Simplified catechin-gallate inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/S0960-894X(01)00577-7

Systematic simplification of the molecular structures of epicatechin gallate and epigallocatechin gallate to determine the minimum structural characteristics necessary for HIV-1 reverse transcriptase inhibition in vitro resulted in several compounds that strongly inhibited the native as well as the A17 double mutant (K103N Y181C) enzyme, which is normally insensitive to most known nonnucleoside inhibitors.

Full text of DOI:10.1016/S0960-894X(01)00577-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2763–2767
Simplified Catechin-Gallate Inhibitors of HIV-1
Reverse Transcriptase
L. M. V. Tillekeratne,a A. Sherette,a P. Grossman,a L. Hupe,b
D. Hupeb and R. A. Hudsona,c,*
aDepartment of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Toledo, OH 43606, USA
bPfizer Global Research and Development, 2800 Plymouth Rd, Ann Arbor, MI 48105, USA
cDepartments of Biology and Chemistry, College of Arts and Sciences, University of Toledo, Toledo, OH 43606, USA
Received 14 March 2001; accepted 9 August 2001
Abstract—Systematic simplification of the molecular structures of epicatechin gallate and epigallocatechin gallate to determine the
minimum structural characteristics necessary for HIV-1 reverse transcriptase inhibition in vitro resulted in several compounds that
strongly inhibited the native as well as the A17 double mutant (K103N Y181C) enzyme, which is normally insensitive to most
known nonnucleoside inhibitors. # 2001 Elsevier Science Ltd. All rights reserved.
The life cycle of the human immunodeficiency virus
type-1 (HIV-1), the primary causative agent of AIDS,
provides a number of targets for potential chemothera-
peutic intervention.1 Most of the chemotherapeutic
agents that are currently in use for HIV treatment target
at reverse transcriptase (HIV-1-RT), a key enzyme that
catalyzes the reverse transcription of viral RNA into a
double-stranded DNA.2,3 More recently introduced
protease inhibitors also have become important com-
ponents in current anti-HIV drug regimens.4
on HIV through a common mode of binding. Their
binding site is closely associated with, but distant from
the substrate binding site.8 Several NNRTIs (nevir-
apine, delaviridine, and efavirenz) have been approved
for clinical use while a number of others (e.g., tivirapine,
loviride, MKC-422, and troviridine) remain in clinical
trials.8
A major impediment to currently available anti-HIV-
RT and other therapies is the emergence of drug-resis-
tant viral variants due to the rapid rate of replication of
HIV and to its inherent genetic variability. NNRTIs in
particular, are known to induce drug-resistant viral
variants, hence compromising clinical effectiveness.9À11
Combined use of NRTIs, NNRTIs, and protease inhi-
bitors have been shown to confer additive/synergistic
anti-HIV activity. The emergence of resistant viral
strains can be prolonged if the NNRTIs, NRTIs, and
protease inhibitors are used in combination.8 However,
current treatment regimens can only suppress viral
replication and control plasma viremia, but do not era-
dicate viral reservoirs. Thus, the discovery of new agents
that act at different sites on HIV-RT, or at other key
viral proteins or enzymes, adds to the anti-HIV drug
armamentarium for effective control of HIV infection.
