New 4-[(1-benzyl-1H-indol-3-yl)carbonyl]-3-hydroxyfuran-2(5H)-ones, β-diketo acid analogs as HIV-1 integrase inhibitors

Article abstract of DOI:10.1002/ardp.200700066

In addition to our recent report on a series of rationally designed benzylindolyldiketo acids acting as potent HIV-1 integrase strand transfer inhibitors, we disclose the results obtained with novel compounds chemically modified on the diketo acid moiety in order to investigate its influence on the biological activity and cytotoxicity. The activity of designed and synthesized 4-[(1-benzyl-1H-indol-3-yl)carbonyl]-3-hydroxyfuran-2(5H)-one derivatives lies in the micromolar range with regard to HIV IN enzymatic activity. The microwave-assisted synthesis was employed in some steps of the chemical procedures.

Full text of DOI:10.1002/ardp.200700066

292
Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
Full Paper
New 4-[(1-Benzyl-1H-indol-3-yl)carbonyl]-3-hydroxyfuran-
2(5H)-ones, b-Diketo Acid Analogs as HIV-1 Integrase Inhibitors
Stefania Ferro1, Maria Letizia Barreca1, Laura De Luca1, Angela Rao1, Anna Maria Monforte1,
Zeger Debyser2, Myriam Witvrouw2, and Alba Chimirri1
1 Dipartimento Farmaco-Chimico, Universitꢀ di Messina, Messina, Italy
2 Molecular Medicine, Katholieke Universiteit Leuven and IRC KULAK, Leuven, Belgium
In addition to our recent report on a series of rationally designed benzylindolyldiketo acids act-
ing as potent HIV-1 integrase strand transfer inhibitors, we disclose the results obtained with
novel compounds chemically modified on the diketo acid moiety in order to investigate its influ-
ence on the biological activity and cytotoxicity. The activity of designed and synthesized 4-[(1-
benzyl-1H-indol-3-yl)carbonyl]-3-hydroxyfuran-2(5H)-one derivatives lies in the micromolar range
with regard to HIV IN enzymatic activity. The microwave-assisted synthesis was employed in
some steps of the chemical procedures.
Keywords: HIV-1 IN inhibitors / Hydroxyfuran-2(5H)-ones / Microwave-assisted synthesis / Parallel synthesis / Phar-
macophore model /
Received: March 28, 2007; accepted: April 27, 2007
DOI 10.1002/ardp.200700066
Integrase inserts a double-stranded DNA copy of the
Introduction
viral RNA genome into the chromosomes of an infected
cell through two separate reactions: “39 processing” and
“strand transfer”.
Despite the considerable success of highly antiretroviral
therapy (HAART) in western countries, AIDS remains one
of the most urgent world health problems. The causative
agent, the human immunodeficiency virus type 1 (HIV-1)
encodes three enzymes which are required for viral repli-
cation: reverse transcriptase (RT), protease (PR), and inte-
grase (IN). Although drugs targeting RT and PR, and
recently a viral entry inhibitor (i.e. enfuvirtide), are
widely used and show effectiveness particularly when
employed in combination, toxicity and development of
resistant strains have limited their usefulness [1, 2].
Therefore, there is ever increasing impetus for the dis-
covery of new agents directed against alternative sites in
the viral life cycle. IN has thus emerged as an attractive
target for anti-HIV therapy, both because it is necessary
for stable infection and because known functional ana-
logues are lacking in the human host [3].
For the integration divalent cations such as Mn2+ or
Mg2+ are required for the catalytic activity. Although a
wide variety of compounds have been reported as IN
inhibitors [4], drugs active against this enzyme have not
yet been approved by the FDA. One of the major leads in
the development of anti-HIV-1 IN drugs is represented by
b-diketo acids (DKAs) [5] and their derivatives [4] which
selectively inhibit the strand-transfer step by sequester-
ing the divalent cations in the active site of the enzyme
[6] and block HIV-1 replication in the infected cells.
