5-Carba-pterocarpens: A new scaffold with anti-HCV activity

Article abstract of DOI:10.1016/j.ejmech.2016.02.010

The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon reporter cells. The best profile in H

Full text of DOI:10.1016/j.ejmech.2016.02.010

Accepted Manuscript
5-Carba-pterocarpens: A New Scaffold with Anti-HCV Activity
Talita de A. Fernandes, Dinesh Manvar, Jorge L.O. Domingos, Amartya Basu, Daniel
Brian Nichols, Neerja Kaushik-Basu, Paulo R.R. Costa
PII:
S0223-5234(16)30080-0
10.1016/j.ejmech.2016.02.010
EJMECH 8361
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 September 2015
Revised Date: 3 February 2016
Accepted Date: 4 February 2016
Please cite this article as: T.d.A. Fernandes, D. Manvar, J.L.O. Domingos, A. Basu, D.B. Nichols, N.
Kaushik-Basu, P.R.R. Costa, 5-Carba-pterocarpens: A New Scaffold with Anti-HCV Activity, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
5-Carba-pterocarpens: A New Scaffold with Anti-HCV Activity
Talita de A. Fernandesa,+, Dinesh Manvar b,+, Jorge L. O. Domingosc, Amartya Basub, Daniel Brian
Nicholsd, Neerja Kaushik-Basub*,1, Paulo R. R. Costaa*
a Laboratório de Química Bioorgânica, Instituto de Pesquisas de Produtos Naturais, Universidade Federal do
Rio de Janeiro, Centro de Ciências da Saúde, Bloco H, Ilha da Cidade Universitária, 21941-590 Rio de
Janeiro, RJ, Brazil
b Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers-
The State University of New Jersey, 185 South Orange Avenue, New Jersey 07103, USA
c Departamento de Química Orgânica, Centro de Tecnologia e Ciências, Universidade do Estado do Rio de
Janeiro, Rua São Francisco Xavier 524, Pav. Haroldo Lisboa da Cunha – s 406 – Maracanã, 20550-900 Rio
de Janeiro, RJ, Brazil
d
Department of Biological Sciences, Seton Hall University, 400 South Orange Avenue, New Jersey,
07079, USA
*Corresponding authors: Neerja Kaushik-Basu, kaushikbasu.neerja@gmail.com
Paulo R. R. Costa, prrcosta2011@gmail.com
+Equal contribution
1 Disclaimer: This work was prepared while Dr. Neerja Kaushik-Basu was an employee at the New Jersey Medical School,
Rutgers University. The opinions expressed in this article are the author's own and do not reflect the view of the National
Institutes of Health, the Department of Health and Human Services, or the United States government.
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Abstract
The synthesis of a series of 5-carba-pterocarpens derivatives involving the cyclization of α-aryl-α-tetralones
is described. Several compounds demonstrated potent activity and selectivity in vitro against HCV replicon
reporter cells. The best profile in Huh7/Rep-Feo1b replicon reporter cells was observed with 2h (EC50 = 5.5
µM/SI = 20), while 2e was the most active in Huh7.5-FGR-JC1-Rluc2A replicon reporter cells (EC50 = 1.5
µM/SI = 70). Hydroxy groups at A- and D-rings are essential for anti-HCV activity, and substitutions in the
A-ring at positions 3 and 4 resulted in enhanced activity of the compounds.
Keywords:
Carba-pterocarpens; Palladium catalyzed arylation; BBr3 mediated demethylation-cyclization; Microwave
irradiation; Hepatitis C virus inhibitors
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1. Introduction
Hepatitis C Virus (HCV) was identified in 1989 as a causative agent of non-A, non-B viral hepatitis
[1]. Chronic HCV infection is associated with serious health conditions including cirrhosis, steatosis, and
increased risk for the development of hepatocellular carcinoma [1-7]. Despite 25 years of HCV research,
the virus remains major public health concern with 130-150 million cases worldwide [8].
