Synthesis and anti-hIV activity of 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole structurally-related 1,2-substituted benzimidazoles.

Article abstract of DOI:10.1016/S0014-827X(02)01300-9

The synthesis of 1,2-substituted benzimidazoles is reported. These novel derivatives share chemical similarities with 1-aryl-1H,3H-thiazolo[3,4-a]benzimidazoles, a class of HIV-1 NNRTIs studied widely. All compounds prepared were tested in MT-4 cells to explore their potential anti-HIV activity and were found to be less potent than 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole (TBZ).

Full text of DOI:10.1016/S0014-827X(02)01300-9

Il Farmaco 57 (2002) 819ꢀ823
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www.elsevier.com/locate/farmac
Synthesis and anti-HIV activity of 1-(2,6-difluorophenyl)-1H,3H-
thiazolo[3,4-a]benzimidazole structurally-related 1,2-substituted
benzimidazoles
Angela Rao a,*, Alba Chimirri a, Erik De Clercq b, Anna Maria Monforte a,
Pietro Monforte a, Christophe Pannecouque b, Maria Zappala` a
a Dipartimento farmaco-chimico, universita` di Messina, viale Annunziata, 98168 Messina, Italy
b Rega Institute for medical research, katholieke universiteit Leuven, 10 Minderbroedersstraat, 3000 Leuven, Belgium
Received 15 April 2002; accepted 1 July 2002
Abstract
The synthesis of 1,2-substituted benzimidazoles is reported. These novel derivatives share chemical similarities with 1-aryl-1H,3H-
thiazolo[3,4-a]benzimidazoles, a class of HIV-1 NNRTIs studied widely. All compounds prepared were tested in MT-4 cells to
explore their potential anti-HIV activity and were found to be less potent than 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-
a]benzimidazole (TBZ).
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 1,2-Substituted benzimidazoles; Anti-HIV agents; NNRTI
1. Introduction
structureꢀactivity relationship (SAR) studies have
/
been performed within this family of compounds and
it has been observed that specific requirements exist with
regard to the structural determinants for optimum anti-
HIV activity. The C-1 substituent plays a decisive and
crucial role in the interaction of these compounds with
the HIV-1 RT: a 2,6-dihalo substituted phenyl ring at C-
1 furnished rewarding results and gave a large improve-
ment in potency. In particular, 1-(2,6-difluorophenyl)-
1H,3H-thiazolo[3,4-a]benzimidazole (TBZ, Fig. 1)
proved to be the most potent inhibitor and has been
selected as lead compound of this series.
We have also demonstrated previously [5] that the
biological activity of TBZ is associated with the ability
to assume a ‘butterfly-like’ conformation, which allows
a binding mode similar to other NNRTIs by means of a
suitable spatial location of lipophilic and electron-rich
groups. From this study, it resulted that the plausible
pharmacophoric elements for TBZ were: the benzene
fused ring, the aryl group at C-1 and the nitrogen atom
of the thiazole nucleus.
Since the beginning of anti-AIDS chemotherapy,
HIV-1 reverse transcriptase (RT) has been the main
target in the attack against this virus and the majority of
drugs under clinical use are inhibitors of such enzyme
[1]. These inhibitors can be classified into two groups:
nucleosides (NRTIs), that as chain terminators block
the growth of the DNA strand, and the non-nucleosides
(NNRTIs), which are a structurally diverse group of
compounds that inhibit the enzyme in an allosteric mode
by binding to a pocket near the polymerase active site.
In contrast to the former group, NNRTIs present low
toxicity and high selectivity. In recent years, three
NNRTIs namely nevirapine, delavirdine and efavirenz
have been approved by FDA for clinical use in
combined therapy [2].
In previous papers [3,4] we reported a series of 1-aryl-
1H,3H-thiazolo[3,4-a]benzimidazoles which proved to
be highly active as HIV-1 NNRTIs. Extensive
Furthermore, the potential therapeutic utility of TBZ
is hampered by the metabolic oxidation of the thiazole
* Corresponding author
E-mail address: a.rao@pharma.unime.it (A. Rao).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 3 0 0 - 9

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A. Rao et al. / Il Farmaco 57 (2002) 819ꢀ823
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The reaction proceeds through the formation of 2-
arylbenzimidazoline, generated in situ from 1,2-pheny-
lenediamine and the appropriate aromatic aldehyde.
Thanks to the well-known reducing ability exhibited by
this compound [7], the aromatic aldehyde is converted
into the corresponding alcohol which in turn alkylates
the 2-arylbenzimidazole formed in this redox process.