Since the discovery of azidothymidine (AZT), other
nucleoside inhibitors of HIV-1-RT have been developed
for clinical use. These include oxathiolanylcytosine
(3TC), dideoxyinosine (ddI), dideoxycytidine (ddC),
and didehydro-dideoxythymidine (d4T).5 These drugs,
after cellular conversion to a 50-triphosphate, compete
with deoxynucleotide triphosphate (dNTP) binding to
HIV-1-RT and also act as competitive substrates that
lead to chain termination of the DNA. More recently,
nonnucleoside inhibitors of reverse transcriptase
(NNRTIs), that act noncompetitively with dNTP sub-
strates, have come to play an increasingly important
role in anti-HIV therapy.6,7 NNRTIs constitute a group
of compounds of very diverse structure. However, they
have been shown to interact with a specific binding site
Nakane and Ono reported that (À)-epicatechin gallate
and (À)-epigallocatechin gallate (Fig. 1), two natural
products isolated from tea (Camellia sinensis), differen-
tially inhibited reverse transcriptase and cellular DNA
*Corresponding author. Fax: +1-419-530-7946; e-mail: rhudson@
uoft02.utoledo.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00577-7

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L. M. V. Tillekeratne et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2763–2767
and RNA polymerases, the strongest inhibition being
observed with HIV-RT.12 They suggested that these
compounds bind directly to the enzyme and interfere
with template-primer binding. Further, the hydrolysis
products [(À)-epicatechin, (À)-epigallocatechin, and
gallic acid] had virtually no reverse transcriptase inhibi-
tory activity in the concentration ranges tested. How-
ever, the natural products tested were both observed to
be cytotoxic in cell culture studies at concentrations at
which they did not inhibit HIV-1-induced cytopatho-
genicity. It was suggested that these natural products
may not penetrate the cell membrane. Here, we describe
efforts to systematically simplify the molecular struc-
tures of epicatechin and epigallocatechin gallates to
determine the minimum structural characteristics neces-
sary for reverse transcriptase inhibition with a view to
developing active inhibitors with enhanced pharmaco-
kinetic properties and reduced toxicity. We investigated
the action of inhibitors on both wild-type and the A17
mutant enzyme, against which NNRTIs are inactive.
where transoid and cisoid differentiation is untenable in
the context of a conformationally flexible tether. By
deleting ring B and changing the number and the nature
of the hydroxyl substituents, a series of compounds 1–9
(Fig. 2) were synthesized and their RT inhibitory activ-
ities evaluated.
The synthesis of the compounds 1–8 is shown in Scheme
1. 3,4-Dihydro-2H-1-benzopyran-3-ols 14 were synthe-
sized by diborane reduction of the corresponding 4H-1-
benzopyran-4-ones (13).13,14 Condensation of o-
hydroxy-acetophenone and its derivatives 11 with ethyl
formate in the presence of sodium powder gave the
hemi-acetal 12, which was easily converted to the corre-
sponding 4H-1-benzopyran-4-one 13 by acid-catalyzed
dehydration.15 For the synthesis of the target molecules 1
and 5–8, 40,60-dibenzyloxy-20-hydroxyacetophenone 11
(R3=R4=–OCH2C6H5) was obtained by selective O-
benzylation of 20,40,60-trihydroxyacetophenone with 2.2
mol equivof benzyl chloride in the presence of anhy-
Initially, we focused on the two general classes of agents
A and B (Fig. 1) to determine whether catechins can be
significantly simplified with retention of reverse tran-
scriptase inhibition. The gallate ester moiety was
retained as its removal has been shown to result in loss
of activity. The two classes A and B consist of two
hydroxylated aromatic ring systems joined together in
tacitly transoid and cisoid arrangements, held together
by a five-bond tether. Assuming that the role of the
cyclic component of the tether is merely to hold the two
aromatic rings in their respective orientations, classes A
and B may be further simplified to types A1 and B1,
Figure 2. Structures of HIV-1-RT inhibitors synthesized.
Scheme 1. Synthesis of compounds 1–8. Reagents: (a) benzyl chloride,
K2CO3, DMF; (b) HCO2Et, Na; (c) H+ (–H2O); (d) BH3, NaOH/
H2O; (e) DCC, DMAP, CH2Cl2; (f) H2/Pd–C.
Figure 1. Simplification of catechin structures.

