Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated RNase H

Article abstract of DOI:10.1016/j.ejmech.2017.09.054

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only HIV enzymatic function not targeted by current antiviral drugs. Although various chemotypes have been reported to inhibit HIV RNase H, few have s

Full text of DOI:10.1016/j.ejmech.2017.09.054

Accepted Manuscript
Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-
diones as inhibitors of HIV reverse transcriptase-associated RNase H
Jayakanth Kankanala, Karen A. Kirby, Andrew D. Huber, Mary C. Casey, Daniel J.
Wilson, Stefan G. Sarafianos, Zhengqiang Wang
PII:
S0223-5234(17)30769-9
10.1016/j.ejmech.2017.09.054
EJMECH 9769
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 August 2017
Revised Date: 13 September 2017
Accepted Date: 25 September 2017
Please cite this article as: J. Kankanala, K.A. Kirby, A.D. Huber, M.C. Casey, D.J. Wilson, S.G.
Sarafianos, Z. Wang, Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-
diones as inhibitors of HIV reverse transcriptase-associated RNase H, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.054.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluations of N-Hydroxy
thienopyrimidine-2,4-diones as Inhibitors of HIV Reverse
Transcriptase-Associated RNase H
1
2,3
2,4
2,3
Jayakanth Kankanala , Karen A. Kirby , Andrew D. Huber , Mary C. Casey , Daniel J.
1
2,3,5
1
Wilson , Stefan G. Sarafianos
and Zhengqiang Wang*
1
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, 65211, USA
2
3
Department of Molecular Microbiology & Immunology, School of Medicine, University of Missouri, Columbia, Missouri, 65211, USA
4
5
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, 65211, USA
Department of Biochemistry, University of Missouri, Columbia, Missouri, 65211, USA
Abstract
Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H
(RNase H) is the only HIV enzymatic function not targeted by current antiviral drugs. Although
various chemotypes have been reported to inhibit HIV RNase H, few have shown significant
antiviral activities. We report herein the design, synthesis and biological evaluation of a novel N-
hydroxy thienopyrimidine-2,3-dione chemotype (11) which potently and selectively inhibited
RNase H with considerable potency against HIV-1 in cell culture. Current structure-activity-
relationship (SAR) identified analogue 11d as a nanomolar inhibitor of RNase H (IC50 = 0.04
µM) with decent antiviral potency (EC50 = 7.4 µM) and no cytotoxicity (CC50 > 100 µM). In
extended biochemical assays compound 11d did not inhibit RT polymerase (pol) while inhibiting
integrase strand transfer (INST) with 53 fold lower potency (IC50 = 2.1 µM) than RNase H

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inhibition. Crystallographic and molecular modeling studies confirmed the RNase H active site
binding mode.
Keywords: N-Hydroxy thienopyrimidine-2,4-diones; Human Immunodeficiency Virus (HIV);
RNase H; integrase strand transfer; RT-Polymerase, molecular modeling and crystallography.
Introduction
HIV infection remains a major epidemic and a global healthcare threat. As of 2015, an estimated
3
6.7 million people worldwide are living with HIV [1]. The standard therapy for HIV infection,
highly active antiretroviral therapy (HAART) [2], consists of inhibitors of all three HIV encoded
enzymes: protease, RT and integrase (IN). While HAART has proven to effectively suppress
viral replication, the required long term HAART treatment can be plagued by adverse effects and
drug resistance. This is precisely why even with a large number of FDA-approved drugs there is
still a pressing need for improved antivirals, particularly those with novel and distinct
mechanisms of action [3, 4] to combat drug resistance. Of all FDA-approved HIV drugs,
inhibitors of RT comprise two distinct classes, nucleoside RT inhibitors (NRTIs) [5] and non-
nucleoside RT inhibitors (NNRTIs) [6], and are of particular importance for HAART. RT has
two distinct active sites: an N-terminal DNA pol active site and a C-terminal RNase H active
site. Interestingly, currently known NRTIs and NNRTIs all target the DNA pol function of RT,
and inhibitors of RT RNase H have yet to enter the development pipeline. Therefore, RT-
associated RNase H represents a novel target for HIV drug development and provides an
attractive approach to treating HIV strains resistant to current drugs [7-9].
The active site of RNase H features four carboxylic residues (DEDD) interacting with two metal
2
+
ions (Mg ) in close proximity. Chemical inhibition of RNase H at the active site thus minimally
2
+
entails a chelating triad capable of chelating the two Mg ions [3]. Several types of active site

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inhibitors of RT-associated RNase H have been reported in the literature (Figure 1A). These
include diketoacids (1) [10], 2-hydroxyisoquinoline-1,3-diones (HID, 2) [11-13], hydroxylated
tropolones (3) [14], pyrimidinol carboxylic acids (4) [3], hydroxypyridone carboxylic acids (5)
[15], hydroxynaphthyridines (6) [16], and pyridopyrimidones (7) [17]. Notably, all these
chemotypes feature a metal chelator and a peripheral hydrophobic moiety. A similar
pharmacophore is required for the inhibition of HIV IN. Therefore, most reported RNase H
inhibitors also tend to inhibit IN, making achievement of selective biochemical inhibition of HIV
RNase H over IN challenging. We have long been interested in designing potent and selective
RNase H inhibitors [12, 13, 15, 18-20] particularly with the HID (2) [12, 13] and 3-
hydroxypyrimidine-2,4-dione (HPD) (8) [18-20] chemotypes. Hybridization of these two
scaffolds led to the design of chemotype 9 (Figure 1B) which is similar to the N-hydroxyurea
reported as an inhibitor type of flap endonuclease-1 [21]. Bioisosteric replacement of the phenyl
ring of 9 with a thiophene ring yielded chemotypes 10 and 11 (Figure 1B). The latter (11) proved
to inhibit RNase H potently and selectively in our biochemical assays and reduce viral
replication significantly in our cell-based assay. We report herein the chemical synthesis,
biochemical, antiviral and structural evaluations of inhibitor type 11.
[
Insert Figure 1 here]
Results and Discussion
Chemistry. The initial HID analogue 14 was prepared in two steps according to reported
procedures (Scheme 1A) starting from iodohomophthalic acid (12) [12, 22, 23]. The introduction
of the phenyl group was achieved via Suzuki coupling. The resulting diacid intermediate 13 was
cyclized by treating with O-tetrahydropyran (THP) protected hydroxylamine in the presence of
carbonyldiimidazole (CDI) followed by the deprotection of THP using p-toluenesulfonic acid (p-