When the aromatic diamine employed is strongly
activated by the presence of a methoxy substituent at
position 4, a FriedelꢀCraft alkylation also occurs and
/
the 2-aryl-1-benzylbenzimidazoles 5 or 6-benzyl substi-
tuted were isolated as the main products of the reaction
(Scheme 2).
Fig. 1.
The structural assignment of compounds 3 and 7ꢀ10
/
ring of TBZ leading to the formation of less potent
sulfoxide and sulfone metabolites [6].
has been carried out on the basis of long-range hetero-
nuclear chemical shift correlation (LR-HETCOR) ex-
periments.
The key entry point is the identification of the C-7a
resonance which is assigned on the basis of its correla-
tion with the N-benzyl methylene protons. In compound
3 this carbon is long-range correlated only to H-4
resonance, so indicating that the carboxylic group is
linked to C-6.
On these bases, we have removed the thiazolidine
nucleus and synthesized a series of 1-benzyl-2-arylben-
zimidazoles (Fig. 1) as new NNRTIs. The elimination of
the thiazole nucleus allowed keeping all key structural
requirements, that is two p-systems and a nitrogen
atom, for enzyme inhibition.
In derivatives 7ꢀ
substituents on the benzene fused ring has been esta-
blished on the basis of the presence, in the spectra of 7ꢀ
8, of cross-peaks between the CH2-6 and C-5 and C-7,
whereas in derivatives 9ꢀ10 the same methylene protons
/
10, the relative position of the two
2. Results and discussion
/
The synthesis of 2-aryl-1-benzylbenzimidazoles was
accomplished by reacting a 1,2-phenylenediamine with
an excess of an aromatic aldehyde in the presence of a
catalytic amount of p-toluenesulfonic acid, as shown in
Scheme 1.
/
are long-range coupled with C-4 and C-6.
The antiviral activities of the synthesized compounds
1ꢀ10 were evaluated in MT-4 cells acutely infected with
/
HIV-1 (IIIB) or HIV-2 (ROD) and compared with TBZ.
Scheme 1.
Scheme 2.

A. Rao et al. / Il Farmaco 57 (2002) 819ꢀ
/823
821
Table 1
congener TBZ here used as reference compound (Table
1).
Anti-HIV activity and cytotoxicity for compounds 1ꢀ
/10
On the basis of our previous modeling studies on TBZ
derivatives and other NNRTIs [5], the 3D structure of
new benzimidazole derivatives was compared with that
of TBZ aiming at the maximum overlap of the most
plausible pharmacophoric elements, with the purpose to
rationalize the lack of activity observed for benzimida-
a
b
Comp.
EC50 (mM)
CC50 (mM)
HIV-1IIIb
HIV-2ROD
1
2.6290.25
2.0190.47
ꢀ116.66
ꢀ6.15
4.8991.32
7.3592.16
31.2292.83
ꢀ18.75
ꢀ5.40
ꢀ23.33
ꢀ33.63
ꢀ116.66
ꢀ6.15
ꢀ31.95
ꢀ30.45
ꢀ31.35
ꢀ18.75
ꢀ5.40
23.3392.81
33.6397.02
116.66912.0
6.1590.39
31.9594.01
30.4593.42
31.3592.75
18.7592.85
5.4092.05
2
3
4
zole derivatives 1ꢀ10.
/
5
The molecular model of compound 6 was constructed
using the SYBYL 6.7 molecular modeling software [8] and
fitted with TBZ. As seen in Fig. 2, considerable
similarity exists between the energy minimized butter-
fly-like shape of TBZ and 6.
The obtained results suggest that also the new
compounds could bind to RT assuming a butterfly-like
conformation essential for enzyme inhibition, but the
extra phenyl ring at C-2 probably enhance too much the
steric hindrance to well accommodate into the apolar
pocket of RT.
6
7
8
9
10
TBZ
ꢀ126.67
ꢀ126.67
ꢀ48.60
126.67933.81
48.6095.5
0.6090.5
a
Concentration required to reduce HIV-induced cytopathic effect
by 50%.
b
Concentration required to reduce MT-4 cell viability by 50%.
The ability of the structural changes introduced in the
benzimidazole system to influence the anti-HIV activity
is shown in Table 1.
Derivatives 1, 2, 5 and 6 were found to selectively
inhibit HIV-1 (IIIB) replication whereas, like other
NNRTIs, none of the compounds inhibited the replica-
tion of HIV-2 (ROD) in MT-4 cells.