HCV is a member of the Flaviviridae family [1] and eleven different genotypes of HCV have been
described [9]. The positive sense, 9.6 kb RNA genome is translated as a single polyprotein that is cleaved
by viral and host proteases into four structural (core, E1, E2, and p7) and six non-structural (NS2, NS3,
NS4A, NS4B, NS5A, and NS5B) proteins [10]. Prior to 2011, HCV infections were treated with a
combination of pegylated interferon α and ribavirin [11]. However, this treatment had many undesirable
side effects and only a 40-50% sustained virological response (SVR) in patients infected with genotypes 1
and 4 [11]. Therefore, the development of direct acting antivirals (DAAs) targeting HCV specific
processes remained a top priority. The majority of HCV DAAs target the NS3/4A protease, NS5A, or the
viral RNA-dependent RNA polymerase NS5B. In 2011, the NS3/4A protease inhibitors boceprivir and
telaprivir were the first to receive approval and greatly improved SVR to 60-80%, but were only capable of
treating a limited number of genotypes [12, 13]. Simeprivir, a second generation protease inhibitor, is
effective against a broader range of HCV genotypes [14-16]. However, in the majority of cases, NS3/4A
protease inhibitors are administered along with ribavirin and peg-IFNα. Therefore, IFN-related side effects
remain. Sofosbuvir is a nucleotide inhibitor of the HCV NS5B and has been successful in IFN-free anti-
HCV therapies [15-17]. Currently, Harvoni (ledispavir and sofosbuvir) and Viekira Pak (ombitasvir,
paritaprevir, ritonavir, and dasabuvir) are available to treat HCV genotype 1 infections and have a SVR of
around 90% [18-20].
Despite the remarkable success of current HCV inhibitors approved within the last five years, a
need remains to identify and develop novel anti-HCV therapies. Current HCV antivirals remain cost
restrictive, thus limiting patients’ ability to receive these drugs. Further, HCV replicates as a quasispecies
of RNA due to the high mutation rate of the viral RNA polymerase [21-23]. Therefore, the emergence of
drug resistant variants remains a major medical concern.
Our group previously reported that coumestans possess anti-HCV NS5B activity [24]. We identified
compound 1 (LQB-34) as having the most potent anti-NS5B activity in our initial screening (Fig. 1) [24]. In
our continuing efforts to discovery new isoflavonoid-based anti-HCV candidates, we have explored this
structure as a starting point for the design of a series of analogues [25, 26]. In the current study, we report 5-
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carba-pterocarpens (2) as a trimmed version of 1, in which the oxygen atom at the B-ring was changed by
an isosteric methylene group and the carbonyl group at the B-ring was removed (Fig. 1) [27]. Our proposal
is based on the absence of interactions of these moieties in 1 with amino acid residues in the active site
pocket of HCV NS5B polymerase [24]. These compounds share a planar structure with coumestans and
different patterns of oxygenation at A- and D-rings. While the preparation of coumestans involves
expensive multi-step synthesis [28], the 5-carba-pterocarpens (2a-h) can be prepared in high yields, in two
steps from available starting materials [26], making easier to introduce chemical diversity. Here we report
on the anti-HCV activity of 5-carba-pterocarpens (2a-h).
Figure 1 – Coumestan (1) and analogues with anti-HCV activity
2. Results and discussion
2.1 Synthesis of 5-carba-pterocarpens
The synthesis of 5-carba-pterocarpens 2 is shown in Scheme 1. Compounds 2c-f were previously
prepared by our group [26] while 2a,b,g are reported for the first time in this work. Palladium catalyzed α-
arylation of α-tetralones (3a-e) with o-bromoarenes (4a,b) in aqueous dioxane under MW irradiation led to
α-aryl-α-tetralones (1-carba-isoflavanones) 5a-g. Compounds previously synthesized were obtained in
similar chemical yield and new products were also obtained in good yields.
Scheme 1- Synthesis of carba-pterocarpens 2a-h
These compounds were allowed to react with BBr3 in CH2Cl2 from 00C to room temperature, leading
to the target compounds (2) in good yields. In the case of 2e [26], in which the demethylation at A-ring did
not occur, the reactions were accomplished at low temperature [26]. Crude products could be easily purified
by filtration through a pad of silica gel.
