6-Cyclohexylmethyl-3-hydroxypyrimidine-2,4-dione as an inhibitor scaffold of HIV reverase transcriptase: Impacts of the 3-OH on inhibiting RNase H and polymerase

Article abstract of DOI:10.1016/j.ejmech.2017.01.041

3-Hydroxypyrimidine-2,4-dione (HPD) represents a versatile chemical core in the design of inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H and integrase strand transfer (INST). We report herein the design, synthesis and biological evaluation of an HPD subtype (4) featuring a cyclohexylmethyl group at the C-6 position. Antiviral testing showed that most analogues of 4 inhibited HIV-1 in the low nanomolar to submicromolar range, without cytotoxicity at concentrations up to 100?μM. Biochemically, these analogues dually inhibited both the polymerase (pol) and the RNase H functions of RT, but not INST. Co-crystal structure of 4a with RT revealed a nonnucleoside RT inhibitor (NNRTI) binding mode. Interestingly, chemotype 11, the synthetic precursor of 4 lacking the 3-OH group, did not inhibit RNase H while potently inhibiting pol. By virtue of the potent antiviral activity and biochemical RNase H inhibition, HPD subtype 4 could provide a viable platform for eventually achieving potent and selective RNase H inhibition through further medicinal chemistry.

Full text of DOI:10.1016/j.ejmech.2017.01.041

European Journal of Medicinal Chemistry 128 (2017) 168e179
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
6-Cyclohexylmethyl-3-hydroxypyrimidine-2,4-dione as an inhibitor
scaffold of HIV reverase transcriptase: Impacts of the 3-OH on
inhibiting RNase H and polymerase
Jing Tang a, Karen A. Kirby b, Andrew D. Huber b, Mary C. Casey b, Juan Ji b,
,
*
Daniel J. Wilson a, Stefan G. Sarafianos b, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Hydroxypyrimidine-2,4-dione (HPD) represents a versatile chemical core in the design of inhibitors of
human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H and integrase
strand transfer (INST). We report herein the design, synthesis and biological evaluation of an HPD
subtype (4) featuring a cyclohexylmethyl group at the C-6 position. Antiviral testing showed that most
analogues of 4 inhibited HIV-1 in the low nanomolar to submicromolar range, without cytotoxicity at
Received 8 December 2016
Received in revised form
25 January 2017
Accepted 27 January 2017
Available online 30 January 2017
concentrations up to 100 mM. Biochemically, these analogues dually inhibited both the polymerase (pol)
and the RNase H functions of RT, but not INST. Co-crystal structure of 4a with RT revealed a non-
nucleoside RT inhibitor (NNRTI) binding mode. Interestingly, chemotype 11, the synthetic precursor of 4
lacking the 3-OH group, did not inhibit RNase H while potently inhibiting pol. By virtue of the potent
antiviral activity and biochemical RNase H inhibition, HPD subtype 4 could provide a viable platform for
eventually achieving potent and selective RNase H inhibition through further medicinal chemistry.
© 2017 Elsevier Masson SAS. All rights reserved.
Keywords:
3-Hydroxypyrimidine-2,4-dione (HPD)
Human immunodeficiency virus (HIV)
RNase H
Polymerase
Inhibitors
1. Introduction
a C-5 carboxamide moiety and replacement of the C-6 aryl with a
small alkyl group resulted in subtype 3, which potently inhibited
HIV-1 infection is clinically managed [1] by highly active anti-
retroviral therapy (HAART) [1] which comprises four major classes
of drugs: the nucleoside RT inhibitors (NRTIs) [2], NNRTIs [3],
protease inhibitors (PIs) [4], and INST inhibitors (INSTIs) [5]. Long
term HAART can be plagued by toxicity and the emergence of drug
resistance. Structurally novel HIV inhibitors, particularly those with
a distinct mechanism of action, could add significantly to the
existing HAART repertoire. We have long been interested in
exploring chemotypes amenable to structural elaboration toward
inhibiting different targets of HIV-1 [6e12]. One such chemotype is
the versatile HPD core (Fig.1) which was initially designed to dually
inhibit RT pol and INST (subtype 1) [12]. Removal of the N-1 and C5
substituents and redesign of the C6 moiety led to subtype 2 which
selectively inhibited the RNase H function of RT [8]. Introduction of
both RNase H and INST [9]. Among other viable modifications for
the HPD core, the replacement of the phenyl ring at the C-6 position
of subtype 1 with a cyclohexyl group generated a new subtype 4
(Fig. 1b) which conferred highly potent antiviral activity with dual
biochemical inhibition against HIV-1 RT pol and RNase H functions.
We report herein the synthesis, antiviral, biochemical and bio-
physical characterizations of subtype 4. The key medicinal chem-
istry observation was that most analogues potently inhibited HIV in
cell culture and that the biochemical inhibitory profile of RT hinges
largely on the 3-OH group. The synthetic precursor of subtype 4,
chemotype 11 which lacks the 3-OH group, did not inhibit the
RNase H function of RT, though the higher potency against RT pol
conferred better antiviral activity against HIV-1 when compared to
4.
* Corresponding author.
E-mail address: wangx472@umn.edu (Z. Wang).
http://dx.doi.org/10.1016/j.ejmech.2017.01.041
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
169
2. Results and discussion
a favorable therapeutic profile in cell culture. Importantly, the
antiviral potency observed from the MAGI assay was confirmed in a
distinct cytoprotection assay [16,17] in which cytopathic effect of
2.1. Chemistry
HIV-1 was measured. In this assay, 4c (EC50 ¼ 0.56
(EC50 ¼ 0.32 M) and 4e (EC50 ¼ 1.8 M) all showed potent antiviral
activity (data not listed in Table 1).
mM), 4d
The synthesis of HPD subtype 4 is described in Scheme 1. The
overall approach was adapted from the route reported for the
m
m
synthesis of subtype 1 [12]. The synthesis entails a
b
-ketoester in-
Subtype 4 dually inhibited RT pol and RNase H. Biochemical
evaluations of our compounds resulted in a few major observations.
First of all, none of the analogues inhibited INST at concentrations
termediate 6 which was previously prepared via a Blaise reaction
between cyclohexylacetonitrile (7) and a bromoester (8) [13].
However, it turned out that the cost of starting material 7 is too
high to allow the preparation of 6 at the scale necessary for
analogue synthesis. In our synthesis, an alternative method starting
from the much cheaper cyclohexylacetic acid (5) was used [14].
Acid 5 was first activated by treating with carbonyldiimidazole
(CDI) and then reacted with the mono potassium salt of a
substituted malonate. This procedure proved to be highly efficient
up to 100 mM (Table 1), whereas subtype 1 was previously reported
to confer low micromolar inhibition against INST [12]. This is not
surprising as inhibition of INST minimally requires a chelating triad
and a hydrophobic aromatic group, typically a fluorobenzyl group
[18]. In subtype 4 the benzyl group is replaced with a cyclo-
hexylmethyl group, thus the lack of anti-INST activity. It is note-
worthy that achieving selective RNase H inhibition over INST
inhibition could be a challenge due to their similarity in active site
fold and mechanism of catalysis [19]. By abrogating the inhibitory
activity against INST, substituting the cyclohexyl group for the
phenyl ring provides a distinct advantage toward achieving selec-
tive RNase H inhibition. Second, all analogues with an isopropyl or
ethyl group at the C5 position (4ae4h) showed dual inhibition
against RT pol and RNase H, except for compound 4f which did not
inhibit RT pol. By contrast, analogues with a methyl substituent
(4ie4m) only inhibited RT RNase H (Table 1). Importantly, with
most compounds the RT pol inhibition appeared to correlate very
well with the observed antiviral activity. As mentioned earlier, the
methyl substitution at the C5 position conferred no antiviral ac-
in delivering b-ketoester intermediate 6 in large scale. Cyclization
of 6 with thiourea yielded the 2-thioxopyrimidine-4-one inter-
mediate 9, which upon S-alkylation and hydrolysis was converted
to the key pyrimidine-2,4-dione intermediate 10. Subsequent se-
lective N-1 alkylation of 10 via a bis-silylated pyrimidine interme-
diate with an in situ generated chloromethyl ether produced 11.