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TSA) to afford the desired product (14). The synthesis of scaffolds 9, 19, and 10 all featured a
key base-promoted cyclization (step c) from an ortho amino ester intermediate (16, 18, 20,
Scheme 1B–D) to construct the hydroxyurea moiety. The cyclization presumably involves three
sequential events: first the amino group of the intermediate (e.g. 16) reacted with CDI to form an
imidazole urea intermediate; second, the imidazole ring of the urea intermediate was replaced by
a protected hydroxylamine to form a hydroxyurea intermediate; and finally upon treatment of
NaOH the deprotonated hydroxyurea reacted with the ortho ester group to produce cyclized
hydroxyureas which upon deprotection afforded desired compounds of chemotypes 9, 19, and 10
(Scheme 1). The key intermediate 16 was synthesized via Suzuki coupling of arylboronic acid
with methyl anthranilate (15). The intermediate 18 [24] was prepared by treating 1-benzyl-3-
oxo-piperidine-4-carboxylic acid ethyl ester hydrochloride (17) with an excess amount of
ammonium acetate in refluxing ethanol. Intermediates 20 are all commercially available.
[
Insert Scheme 1 here]
The synthesis of chemotype 11 also involved a base-promoted cyclization (step b, Scheme 2)
from ortho amino ester intermediates (22, 24 and 25). Intermediates 22 and 24 were prepared via
Gewald reaction [25, 26] which involves condensation of appropriate aldehydes (20) or ketones
(
22) with ethyl cyanoacetate in presence of an elemental sulfur and base (Scheme 2A). All
intermediates 25 are commercially available.
Insert Scheme 2 here]
[
Biology. All final compounds were first tested in a biochemical assay for inhibition against HIV
RT-associated RNase H. HIV RNase H cleaves viral RNA at multiple stages of reverse
transcription with at least three distinct modes [12]: random internal cleavages, DNA 3′ end-
directed and polymerase dependent cleavages, and RNA 5′ end-directed cleavages. In the current

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work our biochemical assay used the HTS-1 RNA/DNA substrate to specifically probe random
internal cleavage, which is believed to be the dominant mode of RNA cutting. Importantly, due
to the spatial and temporal relationship between the RNase H and pol domains [27], inhibition of
RNase H is often accompanied by the inhibition of RT pol. In addition, RNase H shares a similar
active site fold and mechanism of catalysis with HIV IN [28]. Therefore, we have included
biochemical assays measuring the activity of RT pol and INST reactions to gauge the selectivity
of our compounds toward RNase H inhibition. Finally, all analogues were also tested for
cytotoxicity and antiviral potency in a cell-based MAGI assay [29]. Previously reported HIV
RNase H inhibitor trihydroxybenzoyl naphthyl hydrazone (THBNH) [30] was used as control in
all RT assays and the FDA-approved INSTI, dolutegravir (DTG) [31] was used as control in both
the INST assay and the MAGI antiviral assay. The assay results are summarized in Table 1.
The current inhibitors were designed based on 6-phenyl HID (14), which inhibited both HIV
RNase H and INST in low micromolar range without inhibiting RT pol. Replacing the CH at
2
position 4 of compound 14 yielded N-hydroxybenzo pyrimidine-2,4-diones 9a and conferred a
6
.5-fold increase in RNase H inhibition (IC50 = 0.2 µM for 9a vs. 1.3 µM for 14) and a 5.5-fold
decrease in INST inhibition (IC = 11 µM for 9a vs. 2.0 µM for 14). These early data strongly
5
0
suggest that potent and selective biochemical inhibition of RNase H can be achieved with
chemotype 9, though just like HID analogue 14, both 9a and 9b exhibited severe cytotoxic (CC50
=
6.0 µM and 9.9 µM, respectively) which likely accounts for their observed antiviral potencies
(EC50 = 3.8 µM and 4.5 µM, respectively). However, when the fused benzene ring of 9a and 9b
is replaced with a nonaromatic piperidine ring, the resulting compound 19 did not demonstrate
significant antiviral activity at concentrations up to 20 µM, which prompted us to further
diversify the general hydroxyurea chemotypes 9 and 19.