In a previous paper, we reported a 3D pharmacophore
model for DKAs consisting of four features: two hydrogen
bond acceptors (HBA1-2), one hydrogen bond donor
(HBD), and one hydrophobic aromatic region (HyAr) [1].
The resulting pharmacophore model guided the rational
design of benzylindolyldiketo acids as new IN inhibitors
able to map all features of the model (Fig. 1).
Correspondence: Stefania Ferro and Maria Letizia Barreca, Dipartimen-
to Farmaco-Chimico, Universitꢀ di Messina, Viale Annunziata, 98168
Messina, Italy.
E-mail: sferro@pharma.unime.it; barreca@pharma.unime.it
Fax: +39 090355613 (S.F. and M.L.B.)
The designed derivatives were synthesized and proved
to be highly potent IN inhibitors by selectively blocking
the strand-transfer process at nanomolar concentrations
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Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
b-Diketo Acid Analogs as HIV-1 Integrase Inhibitors
293
Figure 2. Compound 29 mapped into the four-feature pharma-
cophore model generated for HIV-1 IN DKA inhibitors (HBD,
hydrogen bond donor, purple; HBA, hydrogen bond acceptor,
green; HyAr, hydrophobic region, cyan). Predicted IC50 value =
0.12.
Figure 1. Benzylindolyl diketoacid lead compound 39 aligned
on the four-feature pharmacophore model generated for HIV-1
IN DKA inhibitors (HBD, hydrogen bond donor, purple; HBA,
hydrogen bond acceptor, green; HyAr, hydrophobic region,
cyan). Predicted IC50 value = 0.02.
considering that even if this molecule belong to a class of
compounds different from previously described DKA ana-
logues, it incorporates the 1,3-diketoacid motif into a
closed system [9].
[1] whereas, generally, they were only slightly inhibiting
the replication of HIV-1 in cell culture.
It is well known that the 1,3-diketo acids enolize at the
a-position forming 2-hydroxy-4-oxo-2-butanoic acids able
to coordinate the two magnesium ions required for IN
activity [6]. Analogously, it is possible to suppose that also
the 4-carbonyl-3-hydroxyfuran-2-one ring presenting the
same chemical functionalities (Fig. 2) could sequester the
Mg2+ ions.
On this basis, we designed and synthesized a series of 4-
[(1-benzyl-1H-indol-3-yl)carbonyl]-3-hydroxyfuran-2(5H)-
ones 29–38, analogues to DKA derivatives 39–48, with
the aim to explore the influence of this chemical manip-
ulation on IN inhibitory activity.
Furthermore, it had been reported both that the pres-
ence of the biologically labile 1,3-diketoacid moiety is a
concern for the development of these compounds as che-
motherapeutic agents [7] and that IN inhibitors of the
diketo acid type would affect the activities of other
enzymes such as RAG1_2 [8].
On these bases and considering that the 1,3-diketo acid
functionality is particularly important for the IN inhibi-
tory activity to capture bivalent cations, our efforts were
directed towards seeking viable replacement for this crit-
ical moiety in a corresponding closed system.
The chemistry sequence leading to derivatives 19–48 is
described in Scheme 1 and was achieved in a multistep
reaction, starting from the commercially available 1H-
indoles 1–4 (Scheme 1). At first, they were 3-acetylated by
reaction with acetyl chloride and a slight excess of dieth-
ylaluminum chloride, then N-alkylated by treatment
with the suitable benzyl bromide and a small amount of
sodium hydride to give intermediates 9–18. Successively,
the coupling with diethyl oxalate in the presence of a cat-
alytic amount of sodium methoxide was very efficiently
performed under microwave irradiation, to obtain inter-
mediates 19–28 with a short reaction time and high
yields. Derivatives 19–28 were converted to the final c-
lactones 29–38 by treatment with water and formalde-
hyde and successively with hydrochloric acid. From the
Results and discussion
With the aim to replace the 1,3-diketo acid group with a
more stable chelating moiety, we describe in this paper
the design, synthesis, and biological activity of a set of
DKA-analogues in which the 4-carbonyl-3-hydroxyfuran-
2(5H)-one moiety was introduced as a suitable replace-
ment for the b-diketo acid motif. This idea stems from
our previous studies in which we used a qualitative phar-
macophore model as a search query to identify structural
templates from 3D small molecule databases [9].