Finally, the key N-3 OH group was introduced via a base-mediated
oxidation with meta-chloroperoxybenzoic acid (mCPBA) to com-
plete the synthesis of subtype 4.
2.2. Biology
tivity at 15 mM and weak or no pol inhibition as observed with
All analogues of subtype 4 were tested in a MAGI antiviral assay
[15,16] in dose response fashion, as well as three biochemical assays
against RT pol, RT RNase H and INST. The results are described in
Table 1.
HPD 4 potently inhibited HIV-1. In the antiviral assays, all ana-
logues with an isopropyl or ethyl group at the C5 position (4ae4h)
showed strong inhibition against HIV-1 in the low nanomolar to
low micromolar range, whereas those with a methyl substituent
compounds 4ie4l, whereas the three most active analogues (4a, 4c
and 4d) from the antiviral assay also exhibited the highest potency
against RT pol. In addition, compound 4a inhibited RT pol with an
IC50 (0.45
m
M) similar to 14 (IC50 ¼ 0.42
mM) and conferred a
comparable antiviral activity (EC50 ¼ 26 nM) to 14 (EC50 ¼ 11 nM).
Furthermore, significant inhibition was not observed with com-
pound 4a at concentrations up to 10
mM against the Y181C or
K103 N mutants of RT (data not shown). All these findings strongly
indicate that the observed antiviral activity is most likely via RT pol
inhibition. The exceptions are compounds 4f and 4m with which
the antiviral activity correlated with only the RNase H inhibition as
no inhibition against RT pol was observed.
(4ie4m) did not inhibit HIV-1 at concentrations up to 15
mM
(Table 1). Among the active analogues, 4a (EC50 ¼ 26 nM), 4c
(EC50 ¼ 59 nM) and 4d (EC50 ¼ 39 nM) exhibited antiviral potencies
comparable to that of 14 (EC50 ¼ 11 nM), a potent NNRTI. In addi-
tion, while 14 showed modest cytotoxicity (CC50 ¼ 58
mM), toxicity
Subtype 4 binds to NNRTI binding pocket. To confirm the antiviral
mechanism of action through RT pol inhibition, we solved a crystal
was not observed with any of the subtype 4 analogues at concen-
trations up to 100 M, suggesting that our new compounds possess
m
Fig. 1. Variants of the HPD chemotype. (a) HPD subtypes 1e3 have been reported to inhibit RT and/or INST of HIV-1; (b) replacement of the C-6 phenyl ring of subtype 1 led to
subtype 4 with substantially different biological activity profiles.

170
J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
Scheme 1. aSynthesis of HPD subtype 4. aReagents and conditions: a) CDI, CH3CN, rt, 30 min, R1CH(CO2Et)(CO2K), Et3NeMgCl2, rt, overnight; b) Zn, THF; c) thiourea, EtONa, EtOH,
refluxing for 8 h; d) ClCH2COOH, AcOH, H2O, refluxing, overnight; e) CH3C(OTMS)¼NTMS (BSA), substituted benzyl alcohol, (HCHO)n, TMSCl, TBAI (cat.), CH2Cl2, rt, 70e90%; f) NaH,
mCPBA, THF, rt.
structure of analogue 4a bound to RT at 2.7 Å. The structure
revealed that analogue 4a binds at the NNRTI binding pocket of RT,
reminiscent to that of the 1-[(2-Hydroxyethoxy)methyl]-6-(phe-
nylthio)thymine (HEPT) type of NNRTIs [20,21]. HEPT (12) [22,23]
was among the earliest NNRTIs and a few variants (Fig. 3), such
as MKC-442 (13) [20,24] and TNK-651 (14) [20] and TNK-6123 (15)
[21], have been reported to inhibit HIV-1 with exceptional
potencies.
The precursor 11 potently inhibited HIV-1 and RT pol. The imme-
diate synthetic precursor of subtype 4, chemotype 11, closely
mimics the HEPT variants, particularly 14. Therefore, we were
prompted to test all analogues of 11. We first tested chemotype 11
in the MAGI antiviral assay. Remarkably, with the single exception
of 11m, all analogues inhibited HIV-1 in low nanomolar to sub-
micromolar range (Table 2). Particularly potent are 11aed, 11f and
11h, which exhibited antiviral potencies (EC50 ¼ 5.2e14 nM)
comparable to or better than that observed with 14 (EC50 ¼ 11 nM).
Since 14 was among the most potent NNRTIs of the HEPT family, the
exceptional antiviral potency may render 11 a suitable chemotype
for antiviral development.
in the NNRTI binding pocket (main chain of K101, Fig. 2), providing
some steric hindrance for inhibitor binding. Compounds without
the 3-hydroxy group, such as 11a, should be able to bind at the
NNRTI binding pocket without this interference, similar to the
previously reported crystal structures of HIV RT in complex with
HEPT compounds 13 [20] and 15 [21]. It is also noteworthy that the
correlation between pol inhibition and antiviral potency of 11
appeared to be as strong as it is with 14, suggesting that the anti-
viral potency of 11 is most likely due to the RT pol inhibition.
3. Conclusion
By replacing the phenyl ring with a cyclohexyl group at the C-6
position of HPD subtype 1, we have generated an HPD subtype 4
which potently inhibited HIV-1. Biochemically, subtype 4 demon-
strated dual inhibition against both the RNase H and the pol
functions of RT without inhibiting INST. The antiviral SAR was
found to better correlate with that of RT pol inhibition. Crystal
structure of 4a bound RT and the lack of biochemical inhibition of
4a against Y181C and K103N RT mutants further corroborate an
antiviral mechanism of action of 4 via RT pol inhibition. Interest-
ingly, the synthetic precursor 11 inhibited HIV-1 and RT pol sub-
stantially better than subtype 4 without inhibiting INST or RNase H.
The crystal structure of 4a bound to RT provided a structural basis
for the observed increase in potency of chemotype 11 over 4. The
best compounds of chemotypes 4 and 11 exhibited antiviral po-
tencies comparable to that of 14, a well-known and highly potent
NNRTI, and hence provide valuable additions to NNRTIs. Most
importantly, the consistent biochemical inhibition against RNase H,
the potent antiviral activity and the close correlation between
antiviral potency and RNase H inhibition with analogues 4f and 4m
render chemotype 4 a valuable platform for achieving potent
antiviral phenotype through selective RNase H inhibition.
