3-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides Potently Inhibit HIV-1 Integrase and RNase H

Article abstract of DOI:10.1021/acs.jmedchem.6b00040

Resistance selection by human immunodeficiency virus (HIV) toward known drug regimens necessitates the discovery of structurally novel antivirals with a distinct resistance profile. On the basis of our previously reported 3-hydroxypyrimidine-2,4-dione (HPD) core, we have designed and synthesized a new integrase strand transfer (INST) inhibitor type featuring a 5-N-benzylcarboxamide moiety. Significantly, the 6-alkylamino variant of this new chemotype consistently conferred low nanomolar inhibitory activity against HIV-1. Extended antiviral testing against a few raltegravir-resistant HIV-1 clones revealed a resistance profile similar to that of the second generation INST inhibitor (INSTI) dolutegravir. Although biochemical testing and molecular modeling also strongly corroborate the inhibition of INST as the antiviral mechanism of action, selected antiviral analogues also potently inhibited reverse transcriptase (RT) associated RNase H, implying potential dual target inhibition. In vitro ADME assays demonstrated that this novel chemotype possesses largely favorable physicochemical properties suitable for further development.

Full text of DOI:10.1021/acs.jmedchem.6b00040

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Article
3-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides
potently inhibit HIV-1 integrase and RNase H
Bulan Wu, Jing Tang, Daniel J. Wilson, Andrew D. Huber, Mary C Casey, Juan Ji,
Jayakanth Kankanala, Jiashu Xie, Stefan G. Sarafianos, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00040 • Publication Date (Web): 10 Jun 2016
Downloaded from http://pubs.acs.org on June 12, 2016
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3
-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides
potently inhibit HIV-1 integrase and RNase H
a
a
a
b
b
Bulan Wu , Jing Tang , Daniel J. Wilson , Andrew D. Huber , Mary C. Casey , Juan
b
a
a
b
Ji , Jayakanth Kankanala , Jiashu Xie , Stefan G. Sarafianos and Zhengqiang Wang*
a
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
b
Department of Molecular Microbiology and Immunology and Department of Biochemistry, University of Missouri School
of Medicine, Christopher S. Bond Life Sciences Center, Columbia, MO 65211, USA
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Abstract
Resistance selection by human immunodeficiency virus (HIV) towards known drug
regimens necessitates the discovery of structurally novel antivirals with a distinct resistance
profile. Based on our previously reported 3-hydroxypyrimidine-2,4-dione (HPD) core we
have designed and synthesized a new integrase strand transfer (INST) inhibitor type
featuring a 5-N-benzylcarboxamide moiety. Significantly, the 6-alkylamino variant of this
new chemotype consistently conferred low nanomolar inhibitory activity against HIV-1.
Extended antiviral testing against a few raltegravir-resistant HIV-1 clones revealed a
resistance profile similar to that of the second generation INST inhibitor (INSTIs)
dolutegravir. Although biochemical testing and molecular modeling also strongly
corroborate the inhibition of INST as the antiviral mechanism of action, selected antiviral
analogues also potently inhibited reverse transcriptase (RT) associated RNase H, implying
potential dual target inhibition. In vitro ADME assays demonstrated that this novel
chemotype possesses largely favorable physicochemical properties suitable for further
development.
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Introduction
HIV infects an estimated 34 million people worldwide. Clinical management of HIV /
AIDS continues to rely on highly active antiretroviral therapy (HAART) that consists of
1
five different classes of FDA-approved drugs : nucleoside reverse transcriptase inhibitors
(NRTIs); nonnucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors
PIs); INSTIs; and entry inhibitors. However, HIV infection remains incurable2-3 due to the
(
persistence of viral reservoirs. The resulting long duration of HAART typically amounts
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to two treatment challenges: toxicities and the selection of resistant viral strains. The
emergence of multidrug resistance is particularly concerning as treatment options will be
severely limited. Therefore, sustained success in HIV HAART requires novel antivirals
with structurally unique pharmacophore and distinct resistance profile. HIV IN plays
critical roles in viral infection and the establishment of proviral latency.4-6 As an antiviral
target IN is particular attractive because 1) there is no host cellular counterpart, hence
specific inhibitors should not interfere with cellular functions; and 2) IN uses the same
active site (DD35E motif) for both the 3’ processing and the ST steps, therefore, inhibitors
could benefit from a potentially high genetic barrier to resistance selection. Specific INSTIs
all feature a diketoacid (DKA) functionality or its heterocyclic bioisosteres7-11 along with
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a hydrophobic terminal benzyl moiety,12-17 as demonstrated by all three FDA-approved
INSTIs (Figure 1): raltegravir (1)18-19, elvitegravir (2),20 and dolutegravir (3).21-22
Particularly significant is the second-generation INSTI 3 which retains potency against
2
3
many raltegravir-resistant HIV strains. We have previously developed a few chemotypes
featuring the HPD core24-27 that effectively inhibited HIV-1 in cell culture. The antiviral
potency associated with these HPD subtypes is likely due to the dual inhibition of RT and
IN as indicated by biochemical assays. However, the IN inhibition was typically much
weaker than the inhibition of RT. Furthermore, cross resistance to 1 was also observed,
suggesting that these early HPD subtypes may have the characteristics of first-generation
INSTIs. Another variant of HPD was recently found to selectively inhibit the RT-
2
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associated RNase H without significantly inhibiting INST. We report herein a rationally
designed new HPD variant (Figure 1, 4) featuring a unique C5 carboxamide moiety to
specifically inhibit INST. Significantly, chemotype 4 has the two structural determinants
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essential for INST binding and inhibition (Figure 1). The overall shape and functionalities
of 4 particularly resemble those of 3, suggesting that our novel inhibitors can be second
generation INSTIs.