Among different identified compounds, 4-(4-chloro-
benzoyl)-3-hydroxy-5-phenyl-furan-2(5H)-one proved to be
an interesting IN inhibitor. We explained this behavior
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S. Ferro et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
Reagents and conditions: i) AcCl, Et2AlCl, CH2Cl2, 08C, 2 h; ii) appropriate benzyl bromide, NaH, DMF, 08C, 30 min; iii) diethyl oxalate,
dry CH3ONa, THF, two separated steps in the same conditions: 508C, 2 min, 250 W, 300 psi; iv) 2 N NaOH, MeOH, rt, 1.5 h; v) CH2O,
Et2O/H2O, rt, 2 h.
Scheme 1. Synthesis route of compounds 1–48.
same derivatives 19–28 the corresponding diketo acids with a low selectivity profile. These results suggest that
39–48 were obtained in alkaline medium.
the replacement of the b-diketo acid motif negatively
Both analytical and spectral data (1H-NMR) of all syn- influences the in-vitro biological activity, probably due to
thesized compounds are in full agreement with the pro- a reduced conformational mobility of a closed system
posed structures.
with respect to the open form.
All new 3-hydroxyfuran-2(5H)-ones 29–38, diketo acids
However, although their biological profile is far from
43, 44 and 47, 48, and esters 23, 24, 27, 28 were evaluated that of clinically useful chemotherapeutic agents, their
for their ability to inhibit IN enzymatic activity both activity values, in a micromolar range, are in accordance
against the overall integration reaction and more specifi- with the potency required for a new lead and as a starting
cally to the strand-transfer step.
point for activity optimization. Therefore, in view of the
Table 1 shows the biological data of the synthesized paucity of specific IN inhibitors up to date approved by
compounds in comparison with those of L870-810, a well- FDA, we consider it useful to continue the examination
known HIV-1 IN inhibitor of the Merck group [7, 10]. As of other possible modifications of the labile diketo acid
can be seen, derivative 33, bearing a chlorine atom at C-5 motif and further investigations are in progress.
of the benzimidazole system and a 2,6-difluorophenyl
group at the 1-position, is a very active compound (IC50
=
Financial support for this research was obtained from the Euro-
4 lM). However, even if the furanone derivatives gener- pean Commission (LSHB-CT-2003-503480). The European TRIoH
ally exhibited good potency in a micromolar range, the Consortium is gratefully acknowledged. We are grateful to
comparison of these derivatives with the corresponding Linda Desender, Martine Michiels, An Nijs, and Barbara Van
diketo acids and the respective esters pointed out a Remoortel for excellent technical assistance.
diminished potency both in enzymatic and cell assays
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Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
b-Diketo Acid Analogs as HIV-1 Integrase Inhibitors
295
Table 1. Inhibition of HIV-1 integrase enzymatic activity, replication of HIV-1, and cytotoxicity in MT-4 cells.
