CCR2B, CXCR3, CCR4, or CCR5. These receptors
were cloned from commercial cDNA libraries by PCR using published
gene sequences and were inserted into the TOPO-pcDNA3.1 vector
(Invitrogen). Cells expressing CCR4, CCR5, and CXCR3 were also
cotransfected with a chimeric Gαqi5 protein to improve signaling.
HEK393F cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum (FBS), 1 mM
sodium pyruvate, 1 mM nonessential amino acids, and 4 mM L-
glutamine. The CHO-Tat-10 cell line was maintained in RPMI 1640
media supplemented with 10% fetal bovine serum, 2 mM glutamine, 1
mM sodium pyruvate, 1× nonessential amino acids, 0.5 mg/mL
Geneticin, and 12 μg/mL puromycin. The HeLa-CD4-LTR-β-gal and
P4-CCR5 cell lines were maintained in Dulbecco’s modified Eagle’s
medium supplemented with 10% FBS, 4 mM glutamine, 1 mM sodium
pyruvate, 1× nonessential amino acids, 0.2 mg/mL Geneticin, and 0.1
mg/mL hygromycin. All cell culture media and supplements were
obtained from Hyclone Inc. with the exceptions of the fetal bovine
serum, which was obtained from Invitrogen Inc., and hygromycin,
which was obtained from Calbiochem. CCRF-CEM cells, which
naturally express CXCR4 and CCR7, were cultured in RPMI 1640
supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine and
10% FBS. CEM.NKR.CCR5 cells were obtained from NIH AIDS
Research and Reagent program and grown in RPMI 1640
supplemented with 10% FBS and 4 mM L-glutamine.
hERG Automated Patch-Clamp Assay. The hERG assay was
performed at Genzyme in Waltham, MA. Chinese hamster ovary cells
overexpressing the human cloned recombinant hERG channel were
patch voltage clamped in the whole cell configuration using the parallel
automated patch-clamp platform QPatch (Sophion Biosciences)
according to the protocol described by the manufacturer. The external
solution was composed of (in mM) 145 NaCl, 4 KCl, 2 CaCl2, 1
MgCl2, 10 HEPES, 10 Glucose, pH 7.4, ∼300 mOsm. The internal
solution was composed of (in mM) 120 KCl, 31.25 KOH, 5.4 CaCl2,
1.75 MgCl2, 10 EGTA, 10 HEPES, 4 Na2-ATP, pH 7.2, ∼290 mOsm.
After a brief voltage step to −50 mV (to measure leak current) from a
holding potential of −80 mV, channels were activated by a
depolarizing voltage pulse to +20 mV for 5 s followed by a return
of the voltage to −50 mV for 2 s. The peak amplitude of the
characteristic deactivating hERG current was measured after
subtracting the leak current measured prior to the step to +20 mV.
The voltage protocol was applied every 10 s. Data was acquired at 1
Calcium Flux Assay. Chemokine-stimulated calcium flux was
assayed as previously described.49 Briefly, cells were washed and
loaded with Fluo-4/AM (4 μM) (Molecular probes, Inc.) and washed
and resuspended in Hanks’ balanced salt solution in 20 mM HEPES,
0.2% BSA, 2.5 mM probenecid, pH 7.4. Cells were then preincubated
with test compound for 15 min, and changes in the intracellular
calcium concentration upon chemokine addition (RANTES (CCL5)
for CCR5, MIP-1α (CCL3) for CCR1, MCP-1 (CCL2) for CCR2b,
TARC (CCL17) for CCR4, MIP-3β (CCL19) for CCR7, SDF-1α
(CXCL12) for CXCR4, and IP10 (CXCL10) for CXCR3) were
monitored using the FLEXstation (Molecular Devices) at 525 nm
(excitation λ = 485 nm). Data were analyzed using the programs
Softmax PRO 4.3.1 (Molecular Devices) and GraphPad Prism 3.0
software.
125I-RANTES Binding Assay. The 125I-RANTES binding assay was
performed with membranes prepared from HEK293F cells expressing
N
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Journal of Medicinal Chemistry
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kHz and was low-pass filtered through a 4-pole Bessel filter at 0.3 kHz.
