Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.042

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

Full text of DOI:10.1016/j.bmcl.2013.02.042

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2775–2780
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series
of HIV-1 nucleotide-competing reverse transcriptase inhibitors
b
b
a
b
Martin Tremblay a, , Richard C. Bethell , Michael G. Cordingley , Patrick DeRoy , Jianmin Duan ,
Martin Duplessis a, Paul J. Edwards a, Anne-Marie Faucher a, Ted Halmos a, Clint A. James a, Cyrille Kuhn a,
Jean-Éric Lacoste a, Louie Lamorte b, Steven R. LaPlante a, Éric Malenfant a, Joannie Minville a,
⇑
Louis Morency a, , Sébastien Morin a, Daniel Rajotte b, Patrick Salois b, Bruno Simoneau a, , Sonia Tremblay ,
b
⇑
Claudio F. Sturino a,à
a Department of Chemistry, Boehringer Ingelheim (Canada) Ltd, 2100 Cunard Street, Laval, Québec, Canada H7S 2G5
b Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd, 2100 Cunard Street, Laval, Québec, Canada H7S 2G5
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-
one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant
improvement in antiviral potency was achieved when substituents were introduced at positions N1,
C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar
enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further
profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its
potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 19 December 2012
Revised 31 January 2013
Accepted 7 February 2013
Available online 16 February 2013
This manuscript is dedicated to the memory
of a respected friend and colleague, Dr. Louis
Morency
Keywords:
Reverse transcriptase
NcRTI
HIV-1
Benzofurano[3,2-d]pyrimidin-2-one
According to the United Nations, 34 million individuals were liv-
ing with the human immunodeficiency virus (HIV) at the end of
2010. In the same year, it was estimated that 2.5 million people con-
tracted HIV and the number of Acquired Immune Deficiency Syn-
drome (AIDS) related deaths worldwide was estimated to be
1.7 million.1 Over 30 years of HIV research has resulted in more than
25 approved antiretrovirals. Additionally, combination antiretrovi-
ral therapy (cART) has significantly improved the life expectancy
of HIV positive patients over the years.2 The currently approved
drugs belong to six classes: nucleoside/nucleotide reverse transcrip-
tase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhib-
itors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5
inhibitors and integrase inhibitors. Almost half of these drugs target
HIV-1 reverse transcriptase (RT), a key multifunctional enzyme in-
volved in post-entry viral replication.3 These functions include both
RNA-dependent DNA polymerase and ribonuclease H strand trans-
fer catalytic activity. The chronic use of RT inhibitors associated with
the high error rate of HIV-1 RT during the replication process has re-
sulted in the emergence of multi-drug resistant viruses.4 Conse-
quently, there is a need for the development of potent and less
toxic5 RT inhibitors that retain high potency against wild type
HIV-1 reverse transcriptase and drug-resistant variants.
A few years ago, two groups identified indolopyridones with
antiretroviral activity from cell-based screening campaigns.6 They
found that these inhibitors targeted the polymerization activity
of RT, with a mechanism of action that involves a competitive
binding with the incoming deoxyribonucleoside triphosphate
(dNTP) but without being chain terminators. The following year a
third group identified a set of 4-dimethylamino-6-vinylpyrimi-
dines which also inhibited the RT polymerization with a related
mechanism.7 The antiviral potency of the indolopyridones re-
mained unaffected by most of the NNRTI and NRTI resistant strains
and could be potentially used both in naïve and in treatment-expe-
rienced patients or combined with other classes of RT inhibitors.8
This novel third class of RT inhibition is now referred as nucleo-
tide-competing reverse transcriptase inhibitors (NcRTIs).9
⇑
Corresponding authors. Tel.: +1 450 682 4640; fax: +1 450 682 4189.
E-mail addresses: martin.tremblay@boehringer-ingelheim.com (M. Tremblay),
bruno.simoneau@boehringer-ingelheim.com (B. Simoneau).
Dr. Louis Morency passed away suddenly on July 24, 2012.
Current address: Vertex Pharmaceuticals (Canada) Inc., Laval, Canada.