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Figure 1. Structures of FDA-approved INSTIs: raltegravir (1), elvitegravir (2),
dolutegravir (3), and our newly designed HPD inhibitor subtype 4. Each approved drug
features a chelating traid (red) and a terminal benzyl group (blue) that constitute the
pharmacophore of HIV-1 INSTIs. Chemotype 4 fits the pharmacophore with the same
two structural features.
Results and Discussion
Chemistry. Our target compounds 4 were prepared via a concise and diverse synthetic
route shown in Scheme 1. The synthesis started from commercially available hydroxyurea
5
which was O-benzylated with BnBr in the presence of KOH under reflux to provide 1-
2
9
(
benzyloxy)urea 6 in high yield. Cyclocondensation of 1-(benzyloxy)urea 2 and diethyl
malonate provided six-membered heterocycle compound 3-(benzyloxy)-6-
hydroxypyrimidine-2,4(1H,3H)-dione 7 in moderate yield under microwave irradiation
3
0
(150 °C, 20 min) or under conventional heating (reflux, overnight). Compound 7 was
converted to key intermediate 3-(benzyloxy)-6-chloropyrimidine-2,4(1H,3H)-dione 8 by
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31
reacting with POCl
3
in the presence of BnEt
3
NCl. Reaction of intermediate chloride 8
with amines in the presence of N,N-dimethylaniline under microwave condition provided
-amination products 9 in moderate to good yield.32 The key C5 carboxamide was
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introduced by reacting 6-amino intermediates 9 with commercial or in situ generated
isocyanates (Scheme 1),33 a highly efficient method for small scale synthesis of
intermediate 10. The final debenzylation was achieved by treating compounds 10 with TFA
3
4
under microwave condition or via catalytic hydrogenation.
a
Scheme 1 Synthesis of chemotype 4
a
Reagents and conditions: a) KOH, BnBr, MeOH, reflux, 6 h, 91%; b) CH
2
(COOEt) ,
2
1
NaOEt, MW, 150 ºC, 20 min, 58%; c) POCl
3
, BnEt
3
NCl, 50 ºC, 6 h, 88%; d) R -NH
2
, N,N-
2
2
dimethylaniline, MW, 170 ºC, 20–25 min, 54–78%; e) R NCO (or R CON
3
), PhNO
2
, MW,
210 ºC, 20–40 min, 60–100%; f) Pd/C, H
MW, 120 ºC, 35–55 min, 50–77%.
2
, 40~50 Psi, MeOH, 4–6 h, 82–97%; g) TFA,
Alternatively, the C5 carboxamidation can be achieved via a two-step reaction sequence
scheme 2) to avoid the use of the unpleasant nitrobenzene. In this case, the amino
(
intermediate 9 was first treated with phenyl chloroformate and a base, such as pyridine, to
give intermediate phenyl ester 11 which was converted to amide 10 upon reacting with a
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primary amine under conventional heating or microwave conditions. Debenzylation with
the same protocol afforded the desired chemotype 4.
a
Scheme 2 Alternative synthesis of chemotype 4
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aReagents and conditions: a) ClCO
Ph, pyridine, rt, 2h, 62%; b) R -NH
2
, MW, dioxane,
2
2
1
1
00 ºC, 50 min, 82–92%; c) Pd/C, H
20 ºC, 35–55 min, 50–77%.
2
, 40~50 Psi, MeOH, 4–6 h, 82–97%; d) TFA, MW,
Meanwhile our analogue synthesis also included a few variants of 4 which entailed slightly
different synthetic routes or further functionalization (Scheme 3). In these events, direct
debenzylation of intermediate 12 afforded compound 51 (Scheme 3, a), whereas
methylation of intermediate 13 produced two regio-isomers 14 and 15, which upon
debenzylation yielded compounds 52 and 53, respectively (Scheme 3, b). Interestingly, a
6
-deamino analogue 54 was also synthesized from intermediate 7 via the carboxamidation
and debenzylation sequence (Scheme 3, c).
a
Scheme 3 Synthesis of compounds 51–54
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aReagents and conditions: a) TFA, MW, 120 ºC, 35–55 min; b) MeI, Cs
CO , DMSO, rt,
2
3
i
2
h; c) benzyl isocyanate, Pr
2
NEt, DCM, rt, 3h.
Finally, synthesis of analogues (55–57) with another six-membered ring fused to the HPD
core via N1-C6 (55) or C6-C5 (56–57) was also attempted. Compound 55 was synthesized
from 6-amino HPD intermediate 17 which was cyclized to 18 upon treating with 1,3-
dibromopropane under basic condition (Scheme 4). The rest of the synthesis involved the
same carboxamidation –debenzylation sequence as used for the synthesis of chemotype 4.
The synthesis of C6-C5 fused analogues (56–57) was attempted based on Scheme 5. While
the two intermediates 22–23 were obtained, the subsequent debenzylation was
unsuccessful (Scheme 5).
a
Scheme 4 Synthesis of compound 55
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aReagents and conditions: a) 1,3-dibromopropane, K
CO , DMF, 60 ºC, 2h, 90%; b) phenyl
2
3
chloroformate, pyridine, rt, 3 h, 86%; c) 4-fluorobenzylamine, MW, dioxane, 100 ºC, 50
min, 91%; d) TFA, MW, 120 ºC, 35 min, 96%.
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a
Scheme 5 Attempted synthesis of compounds 56–57
aReagents and conditions: a) ClCO
Ph, pyridine, rt, 2h; b) BnNH
CO H (cat.), MW 150 ºC, 10 min, 49%; d) BnNCO,
, MW 210 ºC, 10 min, 62%; e) TFA, MW, 120 ºC, 35–55 min; or Pd/C, H , 40~50
, MW, dioxane, 100 ºC,
2
2
5
0 min, 76%; c) HC(OEt)
3
, CF
3
2
PhNO
2
2
Psi, MeOH, 4–6 h.