3. Experimental
3.1. Chemistry
The biological results obtained suggest that in this
series of compounds the anti-HIV activity is maintained
when the benzene-fused ring is not substituted, whereas
the substitution in position 5 and/or 6 with bulky group
negatively influences the biological activity. However,
new tested compounds were found to have low efficacy.
They were much less potent than their structural
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. Elemental analyses (C,
H, N) were carried out on a Carlo Erba Model 1106
elemental analyzer and the results are within 90.4% of
/
the theoretical values. Merck silica gel 60 F254 plates
were used for analytical TLC; column chromatography
was performed on Merck silica gel 60 (230ꢀ400 mesh).
/
1H NMR spectra were recorded in CDCl3 on a Varian
Gemini-300 spectrometer. Chemical shifts were ex-
pressed in d (ppm) relative to TMS as internal standard
and coupling constants (J) in Hz. HETCOR and LR-
HETCOR experiments were carried out by using the
standard software package.
3.1.1. Synthesis of 1-benzyl-2-arylbenzimidazoles (1ꢀ10)
/
To a stirred solution of 1,2-phenylenediamine (8
mmol) in ethanol (80 ml), the appropriate aromatic
aldehyde (16 mmol) and p-toluenesulfonic acid (16
mmol) were added. The reaction mixture was refluxed
for 2 h, then diluted with water and extracted with
chloroform. The collected organic phases were dried
over sodium sulfate and the solvent evaporated under
reduced pressure to give an oily residue which was
powdered by treatment with a mixture of ethanol and
diethyl ether to afford compounds 4, 5 and 7, or
separated by silica gel column chromatography eluting
with cyclohexane/ethyl acetate 4:6. All compounds were
recrystallized from ethanol.
Fig. 2. Superimposition of TBZ (grey) and 6 (black) resulting from
QXP fitting.

822
A. Rao et al. / Il Farmaco 57 (2002) 819ꢀ823
/
3.1.2. 1-(2?-Fluorobenzyl)-2-(2?-fluorophenyl)-
benzimidazole (1)
3.1.9. 1,6-(2?-Chloro-6?-fluorobenzyl)-2-(2?-chloro-6?-
fluorophenyl)-5-methoxybenzimidazole (8)
1
1
Yield 34%, m.p. 86ꢀ
/
88 8C. H NMR: 5.38 (s, 2H,
Yield 23%, m.p. 223ꢀ
/
225 8C. H NMR: 3.94 (s, 3H,
CH2), 6.73ꢀ
/
7.90 (m, 12H, ArH). 13C NMR: 42.05
OCH3), 4.19 (s, 2H, CH2), 5.19 (d, 1H, H2), 5.29 (d, 1H,
(CH2), 110.38 (C-7), 115.23 and 115.91 (C-3?, C-3ƒ),
118.26 and 122.79 (C-1?, C-1ƒ), 119.99 (C-4), 122.50 and
123.13 (C-5, C-6), 124.20, 124.56, 128.20, 129.38, 132.04,
132.17 (C-4?, C-4ƒ, C-5?, C-5ƒ, C-6?, C-6ƒ), 135.06 (C-
7a), 143.13 (C-2), 149.15 (C-3a), 158.29 and 161.58 (C-
2?, C-2ƒ).
H2), 6.59ꢀ7.44 (m, 11H, ArH).
/
3.1.10. 1,5-(2?,6?-Difluorobenzyl)- 2-(2?,6?-
difluorophenyl)-6-methoxybenzimidazole (9)
1
Yield 15%, m.p. 169ꢀ
/
171 8C. H NMR: 3.88 (s, 3H,
OCH3), 4.08 (s, 2H, CH2), 5.29 (s, 2H, CH2), 6.77ꢀ
/
7.45
(m, 11H, ArH). 13C NMR: 22.69 (CH2ꢀ
/
5), 36.29 (CH2ꢀ
/
N), 55.61 (OCH3), 91.27 (C-7), 110.88, 111.12, 111.31,
111.52, 111.67, 111.83 (C-3?, C-3ƒ, C-3§, C-5?, C-5ƒ, C-
5§), 116.10 (C-1?), 119.87 (C-4), 123.53 (C-5), 127.84,
130.68, 132.00 (C-4?, C-4ƒ, C-4§), 133.64 (C-7a), 137.74
(C-3a), 141.80 (C-2), 154.84 (C-6), 160.78,
160.86,161.02, 161.15, 161.20, 161.22 (C-2?, C-2ƒ, C-2§,
C-6?, C-6ƒ, C-6§).