L. M. V. Tillekeratne et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2763–2767
2765
drous K2CO3 in DMF. Also, selective O-benzylation of
20,60-dihydroxy and 20,40-dihydroxy-acetophenones
under similar conditions with 1.2 mol equivof benzyl
chloride gave 60-benzyloxy-20-hydroxyacetophenone 11
(R3=H, R4=–OCH2C6H5) and 40-benzyloxy-20-hy-
droxyacetophenone 11 (R3=–OCH2C6H5, R4=H),
respectively. The 3,4-dihydro-4H-benzopyran-3-ols 14
were esterified with appropriately substituted benzoic
acid derivatives 15 using DCC to obtain the ester 16.16
Hydrogenolysis of the benzyl protecting groups gave the
target compounds 1–8. Esterification and subsequent O-
benzylation of 3,4-dihydroxyphenylacetic acid 17 gave
methyl 3,4-dibenzyloxyphenylacetate 19 that was
reduced with LiBH4 to 2-(3,4-dibenzyloxy-phenyl)ethyl
alcohol 20. The alcohol 20 was esterified with 3,4,5-tri-
benzyloxybenzoic acid using DCC to give the ester 22
(Scheme 2). The benzyl groups were removed by
hydrogenolysis to obtain 9. All compounds were char-
37 ꢀC, the reaction mixture was treated with 13% TCA/
10 mM sodium pyrophosphate and allowed to stand on
ice for 30 min. The acid-precipitable material was col-
lected on filters, rinsed with 1 M HCl acid/10 mM
sodium pyrophosphate and radioactivity was deter-
mined by liquid scintillation counting. Reduction of
initial polymerization rates as a function of inhibitor
concentrations was also determined in this manner and
used to estimate IC50 for polymerization inhibition.
Table 1 records the observed IC50’s for polymerization
inhibition in both wild-type and A17 mutant enzymes
for the reported compounds. Some agents were inhibi-
tors of wild-type HIV-1-RT and, in most cases, com-
parable inhibition was observed in the A17 mutant
enzyme. Compound 1 was the most potent inhibitor
whereas compound 2 showed no activity, indicating that
one or both of the hydroxyl groups on the aromatic ring
of the chromanol moiety were necessary for inhibitory
activity. Compounds 3 and 4 showed significant but
reduced inhibition when compared to 1, suggesting a
cumulative effect of the 5- and 7-hydroxyl groups of the
chromanol moiety on activity. Compound 5 was also
inactive, suggesting one or more of the hydroxyl groups
on the gallate ring were required. Thus, compounds 6–8
were also prepared and examined. These studies showed
that the 3,4,5-trihydroxy substitution of this ring was
important for inhibition. Compound 9 showed activity
comparable to that of 1, indicating that the tether
between the two aromatic rings may be acyclic and that
the central portion of the molecule is not intimately
involved in binding to the enzyme. The lead natural
product molecules (À)-epicatechin gallate and (À)-epi-
gallocatechin gallate were also tested under the assay
conditions used in this study and were found to be more
active than any of the simplified analogues synthesized
(Table 1). The hydrolysis products (À)-epicatechin and
gallic acid did not show any inhibitory activity at con-
centrations below 100 mM.
1
acterized by H NMR and 13C NMR spectroscopy and
by elemental analysis.17
Inhibition of HIV-1-RT polymerization was carried out
using a template primer of poly rC/dG12À18 and [3H]
dGTP.18,19 The purified, wild-type and A17 mutant
HIV-1-RT enzymes used were p66/p51 heterodimers
and were prepared as described elsewhere.20 Reactions
were initiated by the addition of the enzyme (90 nM
final concentration) to the reaction mixture (final total
volume 50 mL) containing 10 mM Tris–HCl (pH 7.6),
1 mM dithiothreitol, 80 mM KCl, 12 mM MgCl2,
0.005% Triton X-100, 60 mg/mL bovine serum albumin,
20 mM [3H]dGTP (5 mCi per assay mixture), 20 mg/mL
poly(rC)/dG12À18 (Pharmacia), and the test compounds
over a range of concentrations in DMSO (1.5 mL, 3%
final DMSO concentration). After 15 min incubation at
The new compounds presented here represent an
attempt to define a limiting structure necessary for
binding to the catechin site in HIV-1-RT. Compounds
Table 1. Inhibition of wild-type and double mutant HIV-1 reverse
transcriptase
IC50 (mM) polymerization inhibition
Compound
Wild-type
A17 double
mutant
1
2
3.83Æ0.41
>100
2.36Æ1.21
>100
3
4
5
36.29Æ7.72
45.50Æ9.65
>100
30.84Æ8.35
64.67Æ15.01
>100
6
7
8
9
>100
ND
>100
ND
37.24Æ10.80
96.30Æ25.95
10.74Æ1.66
0.76Æ0.44
0.73Æ0.30
>100
6.33Æ1.53
(À)-Epicatechin gallate
(À)-Epigallocatechin gallate
(À)-Epicatechin
Gallic acid
ND
ND
ND
ND
Scheme 2. Synthesis of compound 9. Reagents: (a) SOCl2/MeOH; (b)
benzyl chloride, K2CO3, DMF; (c) LiBH4, THF; (d) DCC, DMAP,
CH2Cl2; (e) Pd–C, H2.