Biology. All final compounds (24–55) were first screened in a viral cytopathic effect (CPE)
based antiviral assay against HIV-1IIIB in CEM-SS cells.35-36 In this assay, the reduction
of CPE was used to indicate the antiviral activity of a compound. The screening was
conducted under single concentration (10 M) and the percent inhibition and percent cell
viability were calculated using a virus control and cell control, respectively. Best
compounds from this assay were further evaluated in a single replication cycle MAGI
assay. In addition, all compounds were tested in a biochemical assay with recombinant
HIV-1 IN measuring INST activity. Selected ones were also tested against RT-associated
RNase H activity. Both 3 and 1 were included in all assays as reference compounds. The
results indicate a few prominent structure-activity-relationship (SAR) trends.
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Alkylamino group is required at C6. From the antiviral screening assay, the most striking
SAR was that the two analogues with an aromatic amine at the C6 position (24–25) were
completely inactive and largely cytotoxic in the cytoprotection antiviral assay, whereas a
small aliphatic amine substitution at the same position (26–37, Table 1) conferred excellent
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t
antiviral activity without cytotoxicity, with the lone exception being the butylamino
analogue (42) which was inactive and cytotoxic. The same dramatic SAR was observed
from the INST biochemical assay in which neither of the arylamino analogues (24–25)
showed appreciable inhibition at concentrations up to 100 M whereas the alkylamino
analogues (26–37) all demonstrated nanomolar inhibitory activity, including the
tbutylamino analogue (35). Furthermore, although most alkyl analogues (26–31, 33–34)
produced maximal cytoprotection at 10 M without significant cytotoxicity, linear alkyl
substituted analogues (26–31) are generally favored in the INST biochemical assay over
the unsubstituted analogue (36) and the bulky alkyl substituted ones, such as the isopropyl
(
33) and cyclopropyl (34) groups. Biochemical analysis also showed that the ethyl
substituent (30) confers the best inhibition against INST (IC50 = 33 nM).
Table 1. SAR of the C6 amino group: antiviral screening and biochemical assay of 24–36
a
Antiviral Screening (10 M)
INST IC50
Structure
Compd
b
CPE reduction % viability %
( M)
2
4
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>100
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0.080 ± 0.007
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0.11 ± 0.033
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0.15 ± 0.034
0.21 ± 0.077
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0.90 ± 0.29
99
100
3
--
--
--
--
--
--
0.068 ± 0.01
0.65 ± 0.14
1
a
Cytoprotection assay using HIV-1 IIIB in CEM-SS cells with a viral control for CPE
b
reduction and cell control for cell viability. Concentration inhibiting INST by 50%,
expressed as mean ± standard deviation from at least three independent experiments.
N-Benzyl substitution on the C5 carboxamide is highly desired. To explore the SAR
concerning the C5 carboxamide moiety, analogues of compound 30 were synthesized and
tested in the antiviral screening assay and the INST biochemical assay (Table 2).
Remarkably, all analogues with a benzyl group on the C5 carboxamide (30, 37–46)
demonstrated maximal cytoprotection (92–100%) without considerable cytotoxicity (cell
viability 87–100%), with the only exception being compound 43 which provided 62%
cytoprotection with 73% cell viability at 10 M. Consistent with the maximal
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cytoprotection, exceptional biochemical inhibition (IC50 = 21–230 nM) was also observed
for these analogues. Interestingly, compound with an extra methylene group (47) also
produced maximal cytoprotection at 10 M, though the biochemical inhibition was
substantially reduced (47 vs 30). In stark contrast, replacing the benzyl group with a phenyl
ring (48), alkyl group (50), or removing the N-substituent (49) completely abrogated both
the antiviral activity and biochemical inhibition. Compound with a C5 phenyl ester (51)
was also devoid of biochemical inhibition and yielded 50% cytoprotection at 10 M. All
these SAR observations are well aligned with the known pharmacophore model of INSTIs.
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0
1
2
3
4
5
6
7
8
9
0
Table 2. SAR around the C5 carboxamide: antiviral screening and biochemical assay of
3
0 analogues 47–51
a
Antiviral Screening (10 M)
INST IC50
Structure
Compd
b
CPE reduction %
viability %
( M)
3
0
100
100
0.033 ± 0.004
37
38
39
100
100
92
90
99
87
0.068 ± 0.007
0.049 ± 0.004
0.068 ± 0.011
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1
2
3
4
5
6
100
100
100
62
92
100
100
73
0.048 ± 0.007
0.23 ± 0.062
0.077 ± 0.011
0.095 ± 0.019
0.12 ± 0.021
0.021 ± 0.002
0.051 ± 0.013
0.10 ± 0.03
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100
100
100
87
4
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7
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27
35 ± 17
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1
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4
5
5
9
0
1
--
--
>100
>100
>100
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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9
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9
0
1
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
5.1
50
100
100
3
--
--
--
--
--
--
0.068 ± 0.01
0.65 ± 0.14
1
a
Cytoprotection assay using HIV-1 IIIB in CEM-SS cells with a viral control for CPE
b
reduction and cell control for cell viability. Concentration inhibiting INST by 50%,
expressed as mean ± standard deviation from at least three independent experiments.
Scaffold analogues lack antiviral activity or biochemical INST inhibition. To further
confirm the SAR trends shown in Tables 1–2, we also synthesized a few scaffold analogues
of the new chemotype. Compounds 52–53 were synthesized to explore the impact of
methylation on N1 or the C5 carboxamide. Remarkably, a simple methylation at N1
completely abrogated the antiviral activity, cell viability and the biochemical inhibition
(
52). Methylation of the C5 carboxamide also caused a complete loss of antiviral activity
while substantially attenuating the biochemical inhibition (53 vs 38). Similarly, the 6-
deamino analogue 61 demonstrated drastically reduced antiviral potency and biochemical
inhibition (54 vs 30), confirming the importance of the amino group at C6 for biological
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activities. Finally, cyclized analogue 55 was inactive and cytotoxic in the antiviral
screening assay, though the biochemical inhibition was largely preserved (IC50 = 430 nM).