2.2 Anti-HCV activity of compounds 2a-2h
Compounds 2a-2h were evaluated for anti-HCV activity employing the Huh7/Rep-Feo1b and
Huh7.5-FGR-JC1-Rluc2A replicon system. These cell lines contain the autonomously replicating HCV
RNA of genotypes 1b and 2a origins with firefly and Renilla luciferase reporter genes respectively [29-31]
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. In Huh7/Rep-Feo1b replicon reporter cells, 5-carba-pterocarpen 2a, unsubstituted at A and D-rings,
showed weak anti-HCV activity (Table 1, entry 1). The introduction of a phenol group at the D-ring, 3b
(entry 2) increased activity approximately 3 times the potency. Interestingly the addition of phenol groups
at A and D-rings in compound 2b (entry 3) increased anti-HCV activity ~7 fold when compared with 2a
(entry 3). The presence of phenol groups at both A and D ring led to more active compounds. Our next
goal was to investigate the importance of the pattern of oxygenation in A ring, maintaining the phenol
group at C9 in the D-ring. Carba-pterocarpens 2d and 2e were less active than 2c, due to the change of the
phenol group to the 4-position in the A-ring and also when it was alkylated (entries 4 and 5). Compound 2f,
with phenol group at 3-position in A-ring and unsubstituted at D-ring, was about 1.5 times more active than
the unsubstituted 2a, and the introduction of the phenol group in D-ring led to a compound (2g) active only
in high concentrations (entries 6 and 7). Interestingly, the carba-pterocarpen 2h,with three phenol groups
was the most active and also showed the highest selective index (entry 8).
The replicon derived from HCV 2A origin in Huh7.5-FGR-JC1-Rluc2A reporter cells was more
sensitive to the effects of carba-pterocarpens with higher selective indexes observed in these cells (Table
1). Again, the unsubstituted compound 2a was the less potent of the series, and the introduction of phenol
groups led to compounds from 2.5 to 10 times more potent. In these replicon reporter cells compound 2e
was the most potent (methoxylated at the A-ring and with phenol at D-ring), and also with the highest
selective index (Table 1, entry 5).
Table 1. Inhibition of cell proliferation in Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon
reporter cells
2.3. Evaluation of anti-NS5B activity.
A preliminary study of the potential mechanism of action of the compounds was examined, by measuring
the inhibition of the HCV NS5B RdRp activity in vitro, employing the primer-extension assay as
previously described [24, 32-34]. Next, we evaluated the compounds for their ability to inhibit the cellular
NS5B polymerase employing the BHK-NS5B-FRluc reporter cells [35, 36].This system stably expresses
NS5B and a bicistronic reporter gene, (+)FLuc-(-)UTR-RLuc, and permits the simultaneous measurement
of intracellular HCV NS5B RdRp activity, derived from the ratio of Renilla to firefly luciferase
luminescence, and cellular viability, measured by the firefly luciferase luminescence, thereby facilitating
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the identification of potent nontoxic inhibitors. Interestingly, both methods yielded a similar pattern of
NS5B inhibition (Table 2).This is evident from the 33% to 39% inhibition of HCV NS5B RdRp activity by
compounds 2c, 2e, 2f and 2g, while others displayed no inhibition at this concentration. Notably, all
compounds exhibited greater than 80% cellular viability at 50 ꢀM compound concentration. Because NS5B
RdRp activity was only weakly affected at the tested concentration, the data suggests that the mode of
action of the compounds involves a target other than HCV NS5B. Therefore, NS5B binding studies were
not pursued. Further studies to determine the mechanism of these compounds are ongoing.
Table 2. Effect of compounds on intracellular NS5B RdRp activity
3. Conclusion
In conclusion, we identified the 5-carba-pterocarpens scaffold as possessing anti-HCV activity. 5-
carba-pterocarpens can be prepared in two steps from commercially available starting materials. The
presence of one phenol group at A- and D-ring is essential for anti-viral activity. Substitutions in the A-ring
at positions 3 and 4 led to more active compounds. The moderate inhibitor activity on NS5B RdRp enzyme
suggests that the mechanism of action is independent of inhibition of NS5B polymerase activity and may
involve another HCV or host protein present in the replicon but absent from the in vitro assay. Studies are
ongoing to synthesize of new 5-carba-pterocarpens bearing optimized pattern of substitutions for increased
anti-HCV activity. Further studies to determine the mechanism of action of these compounds are ongoing.