Com-
IN enzymatic activity
Activity in MT-4 cells
pound
Overall
ST
HIV-1
Cytotoxicity
SI*
IC50 (lM)a)
IC50 (lM)b)
EC50 (lM)c)
CC50 (lM)d)
19 £
20 £
21 £
22 £
23
0.76 l 0.10
1.20 l 0.0
5.40 l 4.16
1.50 l 0.57
0.03 l 0.01
0.07 l 0.05
7.03 l 4.55
0.38 l 0.02
0.68 l 0.42
0.80 l 0.7
ND
14.30 l 2.33
9.25 l 3.81
4.50 l 1.00
3.42 l 2.23
4.66 l 2.43
21.84 l 2.54
6.07 l 0.0
14.34 l 7.14
32.46 l 4.67
6.87 l 3.56
A81.8 l 24.2
A66.6 l 40.1
A23.4 l 14.1
A30.5
A10.5 l 3.1
A136.6 l 12.6
A20.5
A48.8 l 17.8
A29.9 l 10.4
A13.6 l 5.5
19.92 l 15.25
5.31 l 0.88
4.78 l 0.98
2.78 l 0.39
1.84 l 0.12
7.89 l 1.11
5.32 l 0.24
5.81 l 1.93
45.90 l 8.97
35.57 l 26.48
0.0047 l 0.0007
119.7 l 66.1
27.2 l 5.4
8
3
8
8
4
4
5
9
1
1
0.22 l 0.04
0.39 l 0.14
8.46 l 0.86
0.95 l 0.33
0.15 l 0.05
0.22 l 0.08
F100
F100
7.5 l 2.4
20.8 l 2.9
22.1 l 5.0
44.7 l 0.4
3.3 l 1.0
71.2 l 5.5
F100
76.3 l 0.3
53.7 l 1.2
54.3 l 18.7
0.02 l 0.0006
0.002 l 0.001
0.010 l 0.003
0.20 l 0.08
0.20 l 0.10
0.13 l 0.03
0.017 l 0.006
0.019 l 0.008
1.81 l 1.12
1.51 l 0.53
0.0005 l 0.0003
34.00 l 6.78
26.53 l 4.24
20.32 l 3.67
69.70 l 12.45
29.04 l 0.0
127.45 l 26.55
32.46 l 4.67
6.873.56
81.8 l 24.2
66.6 l 40.1
23.4 l 14.1
30.5
10.5 l 3.1
136.6 l 12.6
20.5
48.8 l 17.8
29.9 l 10.4
13.6 l 5.5
24
25 £
26 £
27
28
29
30
31
32
33
34
35
ND
276.7 l 21.7
48.7 l 0.3
90.8 l 1.4
46.8 l 1.3
4.0 l 0.3
80.1 l 9.0
ND
69.0 l 1.2
F100
127.0 l 51.6
0.03 l 0.0006
0.015 l 0.003
0.10 l 0.01
0.01 l 0.001
0.10 l 0.10
0.10 l 0.00
0.021 l 0.007
0.004 l 0.001
4.08 l 0.55
3.00 l 0.48
0.0025 l 0.0007
A1
A1
A1
A1
A1
A1
A1
A1
A1
A1
A4
A14
8
19
15
7
16
11
1
36
37
38
39 £
40 £
41 £
42 £
43
A78
A74
37.24 l 1.54
53.42 l 6.69
25.63 l 11.01
52.56 l 14.24
86.38 l 52.79
63.54 l 39.53
45.90 l 8.97
35.57 l 26.48
2.2 l 0.2
44
45 £
46 £
47
48
1
457
L-870, 810
a)
Concentration required to inhibit the in-vitro overall integrase activity by 50%.
Concentration required to inhibit the in-vitro strand-transfer step by 50%.
Effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells.
Cytotoxic concentration to reduce MT-4 cell viability by 50%.
b)
c)
d)
* Selectivity index: ratio CC50/EC50. £ Reference [1].
0,001 mol). Mp. 2098C dec, yield 97%.1H-NMR (d) 2.43 (s, 3H, CH3),
7.16–8.34 (m, 4H, ArH), 12.01 (bs, 1H, NH). Anal. Calcd for
C10H8ClNO: C, 62.03; H, 4.16; N, 7.23. Found: C, 62.17; H, 4.00; N,
7.25.
Experimental
Chemistry
Melting points were determined on a Kofler hot stage apparatus
(C. Reichert, Vienna, Austria) and are uncorrected. Elemental
analyses (C, H, N) were carried out on a C. Erba Model 1106 Ele-
mental Analyzer (Carlo Erba, Milan, Italy) and the results were
within l 0.4% of the theoretical values. Merck silica gel 60 F254
plates were used for TLC (Merck, Darmstadt, Germany). 1H-NMR
spectra were measured with a Varian Gemini 300 spectrometer
(Varian Inc., Palo Alto, CA, USA) in CDCl3 with TMS as internal
standard: chemical shifts are expressed in d (ppm) and coupling
constants (J) in Hertz.