Table 3. Antiviral screening and biochemical assay of scaffold analogues 52–55
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45
46
47
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52
53
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56
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a
Antiviral Screening (10 M)
INST IC50
Structure
Compd
b
CPE reduction %
viability %
( M)
5
2
6.5
10
>100
5
5
3
4
0
26
0
89
100
5.5
7.0 ± 3.3
37 ± 21
55
0.43 ± 0.13
a
Cytoprotection assay using HIV-1 IIIB in CEM-SS cells with a viral control for CPE
b
reduction and cell control for cell viability. Concentration inhibiting INST by 50%,
expressed as mean ± standard deviation from at least three independent experiments.
Dose response antiviral testing. To better gauge the antiviral potency, selected inhibitors
with excellent CPE reduction and cell viability from the antiviral screening assay were
further tested in a dose response fashion, from which the EC50 and CC50 were determined
(
Table 4). All compounds tested showed nanomolar activity and five of the eight analogues
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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
were active in low nanomolar range (EC50 = 15–61 nM). Although these analogues showed
mild cytotoxicity with CC50s in the range of 18–58 M, the selectivity window (Table 4),
particularly that for analogues 40 (TI =1500) and 45 (TI = 1200), is sufficiently large to
warrant their further development. To confirm the observed antiviral potency, these same
analogues were further evaluated in a distinct MAGI assay.36-37 This assay measures HIV
infection in indicator cells (P4R5) through the expression of a Tat-dependent reporter -
galactosidase) and requires only one replication cycle. While the EC50 values were
generally higher in this assay, our compounds still demonstrated nanomolar antiviral
potency with the exception of compounds 26 and 46. Interestingly, compound 45, while
showing the best potency in both the INST assay and the CPE antiviral assay, exhibited a
significantly higher EC50 (0.30 M vs 0.015 M) in the MAGI assay. By comparison, EC50
values for analogues 38 and 40 were comparable to 1 in the CPE assay and only 2-5 fold
higher than 3 and 1 in the MAGI assay. Nevertheless, dose response antiviral testing in two
distinct assays confirmed a highly potent antiviral profile of our newly designed
compounds.
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. Dose-response antiviral results of selected analogues from two different assays
Cytoprotection Assay a
MAGI Assay b
Compound
c
d
TI e
f
EC50 (M)
0.83
CC50 (M)
EC50 (M)
2
3
3
6
0
8
58
23
19
70
3.7 ± 0.3
0.30 ± 0.01
0.090 ± 0.02
0.061
380
580
0.033
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40
42
0.032
0.049
0.19
0.015
0.15
--
47
21
19
18
20
--
1500
430
100
1200
130
--
0.070 ± 0.02
0.30 ± 0.06
0.80 ± 0.2
4
4
4
4
5
6
10
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45
46
47
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52
53
54
55
56
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59
60
0.30 ± 0.09
1.3 ± 0.2
3
0.020 ± 0.002
0.030 ± 0.008
1
0.023
>10
>430
a
c
Concentration inhibiting INST by 50%, Concentration of a compound inhibiting virus
replication by 50%. d Concentration of a compound resulting in 50% cell death. e
Therapeutic index, defined as CC50/EC50. f Expressed as mean ± standard deviation from
two independent experiments.
Antiviral resistance profile. Mutations conferring resistance to INSTIs have been generated
in cell culture and emerged in clinical studies.38-40 To establish the resistance profile of
chemotype 4, a representative compound 45 was evaluated for antiviral potency against
four raltegravir-resistant HIV-1 clones 41 in the CPE assay. These clones contain one or
more major mutations in HIV-1 IN that are associated with raltegravir resistance,23, 24
namely a Y143C or an N155H single mutation; a G140S / Q148H double mutation and a
G140S / Y143H / Q148H triple mutation. AZT, 1 and 3 were included in the study for
comparison purpose. The results are summarized in Table 5. As expected cross-resistance
to 1 was not observed for AZT due to its orthogonal mechanism of action as an NRTI,
whereas the resistance profile observed in our antiviral assay for 3 is largely consistent
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
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3
4
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4
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5
6
23
with the reported profile. Similar to 3, compound 45 retains its potency against clones
with only one major IN mutation (clones 1556-1 and 4736-2) while HIV-1 clones with IN
double mutations (clone 8070-1) and triple mutations (clone 8070-1) demonstrated
moderate resistance to 45, with fold resistance of 13 and 6.3, respectively. The fold-
resistance of 45 is strikingly similar to that observed with 3 for each of the four HIV-1
clones (Table 5). These observations strongly suggest that our chemotype 4 may confer
antiviral activity via inhibiting INST in a manner similar to that of 3, a second generation
INSTI.42
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 5. Resistance profile of 45 against raltegravir-resistant HIV-1 clones
Fold-resistancea
HIV-1
Major Mutations
Clone
4
5
AZT
1.4
1
3
1
4
556-1
736-2
Y143C
N155H
0.4
2.4
170
14
2.0
1.8
0.7
G140S / Y143H /
Q148H
8
8
070-1
070-2
6.3
13
0.9
0.5
220
145
9.6
12
G140S / Q148H
a
Fold-resistance is defined as EC50(mutant) / EC50 (WT)
.