à
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.042

2776
M. Tremblay et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2775–2780
Inspired by these results, we carried out a screen of our com-
on the benzofuranopyrimidone scaffold was found to play a key
role for potency. Unfortunately, no antiviral activity was discern-
able for the compounds in Table 1 due to the poor cytotoxicity in-
dex (CC50/EC50 <5-fold). We thus decided to further evaluate this
series with the primary focus on establishing unambiguous antivi-
ral potency and its potential to yield a lead optimization NcRTI
series.
Using compound 6 as a starting point, we verified if productive
SAR could be obtained upon modifying position C4 (Table 2). Incor-
poration of a small substituent, such as a methyl at the alpha posi-
tion to the carbonyl, was tolerated (cf. 6 and 10). Additionally,
modification of the amino group had a modest impact on potency.
Rigidifying the dimethylamino group to the corresponding pyrrol-
idine containing derivative 11 improved potency by threefold. It
seemed that a hydrogen bond donor was preferred at this position
since replacing the dimethylamino group of 6 with an N-ethyl sub-
stituent (12) improved intrinsic potency to 73 nM. Incorporation of
a chlorine atom at position C8 of inhibitor 12 had a modest impact
on potency (compound 13). Reducing the carbonyl anilide of 11 to
the corresponding aniline 14 improved intrinsic potency by
threefold.
pound collection in order to identify novel compounds displaying
this mode of action.10 A total of 7335 primary hits were identified.
A series of subsequent profiling assays were performed including
counter-screens against sensitivity to well characterized NNRTI
resistance RT mutations and RT enzyme steady state kinetics. As
a result, several benzofuranopyrimidone hits acting as NcRTIs (Ta-
ble 1, inhibitors 1, 2 and 3) were identified.11 Upon testing related
analogues from our compound database, we realized that enzy-
matic activity was lost when the 2-pyrrolidinylethyl chain of inhib-
itor 1 was replaced by a 2-morpholinylethyl (4) or a methyl (5).12
A
greater than fourfold improvement in intrinsic potency was ob-
served when a chlorine atom was introduced at position C8 (cf. 2
and 4). The 2-dimethylaminoacetamide group of 3 seemed impor-
tant because this compound was found to be at least 26-fold more
potent than 5. Upon screening different N1 substituents, we found
that replacing the N1 methyl of 3 by a 2-methoxyethyl (6) resulted
in a small gain in intrinsic potency. Since we observed that inhib-
itor 2 was more potent than compound 4, we prepared inhibitor 7
which has a chlorine atom at C8. In this case, the modification did
improve the IC50 value by sevenfold compared to 1. Keeping a chlo-
rine atom at C8, we further evaluated the impact of modifying the
N1 pyrrolidinylethyl side chain on potency. Even though amide 8
and difluoropyrrolidinyl 9 were found to be equipotent to 7, these
side chains presented different physicochemical properties. For in-
stance, inhibitors 8 and 9 have a reduced pKa compared with 7
Replacing the anilide linker of 6 with ethers (15 and 16) or an
amide (17) was not well tolerated since these modifications re-
sulted in at least threefold antiviral potency loss. Upon screening
other N-linked analogues, we found that N-arylpiperazines were
also favored (18 and 19). As observed earlier, incorporating hydro-
gen bond donors improved antiviral potency (cf. 18, 19 and 20).
Inhibitor 20 was one of the most potent compounds prepared in
this study with an antiviral potency of 87 nM. This example
13
(calcd pKa of 9.9, 8.1 and 5.3 for 7, 8 and 9, respectively) which
might have an impact on in vitro proprieties upon combining with
other appendages. Clearly, substitution at positions N1, C4 and C8
Table 1
Potency and cytotoxicity of compounds identified during uHTS campaign and related analogues
O
R1
1
N
8
X1
N
4
O
R2
a
Compd
R1
R2
H
X1
IC50 (nM)
4200
EC50 (nM)
11,000
CC50 (nM)
N
1
H
18,000
–
N
2
3
H
Cl
6500
3100
–
O
O
O
N
Me
H
1600
7700
H
N
N
4
5
H
H
H
H
>26,000
>83,000
–
–
–
–
O
Me
MeO
>11,000
(15%)
N
H
6
7
H
1200
590
3600
3700
N
>7300
(6%)
N
H
H
Cl
N
>6700
8
Cl
650
320
2600
2900
NH2
(À)
O
F
F
>4500
N
9
H
Cl
(À)
aThe number in parentheses is the percent inhibition at the highest concentration achieved.