3-Acetyl-1-(2,6-difluorobenzyl)-5-chloro-1H-indole 13
According to the procedure reported in reference [1] derivative
13 was obtained starting from 3-acetyl-5-chloro-1H-indole 6
(194 mg, 0.001 mol) and 2,6-fluorobenzyl bromide (311 mg,
0.0015 mol). Mp. 123–1258C, yield 50%; 1H-NMR (d) 2.50 (s, 3H,
CH3), 5.37 (s, 2H, CH2), 6.93–8.36 (m, 7H, ArH). Anal. Calcd for
C17H12ClF2NO: C, 63.86; H, 3.78; N, 4.38. Found: C, 63.92; H, 3.55;
N, 4.60.
3-Acetyl-6-chloro-1H-indole 7
3-Acetyl-1-(2,6-difluorobenzyl)-6-chloro-1H-indole 14
Accordingly, 14 was obtained starting from 3-acetyl-6-chloro-1H-
indole 7 (194 mg, 0.001 mol) and 2,6-difluorobenzyl bromide
According to the procedure reported in reference [1] derivative 7
was obtained starting from 6-chloro-1H-indole
3 (152 mg,
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S. Ferro et al.
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(311 mg, 0.0015 mol). Mp. 181-1838C, yield 49%; 1H-NMR (d) 2.51
(s, 3H, CH3), 5.35 (s, 2H, CH2), 6.96–8.30 (m, 7H, ArH). Anal. Calcd
for C17H12ClF2NO: C, 63.86; H, 3.78; N, 4.38. Found: C, 63.71; H,
3.90; N, 4.42.
4-(1-Benzyl-1H-indol-3-oyl)-3-hydroxyfuran-2(5H)-one 29
To a mixture of 19 (349 mg, 0.001 mol) in diethyl ether (5 mL)
was added a solution of 40% aqueous formaldehyde in water
(4 mL). The stirring was then continued until two clear layers
formed (usually within 1–2 hours). Sometimes, an additional
4 mL of water was added if the reaction was especially thick, or
the solid appeared to react slowly. The clear, aqueous bottom
layer was removed and the organic layer was extracted twice
with 5 mL of water. The combined aqueous extracts were cooled
followed by acidification with 3 mL of concentrated hydro-
chloric acid. The corresponding furanone precipitated; the solu-
tion was then cooled overnight to ensure complete product for-
mation. The resulting solid was collected, dried, and recrystal-
lized from ethanol. Mp. 191–1938C, yield 50%; 1H-NMR (d) 5.13
(s, 2H, CH2), 5.54 (s, 2H, CH2), 7.22–8.65 (m, 10ArH). Anal. Calcd
for C20H15NO4: C, 72.06; H, 4.54; N, 4.20. Found: C, 71.98; H, 4.33;
N, 4.32.
3-Acetyl-5-methoxy-1-(4-(trifluoromethyl)benzyl)-1H-
indole 17
Accordingly, 17 was obtained starting from 3-acetyl-5-methoxy-
1H-indole 8 (189 mg, 0.001 mol) and 4-trifluoromethylbenzyl
1
bromide (359 mg, 0.0015 mol). Mp. 111–1128C, yield 51%; H-
NMR (d) 2.57 (s, 3H, CH3), 3.95 (s, 3H, OCH3), 5.44 (s, 2H, CH2),
6.92–7.98 (m, 8H, ArH). Anal. Calcd for C19H16F3NO2: C, 65.70; H,
4.64; N, 4.03. Found: C, 65.88; H, 4.71; N, 3.81.
3-Acetyl-1-(4-cyanobenzyl)-5-methoxy-1H-indole 18
Accordingly, 18 was obtained starting from 3-acetyl-5-methoxy-
1H-indole 8 (189 mg, 0.001 mol) and 4-cyanobenzyl bromide
(294 mg, 0.0015 mol). Mp. 97-1008C, yield 53%; 1H-NMR (d) 2.52
(s, 3H, CH3), 3.90 (s, 3H, OCH3), 5.40 (s, 2H, CH2), 6.87–7.93 (m,
8H, ArH). Anal. Calcd for C19H16N2O2: C, 74.98; H, 5.30; N, 9.20.