Inhibition against RT-associated RNase H. Consistent with the aforementioned antiviral
profile against raltegravir-resistant HIV-1 clones, which implies an antiviral mechanism of
action (MOA) similar to second generation INSTI 3, biochemical data also closely
correlate with the observed antiviral activity, as each of the selected analogues exhibited a
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biochemical IC50 against INST very similar to the antiviral EC50, particularly from the CPE
assay (Table 6). In addition, none of the inactive compounds (24–25, 48 and 50–54) from
the CPE antiviral assay inhibited INST (Tables 1–3), strongly suggesting that our new
compounds target INST. However, HIV IN and RNase H share a similar active site fold,43
hence their inhibition entails similar pharmacophore elements, mainly a chelating triad and
a hydrophobic aromatic moiety. Therefore, we also tested selected analogues in another
biochemical assay measuring the RNase H function of RT. Remarkably, all these analogues
demonstrated exceptional biochemical inhibition against RNase H with IC50s in the low
nanomolar range (10—61 nM, Table 6), which correlates with the antiviral activity as well
as the INST inhibition does in general. By contrast, the two reference compounds, 3 and 1,
did not inhibit RNase H significantly at concentrations up to 10 M. These observations
indicate that the exceptional antiviral potency of our new chemotype could be due to dual
target inhibition. Further virological characterizations of our compounds, including MOA
studies via viral passage experiments for resistance selection, are currently underway and
will be reported in due course.
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. Biochemical inhibition of RT-associated RNase H and the antiviral correlation
RNase H
INST
CPE
EC50 (M)
0.83
MAGI
EC50 (M)
3.7 ± 0.3
Compound
a
a
b
c
IC50 (M)
IC50 (M)
26
30
0.061 ± 0.004
0.042 ± 0.012
0.011 ± 0.003
0.080 ± 0.007
0.033 ± 0.004
0.049 ± 0.004
0.061
0.30 ± 0.01
0.09 ± 0.02
3
8
0.033
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40
42
0.010 ± 0.006
0.068 ± 0.011
0.077 ± 0.011
0.12 ± 0.021
0.021 ± 0.002
0.051 ± 0.013
0.068 ± 0.01
0.65 ± 0.14
0.032
0.07 ± 0.02
0.30 ± 0.06
0.80 ± 0.2
0.026 ± 0.009
0.022 ± 0.009
0.029 ± 0.015
0.020 ± 0.010
>10
0.049
0.19
0.015
0.15
--
4
4
4
4
5
6
0
1
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0
1
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8
9
0
0.30 ± 0.09
1.3 ± 0.2
3
0.020 ± 0.002
0.030 ± 0.008
1
>10
0.023
a
Concentration with 50% enzyme inhibition, expressed as mean ± standard deviation from
b
three independent experiments. Concentration of a compound inhibiting virus replication
c
by 50%. Concentration of a compound inhibiting virus replication by 50%, expressed as
mean ± standard deviation from three independent experiments.
Mechanism of strand transfer inhibition. HIV IN is a 32-kDa protein encoded by viral
4
4
pol gene consisting of three functional domains : the N-terminal domain (NTD) with a
conserved “HH-CC” zinc-binding motif; the catalytic core domain (CCD) containing the
key D64-D116-E152 catalytic triad; and the C-terminal domain (CTD) important for DNA
binding. Although crystal structures of each single domain as well as double domains had
been reported;45-47 detailed understanding on HIV INST catalysis and inhibition remained
elusive until the disclosure of crystal structures of full-length IN and viral DNA complex
(
intasome) for a homologous prototype foamy virus (PFV). 48-49 Homologous models50-51
constructed based on these PFV intasome crystal structures have provided valuable details
into HIV INST mechanism of action and formed the basis for structure-based INSTI
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design. Significantly these models corroborate the minimal pharmacophore embedded in
major INSTIs as shown in Figure 1.
To illustrate the binding mode of our new chemotype at HIV IN active site, we have
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59
60
performed molecular docking on a representative analogue 45 using a PFV intasome model
50
(
Figure 2). Significantly, 45 fits perfectly into the IN binding site through two major
2
+
binding domains: the 3-N hydroxyl group simultaneously chelates to both Mg ions while
allowing the placement of the benzyl group into the protein-DNA interfacial hydrophobic
pocket involving π-stacking of the i) fluorobenzyl side chain with the deoxycytosine C16
of viral DNA, and ii) π -stacking of the central uracil ring with the terminal 3′-
deoxyadenosine A17 (Figure 2a). In addition, a structure overlay of 45 with 3 reveals a
high degree of similarity between their binding (Figure 2b), suggesting that our new
molecular scaffold can effectively engage with HIV IN for ST inhibition.
Figure 2. a) Predicted binding mode of 45 (yellow) as obtained by molecular docking in
5
0
the PFV intasome catalytic site (PDB code: 3S3M). Magnesium cations depicted in green
spheres, catalytic residues in green sticks and viral DNA in magenta cartoon. b) Overlay
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6
of 45 (yellow) and crystallographic 3 (cyan) in the catalytic active site of PFV intasome
2
+
(
PDB code: 3S3M) with Mg ions in green spheres and viral DNA in magenta cartoon.
Numbering in black is in accordance to the PFV intasome crystal structure (PDB code:
S3M).
0
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9
0
3
To understand the effect of mutations on antiviral profile of 1 and 45, molecular modeling
was performed with these two compounds into the PFV IN Y212C mutant which
corresponds to the Y143C of HIV IN (Figure 3). It was observed that the oxadiazole moiety
of 1 makes a critical π-π stack interaction with Y143 (Figure 3a). This interaction is not
available with the C143 residue and the binding of 1 to the Y143C mutant is significantly
weakened (Figure 3b). On the other hand, 45 lacks the oxadiazole moiety to interact with
Y143 (Figure 3a). Consequently, the Y143C mutation would not impact its binding to IN.
These docking studies are consistent with the observations that Y143 mutation confers
major resistance to 1 and no resistance to 45 (Table 5).