3.1.3. 1-(2?,6?-Difluorobenzyl)-2-(2?,6?-difluorophenyl)-
benzimidazole (2)
1
Yield 20%, m.p. 103ꢀ
/
105 8C. H NMR: 5.38 (s, 2H,
CH2), 6.79ꢀ7.87 (m, 10H, ArH).
/
3.1.4. 6-Carboxy-1-(2?,6?-difluorobenzyl)-2-(2?,6?-
difluorophenyl)-benzimidazole (3)
1
Yield 10%, m.p. 223 8C dec. H NMR: 5.45 (s, 2H,
CH2), 6.80ꢀ
8.39 (m, 9H, ArH). 13C NMR: 36.69 (CH2),
/
3.1.11. 1,5-(2?-Chloro-6?-fluorobenzyl)-2-(2?-chloro-6?-
fluorophenyl)-6-methoxybenzimidazole 10
108.05 and 110.90 (C-1? and C-1ƒ), 111.51 and 111.91
(C-3?, C-5?, C-3ƒ, C-5ƒ), 113.19 (C-7), 120.10 (C-4),
124.56 (C-5), 124.66 (C-2), 130.92 and 132.82 (C-4?, C-
4ƒ), 134.48 (C-7a), 146.18 (C-2), 146.81 (C-3a), 160.97,
161.05, 161.18, 161.27 (C-2?, C-2ƒ, C-6?, C-6ƒ), 171.73
1
Yield 7%, m.p. 195ꢀ
/
199 8C. H NMR: 3.88 (s, 3H,
OCH3), 4.14 (s, 2H, CH2), 5.22 (d, 1H, H2), 5.31 (d, 1H,
H2), 6.63ꢀ7.49 (m, 11H, ArH).
/
3.2. Pharmacology
(CÄ/O).
The antiviral experiments using MT-4 cells and HIV-1
(IIIB) and HIV-2 (ROD) strains were performed follow-
ing procedures that have already been described [9,10].
3.1.5. 1-(2?,6?-Difluorobenzyl)-2-(2?,6?-difluorophenyl)-
5,6-dimethyl-benzimidazole (4)
1
Yield 70%, m.p. 189ꢀ
/
191 8C. H NMR: 2.36 (s, 3H,
CH3), 2.38 (s, 3H, CH3), 5.31 (s, 2H, CH2), 6.76ꢀ
/
7.58
(m, 8H, ArH).
Acknowledgements
3.1.6. 1-(2?,6?-Difluorobenzyl)-2-(2?,6?-difluorophenyl)-
5-methyl-benzimidazole (5)
These investigations were supported in part by the
Ministero dell’Istruzione, dell’Universita` e della Ricerca
(COFIN 2000).
1
Yield 27%, m.p. 136ꢀ
/
138 8C. H NMR: 2.47 (s, 3H,
CH3), 5.33 (s, 2H, CH2), 6.76ꢀ/7.62 (m, 9H, ArH).
3.1.7. 1-(2?,6?-difluorobenzyl)-2-(2?,6?-difluorophenyl)-
6-methyl-benzimidazole (6)
References
1
Yield 34%, m.p. 143ꢀ
/
145 8C. H NMR: 2.48 (s, 3H,
[1] H. Jonckheere, J. Anne´, E. De Clercq, The HIV-1 reverse
transciption (RT) process as target for RT inhibitors, Med. Res.
CH3), 5.33 (s, 2H, CH2), 6.77ꢀ/7.72 (m, 9H, ArH).
Rev. 20 (2000) 129ꢀ
[2] S. Vella, L. Palmisano, Antiretroviral therapy: state of the
HAART, Antiviral Res. 45 (2000) 1ꢀ7.
[3] (a) A. Chimirri, S. Grasso, A.M. Monforte, P. Monforte, M.
Zappala`, Anti-HIV agents II. Synthesis and in vitro anti-HIV
activity of novel 1H,3H-thiazolo[3,4-a]benzimidazoles, Farmaco
/154.