Biochemically, analogues of 11 did not inhibit RNase H at con-
centrations up to 10 mM, presumably due to the lack of chelating
triad required for RNase H active site inhibitors [25]. This is in stark
contrast to subtype 4 with which the 3-OH group forms a chelating
triad with the two flanking carbonyls, resulting in the observed
consistent inhibition of RNase H. However, the precursor scaffold 11
conferred substantially stronger inhibition against RT pol than
chemotype 4. As shown in Table 2, all analogues with an isopropyl
or ethyl group at the C5 position (11ae11h) showed nanomolar
inhibition against RT pol, with the exception of compound 11e
which inhibited RT pol with an IC50 of 1.5 mM. By contrast, ana-
logues with a methyl substituent (11ie11m) did not inhibit RT pol.
This structure-activity-relationship (SAR) trend is similar to that
observed with chemotype 4 and consistent with the binding mode
of HEPT NNRTIs which typically require a bulkier isopropyl or ethyl
group at C5 position [20]. Furthermore, the crystal structure of HIV
RT in complex with 4a provides a structural basis for the observed
higher potency of 11a (5.2 nM) vs. 4a (26 nM). In the structure, the
3-hydroxy group of 4a is in close proximity to the protein backbone
4. Experimental
4.1. General-chemistry
All commercial chemicals were used as supplied unless

J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
171
Table 1
Biochemical and antiviral assay results of subtype 4.
a
a
Compd
Structure
RT IC50
RNase H
5.2 0.8
(m
M)
INST IC50
(m
M)
MAGI antiviral
b
c
Pol
EC50
(
m
M)
CC50 (mM)
4ad
0.45 0.07
>100
0.026 0.002
>100
4b
2.7
2
9.9 0.04
>100
2.4 0.2
>100
4c
5.5 0.2
2.3
2
>100
>100
0.059 0.004
0.039 0.008
>100
>100
4d
5.8 0.2
1.5 0.5
4e
4f
1.1 0.3
6.5 0.2
>100
>100
0.42 0.1
>100
>100
1.7 0.5
>10
4.1 0.5
4g
4h
10
7.0
6.5
2
1
>100
>100
2.4 0.3
>100
>100
2.1
3.1
1
1
0.43 0.06
4i
>10
>10
10
>100
>100
>100
>15
>15
>15
>100
>100
>100
4j
2.2 0.9
4k
4.6 0.4
(continued on next page)

172
J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
Table 1 (continued )
a
a
Compd
Structure
RT IC50
RNase H
(m
M)
INST IC50
(m
M)
MAGI antiviral
b
c
Pol
10
EC50
(
mM)
CC50
(
mM)
4l
7.6 0.6
>100
>15
>100
4m
3.9 0.2
>10
>100
10 1.1
>100
14
e
>10
0.42 0.2
e
0.011 0.0001
58
2
a
Concentration of compound inhibiting the target enzyme by 50%.
Concentration of compound inhibiting virus replication by 50%.
Concentration of compound resulting in 50% cell death. All assay results expressed as mean standard deviation from at least two independent experiments.
b
c
d
No pol inhibition was observed against RT mutants Y181C or K103N at concentrations up to 10 mM.
otherwise indicated. Dry solvents were either purchased (CH3CN
and EtOH) or dispensed under argon from an anhydrous solvent
system with two packed columns of neutral alumina or molecular
sieves (CH2Cl2, THF), Flash chromatography was performed on a
Teledyne Combiflash RF-200 with RediSep columns (silica) and
indicated mobile phase. All moisture sensitive reactions were per-
formed under an inert atmosphere of ultra-pure argon with oven-
dried glassware. 1H and 13C NMR spectra were recorded on a Var-
ian 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
(250 mm ꢀ 4.6 mm). HPLC conditions: solvent A ¼ H2O, solvent
B ¼ MeCN; flow rate ¼ 1.0 mL/min; compounds were eluted with a
gradient of 20% MeCN/H2O to 90% MeCN for 20 min. Purity was
determined by total absorbance at 254 nm. All tested compounds
have a purity ꢁ96%. In addition, all tested compounds were
examined for potential pan assay interference and none was
deemed prone to any of the potential mechanisms of interference:
aggregation, redox activity, fluorescence, protein reactivity, singlet-
oxygen quenching, member disruption, and decomposition in assay
buffer to form reactive compound.
1.22 (d, J ¼ 6.0 Hz, 6H), 1.12 (m, 5H); 13C NMR (150 MHz, CDCl3)
d
162.3, 152.2, 150.0, 137.3, 128.4, 127.9, 127.8, 118.9, 72.2, 71.9, 40.8,
37.1, 35.2, 30.8, 28.5, 28.2, 22.4; HRMS (ESIþ) calcd. for C22H31N2O3
[MþH]þ 371.2356, found 371.2354.
4.1.1.2. 6-(Cyclohexylmethyl)-1-(((4-fluorobenzyl)oxy)methyl)-5-
isopropylpyrimidine-2,4(1H,3H)-dione (11b). This compound was
prepared as white solid following the procedure described for the
preparation of 11a, Yield: 81%; 1H NMR (600 MHz, CDCl3)
d 8.84 (s,
1H), 7.22 (m, 2H), 6.96 (m, 2H), 5.40 (s, 2H), 4.53 (s, 2H), 2.72 (m,
1H), 2.52 (d, J ¼ 6.0 Hz, 2H), 2.20 (m, 5H), 1.40 (m, 1H), 1.23 (d,
J ¼ 6.0 Hz, 6H), 1.12 (m, 3H), 0.98 (m, 2H); HRMS (ESIþ) calcd. for
C
22H30FN2O3 [MþH]þ 389.4723, found 389.4725.
4.1.1.3. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-5-
ethylpyrimidine-2,4(1H,3H)-dione (11c). This compound was pre-
pared as a white solid following the procedure described for the
preparation of 11a. Yield: 73%; 1HNMR (600 MHz, CDCl3)
d 7.34 (m,
5H), 5.43 (s, 2H), 4.64 (s, 2H), 2.58 (d, J ¼ 6.0 Hz, 2H), 2.83 (t,
J ¼ 7.2 Hz, 2H), 2.43 (q, J ¼ 7.2 Hz, 2H),1.72 (m, 6H),1.49 (m,1H),1.27
(m, 4H), 1.06 (t, J ¼ 7.8 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 163.4,
152.3, 150.4, 137.3, 128.4, 127.9, 127.8, 116.2, 72.9, 71.8, 38.5, 35.1,
33.2, 26.2, 26.0, 19.4, 13.6; HRMS (ESIþ) calcd. for C21H29N2O3
[MþH]þ 357.2229, found 357.2228.