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Toward this end, we pursued the replacement of the fused benzene ring with a bioisosteric
thiophene ring. The thiophene replacement is particularly attractive due to the commercial
availability of many ortho amino ester intermediates (25, Scheme 2B) or easy synthetic
accessibility via Gewald reaction (Scheme 2A). The initial thiophene derivative 10a displayed
very similar biological profiles to 9a and 9b with potent RNase H inhibition (IC = 0.10 µM),
5
0
moderate INST inhibition (IC = 4.5 µM), no RT pol inhibition and a moderate antiviral activity
5
0
(
EC50 = 11 µM) that mostly reflects its cytotoxicity (CC50 = 28 µM). Notably, halogen
substitution at the 4′ position of the phenyl ring of 10 largely mitigated the cytotoxicity (10b and
0c). Particularly interesting is compound 10c which exhibited improved RNase H inhibition
IC50 = 0.070 µM) along with substantially decreased INST inhibition (IC50 = 23 µM),
amounting to a more potent and selective RNase H inhibitory profile in vitro when compared to
a and 9b. The lack of antiviral activity, however, remains a challenge. Interestingly, for
1
(
9
bioisosteric replacement the fused thiophene ring can be placed such that the sulfur atom is either
facing up (chemotype 10) or facing down (chemotype 11). When compared to 10a, the isomeric
1
1a displayed similar levels of biochemical inhibition against RNase H and INST. Introducing a
flexible methylene group between the phenyl ring and the hydroxythieno pyrimidine-2,4-dione
core (compound 11b) further improved the RNase H inhibition (IC = 0.043 µM). Significantly,
5
0
when the phenyl ring is placed one position further removed from the sulfur atom (11c vs 11a),
moderate antiviral activity was observed (EC50 = 14 µM) with no cytotoxicity (CC50 > 100 µM).
Biochemically, 11c inhibited RNase H (IC = 0.10 µM) and to a much lesser degree INST (IC
50
5
0
=
1.3 µM) but not RT pol (IC50 > 10 µM). Introducing a chloro substituent at the 4′ position of
the phenyl ring led to compound 11d with exceptional inhibition against RNase H (IC = 0.04
5
0
µM), increased antiviral activity (EC50 = 7.4 µM) and no observed cytotoxicity at 100 µM.

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Although 11d also inhibited INST (IC = 2.1 µM), its inhibition against RNase H was far more
5
0
potent (53-fold) and it did not inhibit RT pol (IC > 10 µM). To further assess the impact of
50
thiophene ring substitutions on biochemical inhibitory profile and antiviral activity, several
analogues involving simple alkyl substituents (11e–g), carboxylic acid (11h), cycloalkyl fused
ring (11i–j) and piperidine fused (11k–m) were synthesized and evaluated. These analogues
generally inhibited RT-RNase H in the nanomolar range (IC50 = 0.10–0.30 µM) without
inhibiting RT pol (IC > 10 µM) and with moderate INST inhibition (IC = 3.6–35 µM), except
5
0
50
for 11k which did not inhibit INST at 100 µM. Importantly, a few of these analogues (11e, 11g,
1i and 11j) also showed significant antiviral activity in micromolar range (EC50 = 7.4–22 µM).
1
Overall, our data suggest that the hydroxythienopyrimidine-2,4-dione chemotype (11) represents
a novel scaffold for designing potent and selective HIV RNase H inhibitors with antiviral
activity. Compared to previously reported HID 14, our best compound 11d exhibited
substantially improved potency and selectivity inhibiting RNase H in vitro and a much more
favorable antiviral profile.
[
Insert Table 1 here]
Molecular Modeling. Docking analysis was performed with Glide XP (version 6.4) [32, 33]
using two metal sites as a constraint. The predicted binding mode of compound 11a within the
active site of RNase H suggests a potential interaction between the N-hydroxypyrimidine-2,4-
2
+
dione (chelating triad) and the two metal cofactors (Mg ), which are coordinated to the
conserved active site acidic residues D443, E478, D498, and D549. A similar binding mode was
postulated for compound 11d. Compound 11d was predicted to bind in the active site of RNase
H with two possible binding modes (Figure 2B and 2C). In both binding modes, an interaction
between the N-hydroxy-pyrimidine-2,4-dione (chelating triad) and the two active site metal

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cations (coordinated to essential active site residues D443, E478, D498, and D549) was
predicted. The keto group at the 4-position of 11d appears to form H-bonding interactions with
H539, which would stabilize inhibitor positioning near the active site metal-chelating residues
D443, E478, D498, and D549, all of which are crucial for RT-RNase H activity. In the top
scoring pose of 11d (magenta, Figure 2B), the 4-chlorophenyl at 5-position of the N-hydroxy-
pyrimidine-2,4-dione core was predicted to have an edge-on π-stacking interaction between the
chlorophenyl ring of the ligand and RNase H residue H539. This interaction may limit the
conformational flexibility of the loop with H539 and thus impact the efficiency of RNase H
catalytic activity. Notably, a similar binding mode was observed for RT complexes with dsDNA
or RNA−DNA hybrids as reported by Harrison et.al [34] and Sarafianos et.al [35, 36]. In an
alternate predicted binding mode of 11d (blue, Figure 2C), the interaction of the chelating triad
with the metal ions is intact but the 4-chlorophenyl ring flips to the other side of the binding
pocket resulting in the loss of key interaction with H539. The Glide XP scores for the predicted
binding modes of 11a and 11d was shown in Figure 2D. Ligands with the low score is predicted
to have a better binding with the protein.
[
Insert Figure 2 here]
Crystal structure of HIV RT in complex with 11b. In order to further understand the
molecular details of HIV RT-associated RNase H inhibition by compounds of the N-hydroxy
thienopyrimidine-2,3-dione chemotype, we solved the crystal structure of HIV RT in complex
with analogue 11b, at 1.8 Å resolution. The asymmetric unit comprises two RT molecules
(
Figure 3, middle panel), and therefore we were able to capture two distinct RNase H active sites
in the same crystal structure, similar to previous reports [15, 38]. Importantly, analogue 11b is
bound at both of these sites and adopts a different conformation in each active site (Figure 3, top