Found: C, 74.87; H, 5.49; N, 9.01.
4-[1-(4-Fluorobenzyl)-1H-indol-3-oyl]-3-hydroxyfuran-
2(5H)-one 30
Accordingly, 30 was obtained starting from 20 (367 mg,
1
0.001 mol). Mp. 199–2018C, yield 55%; H-NMR (d) 5.13 (s, 2H,
CH2), 5.23 (s, 2H, CH2), 7.13–8.62 (m, 9ArH). Anal. Calcd for
C20H14FNO4: C, 68.37; H, 4.02; N, 3.99. Found: C, 68.23; H, 4.33; N,
4.02.
Ethyl-4-[1-(2,6-difluorobenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate 23
According to the procedure reported in reference [1] derivative
23 was obtained starting from 13 (320 mg, 0.001 mol).1H-NMR (d)
1.45 (t, J = 7.1, 3H, CH3), 3.94 (q, J = 7.1, 2H, CH2), 5.52 (s, 2H, CH2),
6.12 (bs, 1H, OH), 7.13–8.39 (m, 8H, 7ArH and CH). Anal. Calcd
for C21H16CLF2NO4: C, 60.08; H, 3.84; N, 3.34. Found: C, 60.19; H,
3.56; N, 3.48.
4-(1-Benzyl-5-chloro-1H-indol-3-oyl)-3-hydroxyfuran-
2(5H)-one 31
Accordingly, 31 was obtained starting from 21 (384 mg,
1
0.001 mol). Mp. 196–1988C, yield 69%; H-NMR (d) 5.15 (s, 2H,
CH2), 5.59 (s, 2H, CH2), 7.30–8.76 (m, 9ArH). Anal. Calcd for
C20H14ClNO4: C, 65.31; H, 3.84; N, 3.81. Found: C, 65.49; H, 3.73;
N, 3.72.
Ethyl-4-[1-(2,6-difluorobenzyl)-6-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate 24
Accordingly, 24 was obtained starting from 14 (320 mg,
0.001 mol). Mp. 2038C dec, yield 99%; 1H-NMR (d) 1.16 (t, J = 7.1,
3H, CH3), 4.10 (q, J = 7.1, 2H, CH2), 5.55 (s, 2H, CH2), 6.14 (bs, 1H,
OH), 7.15–8.33 (m, 9H, 8ArH, and CH). Anal. Calcd for
C21H16CLF2NO4: C, 60.08; H, 3.84; N, 3.34. Found: C, 60.01; H, 3.93;
N, 3.40.
4-[1-4-Fluorobenzyl)-5-chloro-1H-indol-3-oyl]-3-
hydroxyfuran-2(5H)-one 32
Accordingly, 32 was obtained starting from 22 (401 mg,
1
0.001 mol). Mp. 146–1488C, yield 25%; H-NMR (d) 5.12 (s, 2H,
CH2), 5.54 (s, 2H, CH2), 7.13–8.70 (m, 8H, ArH), 11.80 (bs, 1H, OH).
Anal. Calcd for C20H13ClFNO4: C, 62.27; H, 3.40; N, 3.63. Found: C,
62.35; H, 3.28; N, 3.68.
Ethyl-4-[5-methoxy-1-(4-(trifluoromethyl)benzyl)-1H-
indol-3-yl]-2-hydroxy-4-oxobut-2-enoate 27
4-[1-(2,6-Difluorobenzyl)-5-chloro-1H-indol-3-oyl]-3-
Accordingly, 27 was obtained starting from 17 (347 mg,
0.001 mol). Mp. 268–2708C dec, yield 100%;1H-NMR (d) 1.24 (t, J =
7.1, 3H, CH3), 3.74 (s, 3H, OCH3), 4.12 (q, J = 7.1, 2H, CH2), 5.54 (s,
2H, CH2), 6.28 (bs, 1H, OH), 6.74–8.16 (m, 9H, 8ArH, and CH).