M. Tremblay et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2775–2780
2777
Table 2
Impact of C4 modifications on potency and cytotoxicity
O
O
N
X1
N
O
C4
a
a
Compd X1 C4
IC50 (nM) EC50 (nM) CC50 (nM) Compd X1 C4
IC50 (nM) EC50 (nM) CC50 (nM)
O
O
O
O
O
>11,000
(15%)
>4500
(18%)
N
N
N
6
H
H
H
H
1200
1400
390
73
3600
4400
1100
680
18
19
20
21
Cl
Cl
Cl
Cl
24
10
10
75
430
175
87
N
H
N
N
>11,000
(14%)
10
11
12
2700
N
H
N
NH
NH
N
5950
5100
3000
960
N
H
NH
N
N
N
H
N
>5800
(37%)
890
N
H
H
N
>9500
(40%)
13
14
Cl
H
46
550
22
23
H
155
40
690
210
N
N
N
H
N
N
N
>7400
(À)
120
2400
Cl
4300
–
N
N
H
N
H
N
N
N
15
16
H
H
630
818
14,000
–
–
24
25
Br
Br
>5000
42
–
O
N
N
N
>4400
(20%)
>11,000
540
O
N
N
N
H
H
N
17
H
945
>9300
–
O
a
The number in parentheses is the percent inhibition at the highest concentration achieved.
showed the synergistic effect on potency of reducing the anilide
carbonyl (cf. 6), incorporating two hydrogen bond donors (cf. 18)
and having an optimal N-alkyl substituent (cf. 14). Knowing the
risks associated with potential bioactivation of anilines14 (e.g., 18
and 19), we prepared the corresponding 2-piperazinylpyridine
and the 2-piperazinylpyrimidine analogues (21 and 22, respec-
tively). These compounds were found to be equipotent with 18
in our cell-based assay. Antiviral potency could be further im-
proved when different amines were incorporated on the pyridine
(e.g., 23). para substituted piperazinylpyridines were favored over
the meta isomer (cf. 21 and 24).15 Bicyclic heterocycles could also
be introduced at position C4. For example, rigidification of the 2-
ethylaminoacetamide of 13 to the corresponding benzimidazole
25 was well tolerated. From this study, we established that consid-
erable improvement in potency and cytotoxicity index could be
achieved when modifications (e.g., increasing hydrogen bonds
and reducing anilide carbonyl) were performed at position C4.
After studying the effect of C4 and N1 modifications on potency
and cytotoxicity index, we turned our attention toward evaluating
the impact of C7/C8 substitution on potency (Table 3). The intro-
duction of a phenyl group (26) or a 3-pyridyl substituent (27) at
position C8 resulted in a 2- to 3-fold improvement in intrinsic
and antiviral potencies compared to 12. Incorporation of
a
hydroxymethyl appendage on the C8 phenyl (28) had no impact
on potency, but introduction of a dimethylamino group (29) was
beneficial. At this point, we investigated whether rigidification of
the dimethylamino functionality would improve potency further.
It was gratifying to obtain an IC50 value of 4.2 nM for the N-meth-
yltetrahydroisoquinoline derivative 30. In addition, 30 turned out
to be the most potent analogue of this series with an EC50 of
30 nM. Interestingly, we realized that some of the most potent
C8 substituents were also tolerated at C7 (e.g., 31 and 32). A similar
exercise was performed at position C6, but this did not lead to a
significant improvement in potency. From these results, it seemed

2778
M. Tremblay et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2775–2780
Table 3
Impact of C8 and C7 modifications on potency and cytotoxicity
O
O
N
C8
C7
N
O
6
O
N
H
NHEt
a
Compd
C8
H
C7
H
IC50 (nM)
73
EC50 (nM)
680
CC50 (nM)
12
5100
26
27
H
H
H
H
H
33
27
360
200
2300
>5100
(33%)
N
HO
>1300
(À)
28
29
30
31
32
34
300
94
30
N
N
12
3900
4800
1000
2100
4.2
7.4
8.6
H
H
53
88
N
N
a
The number in parentheses is the percent inhibition at the highest concentration achieved.