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2
+
and bottom panels). In both RNase H active sites, two Mn ions are bound by conserved active
2
+
site residues D443, E478, D498, and D549. Analogue 11b chelates both Mn ions through the
hydroxyl and carbonyl groups of the pyrimidine ring. 11b also interacts directly with RT in both
RNase H active sites through interactions of a carbonyl group on the pyrimidine ring with RT
residues H539 and D549 (Figure 3, top and bottom panels). In RNase H active site 1 (Figure 3,
top panel), the thieno group points in the direction of H539, while in RNase H active site 2
(
Figure 3, bottom panel), the same group points in the opposite direction. Multiple binding
modes of an inhibitor at the HIV-1 RT RNase H active site have also recently been observed for
a 2-hydroxyisoquinoline-1,3-dione analogue [38]. The lack of additional protein-inhibitor
interactions and the observed multiple modes of inhibitor binding at the RNase H active site may
contribute to the decreased antiviral activity of the N-hydroxy thienopyrimidine-2,3-dione
compounds.
[
Insert Figure 3 here]
Conclusions
Herein we report a series of novel N-hydroxy thienopyrimidine-2,3-diones, which potently and
selectively inhibited RNase H in low sub-micromolar range and with considerable potency
against HIV-1 in cell culture. While some of the analogues also inhibited INST, this inhibition
was substantially less than that for RT RNase H inhibition, suggesting that the N-hydroxy
thienopyrimidine-2,3-diones chemotype may represent an interesting scaffold for developing
RNase H-specific inhibitors. Interestingly, many of these new analogues inhibited HIV-1 in cell
culture at low micromolar concentrations. The best compound 11d exhibited low nanomolar
inhibition of RNase H (IC50 = 0.04 µM) with considerable antiviral activity (EC50 = 7.4 µM) and

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no cytotoxicity (CC50 > 100 µM). The active site-directed RNase H inhibition was further
corroborated with molecular docking and crystallographic studies.
Experimental
Chemistry. General Procedures. All commercial chemicals were used as supplied unless
otherwise indicated. Flash chromatography was performed on a Teledyne Combiflash RF-200
with RediSep columns (silica) and indicated mobile phase. All moisture sensitive reactions were
1
13
performed under an inert atmosphere of ultrapure argon with oven-dried glassware. H and C
NMR spectra were recorded on a Varian 600 MHz and Bruker 400 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 µm C18 column (250 mm × 4.6 mm). HPLC conditions: solvent A =
H2O, solvent B = MeCN; flow rate = 1.0 mL/min; compounds were eluted with a gradient of 5%
MeCN/H O for 0−5 min and to 95% MeCN/H O from 5 to 30 min followed by 100% MeCN
2
2
from 35 to 40 min. Purity was determined by total absorbance at 254 nm. The melting points
m.p.) were determined using an electro thermal Mel-Temp capillary melting point apparatus.
All tested compounds have a purity ≥98%.
-Hydroxy-6-phenylisoquinoline-1,3(2H,4H)-dione (14) [12, 23]. A solution of homophthalic
acid (13, 1.0 equiv) and NH OTHP (1.2 equiv) in toluene (10 mL) was refluxed for 5 min. To the
(
2
2
mixture, a solution of CDI (1.0 equiv) in DCM was added dropwise. The suspension turned clear
and stirred at reflux for 12 h, a black solid separated from solution and the TLC indicated the
disappearance of starting material. The reaction mixture was cooled and passed through a short
pad of silica gel which was then rinsed with (EtOAc/ hexane, 1:3), the combined filtrate was

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evaporated to dryness. The product was used for the next reaction without further purification.
The cyclized product was dissolved in MeOH (5.0 mL) and treated with pTSA hydrate (1.0
equiv) and stirred at room temperature for 2−3 h. Upon the disappearance of starting material by
TLC, the mixture was evaporated to dryness to get pale-yellow solid. The solid obtained was
triturated with water and then with ether and dried at room temperature to afford the desired
1
product (14) as a colorless solid. Yield: 69%; m.p. 181-183 °C. H NMR (600 MHz, DMSO-d )
6
δ 10.40 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.72
1
3
(
s, 1H), 7.52 (t, J = 7.6 Hz, 2H), 7.45 (t, J = 7.3 Hz, 1H), 4.32 (s, 2H). C NMR (150 MHz,
DMSO-d ) δ 166.5, 161.7, 144.8, 138.6, 135.5, 129.3, 128.8, 128.5, 127.1, 125.9, 125.8, 124.1,
7.2. HRMS-ESI (−) m/z calculated for C H NO -, 252.0666 [M-H]-; found: 252.0667.
6
3
1
5
10
3
General procedure for the cyclization of ortho amino esters and deprotection (9a-b, 19, 10a-
c, 11a-m) [21]. To a solution of ortho amino ester (1.0 eq) in toluene (0.1 M) was added CDI
(1.25 equiv) and refluxed for 2 h. The THP or benzyl protected hydroxylamine (1.5 equiv) was
then added and refluxing was continued for 1 h. The solution was concentrated, suspended in
EtOH (5 mL) and treated with 2 M NaOH (5 mL) and refluxed for 1 h. The resultant solution
was cooled and acidified with AcOH to precipitate the O-protected imide. The product was
filtered, washed with water and ether and air dried. The product was sufficiently pure without
any further purification for the next reaction. The deprotection of O-protected imide was carried
out using standard procedures. The cyclized product was dissolved in MeOH (5.0 mL) and
treated with pTSA hydrate (1.0 equiv) and stirred at room temperature for 2−3 h. The mixture
was evaporated to dryness to get pale-yellow solid and the solid obtained was triturated with
water and then with ether/methanol and dried at room temperature to afford the desired product