3.1.8. 1,6-(2?,6?-Difluorobenzyl)- 2-(2?,6?-
difluorophenyl)-5-methoxybenzimidazole (7)
/
1
Yield 14%, m.p. 192ꢀ
/
196 8C. H NMR: 3.94 (s, 3H,
OCH3), 4.13 (s, 2H, CH2), 5.24 (s, 2H, CH2), 6.65ꢀ7.56
/
46 (1991) 925ꢀ933;
/
(m, 11H, ArH). 13C NMR: 22.78 (CH2-6), 36.37 (CH2-
N), 52.72 (OCH3), 91.42 (C-7), 111.01, 111.24, 111.40,
111.56, 111.75, 111.84 (C-3?. C-5?, C-3ƒ, C-5ƒ, C-3§, C-
5§), 116.08 (C-1?, C-1ƒ, C-1§), 120.03 (C-4), 123.70 (C-
6), 127.84, 130.67, 132.00 (C-4?, C-4ƒ, C-4§), 133.79 (C-
7a), 137.23 (C-3a), 141.51 (C-2), 155.03 (C-5), 159.96,
160.54, 160.85, 161.40, 161.58, 161.86 (C-2?, C-2ƒ, C-2§,
C-6?, C-6ƒ, C-6§).
(b) A. Chimirri, S. Grasso, C. Molica, A.M. Monforte, P.
Monforte, M. Zappala`, Anti-HIV agents. IV. Synthesis and in
vitro anti-HIV activity of novel 1-(2,6-difluorophenyl)-
1H,3H-thiazolo[3,4-a]benzimidazoles, Farmaco 51 (1996) 279ꢀ
282.
/
[4] (a) A. Chimirri, S. Grasso, C. Molica, A.M. Monforte, P.
Monforte, M. Zappala`, G. Bruno, F. Nicolo`, M. Witvrouw, H.
Jonckeere, J. Balzarini, E. De Clercq, Structural features and anti-
human immunodeficiency virus (HIV) activity of the isomers of 1-

A. Rao et al. / Il Farmaco 57 (2002) 819ꢀ
/823
823
(2?,6?-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole, a po-
tent non-nucleoside HIV-1 reverse transcriptase inhibitor, Anti-
[6] R.J. Schultz, J.P. Bader, A. Chimirri, J.M. Covey, D.L. Hill, R.D.
Haugwitz, F.S. Guziec, V.L. Narayanan, Thiazolobenzimida-
viral Chem. Chemother. 8 (1997) 363ꢀ370;
/
zoles*a new class of anti-HIV agents, Proc. Am. Assoc. Cancer
/
(b) A. Chimirri, S. Grasso, A.M. Monforte, P. Monforte, A. Rao,
M. Zappala`, G. Bruno, F. Nicolo`, C. Pannecouque, M. Witv-
rouw, E. De Clercq, Synthesis, structure and in vitro anti-human
immunodeficiency virus activity of novel 3-methyl-1H,3H-thia-
zolo[3,4-a]benzimidazoles, Antiviral Chem. Chemother. 9 (1998)
Res. 33 (1992) 517.
[7] F. Risitano, G. Grassi, F. Caruso, F. Foti, C,C- and C,N-linked
dimers and 4-arylmethyl derivatives from 4-arylmethylene pyr-
azol-5-ones and isoxazol-5-ones with 2-arylbenzimidazolines,
Tetrahedron 52 (1996) 1443ꢀ1450.
/
431ꢀ438;
/
[8] SYBYL 6.7, Tripos Associates Inc., St. Louis, Missouri, MO, 2000.
[9] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P.
Herdewijin, J. Desmyter, E. De Clercq, Rapid and automated
tetrazolium-based colorimetric assay for the detection of anti-HIV
(c) A. Chimirri, S. Grasso, P. Monforte, A. Rao, M. Zappala`,
A.M. Monforte, C. Pannecouque, M. Witvrouw, J. Balzarini, E.
De Clercq, Synthesis and biological activity of novel 1H,3H-
thiazolo[3,4-a]benzimidazoles: non-nucleoside human immuno-
deficiency virus type 1 reverse transcriptase inhibitors, Antiviral
compounds, J. Virol. Methods 20 (1988) 309ꢀ321.
/
[10] M. Witvrouw, D. Schols, G. Andrei, R. Snoeck, M. Hosoya, R.
Pauwels, J. Balzarini, E. De Clercq, Antiviral activity of low-MW
dextran sulfate (derived from dextran MW 1000) compared to
dextran sulfate samples of higher MW, Antiviral Chem. Che-
Chem. Chemother. 10 (1999) 211ꢀ217.
/
[5] M.L. Barreca, A. Chimirri, A. Carotti, A. Carrieri, A.M.
Monforte, M. Pellegrini Calace, A. Rao, Comparative molecular
field analysis (CoMFA) and docking studies of non-nucleoside
HIV-1 RT inhibitors (NNRTIs), Bioorg. Med. Chem. 7 (1999)
mother. 2 (1991) 171ꢀ179.
/
2283ꢀ2292.
/