>100
ND, not determined.

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L. M. V. Tillekeratne et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2763–2767
1, 3, 4, and 9 were distinct in their ability to inhibit both
wild-type and A17 double mutant HIV-1-RT. In pre-
liminary cell culture studies with compound 1 antiviral
activity was not observed. Although this compound is
considerably less hydrophilic compared to the lead
catechins, it may still be unable to penetrate the cell
membrane. We have focused here on substitution pat-
terns in the two phenyl groups and retaining the struc-
ture of the tether which joins the catechin and gallate
segments. These are significant structural changes that
allow retention of much of the HIV-RT inhibitory
character of the catechins.
acetone-d6) d 2.96 (d, 1H, J=16.9 Hz), 3.27 (dd, 1H, J=16.9,
4.63 Hz), 4.24 (m, 1H), 4.31 (m, 1H), 5.41 (brm, 1H), 6.84 (m,
2H), 7.08 (s, 2H), 7.11 (d, 2H, J=6.87 Hz); 13C NMR
(75.4 MHz, acetone-d6) d 30.62 (C-4), 66.6 (C-3), 67.55 (C-2),
109.9, 117.12 (ArCH), 120.32 (ArC), 121.56 (ArCH), 121.64
(ArC), 128.21, 130.93 (ArCH), 138.94, 146.01, 154.99 (ArC),
166.22 (C¼O). Anal. calcd for C16H14O6: C, 63.56; H, 4.67.
Found: C, 63.06; H, 4.60.
3,4-Dihydro-7-hydroxy-2H-1-benzopyran-3-yl 3,4,5-trihydr-
oxybenzoate (3). Overall yield 5%; mp 234–235 ꢀC; H NMR
1
(300 MHz, acetone-d6) d 2.85 (dd, 1H, J=16.85, 2.38 Hz), 3.15
(dd, 1H, J=16.85, 4.87 Hz), 4.18 (dd, 1H, J=11.54, 1.90 Hz),
4.26 (m, 1H), 5.37 (brm, 1H), 6.30 (d, 1H, J=2.41 Hz), 6.39
(dd, 1H, J=8.23, 2.41 Hz), 6.89 (d, 1H, J=8.23 Hz), 7.04 (s,
2H); 13C NMR (75.4 MHz, MeOH-d4) d 30.57 (C-4), 67.56 (C-
3), 67.98 (C-2), 104.02, 109.89, 110.23 (ArCH), 111.39, 121.51
(ArC), 131.58 (ArCH), 140.0, 146.54, 156.01, 158.01 (ArC),
167.83 (C¼O). Anal. calcd for C16H14O7 (+0.5 H2O): C,
58.72; H, 4.62. Found: C, 58.81; H, 4.26.
Acknowledgements
The authors thank the De Arce Foundation and the
Ohio Board of Regents Research Challenge Fund for
financial assistance.