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as a colorless solid. Benzyl deprotection was carried out using HBr in acetic acid and compound
was isolated as described above.
1
3
-Hydroxy-7-phenylquinazoline-2,4(1H,3H)-dione (9a). Yield: 52%; m.p. >250 °C. H NMR
(
600 MHz, DMSO-d ) δ 11.61 (s, 1H), 10.59 (s, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.4
6
1
3
Hz, 2H), 7.55-7.52 (m, 3H), 7.47 (t, J = 7.3 Hz, 1H), 7.41 (s, 1H). C NMR (100 MHz, DMSO-
d
6
) δ 159.07, 148.68, 146.12, 138.66, 138.47, 129.07, 128.62, 127.70, 126.82, 121.17, 112.98,
1
12.75. HRMS-ESI (−) m/z calculated for C H N O -, 253.0619 [M-H]-; found: 253.0617.
1
4
9
2
3
7
-(4-Chlorophenyl)-3-hydroxyquinazoline-2,4(1H,3H)-dione (9b). Yield: 60%; m.p. >250 °C.
1
H NMR (600 MHz, DMSO-d ) δ 11.63 (s, 1H), 10.61 (s, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.70 (d,
6
1
3
J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.39 (s, 1H). C NMR (150
MHz, DMSO-d ) δ 159.3, 148.9, 145.0, 139.0, 137.6, 133.8, 129.4, 129.3, 128.9, 121.4, 113.5,
12.9. HRMS-ESI (−) m/z calculated for C H ClN O -, 287.0229 [M-H]-; found: 287.0228.
6
1
1
4
8
2
3
7
-Benzyl-3-hydroxyhexahydropyrido[3,4-d]pyrimidine-2,4(1H,3H)-dione
hydrochloride
1
(
19). Yield: 40%; m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 11.41 (s, 1H), 11.35 (s, 1H),
6
1
0.55 (s, 1H), 7.62-7.48 (m, 5H), 4.43 (s, 2H), 3.92-3.46 (m, 4H), 2.60 (s, 2H). HRMS-ESI (−)
m/z calculated for C14 -, 272.1041 [M-H]-; found: 272.1040.
-Hydroxy-6-phenylthieno [3,2-d]pyrimidine-2,4(1H,3H)-dione (10a). Yield: 75%; m.p. >250
14 3 3
H N O
3
1
°
C. H NMR (600 MHz, DMSO-d ) δ 12.06 (s, 1H), 10.55 (s, 1H), 7.76 (d, J = 7.3 Hz, 2H),
6
1
3
7
1
2
.51-7.46 (m, 3H), 7.22 (s, 1H). C NMR (150 MHz, DMSO-d ) δ 155.9, 151.5, 149.6, 143.7,
6
32.0, 129.9, 129.4, 126.1, 112.8, 109.4. HRMS-ESI (−) m/z calculated for C12
H N O S-,
7 2 3
59.0183 [M-H]-; found: 259.0180.

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6
-(4-Fluorophenyl)-3-hydroxythieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (10b). Yield: 44%;
1
m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.07 (s, 1H), 10.54 (s, 1H), 7.90 – 7.78 (m,
6
1
3
2
1
H), 7.34 (t, J = 8.6 Hz, 2H), 7.20 (s, 1H). C NMR (100 MHz, DMSO-d ) δ 155.6, 150.1,
6
49.4, 143.6, 128.4, 128.3, 116.3, 116.1, 112.9, 109.4. HRMS-ESI (−) m/z calculated for
C H FN O S-, 277.0089 [M-H]-; found: 277.0088.
12
6
2
3
6
-(4-Chlorophenyl)-3-hydroxythieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (10c). Yield: 50%;
1
m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.10 (s, 1H), 10.56 (s, 1H), 7.80 (d, J = 8.3
6
1
3
Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.25 (s, 1H). C NMR (150 MHz, DMSO-d ) δ 155.8, 150.0,
6
1
49.6, 143.8, 131.0, 129.4, 127.9, 121.2, 113.5, 109.8. HRMS-ESI (−) m/z calculated for
C H ClN O S-, 292.9793 [M-H]-; found: 292.9793.
12
6
2
3
3
-Hydroxy-6-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11a). Yield: 71%; m.p. >250
1
°
C. H NMR (600 MHz, DMSO-d ) δ 12.44 (s, 1H), 10.51 (s, 1H), 7.66 (d, J = 7.5 Hz, 2H), 7.61
6
1
3
(
s, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 1H). C NMR (150 MHz, DMSO-d ) δ
6
1
56.3, 148.8, 148.2, 134.2, 132.6, 129.3, 128.1, 125.3, 118.0, 115.9. HRMS-ESI (−) m/z
calculated for C H N O S-, 259.0183 [M-H]-; found: 259.0185.
1
2
7
2
3
6
-Benzyl-3-hydroxythieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11b). Yield: 79%; m.p. >250
1
°
C. H NMR (600 MHz, DMSO-d ) δ 12.20 (s, 1H), 10.40 (s, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.28
6
1
3
(
d, J = 7.5 Hz, 2H), 7.24 (t, J = 7.1 Hz, 1H), 6.94 (s, 1H), 4.08 (s, 2H). C NMR (150 MHz,
DMSO-d ) δ 156.3, 148.8, 148.1, 139.8, 135.9, 128.8, 128.7, 126.8, 119.3, 114.2, 35.1. HRMS-
6
ESI (−) m/z calculated for C H N O S-, 273.0339 [M-H]-; found: 273.0341.
1
3
9
2
3
3
2
7
-Hydroxy-5-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11c). Yield: 67%; m.p. 239-
1
41 °C. H NMR (600 MHz, DMSO-d ) δ 12.40 (s, 1H), 10.36 (s, 1H), 7.45 (d, J = 6.7 Hz, 2H),
6
1
3
.37-7.35 (m, 3H), 7.04 (s, 1H). C NMR (150 MHz, DMSO-d ) δ 155.6, 150.3, 147.8, 138.3,
6