4. Experimental
4.1. Biological activity
4.1.1. HCV NS5B Polymerase Inhibitory Activity
All synthesized compounds were evaluated for their effect on the polymerase activity of recombinant HCV
NS5B by primer dependent elongation assays as previously described [24, 32-34]. The amount of radio
labeled UTP incorporated into the nascent RNA product in the presence of the compound or DMSO was
counted in a liquid scintillation counter.
4.1.2. Cell Culture
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BHK-NS5B-FRLuc reporter cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10%
heat inactivated fetal bovine serum, 5% Penicillin-streptomycin antibiotic, 5% nonessential amino acid, 1
mg/mL G418 and 10 mg/mL blasticidin. The reporter cell line was cultured at 37oC in the presence of 5%
CO2.
4.1.3. BHK-NS5B-FRLuc Reporter Assay
The effect of the compounds on the intracellular HCVNS5B polymerase activity was investigated
employing the BHK-NS5BFRLuc reporter system as previously described [35, 36]. Briefly, BHKNS5B-
FRLuc reporter cells were treated with 50 ꢀM compounds or equivalent concentration of DMSO (1%).
After incubation at 37oC for 40 h, cell lysates were harvested and reporter genes expressions were
measured using the Dual-Luciferse reporter kit (Promega Corporation). The effect of the compounds on
cell viability was estimated as the relative levels of Firefly luciferase expression
4.1.4. Anti-HCV Activity and Cytotoxicity in Cell-Based Assays
Anti-HCV activity of compounds in cell-based assays were evaluated in the Huh7/Rep-Feo1b cells
replicating the subgenomic HCV genotype 1b replicon RNA carrying the firefly luciferase reporter, or the
Huh7.5-FGR-JC1-Rluc2A reporter cells bearing the HCV genotype 2a replicon RNA and the Renilla
reporter as previously described [29-31].The evaluation of compounds was performed similarly to that
described earlier [29-31] . In brief, the cells were grown in 96-well plates and treated with the compound
(50 ꢀM) or equal amount of DMSO (control). The final concentration of DMSO was maintained at 1.0%.
After 48h of treatment, cells were washed with phosphate buffered saline, and the inhibitory effect of
compounds on HCV replication was measured as the relative levels of the luciferase signals in compound
treated cells versus DMSO controls employing luciferase assay system (Promega).The effect of the
compounds on cell viability was investigated in Huh7.5 parental cells under conditions similar to the
antiviral assays. Briefly, cells plated in 96-well plates were treated with compounds or DMSO (control) for
48 h. Cell viability was assessed employing Cell Titer 96 AQueous One solution in accordance with the
manufacturer’s protocol (Promega, U.S.).
4.2. Synthesis
4.2.1. Materials and methods
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1H NMR and 13C NMR spectra were obtained using a Varian 400-MR or a Varian 500-NMR with CDCl3 or
MeOD as solvent and TMS as internal standard. Low-resolution electron impact (GC-EI) mass spectra
were obtained at 70 eV using a Shimadzu QP-5000, and HRMS (GC-EI) were recorded using a Finnigan
MAT 95S instrument. Analytical TLC was performed using Schleicher & Schuell F1400/LS silica gel
plates, and the spots were visualized under UV light (λ=254 nm). For flash chromatography, we employed
Merck silica gel 60 (0.040–0.063 mm). CEM Discover and Explorer-Coolmate accessory were employed
in the microwave-assisted reactions for the generation of α-aryl-tetralones.
4.2.2. General procedure for the microwave assisted synthesis of α-aryl-α-tetralones (5)
A suspension of Pd2(dba)3 (6.9 mg, 0.0075 mmol), tBu3PHBF4 (8.7 mg, 0.03 mmol), KOH (42 mg, 0.75
mmol), aryl bromide 4 (0.36 mmol) and tetralone 3 (0.3 mmol) in a mixture of dioxane/water (4:1, v/v, 3
mL) was degassed and heated under Ar and microwave irradiation (80W of initial power, 100 ºC, 40 min,
infrared probe). Then, the mixture was allowed to cool to rt, diluted in AcOEt, washed with saturated
NH4Cl solution, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude
material was purified by silica gel chromatography. Dioxane quality is extremely important for the success
of the reaction. Characterization of α-aryl-α-tetralones is given in reference 26.