3,4-Dihydro-5-hydroxy-2H-1-benzopyran-3-yl 3,4,5-trihydr-
oxybenzoate (4). Overall yield 8%; mp 260–261 ꢀC; H NMR
1
(300 MHz, acetone-d6) d 2.87 (dd, 1H, J=17.67, 1.85 Hz), 3.03
(dd, 1H, J=17.67, 5.21 Hz), 4.18 (dd, 1H, J=11.43, 0.74 Hz),
4.26 (m, 1H), 5.41 (m, 1H), 6.34 (d, 1H, J=8.1 Hz), 6.43 (d,
1H, J=8.1 Hz), 6.92 (t, 1H, J=8.1 Hz), 7.06 (s, 2H); 13C
NMR (75.4 MHz, acetone-d6) d 25.8 (C-4), 66.42 (C-3), 67.14
(C-2), 107.73 (ArCH), 108.02 (ArC), 108.54, 109.87 (ArCH),
121.70 (ArC), 127.85 (ArCH), 138.87, 145.97, 156.08, 156.87
(ArC), 166.23 (C¼O). Anal. calcd for C16H14O7 (+0.5 H2O):
C, 58.72; H, 4.62. Found: C, 59.00; H, 4.43.
3,4-Dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl benzoate
(5). Overall yield 3%; mp 193–194 ꢀC; 1H NMR (300 MHz,
CDCl3-acetone-d6) d 2.88 (dd, 1H, J=17.06, 2.87 Hz), 3.03
(dd, 1H, J=17.06, 5.35 Hz), 4.18 (d, 1H, J=11.32 Hz), 4.29
(m, 1H), 5.51 (m, 1H), 6.04 (s, 2H), 7.41 (t, 2H, J=8.0 Hz),
7.54 (t, 1H, J=8.0 Hz), 8.0 (d, 2H, J=8.0 Hz); 13C NMR
(75.4 MHz, CDCl3-acetone-d6) d 24.73 (C-4), 65.97 (C-3),
66.70 (C-2), 95.95 (ArCH), 99.19 (ArC), 128.34, 129.77
(ArCH), 129.87 (ArC), 133.17 (ArCH), 155.38, 155.43, 155.51
(ArC), 166.22 (C¼O). Anal. calcd for C16H14O5 (+0.5 H2O):
C, 65.08; H, 5.12. Found: C, 65.62; H, 5.22.
3,4-Dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl 4-hydroxy-
benzoate (6). Overall yield 7%; mp 236–237 ꢀC; 1H NMR
(300 MHz, acetone-d6–MeOH-d4) d 2.79 (dd, 1H, J=17.03,
4.24 Hz), 2.96 (dd, 1H, J=17.03, 5.00 Hz), 4.15 (dd, 1H,
J=11.39, 2.21 Hz), 4.18 (dd, 1H, J=11.39, 1.66 Hz), 5.39
(brm, 1H), 5.87 (d, 1H, J=2.32 Hz), 6.0 (d, 1H, J=2.32 Hz),
6.86 (dd, 2H, J=9.5, 2.7 Hz), 7.84 (dd, 2H, J=9.5, 2.7 Hz);
13C NMR (75.4 MHz, acetone-d6) 29.8 (C-4), 70.87 (C-3),
71.52 (C-2), 99.88, 100.60 (ArCH), 103.41 (ArC), 120.25
(ArCH), 126.65 (ArC), 136.75 (ArCH), 160.66, 161.59, 161.94,
166.98 (ArC), 170.32 (C¼O), Anal. calcd for C16H14O6
(+0.25 H2O): C, 62.64; H, 4.76. Found: C, 62.95; H 4.74.
3,4-Dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl 3,4-dihy-
droxybenzoate (7). Overall yield 4%; mp 218–219 ꢀC; 1H
NMR (300 MHz, acetone-d6) d 2.80 (m, 1H), 2.97 (dd, 1H,
J=17.09, 5.21 Hz), 4.15 (dd, 1H, J=11.36, 1.88 Hz), 4.22 (m,
1H), 5.38 (brm, 1H), 5.89 (d, 1H, J=2.29 Hz), 6.04 (d, 1H,
J=2.29 Hz), 6.86 (d, 1H, J=8.27 Hz), 7.40 (dd, 1H, J=8.27,
2.05 Hz), 7.44 (d, 1H, J=2.05 Hz); 13C NMR (75.4 MHz, ace-
tone-d6) d 25.52 (C-4), 66.62 (C-3), 67.27 (C-2), 95.64, 96.41
(ArCH), 99.16 (ArC), 115.74, 117.14 (ArCH), 122.74 (ArC),
123.47 (ArCH), 145.56, 150.98, 156.39, 157.39, 157.69 (ArC),
166.19 (C¼O). Anal. calcd for C16H14O7 (+0.5 H2O): C,
58.72; H, 4.62. Found: C, 58.95; H, 4.52.