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1
2
7 2 3
34.4, 131.9, 128.7, 127.1, 114.6, 110.9. HRMS-ESI (−) m/z calculated for C12H N O S-,
59.0183 [M-H]-; found: 259.0181.
5
-(4-Chlorophenyl)-3-hydroxythieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11d). Yield: 60%;
1
m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.42 (s, 1H), 10.38 (s, 1H), 7.48 (d, J = 8.3
6
1
3
Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 7.09 (s, 1H). C NMR (100 MHz, DMSO-d
6
) δ 155.9, 150.6,
1
48.2, 137.1, 133.4, 132.1, 130.8, 127.4, 115.4, 111.0. HRMS-ESI (−) m/z calculated for
C H ClN O S-, 292.9793 [M-H]-; found: 292.9795.
12
6
2
3
1
3
-Hydroxythieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11e). Yield: 85%; m.p. >250 °C. H
1
3
NMR (600 MHz, DMSO-d ) δ 12.30 (s, 1H), 10.43 (s, 1H), 7.16-7.13 (m, 2H). C NMR (150
6
MHz, DMSO-d
S-, 182.9870 [M-H]-; found: 182.9874.
-Hydroxy-5-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11f). Yield: 80%; m.p. >250
6
) δ 156.0, 148.6, 148.4, 121.8, 117.5, 114.5. HRMS-ESI (−) m/z calculated for
6 3 2 3
C H N O
3
1
13
°
C. H NMR (600 MHz, DMSO-d ) δ 12.23 (s, 1H), 10.34 (s, 1H), 6.71 (s, 1H), 2.35 (s, 3H). C
6
NMR (150 MHz, DMSO-d ) δ 156.60, 149.48, 148.39, 133.68, 112.64, 112.10, 15.4. HRMS-ESI
6
(
−) m/z calculated for C
H
5
N
2
O
3
S-, 197.0026 [M-H]-; found: 197.0027.
7
3
-Hydroxy-5,6-dimethylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (11g). Yield: 77%; m.p.
1
>
250 °C. H NMR (600 MHz, DMSO-d ) δ 12.12 (s, 1H), 10.29 (s, 1H), 2.26 (s, 3H), 2.25 (s,
6
1
3
3
H). C NMR (150 MHz, DMSO-d ) δ 156.8, 148.5, 146.7, 128.6, 122.9, 113.1, 12.6, 11.9.
6
HRMS-ESI (−) m/z calculated for C H N O S-, 211.0183 [M-H]-; found: 211.0182.
8
7
2
3
3
-Hydroxy-5-methyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-6-carboxylic acid
1
(
11h). Yield: 52%; m.p. 237-239 °C. H NMR (600 MHz, DMSO-d ) δ 13.23 (s, 1H), 12.50 (s,
6
1
3
1
H), 10.47 (s, 1H), 2.72 (s, 3H). C NMR (150 MHz, DMSO-d ) δ 162.8, 156.4, 151.2, 147.8,
6

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1
8 5 2 5
43.0, 117.9, 113.6, 13.8. HRMS-ESI (−) m/z calculated for C H N O S-, 240.9925 [M-H]-;
found: 240.9927.
3
-Hydroxy-1,5,6,7-tetrahydro-2H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-2,4(3H)-dione
1
(
11i). Yield: 64%; m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.19 (s, 1H), 10.35 (s, 1H),
6
1
3
2
1
.81-2.79 (m, 4H), 2.42 – 2.28 (m, 2H). C NMR (100 MHz, DMSO-d
6
) δ 156.1, 151.7, 148.6,
S-, 223.0183
39.7, 130.8, 109.5, 28.2, 28.1, 27.3. HRMS-ESI (−) m/z calculated for C
9
7 2 3
H N O
[M-H]-; found: 223.0185.
3
-Hydroxy-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione
(11j).
1
Yield: 69%; m.p. 241-243 °C. H NMR (600 MHz, DMSO-d ) δ 12.14 (s, 1H), 10.30 (s, 1H),
6
1
3
2
1
.76-2.75 (m, 2H), 2.61-2.60 (m, 2H), 1.76-1.71 (m, 4H). C NMR (150 MHz, DMSO-d
6
) δ
56.3, 148.3, 147.0, 130.6, 125.7, 112.1, 24.7, 23.6, 22.4, 21.3. HRMS-ESI (−) m/z calculated
for C H N O S-, 237.0339 [M-H]-; found: 237.0338.
1
0
9
2
3
7
-Ethyl-3-hydroxy-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidine-2,4(1H,3H)-
1
dione hydrochloride (11k). Yield: 48%; m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.48
6
(s, 1H), 10.98 (s, 1H), 10.46 (s, 1H), 4.50-4.49 (m, 1H), 4.20-4.19 (m, 1H), 3.67-3.65 (m, 1H),
1
3
3
1
.25-3.15 (m, 5H), 1.31 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, DMSO-d
6
) δ 156.9, 149.9,
49.0, 129.0, 118.5, 111.9, 50.7, 48.4, 48.3, 22.9, 9.7. HRMS-ESI (−) m/z calculated for
C H N O S-, 266.0605 [M-H]-; found: 266.0606 (free base).
11
12
3
3
3
-Hydroxy-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione
1
hydrobromide (11l). Yield: 69%; m.p. >250 °C. H NMR (600 MHz, DMSO-d
6
) δ 12.34 (s,
1
3
1
H), 10.45 (s, 1H), 9.17 (s, 2H), 4.26 (s, 2H), 3.39-3.37 (m, 2H), 3.05-3.04 (m, 2H). C NMR
(
150 MHz, DMSO-d ) δ 156.8, 149.4, 148.9, 128.9, 118.6, 111.9, 41.6, 40.6, 22.6. HRMS-ESI
6
(
−) m/z calculated for C H N O S-, 238.0292 [M-H]-; found: 238.0288 (free base).
9
8
3
3