4.1.1. General procedures for synthesis of 11ae11m
to a suspension of paraformaldehyde (17 mg, 0.57 mmol, 1.3
equiv) in TMSCl (1.0 mL) was added substituted benzyl alcohol
(0.57 mmol, 1.3 equiv) at room temperature. The resulting sus-
pension was stirred until a clear solution was achieved, removed
solvent to give substituted aryl chloromethyl ether as oil, which can
be used for next step directly. To the solution of 10a (110 mg,
0.44 mmol, 1.0 equiv) in anhydrous DCM (5 mL), was added BSA
4.1.1.4. 6-(Cyclohexylmethyl)-5-ethyl-1-(((4-fluorobenzyl)oxy)
methyl)pyrimidine-2,4(1H,3H)-dione (11d). This compound was
prepared as a white solid following the procedure described for the
preparation of 11a. Yield: 63%; 1H NMR (600 MHz, CDCl3)
d 8.76 (s,
1H), 7.15 (t, J ¼ 8.4 Hz, 2H), 6.88 (t, J ¼ 8.4 Hz, 2H), 5.25 (s, 2H), 4.45
(s, 2H), 2.42 (d, J ¼ 7.2 Hz, 2H), 2.27 (q, J ¼ 6.6 Hz, 2H), 1.59 (m, 6 H),
1.38 (m, 1H), 1.03 (m, 4H), 0.91 (t, J ¼ 6.6 Hz, 3H); 13C NMR
(235 mM, 0.96 mmol, 2.2 equiv). The resulting reaction mixture was
stirred at rt for 1 h. Then the fresh made substituted chloro-methyl-
pheny ether and catalytic amount TBAI was added. This reaction
was stirred at rt overnight, quenched by adding saturated NaHCO3
solution and extracted with CH2Cl2 (3 ꢀ 10 mL). Combined the
organic phase, dried over Na2SO4, removed the solvent to give the
crude product which purified by combiflash (Hex/EtOAc, 3:1) to
give compound as white solid.
(150 MHz, CDCl3)
d 163.1, 152.1, 150.4, 133.0, 129.6, 116.2, 115.4,
115.2, 72.8, 71.0, 38.6, 35.1, 33.2, 26.3, 26.0, 19.4, 13.6; HRMS (ESIþ)
calcd. for C21H28FN2O3 [MþH]þ 375.2110, found 375.2109.
4.1.1.5. 6-(Cyclohexylmethyl)-1-((4-fluorophenethoxy)methyl)-3-
hydroxy-5-methylpyrimidine-2,4(1H,3H)-dione (11e). This com-
pound was prepared as a white solid following the procedure
described for the preparation of 11a. Yield: 53%; 1HNMR (600 MHz,
4.1.1.1. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-5-
CDCl3)
d
8.60 (s, 1H), 7.29 (t, J ¼ 6.6 Hz, 1H), 7.21 (m, 1H), 7.01 (t,
isopropylpyrimidine-2,4(1H,3H)-dione (11a). Yield: 90%; 1H NMR
J ¼ 7.8 Hz, 1H), 5.37 (s, 2H), 4.63 (s, 2H), 2.51 (d, J ¼ 6.0 Hz, 2H), 2.36
(q, J ¼ 7.2 Hz, 2H), 1.67 (m, 5H), 1.41 (m, 1H), 1.12 (m, 3H), 0.99 (m,
(600 MHz, CDCl3)
d 8.77 (s, 1H), 7.30 (m, 5H), 5.36 (s, 2H), 4.57 (s,
2H), 2.72 (m, 1H), 2.52 (d, J ¼ 7.2 Hz, 2H), 1.66 (m, 5H), 1.38 (m, 1H),
5H); 13C NMR (150 MHz, CDCl3)
d 162.1, 154.5, 151.1, 149.4, 129.5,

J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
173
Table 2
Biochemical and antiviral assay results of 3-deoxy precursor 11.
a
Compd
Structure
RT IC50
RNH
(m
M)
MAGI antiviral
b
c
Pol
EC50
(m
M)
CC50
(mM)
11a
>10
0.085 0.034
0.0052 0.0024
23 0.74
11b
>10
0.30 0.034
0.0072 0.0029
31 1.4
11c
11d
>10
>10
0.22 0.12
0.0062 0.00020
>50
0.48 0.089
0.0075 0.0010
42 2.7
11e
11f
>10
>10
1.5 0.50
0.048 0.029
32 0.67
0.82 0.014
0.014 0.0058
29 0.78
11g
11h
>10
>10
0.81 0.59
0.039 0.012
28 0.81
0.36 0.17
0.011 0.0035
30 1.30
11i
11j
>10
>10
6.8 2.0
0.065 0.017
>100
10
0.83 0.020
38 3.2
(continued on next page)

174
J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
Table 2 (continued )
a
Compd
Structure
RT IC50
RNH
(
m
M)
MAGI antiviral
b
c
Pol
10
EC50
(
m
M)
CC50 (mM)
11k
11l
>10
0.21 0.062
65 3.5
>10
10
0.80 0.11
>100
11m
>10
>10
>10
5.3 0.43
56 0.83
14
e
0.42 0.24
0.011 0.00010
58 2.4
a
Concentration of compound inhibiting the target enzyme by 50%.
Concentration of compound inhibiting virus replication by 50%.
Concentration of compound resulting in 50% cell death. All assay results expressed as mean standard deviation from at least two independent experiments.
b
c
Fig. 2. Crystal structure of HIV-1 RT in complex with 4a (PDB ID: 5TUQ). Analogue 4a (cyan sticks) binds at the NNRTI binding pocket of RT (Fingers, blue; Palm, red; Thumb, green;
Connection, yellow; RNase H, orange; p51, gray). Inset: cross-eyed stereo view of 4a at the NNRTI binding pocket with a 2.7 Å 2Fo-Fc electron density map at
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
s
¼ 1.0 in light blue. (For
127.5, 125.1, 123.5, 120.2, 115.3, 72.0, 64.5, 37.6, 34.1, 32.2, 25.3, 25.0,
18.5, 12.6; HRMS (ESIþ) calcd. for C21H27FClN2O3 [MþH]þ 409.1727,
found 409.1725.
4.1.1.7. 6-(Cyclohexylmethyl)-5-ethyl-1-(((4-(trifluoromethyl)benzyl)
oxy)methyl)-pyrimidine-2,4(1H,3H)-dione (11g). This compound
was prepared as a white solid following the procedure described for
the preparation of 11a. Yield: 47%; 1H NMR (600 MHz, CDCl3 d 8.22
(s, 1H), 7.53(d, J ¼ 8.4 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 5.36 (s, 2H),
4.63 (s, 2H), 2.52 (d, J ¼ 7.2 Hz 2H), 2.53 (q, J ¼ 7.2 Hz, 2H), 1.67 (m,
5H), 1.44 (m, 1H), 1.12 (m, 5H), 0.98 (t, J ¼ 7.8 Hz, 3H); 13C NMR
4.1.1.6. 6-(Cyclohexylmethyl)-1-(((3,4-difluorobenzyl)oxy)methyl)-5-
ethylpyrimidine-2,4(1H,3H)-dione (11f). This compound was pre-
pared as a white solid following the procedure described for the
(150 MHz, CDCl3) d 162.3,151.4,149.2,140.4,126.8,124.3,115.4, 71.9,
preparation of 11a. Yield: 70%; 1H NMR (600 MHz, CDCl3)
d
8.43 (s,
70.0, 37.6, 34.1, 25.3, 25.0, 18.4, 12.6; HRMS (ESIþ) calcd. for
C
22H28F3N2O3 [MþH]þ 425.2117, found 425.2115.
1H),
d 7.09 (m, 2H), 6.96 (m, 1H), 5.34 (s, 2H), 4.52 (s, 2H), 2.52 (d,
J ¼ 7.2 Hz, 2H), 2.37 (q, J ¼ 7.2 Hz, 2H), 1.68 (m, 5H), 1.43 (m,1H), 1.13
(m, 5H), 1.09 (t, J ¼ 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 163.3,
152.4, 150.2, 134.5, 123.6, 117.2, 117.1, 116.7, 116.5, 116.4, 72.7, 70.4,
4.1.1.8. 6-(Cyclohexylmethyl)-5-ethyl-1-(((4-methylbenzyl)oxy)
methyl)pyrimidine-2,4(1H,3H)-dione (11h). This compound was
prepared as a white solid following the procedure described for the
38.5, 35.1, 33.2, 26.3, 26.0, 19.4, 13.5; HRMS (ESIþ) calcd. for
C
21H27F2N2O3 [MþH]þ 393.2019, found 393.2022.