4.2.3. General procedure for the synthesis of 5-carba-pterocarpens (2)
The aryltetralone 5 (0.16 mmol) was dissolved in anhydrous CH2Cl2 (3 mL) and stirred under Ar at 0 ºC for
15 min. BBr3 (226µL, 2.4 mmol) was then added dropwise with vigorous stirring. The mixture was stirred
for 90 min at 0 ºC (2e) or RT (2a-d, 2f-h). After that, the reaction mixture was carefully added to water (20
mL) at 0 ºC with continuous stirring over 20 min. The resulting suspension was extracted with EtOAc (4 x
15 mL). The combined organic extracts were washed with brine, then dried over anhydrous Na2SO4 and
filtered. The solvent was removed under reduced pressure to afford a pure product as a solid.
Characterization of 5-carba-pterocarpens 2c-2g is given in reference 26.
1
4.2.3.1. 5,6-dihydronaphtho[1,2-b]benzofuran (2a). H NMR (400 MHz,CDCl3) δ 7.65 (d, J = 7.5 Hz,
13
1H), 7.50 (td, J = 7.1, 1.5 Hz, 2H), 7.31 – 7.17 (m, 5H), 3.10 (t, J = 7.9 Hz, 2H), 2.98 – 2.92 (m, 2H). C
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NMR (101 MHz, CDCl3) δ 155.4 151.9, 136.1, 128.4, 128.1, 127.9, 127.7, 126.9, 124.2, 122.8, 120.7,
119.3, 114.2, 111.5, 28.8, 19.4. HRMS (APCI) calcd for C16H13O (M+H+): 221.0961, found: 221.0961.
4.2.3.2. 5,6-dihydronaphtho[1,2-b]benzofuran-9-ol (2b). 1H NMR (400 MHz, CD3OD) δ 7.49 (d, J = 7.4
Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.25 – 7.18 (m, 2H), 7.13 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 1.7 Hz, 1H),
6.75 (dd, J = 8.4, 1.8 Hz, 1H), 3.02 (t, J = 7.9 Hz, 2H), 2.85 (t, J = 7.9 Hz, 2H). 13C NMR (101 MHz,
CD3OD) δ 156.4, 155.4, 150.1, 135.0, 127.8, 127.5, 126.6, 126.3, 120.7, 119.1, 118.9, 114.0, 111.5, 97.5,
28.2, 18.8. HRMS (ESI) calcd for C16H11O2 (M-H+): 235.0765, found: 235.0768.
1
4.2.3.3. 5,6-dihydronaphtho[1,2-b]benzofuran-2,3,9-triol (2h). H NMR (400 MHz, CD3OD) δ 7.23 (d,
J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.4, 2.1 Hz, 1H), 6.70 (s, 1H), 2.92 –
2.86 (m, 2H), 2.82 – 2.76 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 157.4, 156.1, 152.2, 145.7, 144.9,
128.7, 122.6, 121.3, 119.7, 116.8, 116.7, 112.6, 108.6, 98.9, 29.2, 20.6. HRMS (ESI) calcd for C16H11O4
(M-H+): 267.0663, found: 267.0670.
Acknowledgments
We thank Drs. Naoya Sakamoto and Hengli Tang for the Huh7/ Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A
replicon reporter cells. We are grateful to the Brazilian agencies CAPES, CNPq and FAPERJ for
supporting (PRRC) and Fellowships (TAF and JLOD). We also acknowledge grant support (105110) from
New Jersey Medical School, Rutgers University to N.K-B.
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11

ACCEPTED MANUSCRIPT
List of Captions
Figure 1 – Coumestan (1) and analogues with anti-HCV activity
Scheme 1- Synthesis of carba-pterocarpens 2a-h
Table 1. Inhibition of HCV RNA replication in Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon
reporter cells
Table 2. Effect of compounds on intracellular NS5B RdRp activity
12

ACCEPTED MANUSCRIPT
Table 1. Inhibition of HCV RNA replication in Huh7/Rep-Feo1b and Huh7.5-
FGR-JC1-Rluc2A replicon reporter cells
Huh7/Rep-Feo1b
Huh7.5-FGR-JC1-Rluc2A
Entry
Comp.