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7
-Benzyl-3-hydroxy-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidine-2,4(1H,3H)-
1
dione hydrochloride (11m). Yield: 79%; m.p. >250 °C. H NMR (600 MHz, DMSO-d ) δ 12.47
6
(s, 1H), 11.51 (s, 1H), 10.46 (s, 1H), 7.65-7.62 (m, 2H), 7.47-7.44 (m, 3H), 4.50-4.44 (m, 2H),
1
3
4
1
.22 (s, 2H), 3.66-3.64 (m, 2H), 3.18-3.17 (m, 2H). C NMR (150 MHz, DMSO-d ) δ 156.1,
6
49.0, 148.2, 131.1, 129.3, 128.6, 128.2, 117.3, 111.0, 57.6, 47.8, 47.7, 22.0. HRMS-ESI (−) m/z
calculated for C H N O S-, 328.0761 [M-H]-; found: 328.0762 (free base).
1
6
14
3
3
Biology
Reagents
Biologicals. Recombinant HIV-1 reverse transcriptase (RT) was expressed and purified as
previously described [39]. P4R5 HIV infection indicator cells were obtained from the NIH AIDS
Reagent Program, Division of AIDS, NIAID, NIH (p4R5.MAGI from Dr. Nathaniel Landau).
These cells express CD4, CXCR4 and CCR5 as well as a β-galactosidase reporter gene under the
control of an HIV LTR promoter.
Chemicals. DNA and RNA oligonucleotides for the preparation of RNA/DNA duplexes for
assay of RNase H activity were purchased from Trilink (San Diego, CA).
RNH assay. RNH activity was measured essentially as previously described [40]. Full-length
HIV RT was incubated with the RNA/DNA duplex substrate HTS-1 (RNA 5’-
gaucugagccugggagcu-3’-fluorescein annealed to DNA 3’-CTAGACTCGGACCCTCGA-5’-
Dabcyl), a high sensitivity duplex that assesses non-specific internal cleavage of the RNA strand,
in the presence of various concentrations of compound. Results were analyzed using Prism

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software (GraphPad Software, San Diego, CA) for nonlinear regression to fit dose-response data
to logistic curve models.
RT polymerase assay. RT pol assays were carried out in 96-well plates by measuring the
extension of an 18 nucleotide DNA primer (5'-GTCACTGTTCGAGCACCA-3') annealed to a
1
00
nucleotide
DNA
template
(5'-
ATGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTA
GTCAGTGTGGAATATCTCATAGCTTGGTGCTCGAACAGTGAC-3'). Reactions containing
2
0 nM RT, 40 nM template/primer, and 10 µM 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
MgCl . Reactions contained 1% DMSO and increasing concentrations of compounds. DNA
2
synthesis was carried out for 30 min at 37 °C, 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 [20, 41].
HIV IN assay. HIV integrase was expressed and purified as previously reported [42]. Inhibition
assays were performed using a modified protocol of our reported method [42]. Briefly, 2.1 µL of
compound suspended in DMSO was placed in duplicate into a black 96 well non-binding plate
(
corning 3991). Compounds were plated in duplicate to a final concentration of 0.13 — 100 µM.
To each well of the plate, 186.9 µL of reaction mixture without DNA substrate was added (10
mM HEPES pH 7.5, 10 % glycerol w/v, 10 mM MnCl , 1 mM DTT, 1 µM integrase). The
2

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enzyme was incubated with inhibitor for 10 min at 25 °C after which the reaction was initiated
by the addition of 21 µL of 500 nM oligo (5’-biotin-ATGTGGAAAATCTCTAGCA annealed
with ACTGCTAGAGATTTTCCACAT-3’-Cy5). Reactions were incubated at 37 °C for 30 min
and then quenched by the addition of 5.2 µL 500 mM EDTA. Each reaction was moved (200
µL) to a MultiScreen HTS PCR plate (Millipore MSSLBPC10) containing 20 µL streptavidin
agarose beads (Life Technologies S951) 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 (.05% SDS, 1 mM EDTA in PBS). The plates were further washed 3 times
with 200 µL 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 °C for 5 min and the
NaOH was removed by centrifugation at 1000 g for 1 min. The reaction products were eluted
from the beads by the addition of 150 µL formamide. The plate was incubated at 25 °C for 10
min and read directly at 635/675 in a SpectraMax i3 plate reader (Molecular Devices).
Antiviral assays. MAGI assays were carried out using P4R5 indicator cells essentially as
3
previously described [29]. P4R5 cells were cultured in 96-well microplates with 4 × 10 cells per
well and maintained in DMEM/10% FBS supplemented with puromycin (1 µg/mL). Cells were
incubated with either 1% DMSO or varying concentrations of the drugs for 24 h and then
exposed to HIV-1 (MOI of 1.25) followed by an additional incubation period of 48 h. The extent
of infection was assessed using a fluorescence-based β-galactosidase detection assay, as
previously described with minor modifications [43]. After the 48 h incubation period, cells were
lysed and 4-methylumbelliferylgalactoside (MUG) substrate was added. The β-galactosidase
produced during infection acts on the MUG substrate and yields a fluorescent product, 4-