Figure 3. a) Overlay of 1 (magenta) and 45 (yellow) within the PFV IN active site in
complex with magnesium (green spheres) (PDB code: 3OYA). π-π stack interaction
between oxadiazole moiety within 1 and Y212 is shown by black double headed arrow. b)
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Overlay of 1 (magenta) and 45 (yellow) within the active site of PFV IN Y212C
(corresponding to HIV IN Y143C) mutant. Key residues (D128, D185, E221 andY212) are
represented as green sticks. Numbering in black is in accordance to the PFV intasome
crystal structure (PDB code: 3S3M). The corresponding numbering for HIV IN is
highlighted in red.
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Physicochemical and in vitro ADME. To assess drug-like properties of chemotype 4,
selected analogues were tested in various physicochemical and in vitro ADME studies
(Table 7). First, aqueous stability and solubility were evaluated in Dulbecco's Phosphate-
Buffered Saline (DPBS). The selected compounds showed excellent stability and good
solubility in DPBS with the exception of 45 showed a lower but still acceptable solubility.
Second, the selected compounds were found to be stable in both human and mouse plasma.
Third, all compounds showed a higher protein binding in human plasma (98-99%) than
that in mouse plasma (71-96%). Finally, while the selected compounds exhibited excellent
phase I stability in both human and mouse liver microsomes, they were much less stable
against phase II glucuronidation in microsomes from both species (Table 7). Taken
together, although the selected compounds still need improvement in their phase II
glucuronidation stability, they possess largely favorable physicochemical and in vitro
ADME properties. It is also noteworthy that our chemotype has a significant smaller size
(MW for 45 = 322; the MW for drugs 1–3 is in the range of 419–448) while supporting
antiviral activities comparable to 1 (Table 4). The small size of our chemotype would allow
significant room for further optimization.
Table 7. Physicochemical and in vitro ADME profile of selected analogues
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Microsomal
Stability
Plasma Protein
Binding
Aqueous Aqueous Plasma Stability
Compd Solubility Stability
t
1/2 (h)
(Phase I / Phase
a
(%)
b
(µM)
t
1/2 (h)
II, CLint)
Human Mouse Human Mouse Human Mouse
<
5
0.1 /
00
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
26
138
1091
150
11
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
> 24
99.2
99.1
99.4
98.4
98.9
70.9
83.9
86.5
94.0
95.9
0.8 / 51
<
<
0.1 /
< 0.1 /
732
3
3
4
4
0
8
5
6
3
55
0.1 /
336
< 0.1 /
1240
< 0.1 /
1065
0.4 /
< 0.1 /
850
<
0.1 /
57
81
3
1342
a
Percent of fraction bound.
CLint: intrinsic clearance, µl/min/mg protein.
b
Conclusion. A new HPD subtype was designed and synthesized. Key to the design is the
introduction of an N-benzyl-5-carboxamide moiety to mimic the second-generation INSTI
3
. SAR revealed the requirement of an alkylamino group at C6 and an N-benzyl group on
the C5 carboxamide for INST inhibition and antiviral activity. Numerous analogues
demonstrated low nanomolar potencies against HIV-1 in cell culture and low nanomolar
biochemical inhibition against INST. Molecular modeling showed that the representative
inhibitor 45 binds to IN with a mode similar to 3. Antiviral testing of 45 against a panel of
raltegravir-resistant HIV-1 clones showed a resistance profile similar to that of 3. However,
analogues of our new chemotype also potently inhibited the RNase H function of RT,
suggesting that the antiviral potency could be due to dual target inhibition. in vitro ADME
assays also demonstrated largely favorable physicochemical properties for the new
chemotype.
Experimental
Chemistry
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General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents were either purchased (dioxane and MeOH) or dispensed under
argon from an anhydrous solvent system with two packed columns of neutral alumina or
molecular sieves. Flash chromatography was performed on a Teledyne Combiflash RF-
200 with RediSep columns (silica) and indicated mobile phase. All moisture sensitive
reactions were performed under an inert atmosphere of ultra-pure argon with oven-dried
10
11
12
13
14
15
16
17
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24
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29
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32
33
34
35
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38
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
glassware. H and C NMR spectra were recorded on a Varian 600 MHz spectrometer.
Mass data were acquired on an Agilent TOF II TOS/MS spectrometer capable of ESI and
APCI ion sources. Analysis of sample purity was performed on a Varian Prepstar SD-1
HPLC system with a Phenomenex Gemini, 5 micron C18 column (250mm x 4.6 mm).
HPLC conditions: solvent A = H
2
O containing 0.1% TFA, solvent B = MeCN; flow rate =
O to 100% MeCN
1
.0 mL/min; compounds were eluted with a gradient of 20% MeCN/H
2
for 30 min. Purity was determined by total absorbance at 254 nm. All tested compounds
have a purity ≥ 96%.
General method of 3-O-benzyl deprotection for the synthesis of final compounds 24–
5
5.
Method A: catalytic hydrogenation. To a solution of 100 mg of 3-(benzyloxy)-
pyrimidine-2,4(1H,3H)-dione intermediate (10, 12, 14–16, 20) in 7.0 mL MeOH was added
2
0 mg Pearlman’s catalyst (Pd-C, 20%). The reaction mixture was degassed using vacuum
and refilling with H (40-50 psi) for three times. Then keep the reaction mixture being
shaken under H (40-50 psi) atmosphere for appropriate time. The reaction was monitored
by both TLC and LC-MS. The reaction mixture was filtered through a short celite column
2
2
2
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5
5
5
5
5
5
5
5
5
6
and then removed the solvent. Trituration with MeOH, ethyl acetate and DCM provided
desired final compounds 24–55 in 82-97% yield.