J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
175
Fig. 3. Structures of reported HEPT-based NNRTIs 12e15. Precursor 11 closely mimics 14 and could be a scaffold for novel NNRTIs.
preparation of 11a. Yield: 65%; 1HNMR (600 MHz, CDCl3)
d
8.01 (s,
4.1.1.12. 6-(Cyclohexylmethyl)-5-methyl-1-(((4-(trifluoromethyl)
benzyl)oxy)methyl) pyrimidine-2,4(1H,3H)-dione (11l). Yield: 43%;
This compound was prepared as a white solid following the pro-
cedure described for the preparation of 11a; 1HNMR (600 MHz,
1H), 7.13 (d, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz, 2H), 5.31 (s, 2H), 4.52
(s, 2H), 2.49 (d, J ¼ 7.2 Hz, 2H), 2.32 (q, J ¼ 6.6 Hz, 2H), 2.26 (s, 3H),
1.65 (m, 6H), 1.41 (m, 1H), 1.09 (m, 4H), 0.99 (t, J ¼ 7.2 Hz, 3H); 13
C
NMR (150 MHz, CDCl3)
d
163.4, 152.3, 150.4, 137.7, 134.2, 129.1,
CDCl3)
d
8.42 (s, 1H), 7.54 (d, J ¼ 8.4 Hz, 2H), 7.37 (d, J ¼ 7.2 Hz, 2H),
128.0, 116.2, 72.7, 71.6, 38.6, 35.0, 33.1, 26.3, 26.0, 21.1, 19.4, 13.6;
HRMS (ESIþ) calcd. for C22H31N2O3 [MþH]þ 371.2390, found
371.2387.
5.36 (s, 2H), 4.62 (s, 2H), 2.54 (d, J ¼ 7.2 Hz, 2H), 1.87 (s, 3H), 1.67 (m,
5H),1.42 (m,1H), 1.13 (m, 5H); HRMS (ESIþ) calcd. for C21H26F3N2O3
[MþH]þ 411.1955, found 411.1954.
4.1.1.9. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (11i). This compound was pre-
pared as a white solid following the procedure described for the
preparation of 11a. Yield: 33%; 1HNMR (600 MHz, CDCl3) 8.58 (s,
1H), 7.27 (m, 5H), 5.34 (s, 2H), 4.56 (s, 2H), 2.53 (d, J ¼ 7.2 Hz, 2H),
4.1.1.13. 6-(Cyclohexylmethyl)-1-((4-fluorophenethoxy)methyl)-5-
methylpyrimidine-2,4(1H,3H)-dione (11m). Yield: 50%; This com-
pound was prepared as a white solid following the procedure
described for the preparation of 11a; 1HNMR (600 MHz, CDCl3)
d
8.20 (s, 1H), 7.18 (t, J ¼ 7.2 Hz, 2H), 6.95 (J ¼ 8.4 Hz, 2H), 5.24 (s,
1.85 (s, 3H), 1.65 (m, 7H), 1.43 (m, 1H), 1.11 (m, 3H), 0.98 (m, 3H); 13
C
2H), 3.70 (t, J ¼ 6.0 Hz, 2H), 2.75 (t, J ¼ 6.0 Hz, 2H), 2.40 (d,
J ¼ 6.6 Hz, 2H), 1.84 (s, 3H), 1.67 (m, 2H), 1.61 (m, 4H), 1.41 (m, 1H),
1.15 (m, 2H), 0.99 (m, 2H); HRMS (ESIþ) calcd. for C21H28FN2O3
[MþH]þ 375.2140, found 375.2141.
NMR (150 MHz, CDCl3)
d 160.8, 149.2, 148.4, 134.6, 125.8, 125.4,
125.2, 107.6, 70.1, 69.0, 35.9, 33.1, 30.7, 23.6, 23.4, 9.2; HRMS (ESIþ)
calcd. for C20H27N2O3 [MþH]þ 343.2059, found 343.2066.
4.1.1.10. 1-(((3-Chloro-2-fluorobenzyl)oxy)methyl)-6-(cyclo-
4.1.2. General procedures for synthesis of 4ae4m
hexylmethyl)-5-methylpyrimidine-2,4(1H,3H)-dione
(11j). This
To a solution of 11a (60 mg, 0.162 mmol) in 3 mL of THF was
added NaH (60% dispension in merinal oil) (65 mg, 1.62 mmol) at
compound was prepared as a white solid following the procedure
described for the preparation of 11a. Yield: 62%; 1HNMR (600 MHz,
0
ꢂC. This reaction mixture was allowed to warm to room tem-
CDCl3)
d
8.10 (s, 1H), 7.29 (m, 1H), 7.21 (m, 1H), 7.01 (t, J ¼ 7.2 Hz,
perature over 1 h., and then cooled to 0 ꢂC followed by the addition
of m-CPBA (223 mg, 1.3 mmol). After stirring for 0.5 h, this reaction
mixture was stirred at rt overnight. The reaction was quenched by
adding 10 mL of H2O, the aqueous phase was then extracted with
ethyl acetate (10 mL ꢀ 3). The combined organic extracts were
dried over Na2SO4 and concentrated under reduced pressure. The
resultant residue was subjected to combiflash (Hex/EtOAc, 1:1) to
give compound 4a (42 mg, 70%).
1H), 5.37 (s, 2H), 4.62 (s, 2H), 2.53 (d, J ¼ 7.2 Hz, 2H), 1.87 (s, 3H),
1.67 (m, 5H), 1.44 (m, 1H), 1.12 (m, 3H), 1.00 (m, 2H); 13C NMR
(150 MHz, CDCl3)
d 162.7, 151.2, 149.8, 129.4, 127.5, 125.0, 123.5,
120.0109.4, 71.8, 64.4, 37.4, 34.6, 32.2, 25.2, 25.0, 10.8; HRMS (ESIþ)
calcd. for C20H25FClN2O3 [MþH]þ 395.1599, found 395.1589.
4.1.1.11. 6-(Cyclohexylmethyl)-1-(((3,4-difluorobenzyl)oxy)methyl)-
5-methylpyrimidine-2,4(1H,3H)-dione (11k). This compound was
prepared as a white solid following the procedure described for the
4.1.2.1. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-3-hydroxy-5-
preparation of 11a. Yield: 52%; 1HNMR (600 MHz, CDCl3)
d8.82 (s,
isopropylpyrimidine-2,4(1H,3H)-dione (4a). Yield: 70%; 1H NMR
1H), 7.08 (m, 2H), 6.97 (s, 1H), 5.35 (s, 2H), 4.41 (s, 2H), 2.54 (d,
J ¼ 7.2 Hz, 2H), 1.89 (s, 3H), 1.67 (m, 6H), 1.48 (m, 1H), 1.13 (m, 4H);
HRMS (ESIþ) calcd. for C20H25F2N2O3 [MþH]þ 379.1888, found
379.1880.