Table 4
Cross-resistance profile of selected C8 N-methyltetrahydroisoquinolinyl derivativesa
NH2
F
F
O
O
N
N
O
N
N
O
N
O
N
N
N
N
N
N
O
O
O
O
N
H
NHEt
30
33
34
IC50 (nM)
EC50 WT (nM)
4.2
30
20
25
5.9
52
CC50/EC50
137
1.5
0.1
2
1.1
3.3
1.1
>46
7.3
0.3
–
4.8
14.8
1.7
>127
8.4
0.3
–
5.8
14.2
1.7
Serum shifted EC50 (fold)
EC50 K65R (fold)
EC50 Y115F (fold)
EC50 M184V (fold)
EC50 Y115F/M184V (fold)
EC50 K103N/Y181C (fold)
a
See Supplementary data for assay details.
potency can be improved when basic groups were incorporated at
various positions around the core.
Having discovered the impact of the presence of an N-methyl-
tetrahydroisoquinolinyl substituent at C8 on potency, we intro-
duced this group on previously prepared inhibitors with different
pyrrolidinylethyl N1 side chains (Table 4). Interestingly, the C4 2-
ethylaminoacetamidylphenyl group was not required to provide
similar level of antiviral potency for 33 and 34 compared to 30.

M. Tremblay et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2775–2780
2779
Table 5
In vitro profile of selected C8 N-methyltetrahydroisoquinolinyl derivativesa
NH2
O
Br
O
CN
OH
Br
a
Br
+
30
33
34
O
77%
Sol pH 2.0/6.8 (
lg/mL)
>822/650
<0.1
93/140
2.4
>1000/<0.1
0.55
19/6
781/725
0.17
73/60
3.0
NO2
Caco-2 (Â10À6 cm/s)
HLM/RLM t1/2 (min)
Log D
35
36
37
NO2
O
3.9
MeO
O
MeO
Br
a
N
O
O
See Supplementary data for assay details.
N
b, c
60%
d, e
Br
N
Although these compounds were equipotent, the antiviral potency
of 30 was less shifted in the presence of 50% human serum. This
might be related to the higher lipophilicity (logD, see Table 5) of
the latter analogues. Inhibitors 30, 33 and 34 were further profiled
against recombinant viruses expressing clinically relevant RT resis-
tance mutations. As observed with other NcRTIs, an NRTI resistant
virus bearing the RT K65R mutation showed a 3- to 10-fold hyper-
susceptibility to our compounds.6 NRTI drug-resistant viruses
(having Y115F, M184V and Y115F/M184V mutations) exhibited
minimal to no cross-resistance to compound 30. The potency of
33 and 34 decreased 5- to 6-fold and 14- to 15-fold against recom-
binant viruses carrying the M184V or Y115F/M184V mutations,
respectively. Consistent with their NcRTI mode of action, the po-
tency of the tested compounds was not affected by NNRTI drug-
resistant mutations, such as K103N/Y181C. We also confirmed a
competitive mode of action with the incoming dNTP upon per-
forming kinetic studies with inhibitors 12, 30 and 33.16
74%
O
O
38
39
NH2
NO2
N
f, g
44%
30
O
B
O
40
Scheme 1. Reagents and conditions: (a) Na2CO3, acetone, reflux; (b) ethyl chloro-
formate, K2CO3, toluene, 100 °C; (c) NaH, CH3OCH2CH2Br, DMF, 90 °C; (d) NH4OAc,
130 °C; (e) Fe, EtOH, HCl, H2O, 90 °C; (f) bromoacetyl bromide, CH2Cl2,then 2 M
ethylamine in THF, DMF; (g) boronic ester 40, K2CO3, CsF, Pd(dppf)Cl2, dioxane, H2O,
microwave, 120 °C.