CC50 (µM)a
Inhibition
Inhibition
(%)b
EC50
EC50(µM)c SId
SId
(%)b
(µM) c
15.3 ±
2.0
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
>200
50.7 ± 6.5
90.1 ± 7.5
96.7 ± 0.7
98.1 ± 0.1
97.9 ± 0.2
95.1 ± 2.8
49.3 ± 6.8
98.6 ± 0.7
40.7 ± 5.1 >4.9 86.5 ± 2.5
>13
99.9 ± 4.0
59.5 ± 6.5
84.5 ± 5.6
105.9 ± 6.8
96.8 ± 4.8
157.1 ± 12.1
115.0 ± 5.9
13.9 ± 2.4 7.2
97.2 ± 0.4 5.7 ± 1.9 15.8
5.9 ± 2.3 10.1 99.7 ± 0.3 2.4 ± 1.9 24.7
12.3 ± 3.0 6.8
19.9 ± 1.1 5.3
27.9 ± 2.1 3.5
99.6 ± 0.4 1.9 ± 1.7 44.4
99.8 ± 0.2 1.5 ± 1.5 70.6
98.4 ± 0.1 2.3 ± 0.5 42.1
97.8 ± 1.5 6.0 ± 1.3 26.1
2g
ND
ND
2h
5.5 ± 1.5 20.9 96.9 ± 0.2 6.1 ± 1.9 18.8
aCC50 values were evaluated in Huh7.5 parental cells by the MTS assay (CellTiter 96®
AQueous One Solution Cell Proliferation kit, Promega). Cells treated with equal
b
amounts of DMSO served as viability control. Compounds were screened at 50 µM
concentrations against Huh7.5/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2Ab replicon
reporter cells. Cells treated with equal amounts of DMSO served as control.cEC50
values were computed from dose-response curves using half-log dilutions of the
compounds employing CalcuSyn V2 software. dSI: selectivity index represents the ratio
of CC50 to EC50.

ACCEPTED MANUSCRIPT
Table 2. Effect of compounds on intracellular NS5B RdRp activity
BHK-NS5B-FRLuc
Inhibition (%)b
NS5B RdRp
Inhibition (%) in
vitroa
Entry
Comp.
2a
1
2
3
4
5
6
7
8
NI
NI
NI
NI
2b
2c
38.0 ± 2.4
NI
33.3 ± 4.0
NI
2d
2e
36.2±4.1
38.5 ± 2.5
33.1 ± 1.3
NI
37 ±3.0
39.4 ± 6.5
36.1 ± 5.4
NI
2f
2g
2h
aPercent inhibition was determined at 50 ꢀM concentration of
the indicated compound on poly rA-U12 template-primer and
with 1 mM Mn as the divalent cation. NS5B RdRp activity in
the absence of the inhibitor was taken as 100 percent after
subtraction of residual background activity. Data represents
an average of at least two independent experiments in
duplicate.
bBHK-NS5B-FRLuc reporter cells were treated with the
indicated compounds at 50 ꢀM concentration for 40 h. Percent
viability was estimated as the relative levels of Firefly
luciferase in compound treated cells versus DMSO controls,
while percent inhibition of intracellular NS5B RdRp activity
was evaluated from the percent reduction in RLuc to Fluc
luminescence signal in compound treated cells versus DMSO
controls. Percent viability was ≥80% at 50 µM compound
concentration. Data represents an average of at least three
independent experiments in duplicate.

ACCEPTED MANUSCRIPT
Figure 1 – Coumestan (1) and analogues with anti-HCV activity

ACCEPTED MANUSCRIPT
Scheme 1- Synthesis of carba-pterocarpens 2a-h

ACCEPTED MANUSCRIPT
Highlights
1. 5-carba-pterocarpens were prepared in two steps from α-tetralones and o-bromo-
anisoles
2. The anti-HCV activity of 5-carba-pterocarpens is reported.
3. Compound 2h exhibited the best profile against HCV genotype 1b (EC50 = 5.5
µM/SI = 20)
4. 2e showed the best profile against HCV genotype 2a (EC50 = 1.5 µM/ SI = 70)
5. The presence of one phenol group at A- and D-ring is essential for anti-HCV
activity