No compensation for series resistance was applied; however, on the
basis of the low access resistances and current amplitudes, the voltage
errors were negligible. The experiment protocol for control buffer
(external solution to measure baseline current) or drug addition was
preprogrammed on the QPatch software, and the assay proceeded
automatically without user intervention. Each cell received, in
increasing doses, six concentrations of the test compound. Each
concentration was exposed to the cell for 120 s (12 current recordings
per concentration), and the steady-state current levels after each
concentration application were compared to the baseline values to
generate the percent inhibition. Each test compound was tested in at
least four different cells (n ≥ 4). Data was fit to a Boltzmann function
provided in the assay software.
AUTHOR INFORMATION
Corresponding Author
*Phone 617-610-8624; E-mail: skere01@gmail.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the KU Leuven GOA nos. 10/014
and PF/10/018. We thank all of our past colleagues in the
Chemistry and Biology Departments of AnorMED Inc. (now
Genzyme Corporation, a Sanofi company) for many fruitful
discussions. We are grateful to Becky Provinciael, Sandra Claes,
and Eric Fonteyn for excellent technical assistance.
Purkinje Fiber Action Potential. The hERG assay was used to
predict toxicity as a result of cardiac arrhythmias caused by QT interval
prolongation. However, the electrophysiology of the cardiac action
potential (AP) involves several ion channels other than hERG;
therefore, other techniques, such as the evaluation of AP in cardiac
Purkinje fibers, may be required for evaluating proarrythmic risk. The
Purkinje fiber action potential assay was performed at ChanTest using
isolated canine Purkinje fibers. Compounds were tested at a
concentration of 10 μM (n = 4). Action-potential parameters were
recorded at three different stimulation frequencies representing three
basic cycle lengths (time between stimulating pulses) of 0.5, 1, and 2 s
designed to simulate tachycardia (fast heart rate), normocardia
(normal heart rate), and bradycardia (slow heart rate). The following
parameters were recorded from the action potential trace: resting
membrane potential (mV) (RMP), action-potential amplitude (mV)
(APA), maximum rate of rise (V/s) (Vmax), and action-potential
duration at 60 and 90% repolarization (ms) (APD60 and APD90).
APD60, APD90, and Vmax at each stimulus frequency are presented as
percent change (Δ%) from baseline following application of the test
article. RMP and APA data are presented as the change in membrane
potential (ΔmV). Data are reported as the mean SEM. Pooled data
was tabulated for each condition: control baseline, test article
concentration, and stimulus frequency. Changes in action-potential
parameters were evaluated using one-way ANOVA followed by
Dunnett’s multiple comparison test (JMP Version 5, SAS Institute) to
determine whether the change from baseline observed after
equilibration with the test article concentration is significantly different
(P < 0.05) from that observed in the vehicle control group.
PAMPA Assay. The parallel artificial membrane-permeation assay
was used to assess the potential for oral absorption.50 The principle of
the assay is the passive diffusion of test compound through an artificial
phospolipid bilayer on a filter support into an acceptor compartment.
A 96-well acceptor plate was filled with 300 μL of 50 mM sodium
phosphate, 5% DMSO. A filter plate was then placed on top of the
acceptor plate, with the filters touching the liquid in the acceptor plate.
Five microliters per well of lipid mixture consisting of 0.8% L-α-
phosphatidylcholine, 0.8% L-α-phopshatidylethanolamine, 0.2% L-α-
phosphatidylserine, 0.2% L-α-phosphatidylinositol, and 1% cholesterol
in 1,7-octadiene was pipetted on to filter plate, and 150 μL of a 200
μM solution of test compound was added on top of the lipid layer.
The plates were incubated for 16 h at 30 °C. The compound
concentration in the acceptor compartment was quantified by UV
absorption and was used to calculate the apparent permeability, Pe.
ABBREVIATIONS USED
■
APD, action potential duration; AGP, α-1-acid glycoprotein;
CC50, concentration required to reduce the cell number by 50%
compared to that for the untreated controls; CCR5, C-C-R
Chemokine receptor 5; ECL 2, extracellular loop 2; gp120,
envelope glycoprotein 120; HIV-1, Human immunodeficiency
virus; IC90, concentration at 90% inhibition; PBMC, peripheral
blood mononuclear cell; RANTES, Regulated upon Activation,
Normal T-cell Expressed, and Secreted
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ASSOCIATED CONTENT
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S
* Supporting Information
Experimental procedures for all intermediates and select
compounds 2c, 3m, 4c, 4e−g, 4p, 5b, 5d, 5j, 5p−q, 6a−c,
1
6f, 6j, 6l−n, including H NMR, 13C NMR, and elemental
analysis results. This material is available free of charge via the
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O
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