(600 MHz, CDCl3) d 7.22 (m, 5H), 5.41 (s, 2H), 4.57 (s, 2H), 2.72 (m,
1H), 2.53 (d, J ¼ 6.0 Hz, 2H), 1.63 (m, 5H), 1.37 (m, 1H), 1.24 (d,
J ¼ 6.0 Hz, 6H), 1.39 (m, 3H), 0.94 (m, 2H); 13C NMR (150 MHz,
CDCl3)
d 162.2, 152.2, 150.0, 137.3, 128.4, 127.9, 127.8, 118.9, 72.9,

176
J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
71.7, 38.6, 34.9, 33.0, 28.6, 26.3, 26.0, 20.2; HRMS (ESIþ) calcd. for
CDCl3) d 160.2, 148.3, 137.5, 134.3, 133.6, 129.0, 128.0, 114.5, 73.7,
C
22H31N2O4 [MþH]þ 387.2269, found 387.2272.
71.6, 38.4, 35.0, 33.1, 26.2, 26.0, 21.1, 19.9, 13.2; HRMS (ESIþ) calcd.
for C22H31N2O4 [MþH]þ 387.4846, found 387.4852.
4.1.2.2. 6-(Cyclohexylmethyl)-1-(((4-fluorobenzyl)oxy)methyl)-3-
hydroxy-5-isopropylpyrimidine-2,4(1H,3H)-dione (4b). This com-
pound was prepared following the procedure described for the
4.1.2.9. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-3-hydroxy-5-
methylpyrimidine-2,4(1H,3H)-dione (4i). This compound was pre-
pared following the procedure described for the preparation of 4a.
Yield: 77%; 1HNMR (600 MHz, CDCl3) 7.23 (m, 5H), 5.43 (s, 2H), 4.57
(s, 2H), 2.53 (d, J ¼ 6.0 Hz, 2H),1.90 (s, 3H),1.64 (m, 5H),1.41 (m,1H),
1.10 (m, 3H), 0.96 (m, 2H); HRMS (ESIþ) calcd. for C20H27N2O4
[MþH]- 358.2022, found 358.2025.
preparation of 4a. Yield: 70%; 1H NMR (600 MHz, CDCl3)
d 7.31 (t,
J ¼ 6.6 Hz, 2H), 7.03 (t, J ¼ 8.4 Hz, 2H), 5.50 (s, 2H), 4.62 (s, 2H), 2.88
(m, 1H), 2.63 (d, J ¼ 7.2 Hz, 2H), 1.75 (m, 2H), 1.68 (m, 3H), 1.46 (m,
1H), 1.31 (d, J ¼ 6.6 Hz, 6H), 1.20 (m, 3H), 1.06 (m, 2H); HRMS (ESIþ)
calcd. for C22H30FN2O4 [MþH]þ 405.2227, found 405.2237.
4.1.2.3. 1-((Benzyloxy)methyl)-6-(cyclohexylmethyl)-5-ethyl-3-
hydroxypyrimidine-2,4(1H,3H)-dione (4c). This compound was
prepared following the procedure described for the preparation of
4.1.2.10. 1-(((3-Chloro-2-fluorobenzyl)oxy)methyl)-6-(cyclo-
hexylmethyl)-3-hydroxy-5-methylpyrimidine-2,4(1H,3H)-dione (4j).
This compound was prepared following the procedure described
for the preparation of 4a. Yield: 62%; 1HNMR (600 MHz, CDCl3)
4a. Yield: 74%; 1HNMR (600 MHz, CDCl3)
d 7.22 (m, 5H), 5.39 (s, 2H),
4.57 (s, 2H), 2.51 (d, J ¼ 6.6 Hz, 2H), 2.34 (q, J ¼ 7.2 Hz, 2H), 1.64 (m,
d
7.29 (t, J ¼ 7.2 Hz, 1H), 7.21 (m, 1H), 6.99 (t, J ¼ 7.2 Hz, 1H), 5.44 (s,
2H), 4.64 (s, 2H), 2.54 (d, J ¼ 6.6 Hz, 2H), 1.93 (s, 3H), 1.65 (m, 5H),
1.42 (m, 1H), 1.11 (m, 5H); 13C NMR (150 MHz, CDCl3)
159.2, 157.2,
6H), 1.42 (m, 1H), 1.09 (m, 3H), 0.98 (m, 4H); HRMS (ESIþ) calcd. for
C
21H29N2O4 [MþH]þ 373.4580, found 373.4586.
d
155.5, 148.7, 148.3, 130.6, 128.5, 125.9, 124.5, 108.3, 73.7, 65.9, 38.5,
35.5, 33.2, 26.2, 25.9, 12.1; HRMS (ESIþ) calcd. for C20H25ClFN2O4
[MþH]þ 411.1542, found 411.1550.
4.1.2.4. 6-(Cyclohexylmethyl)-5-ethyl-1-(((4-fluorobenzyl)oxy)
methyl)-3-hydroxypyrimidine-2,4(1H,3H)-dione (4d). This com-
pound was prepared following the procedure described for the
preparation of 4a. Yield: 66%; 1H NMR (600 MHz, CDCl3)
d
8.84 (s,
4.1.2.11. 6-(Cyclohexylmethyl)-1-(((3,4-difluorobenzyl)oxy)methyl)-
3-hydroxy-5-methyl pyrimidine-2,4(1H,3H)-dione (4k). This com-
pound was prepared following the procedure described for the
1H), 7.22 (t, J ¼ 6.6 Hz, 2H), 6.94 (m, 2H), 5.39 (s, 2H), 4.53 (s, 2H),
2.50 (d, J ¼ 6.0 Hz, 2H), 2.36 (m, 2H), 1.65 (m, 5H), 1.40 (m, 1H), 1.12
(m, 5H), 0.98 (t, J ¼ 7.2 Hz, 3H); HRMS (ESIþ) calcd. for C21H28FN2O4
[MþH]þ 391.4485, found 391.4469.
preparation of 4a. Yield: 65%; 1HNMR (600 MHz, CDCl3)
d 7.10 (m,
2H), 6.97 (m, 2H), 5.41 (s, 2H), 4.53 (s, 2H), 2.54 (d, J ¼ 6.0 Hz, 2H),
1.94 (s, 3H), 1.67 (m, 6H), 1.44 (m, 1H), 1.18 (m, 4H); HRMS (ESIþ)
calcd. for C20H25F2N2O4 [MþH]þ 395.1832, found 395.1838.
4.1.2.5. 1-(((3-Chloro-2-fluorobenzyl)oxy)methyl)-6-(cyclo-
hexylmethyl)-5-ethyl-3-hydroxypyrimidine-2,4(1H,3H)-dione (4e).
This compound was following the procedure described for the
4.1.2.12. 6-(Cyclohexylmethyl)-3-hydroxy-5-methyl-1-(((4-(tri-
fluoromethyl)benzyl)oxy) methyl)pyrimidine-2,4(1H,3H)-dione (4l).