Method B: acid hydrolysis. To a microwave reaction vessel were added 0.26 mmol of a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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5
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7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
-(benzyloxy)-pyrimidine-2,4(1H,3H)-dione intermediate (10, 12, 14–16, 20) and 6~10
mL of TFA. The reaction vessel was irradiated at 120 °C for the appropriate time. The
reaction was monitored by both TLC and LC-MS. The reaction mixture was transferred to
a round-bottom flask to remove the solvent under reduced pressure. Then the residue was
purified by flash chromatography on C18 reverse phase column (H
MeOH, Ethylacetate and DCM) to provide the desired compounds 24–55 as solid in 50-
7%.
2
O-MeOH) or trituration
(
7
N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(phenylamino)pyrimidine-5-
1
carboxamide (24). White solid, 50% yield; H NMR (600 MHz, DMSO-d6): δ 12.72 (s,
1
H), 11.26 (s, 1H), 10.32 (s, 1H), 10.03 (t, J = 2.4, 1H), 7.43-7.26 (m, 10H), 4.49 (s, 2H);
13C NMR (150 MHz, DMSO-d6): δ 167.7, 161.6, 154.0, 147.0, 139.3, 135.8, 129.5, 128.4,
1
27.2, 126.9, 126.5, 124.9, 81.7, 41.9; HRMS-ESI(-) m/z calcd for C18
H N
16 4
4
O 351.1099
-
[
M-H] , found 351.1103.
N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(biphenylamino)pyrimidine-5-
1
carboxamide (25). White solid, 77% yield; H NMR (600 MHz, DMSO-d6): δ 12.76 (s,
1
H), 11.33 (s, 1H), 10.34 (s, 1H), 10.04 (dd, J = 6.0, 4.8 1H), 7.74-7.20 (m, 14H), 4.50 (d,
1
3
J = 5.4, 2H); C NMR (150 MHz, DMSO-d6): δ 167.7, 161.6, 154.0, 147.0, 139.3, 138.1,
1
35.2, 129.0, 128.4, 127.7, 127.5, 127.2, 126.9, 126.5, 125.4, 80.9, 41.9; HRMS-ESI(-)
-
m/z calcd for C24
H
20
N
4
O
4
427.1412 [M-H] , found 427.1414.
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N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-4-(methylamino)-2,6-dioxopyrimidine-5-
1
carboxamide (26). White solid, 39% yield; H NMR (600 MHz, DMSO-d6): δ 11.05 (s,
1
4
H), 10.88 (q, J = 4.8 Hz, 1H), 10.20 (s, 1H), 9.90 (t, J = 6.0 Hz, 1H), 7.33-7.22 (m, 5H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
.43 (d, J = 6.0 Hz, 2H); 2.94 (d, J = 5.4 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ
167.7, 161.3, 155.7, 147.3, 139.7, 128.3, 127.1, 126.7, 79.0, 41.7, 28.4; HRMS-ESI(-) m/z
-
calcd for C13
H
14
N
4
O
4
289.0942 [M-H] , found 289.0939.
N-(3-Fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-4-(methylamino)-2,6-
1
dioxopyrimidine-5-carboxamide (27). White solid, 20% yield; H NMR (600 MHz,
DMSO-d6): δ 11.05 (s, 1H), 10.80 (q, J = 4.8 Hz, 1H), 10.18 (s, 1H), 9.94 (t, J = 6.0 Hz,
1
H), 7.35 (ddd, J = 7.8, 7.2, 6.0 Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 7.06 (m, 2H), 4.44 (d, J
1
3
= 6.0 Hz, 2H); 2.93 (d, J = 4.2 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 167.8, 162.2
d, JCF = 241.5 Hz), 161.3, 155.9, 147.4, 143.0 (d, JCF = 6.9 Hz), 130.2 (d, JCF = 8.1 Hz),
(
1
23.1 (d, JCF = 2.4 Hz), 113.7 (d, JCF = 21.9 Hz), 113.4 (d, JCF = 20.0 Hz), 79.0, 41.2, 28.4;
-
HRMS-ESI(-) m/z calcd for C13
H13FN
4
O
4
307.0848 [M-H] , found 307.0852.
N-(4-Fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-4-(methylamino)-2,6-
1
dioxopyrimidine-5-carboxamide (28). White solid, 20% yield; H NMR (600 MHz,
DMSO-d6): δ 11.05 (s, 1H), 10.86 (q, J = 5.4 Hz, 1H), 10.19 (s, 1H), 9.87 (t, J = 6.0 Hz,
1
H), 7.31 (dd, J = 8.4, 5.4 Hz, 2H), 7.14 (t, J = 9.0 Hz, 2H), 4.41 (d, J = 5.4 Hz, 2H); 2.94
1
3
(
d, J = 4.8 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 167.7, 163.7, 161.1 (d, JCF = 241.5
Hz), 161.2, 155.7, 147.3, 136.0 (d, JCF = 2.4 Hz), 129.1 (d, JCF = 8.1 Hz), 115.0 (d, JCF
=
307.0848 [M-H]-,
2
0.7 Hz), 79.0, 40.9, 28.4; HRMS-ESI(-) m/z calcd for C13
H13FN
4
O
4
found 307.0850.
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N-(3-Chloro-2-fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-4-(methylamino)-2,6-
1
dioxopyrimidine-5-carboxamide (29). White solid, 90 % yield; H NMR (600 MHz,
DMSO-d6): δ 11.06 (s, 1H), 10.76 (q, J = 4.2 Hz, 1H), 10.22 (s, 1H), 9.95 (t, J = 6.0 Hz,
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
1
H), 7.47 (t, J = 7.2 Hz, 1H), 7.27 (dd, J = 7.8, 6.0 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 4.50
1
3
(d, J = 4.8 Hz, 2H), 2.93 (d, J = 4.2 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 167.8,
61.3, 155.7, 155.2 (d, JCF = 246 Hz), 147.3, 129.0, 128.7 (d, JCF = 13.8 Hz), 128.2 (d, JCF
1
=
4.5 Hz), 125.2 (d, JCF = 4.7 Hz), 119.4 (d, JCF = 17.3 Hz), 79.0, 35.9 (d, JCF = 3.5 Hz),
-
28.4; HRMS-ESI(-) m/z calcd for C13
H12ClFN
4
O
4
341.0458 [M-H] , found 341.0462.