Further in vitro profiling of compounds 30, 33 and 34 showed
excellent aqueous solubility at pH 2.0 (Table 5) but poor caco-2
permeability. This could be rationalized considering the basicity
(calculated pKa: ꢀ8.8) of the N-methyltetrahydroisoquinoline moi-
ety and that molecules carrying a charge at physiological pH are
known to show poor permeability in the caco-2 assay.17 Inhibitors
30 and 34 were reasonably stable in the presence of human (HLM)
and rat (RLM) liver microsomes. Presumably as a result of its high-
er logD, compound 33 was much less stable with a short t1/2 of 19
and 6 min in the same in vitro assays. Metabolite determination
identified the difluoropyrrolidine as the major site of oxidative
metabolism.
The synthesis of inhibitor 30 started with the alkylation of 4-
bromo-2-cyanophenol (35) with alpha-bromoketone 36 to give
the aminobenzofuran analogue 37 (Scheme 1). The primary amine
was carbamoylated and the resulting carbamate was N-alkylated
with 1-bromo-2-methoxyethane to give compound 38. The urea
ring was formed by heating 38 in melted ammonium acetate and
the nitro group was reduced to aniline 39 upon treatment with
iron in HCl/EtOH at reflux. The resulting amine was acylated with
bromoacetyl bromide followed by displacement with ethylamine
and subsequent Suzuki–Miyaura18 cross-coupling with boronic es-
ter 40 provided inhibitor 30. The other inhibitors described in this
Letter were prepared in a similar fashion.10
ure of the polymerase active site and the requirement of the ATP
moiety, we judged that docking models would not be reliable en-
ough to support a structure-based rational design approach. Fur-
ther profiling of inhibitor 30 showed desirable lipophilicity, liver
microsome stability and aqueous solubility. Additionally, it main-
tained its potency against other classes of RT mutant viruses. Our
efforts aimed at improving the permeability and pharmacokinetic
profile will be reported in subsequent publications.
Acknowledgments
The authors would like to acknowledge the work of Alexandre
Pelletier for IC50 value determinations along with Lise Bourgon
for EC50 value determinations. We are also grateful to Normand
Aubry, Steven Laplante, René Coulombe, Araz Jakalian, Serge Va-
lois, Colette Boucher, Michael Little, Sylvain Bordeleau and Angelo
Filosa for structural research and analytical support. We further
acknowledge Hugo Poirier for Caco-2 permeability data, Josie DeM-
arte and Federico Colombo for microsomal stability data, Laibin
Luo, Danhui Sun and Eduard Bugan for solubility and Log D deter-
minations. Finally, we extend our gratitude to High-Throughput
Biology Group at Boehringer Ingelheim Pharmaceuticals (Ridge-
field, CT) for the HIV-1 RT HTS.
In conclusion, we have identified a novel NcRTI series displaying
excellent antiviral potency. This was accomplished by optimizing
an uHTS hit from micromolar enzymatic activity lacking antiviral
activity to inhibitors with low nanomolar antiviral potency. This
work demonstrated the optimization potential of the scaffold by
identifying four points of diversity where potency and in vitro/
in vivo parameters could potentially be improved. Modification
of the C4 position had a profound impact on antiviral potency
when N-substituted 4-aminoaryls were investigated. We also
found that aromatic basic groups were preferred at positions C8
and C7. This portion of the RT binding site can accommodate a vari-
ety of substituents with different sizes. We discovered that an N-
methyltetrahydroisoquinolinyl substituent at C8 provided inhibi-
tors with the best antiviral and intrinsic potencies. We did attempt
to model the binding mode of our inhibitors using the available
structural information19 on HIV RT. Due to the highly flexible nat-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
042.
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r
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dTTP concentration to 1 lM in order to match its km value in our primer
extension assay and (2) addition of 2 mM ATP to the assay mixture. We
evaluated over 106 compounds, in HTS mode, using this optimized assay
format. See Supplementary data for assay details.
11. IC50 represents the concentration of a drug that is required for 50% inhibition
in vitro and EC50 corresponds to the concentration required for obtaining 50%
of a maximum effect in vivo. CC50 is defined as the concentration of a drug
resulting in the death of 50 percent of the host cells.
12. Sturino, C.; DeRoy, P.; Duplessis, M.; Edwards, P. J.; Faucher, A. -M.; Halmos, T.;
James, C.; Lacoste, J. -E.; Malenfant, E.; Minville, J.; Morency, L.; Morin, S.;
Tremblay, M.; Yoakim, C. PCT Int. Appl. WO 2010/115264, 2010.