This compound was prepared following the procedure described
for the preparation of 4a. Yield: 58%; 1HNMR (600 MHz, CDCl3)
preparation of 4a. Yield: 68%; 1HNMR (600 MHz, CDCl3)
d 7.26 (m,
1H), 7.19 (m, 1H), 6.98 (m, 1H), 5.42 (s, 2H), 4.63 (s, 2H), 2.51 (d,
J ¼ 7.2 Hz, 2H), 2.36 (m, 2H), 1.64 (m, 5H), 1.41 (m, 1H), 1.11 (m, 3H),
0.99 (m, 5H); 13C NMR (150 MHz, CDCl3)
d
162.0, 156.1, 151.0, 149.4,
d
8.64 (s, 1H), 7.52 (d, J ¼ 7.2 Hz, 2H), 7.37 (d, J ¼ 7.8 Hz, 2H), 5.45 (s,
129.5, 127.5, 125.1, 123.5, 120.1, 115.3, 72.0, 64.5, 37.6, 34.0, 32.2,
25.3, 25.0,18.5, 12.6, HRMS (ESIþ) calcd. for C21H27FClN2O4 [MþH]þ
425.1680, found 425.1681.
2H), 4.64 (s, 2H), 2.56 (d, J ¼ 6.6 Hz, 2H), 1.94 (s, 3H), 1.65 (m, 2H),
1.61 (m, 2H), 1.46 (m, 1H), 1.11 (m, 4H), 0.99 (m, 2H); HRMS (ESIþ)
calcd. for C21H26FN2O4 [MþH]þ 427.1733, found 427.1735.
4.1.2.6. 6-(Cyclohexylmethyl)-1-(((3,4-difluorobenzyl)oxy)methyl)-5-
ethyl-3-hydroxypyrimidine-2,4(1H,3H)-dione (4f). This compound
was prepared as a white solid following the procedure described for
4.1.2.13. 6-(Cyclohexylmethyl)-1-((4-fluorophenethoxy)methyl)-3-
hydroxy-5-methyl pyrimidine-2,4(1H,3H)-dione (4m). This com-
pound was prepared following the procedure described for the
the preparation of 4a. Yield: 63%; 1HNMR (600 MHz, CDCl3)
d
7.03
preparation of 4a. Yield: 68%; 1HNMR (600 MHz, CDCl3)
d 7.03 (m,
(m, 2H), 6.96 (m,1H), 5.41 (s, 2H), 4.54 (s, 2H), 2.52 (m, 2H), 2.39 (m,
2H), 1.65 (m, 2H), 1.60 (m, 3H), 1.42 (m, 1H), 1.09 (m, 3H), 1.00 (m,
5H); HRMS (ESIþ) calcd. for C21H27F2N2O4 [MþH]þ 409.4389, found
409.4394.
2H), 6.87 (t, J ¼ 7.2 Hz, 2H), 5.32 (s, 2H), 3.72 (t, J ¼ 6.0 Hz, 2H), 2.73
(t, J ¼ 6.0 Hz, 2H), 2.41 (d, J ¼ 6.0 Hz, 2H), 1.91 (s, 3H), 1.65 (m, 5H),
1.40 (m, 1H), 1.06 (m, 3H), 0.96 (m, 2H); HRMS (ESIþ) calcd. for
C
21H28FN2O4 [MþH]þ 391.2089, found 391.2093.
4.1.2.7. 6-(Cyclohexylmethyl)-5-ethyl-3-hydroxy-1-(((4-(tri-
fluoromethyl)benzyl)oxy) methyl)pyrimidine-2,4(1H,3H)-dione (4g).
This compound was prepared following the procedure described
for the preparation of 4a. Yield: 60%; 1H NMR (600 MHz, CDCl3)
4.2. Biology
4.2.1. RNase H assay
RNase
H activity was measured essentially as previously
d
7.52 (d, J ¼ 7.8 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 5.45 (s, 2H), 4.66 (s,
described [26]. Full-length HIV RT was incubated with the RNA/
DNA duplex substrate HTS-1 (RNA 50-gaucugagccugggagcu-30-
fluorescein annealed to DNA 30-CTAGACTCGGACCCTCGA-50-
Dabcyl), a high sensitivity duplex that assesses non-specific inter-
nal cleavage of the RNA strand. Results were analyzed using
GraphPad Prism (GraphPad Software, San Diego, CA) for nonlinear
regression to fit dose-response data to logistic curve models.
2H), 2.54 (d, J ¼ 7.2 Hz 2H), 2.41 (q, J ¼ 7.8 Hz, 2H), 1.66 (m, 5H), 1.41
(m, 1H), 1.09 (m, 5H), 0.98 (t, J ¼ 7.8 Hz, 3H); HRMS (ESI-) calcd. for
C
22H26F3N2O4 [M-H]- 439.1744, found 439.1745.
4.1.2.8. 6-(Cyclohexylmethyl)-5-ethyl-3-hydroxy-1-(((4-
methylbenzyl)oxy)methyl) pyrimidine-2,4(1H,3H)-dione (4h).
This compound was prepared following the procedure described
for the preparation of 4a. Yield: 71%; 1HNMR (600 MHz, CDCl3)
4.2.2. RT pol assay
d
7.11 (d, J ¼ 7.2 Hz, 2H), 7.03 (d, J ¼ 7.2 Hz, 2H), 5.36 (s, 2H), 4.52 (s,
RT pol assays were carried out in 96-well plates by measuring
the extension of an 18 nucleotide DNA primer (50-GTCACTGTTC-
GAGCACA-30) annealed to a 100 nucleotide DNA template (50-
2H), 2.45 (d, J ¼ 7.2 Hz, 2H), 2.28 (q, J ¼ 6.6 Hz, 2H), 2.23 (s, 3H), 1.65
(m, 6H), 1.35 (m, 1H), 1.06 (m, 3H), 0.91 (m, 4H); 13C NMR (150 MHz,

J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
177
ATGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCA-
4.2.4. Antiviral MAGI assays
GACCCTTTTAGTCAGTGTGGAATATCTCATAGCTTGGTGCTCGAA-
MAGI assays were carried out using P4R5 indicator cells
essentially as previously described [16]. P4R5 cells were cultured in
96-well microplates with 4 ꢀ 103 cells per well and maintained in
CAGTGAC-30). Reactions containing 20 nM RT, 40 nM template/
primer, and 10
mM deoxynucleotide triphosphates (dNTPs) in a
buffer containing 50 mM Tris (pH 7.8) and 50 mM NaCl were
initiated by the addition of 6 mM MgCl2. Reactions contained 1%
DMSO and increasing concentrations of compounds. DNA synthesis
was carried out for 30 min at 37 ꢂC, and reactions were arrested by
the addition of 100 mM EDTA. The QuantiFluor dsDNA System
(Promega) was used to quantify the amount of formed double-
stranded DNA. Reactions were read at ex/em 504/531 nm in a
PerkinElmer EnSpire Multilabel plate reader. Results were analyzed
using “Prism” software (GraphPad Software, San Diego, CA) for
nonlinear regression to fit dose-response data to logistic curve
models.
DMEM/10% FBS supplemented with puromycin (1 mg/mL). Cells
were incubated with either 1% DMSO or varying concentrations of
the drugs for 24 h and then exposed to HIV (MOI of 1.25) followed
by an additional incubation period of 48 h. The extent of infection
was assessed using a fluorescence-based b-galactosidase detection
assay, as previously described with minor modifications [30]. After
the 48 h incubation period, cells were lysed and substrate 4-
methylumbelliferyl-galactoside (MUG) was added. The b-galacto-
sidase produced during infection acts on the substrate MUG and
yields a fluorescent product 4-methylumbelliferone (4-MU) that
could be detected fluorimetrically with excitation wavelength
365 nm and emission wavelength 446 nm.