N-Benzyl-1-(hydroxy)-4-(ethylamino)-1,2,3,6-tetrahydro-2,6-dioxopyrimidine-5-
1
carboxamide (30). White solid, 42% yield; H NMR (600 MHz, DMSO-d6): δ 11.11 (s,
1H), 11.04 (t, J = 5.4 Hz, 1H), 10.20 (s, 1H), 9.93 (t, J = 6.0 Hz, 1H), 7.34-7.24 (m, 5H),
13
4
.43 (d, J = 6.0 Hz, 2H); 3.36 (td, J = 6.6, 6.0 Hz, 2H ), 1.14 (t, J = 6.6 Hz, 3H); C NMR
(150 MHz, DMSO-d6): δ 167.8, 161.4, 154.7, 147.3, 139.6, 128.4, 127.1, 126.8, 78.8, 41.7,
-
36.0, 14.4; HRMS-ESI(-) m/z calcd for C14
H
16
N O
4 4
303.1099 [M-H] , found 303.1098.
N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-(propylamino)pyrimidine-5-
1
carboxamide (31). White solid, 79% yield; H NMR (600 MHz, DMSO-d6): δ 11.16 (s,
1H), 11.13 (s, 1H), 10.20 (s, 1H), 9.94 (s, 1H), 7.32-7.24 (m, 5H), 4.44 (d, J = 4.8 Hz, 2H);
13
3
1
.30 (m, 2H ), 1.54 (m, 2H), 0.91 (t, J = 6.6 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ
67.9, 161.4, 154.9, 147.3, 139.6, 128.4, 127.1, 126.8, 78.8, 42.6, 41.7, 22.1, 11.0; HRMS-
-
ESI(-) m/z calcd for C15
H
18
N
4
O
4
317.1255 [M-H] , found 317.1253.
N-(3-Chloro-2-fluorobenzyl)-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxo-4-
1
(
propylamino)pyrimidine-5-carboxamide (32). White solid, 73 % yield; H NMR (600
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MHz, DMSO-d6): δ 11.15 (s, 1H), 11.02 (s, 1H), 10.23 (s, 1H), 9.99 (s, 1H), 7.47-7.18 (m,
3
H), 4.51 (d, J = 4.2 Hz, 2H), 3.30 (m, 2H), 1.52 (m, 2H), 0.89 (dd, J = 7.8, 6.0 Hz, 3H);
13C NMR (150 MHz, DMSO-d6): δ 168.0, 161.4, 155.2 (d, J
CF = 246 Hz), 154.9, 147.3,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
29.1, 128.6 (d, JCF = 14.9 Hz), 128.2 (d, JCF = 4.5 Hz), 125.3 (d, JCF = 3.5 Hz), 119.5 (d,
CF = 17.3 Hz), 78.8, 42.6, 35.9 (d, JCF = 3.5 Hz), 22.0, 11.0; HRMS-ESI(-) m/z calcd for
369.0771 [M-H]-, found 369.0768.
J
C
15
H
16ClFN
4
O
4
N-Benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-4-(isopropylamino)-2,6-dioxopyrimidine-5-
1
carboxamide (33). White solid, 92% yield; H NMR (600 MHz, DMSO-d6): δ 11.18 (d,
J = 8.4 Hz 1H), 11.14 (s, 1H), 10.21 (s, 1H), 9.96 (d, J = 5.4 Hz 1H ), 7.34-7.23 (m, 5H),
1
3
4
.43 (d, J = 5.4 Hz, 2H); 4.08 (m, 1H ), 1.16 (d, J = 6.0 Hz, 6H); C NMR (150 MHz,
DMSO-d6): δ 167.9, 161.4, 153.8, 147.3, 139.6, 128.4, 127.1, 126.8, 78.7, 42.6, 41.7, 22.8;
-
HRMS-ESI(-) m/z calcd for C15
H
18
N
4
O
4
317.1255 [M-H] , found 317.1261.
N-Benzyl-1-(hydroxy)-4-(cyclopropylamino)-1,2,3,6-tetrahydro-2,6-
1
dioxopyrimidine-5-carboxamide (34). White solid, 82% yield; H NMR (600 MHz,
DMSO-d6): δ 11.17 (s, 1 H), 11.01 (s, 1H), 10.24 (s, 1H), 9.87 (t, J = 6.0 Hz 1H ), 7.31-
13
7
.21 (m, 5H), 4.39 (d, J = 5.4 Hz, 2H); 2.71 (m, 1H ), 0.83 (m, 2H), 0.59 (m, 2 H); C
NMR (150 MHz, DMSO-d6): δ 167.6, 161.2, 156.5, 147.1, 139.5, 128.4, 127.1, 126.8,
-
7
3
9.2, 41.7, 23.0, 7.7; HRMS-ESI(-) m/z calcd for C15
15.1101.
H
16
N
4
O
4
315.1099 [M-H] , found
4-(Tert-butylamino)-N-benzyl-1-(hydroxy)-1,2,3,6-tetrahydro-2,6-dioxopyrimidine-
1
5
-carboxamide (35). White solid, 59% yield; H NMR (600 MHz, DMSO-d6): δ 11.82 (s,
H), 10.24 (s, 1H), 10.10 (t, J = 6.0 Hz, 1H), 10.03 (brs, 1H), 7.33-7.25 (m, 5H), 4.43 (d,
1
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