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methylumbelliferone (4-MU) that can be detected fluorimetrically with excitation wavelength
3
65 nm and emission wavelength 446 nm.
Modeling and docking analysis. Molecular modeling was performed using the Schrödinger
small molecule drug discovery suite 2014-3. The crystal structure of a hydroxypyridone
carboxylic acid active-site RNase H inhibitor in complex with HIV Reverse Transcriptase was
extracted from the Protein Data Bank (PDB code 5J1E) [37] as reported by Kankanala et.al [15].
The above structure was subjected to analysis and found that the native ligand, hydroxypyridone
carboxylic acid was bound to the active site of RNase H. This model was subjected to Protein
Preparation Wizard (Schrödinger Inc.) [44, 45] in which missing hydrogens atoms were added,
zero-order bonds to metals were created followed by the generation of metal binding states. The
structure of protein was minimized using OPLS 2005 force field [46] to optimize hydrogen
bonding network and converge heavy atoms to an rmsd of 0.3 Å. The processed model indicates
that the interaction between the hydroxypyridone carboxylic acid and RNase H is mediated by
2
+
two metal cations (Mg ) coordinated by the active site residues D443, E478, D498, and D549.
The receptor grid generation tool in Maestro (Schrödinger Inc.) was used to define an active site
around the native ligand to cover all the residues within 12 Å from it with both the metal
2
+
cofactors (Mg ) as a constraint to identify the chelating triad during docking. Compounds 11a
and 11d were drawn using Maestro and subjected to Lig Prep [47] to generate conformers,
possible protonation at pH of 7 ± 3, and metal binding states that serve as an input for docking
process. All the dockings were performed using Glide XP (Glide, version 6.4) [32, 33] mode
2
+
with both the Mg metal cofactors as a constraint. The van der Waals radii of nonpolar atoms for
each of the ligands were scaled by a factor of 0.8. The predicted binding mode of compounds
1
1a and 11d features the critical interaction between the chelating triad and to the divalent metal

ACCEPTED MANUSCRIPT
cofactors. All the ligands within the active site of RNase H were further refined post docking by
minimizing under implicit solvent to account for the local protein flexibility.
HIV-1 RT crystallization and data collection. HIV-1 RT containing C280S mutations in the p66
and p51 subunits was expressed and purified as previously described [48, 49]. Co-crystallization
of HIV-1 RT with 11b was accomplished by mixing a solution of RT at a final concentration of
1
1 mg/ml, 10 mM MnCl , 5 mM tris(2-carboxyethyl)phosphine (TCEP) HCl, 0.5% β-
2
octylglucoside (BOG), and 1 mM 11b 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 Tris pH 6.5. Large, blocky crystals
grew by hanging drop vapor diffusion at 18 °C in 2-3 days.
HIV-1 RT/11b co-crystals were additionally soaked with 1.5 mM 11b, 5 mM TCEP HCl,
0
2
.5% BOG, 8% ethylene glycol, and 10 mM MnCl for 20 minutes before brief cryoprotection in
a solution containing 23.5% ethylene glycol and 4% trimethylamine N-oxide, followed by fast
cryo-cooling in liquid nitrogen. Two data sets collected at Beamline 23-ID-D of the Advanced
Photon Source at Argonne National Laboratory were processed, scaled, and merged to 1.8 Å
resolution using xia2 [50, 51]. The HIV-1 RT/11b crystals were of space group P1, and had unit
cell dimensions of a = 70.4 Å, b = 89.1 Å, c = 112.7 Å and α = 105.7°, β = 95.0°, γ = 110.7°.
Two RT molecules were present in the asymmetric unit, and the Matthews coefficient was 2.7
3
Å /Da, corresponding to a solvent content of 53.8% [52]. Crystal data and statistics are listed in
SI Table 1.
Phase determination and structure refinement. The structure was determined by molecular
replacement using the MrBump pipeline [53, 54], which used PDB ID 4KFB as the initial search
model [55]. Buccaneer was used for initial model building [56, 57], and several cycles of model
building and refinement were carried out using Coot [58] and Phenix [59] or Refmac [60, 61]

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respectively. The final RT/11b structure was validated using MolProbity [62] Final refinement
statistics are listed in SI Table 1.
Acknowledgements
This research was supported in part by the National Institutes of Health (AI100890 to SGS and
ZW), and by the Research Development and Seed Grant Program of 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 supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. We also
thank Drs. Michail Isupov and Ronan Keegan of BioMEX Solutions, Inc. for assistance with
structure solution and refinement.
Appendix A. Supplementary data
1
Chemical synthesis, general procedures, spectral data, including H NMR of intermediates (13,
1
6a-b, 18, 22a-b, 24a-b, 25i), and crystallographic data and refinement statistics are available in
the supporting information. This material is available free of charge via the Internet at
http://dx.doi.org/10.1016/j.ejmech.2016.12.030.
PDB ID. The atomic coordinates and structure factors have been deposited in the RCSB Protein
Data Bank (PDB ID: 6AOC). Authors will release the atomic coordinates and experimental data
upon article publication.
AUTHOR INFORMATION
Corresponding Author

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Email: wangx472@umn.edu; Phone: +1 (612) 626-7025.
Abbreviations
HIV
RT
human immunodeficiency virus
reverse transcriptase
RNase H ribonuclease H
SAR
INST
Pol
structure-activity-relationship
integrase strand transfer
polymerase
NNRTI nonnucleoside RT inhibitor
HAART highly active antiretroviral therapy
NRTI
HID
nucleoside reverse transcriptase inhibitor
2-hydroxyisoquinoline-1,3-dione
3-Hydroxypyrimidine-2,4-dione
carbonyldiimidazole
HPD
CDI
MW
microwave
dNTP
MUG
deoxynucleotide triphosphate
4-methylumbelliferyl-galactoside
4-methylumbelliferone
4
-MU

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TCEP
tris(2-carboxyethyl)phosphine
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