4.2.5. HIV-1 cytoprotection assay [17]
4.2.3. INST assay
The HIV cytoprotection assay used CEM-SS cells and the IIIB
strain of HIV-1. Briefly, virus and cells were mixed in the presence of
test compound and incubated for 6 days. The virus was pre-titered
such that control wells exhibit 70e95% loss of cell viability due to
virus replication. Therefore, antiviral effect or cytoprotection was
observed when compounds prevent virus replication. Each assay
plate contained cell control wells (cells only), virus control wells
(cells plus virus), compound toxicity control wells (cells plus
compound only), compound colorimetric control wells (compound
only) as well as experimental wells (compound plus cells plus vi-
rus). Cytoprotection and compound cytotoxicity were assessed by
MTS (CellTiter® 96 Reagent, Promega) and the EC50 (concentration
inhibiting virus replication by 50%), CC50 (concentration resulting
HIV integrase was expressed and purified as previously reported
[10]. Inhibition assays were performed using a modified protocol of
our reported method 1[10]. Briefly, 2.1
mL of compound suspended
in DMSO was placed in duplicate into a Black 96 well non-binding
plate (Corning). Compounds were plated in duplicate to a final
concentration of 0.13e100
reaction mixture without DNA substrate was added (10 mM HEPES
pH 7.5, 10% glycerol w/v, 10 mM MnCl2, 1 mM DTT, 1 M integrase).
The enzyme was incubated with inhibitor for 10 min at 25 ꢂC after
mM. To each well of the plate 186.9 mL of
m
which the reaction was initiated by the addition of 21 mL of 500 nM
oligo (50 biotin ATGTGGAAAATCTCTAGCA annealed with ACTGC-
TAGAGATTTTCCACAT 30 Cy5). Reactions were incubated at 37 ꢂC for
30 min and then quenched by the addition of 5.2
Each reaction was moved (200 L) to a MultiScreen HTS PCR plate
(Millipore) containing 20 L streptavidin agarose beads (Life
m
L 500 mM EDTA.
in 50% cell death) and a calculated TI (therapeutic index CC50/EC50
were provided. Each assay included AZT as a positive control.
)
m
m
Technologies) and incubated with shaking for 30 min. A vacuum
manifold was used to remove the reaction mixture and the beads
were similarly washed 3 times with wash buffer (0.05% SDS, 1 mM
4.2.6. HIV-1 RT/4a crystallization and data collection
HIV-1 RT containing C280S mutations in the p66 and p51 sub-
units was expressed and purified as previously described [31,32].
Co-crystallization of HIV-1 RT with 4a was achieved by mixing a
solution of RT at a final concentration of 11 mg/mL, 50 mM MgCl2,
EDTA in PBS). The plates were further washed 3 times with 200 mL
50 mM NaOH to denature DNA not covalently linked to the biotin
modification. For each denaturation step the plate was incubated
with shaking at 25 ꢂC for 5 min and the NaOH was removed by
centrifugation at 1000g for 1 min. The reaction products were
5 mM tris(2-carboxyethyl)phosphine (TCEP) HCl, 0.5% b-octylglu-
coside, and 1 mM 4a in a 1:1 ratio with a solution of 15% PEG 3500,
0.1 M sodium potassium phosphate, 5% ethylene glycol, and 0.1 M
MES pH 6.1. Large, blocky crystals grew by hanging drop vapor
diffusion at 18 ꢂC in 2e3 days.
eluted from the beads by the addition of 150 mL formamide. The
plate was incubated at 25 ꢂC for 10 min and read directly at 635/675
in a SpectraMax i3 plate reader (Molecular Devices).
HIV-1 RT/4a co-crystals were additionally soaked with 1 mM 4a,
Expression and purification of the recombinant IN in Escherichia
coli were performed as previously reported [27,28] with addition of
10% glycerol to all buffers. Preparation of oligonucleotide substrates
has been described [29]. Integrase reactions were performed in
5 mM TCEP HCl, 0.5% b-octylglucoside, 8% ethylene glycol, and
50 mM MgCl2 for 20 min before brief cryoprotection in a solution
containing 23% ethylene glycol and 4% trimethylamine N-oxide,
followed by fast cryo-cooling in liquid nitrogen. Data collected at
Beamline 23-ID-B of the Advanced Photon Source at Argonne Na-
tional Laboratory was processed and scaled to 2.7 Å resolution
using XDS [33]. The HIV-1 RT/4a crystals were of space group C2,
and had unit cell dimensions of a ¼ 161.9 Å, b ¼ 72.4 Å, c ¼ 107.3 Å
10 m
L with 400 nM of recombinant IN, 20 nM of 50-end [32P]-labeled
oligonucleotide substrate and inhibitors at various concentrations.
Solutions of 10% DMSO without inhibitors were used as controls.
Reactions were incubated at 37 ꢂC (60 min) in buffer containing
50 mM MOPS pH 7.2, 7.5 mM MgCl2, and 14.3 mM 2-
and
b
¼ 99.9ꢂ. One RT molecule was present in the asymmetric unit,
mercaptoethanol. Reactions were stopped by addition of 10
mL of
and the Matthews coefficient was 2.7 Å3/Da, corresponding to a
solvent content of 54.8% [34]. Crystal data and statistics are listed in
SI Table 1.
loading dye (10 mM EDTA, 98% deionized formamide, 0.025%
xylene cyanol and 0.025% bromophenol blue). Reactions were then
subjected to electrophoresis in 20% polyacrylamidee7 M urea gels.
Gels were dried and reaction products were visualized and quan-
titated with a Typhoon 8600 (GE Healthcare). Densitometric ana-
lyses were performed using ImageQuant from Molecular Dynamics
Inc. The concentrations at which enzyme activity was reduced by
50% (IC50) were determined using “Prism” software (GraphPad
Software) for nonlinear regression to fit dose-response data to lo-
gistic curve models.
4.3. Phase determination and structure refinement
The structure was determined by molecular replacement using
Phaser [35], using a high resolution crystal structure of an HIV-1 RT/
NNRTI complex (Protein Data Bank code 4G1Q) as the initial search
model [36]. Rigid-body, simulated annealing (to remove bias),
atomic displacement parameter (ADP), real-space and restrained

178
J. Tang et al. / European Journal of Medicinal Chemistry 128 (2017) 168e179
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Acknowledgments
This research was supported by the National Institutes of Health
(AI100890) and partially by the Center for Drug Design, University
of Minnesota. Use of the Advanced Photon Source, an Office of
Science User Facility operated for the U.S. Department of Energy
(DOE) Office of Science by Argonne National Laboratory, was sup-
ported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.01.041.
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Abbreviations
HPD
HIV
RT
3-Hydroxypyrimidine-2,4-dione
human immunodeficiency virus
reverse transcriptase
INST
Pol
integrase strand transfer
polymerase
NNRTI nonnucleoside RT inhibitor
HAART highly active antiretroviral therapy
NRTI
PIs
nucleoside reverse transcriptase inhibitor
protease inhibitors
CDI
carbonyldiimidazole
mCPBA meta-chloroperoxybenzoic acid
HEPT
SAR
1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine
structure-activity-relationship
dNTP
MUG
4-MU
TCEP
ADP
deoxynucleotide triphosphate
4-methylumbelliferyl-galactoside
4-methylumbelliferone
tris(2-carboxyethyl)phosphine
atomic displacement parameter
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small molecule HIV-1 RNase H inhibitors, Curr. Med. Chem. 21 (17) (2014)
1956e1967.
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