References and Notes
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17, 522.
17. Physical data for the compounds 1–9:
3,4-Dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl 3,4,5-tri-
hydroxybenzoate (1). Overall yield 3%; mp 242–243 ꢀC, 1H
NMR (300 MHz, acetone-d6) 2.79 (dd, 1H, J=17.1, 2.63 Hz),
2.97 (dd, 1H, J=17.1, 5.2 Hz), 4.14 (d, 1H, J=11.34 Hz), 4.22
(dd, 1H, J=11.34, 4.34 Hz), 5.36 (m, 1H), 5.88 (d, 1H,
J=2.2 Hz), 6.04 (d, 1H, J=2.2 Hz), 7.06 (s, 2H); 13C NMR
(75.4 MHz, acetone-d6) 25.46 (C-4), 66.57 (C-3), 67.25 (C-2),
95.66, 96.37 (ArCH), 99.18 (ArC), 109.87 (ArCH), 121.71,
138.89, 145.98, 156.42, 157.35, 157.66 (ArC), 166.25 (C¼O).
Anal. calcd for C16H14O8: C, 57.49; H 4.22. Found: C, 57.03;
H 4.52.
3,4-Dihydro-5,7-dihydroxy-2H-1-benzopyran-3-yl 3,5-dihy-
droxybenzoate (8). Overall yield 8%; mp 245–246 ꢀC; 1H
NMR (300 MHz, MeOH-d4) d 2.77 (dd, 1H, J=17.04,
2.69 Hz), 2.92 (dd, 1H, J=17.04, 5.15 Hz), 4.11 (d, 1H,
3,4-Dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate
(2). Overall yield 11%; mp 221–223 ꢀC; H NMR (300 MHz,
1

L. M. V. Tillekeratne et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2763–2767
2767
J=11.09 Hz), 4.21 (m, 1H), 5.37 (brm, 1H), 5.83 (d, 1H,
J=2.24 Hz), 5.93 (d, 1H, J=2.24 Hz), 6.43 (t, 1H,
J=2.24 Hz), 6.89 (d, 2H, J=2.24 Hz); 13C NMR (75.4 MHz,
MeOH-d4) d 25.75 (C-4), 67.52 (C-2), 67.72 (C-3), 95.71,
96.49 (ArCH), 99.52 (ArC), 108.38, 108.86 (ArCH), 133.08,
156.6, 157.79, 159.72 (ArC), 167.49 (C¼O). Anal. calcd for
C16H14O7 (+1 H2O): C, 57.14; H, 4.8. Found: C, 57.45; H,
4.65.
2-(3,4-dihydroxyphenyl)ethyl 3,4,5-trihydroxybenzoate (9).
Overall yield 5%; mp 189–191 ꢀC, 1H NMR (300 MHz, ace-
tone-d6) d 2.87 (t, 2H, J=6.92 Hz), 4.33 (t, 2H, J=6.92 Hz),
6.12 (dd, 1H, J=7.99, 2.01 Hz), 6.74 (d, 1H, J=7.99 Hz), 6.79
(d, 1H, J=2.01 Hz), 7.10 (s, 2H); 13C NMR (75.4 MHz, ace-
tone-d6) d 35.2 (C-2), 66.03 (C-1), 109.78, 116.03, 116.75,
121.07 (ArCH), 121.97, 130.76, 138.65, 144.42, 145.8, 145.97
(ArC), 166.64 (C¼O). Anal. calcd for C15H14O7 (+0.5 H2O):
C, 57.14; H, 4.80. Found: C, 57.19; H, 4.60.
18. Goldman, M. E.; Nunberg, J. H.; O’Brien, J. A.; Quin-
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