Anal. Calcd for C23H20F3NO5: C, 61.75; H, 4.51; N, 3.13. Found: C,
61.88; H, 4.39; N, 3.17.
hydroxyfuran-2(5H)-one 33
Accordingly, 33 was obtained starting from 23 (420 mg,
1
0.001 mol). Mp. 182–1848C, yield 42%; H-NMR (d) 5.05 (s, 2H,
CH2), 5.60 (s, 2H, CH2), 7.14–8.70 (m, 7ArH). Anal. Calcd for
C20H12ClF2NO4: C, 59.49; H, 3.00; N, 3.47. Found: C, 59.58; H, 3.12;
N, 3.61.
Ethyl-4-[1-(4-cyanobenzyl)-5-methoxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate 28
4-[1-(2,6-Difluorobenzyl)-6-chloro-1H-indol-3-oyl]-3-
hydroxyfuran-2(5H)-one 34
Accordingly, 28 was obtained starting from 18 (304 mg,
0.001 mol). Mp. 248–2508C dec, yield 98%; 1H-NMR (d) 1.24 (t, J =
6.8, 3H, CH3), 3.74 (s, 3H, OCH3), 4.12 (q, J = 6.8, 2H, CH2), 5.53 (s,
2H, CH2), 6.23 (bs, 1H, OH), 6.73–8.43 (m, 9H, 8ArH and CH).
Anal. Calcd for C23H20N2O5: C, 68.31; H, 4.98; N, 6.93. Found: C,
68.54; H, 5.00; N, 6.71.
Accordingly, 34 was obtained starting from 24 (419 mg,
1
0.001 mol). Mp. 183-1858C, yield 44%; H-NMR (d) 5.04 (s, 2H,
CH2), 5.60 (s, 2H, CH2), 7.16–8.58 (m, 7ArH). Anal. Calcd for
C20H12ClF2NO4: C, 59.49; H, 3.00; N, 3.47. Found: C, 59.36; H, 3.21;
N, 3.35.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
b-Diketo Acid Analogs as HIV-1 Integrase Inhibitors
297
4-(1-Benzyl-5-methoxy-1H-indol-3-oyl)-3-hydroxyfuran-
4-[1-(4-Cyanobenzyl)-5-methoxy-1H-indol-3-yl]-2-
2(5H)-one 35
hydroxy-4-oxobut-2-enoic acid 48
Accordingly, 35 was obtained starting from 25 (378 mg,
0.001 mol). Mp. 1928C dec., yield 43%;1H-NMR (d) 3.76 (s, 3H, CH3)
5.13 (s, 2H, CH2), 5.48 (s, 2H, CH2), 6.89–8.62 (m, 9H, ArH), 11.73
(bs, 1H, OH). Anal. Calcd for C21H17NO5: C, 69.41; H, 4.72; N, 3.85.
Found: C, 69.37; H, 4.88; N, 3.77.
Accordingly, 48 was obtained starting from 28 (404 mg,
1
0.001 mol). Mp. 176–1788C, yield 57%; H-NMR (d) 3.78 (s, 3H,
OCH3), 5.59 (s, 2H, CH2), 6.87–8.93 (m, 9H, ArH and CH). Anal.
Calcd for C21H16N2O5: C, 67.02; H, 4.28; N, 7.44. Found: C, 67.19;
H, 4.41; N, 7.12.
Pharmacology
Overall integrase assay using an enzyme-linked immunosorbent
assay (ELISA).
4-[1-(4-Fluorobenzyl)-5-methoxy-1H-indol-3-oyl]-3-
hydroxyfuran-2(5H)-one 36
Accordingly, 36 was obtained starting from 26 (397 mg,
To determine the susceptibility of the HIV-1 integrase enzyme
towards different compounds, we optimized enzyme-linked
immunosorbent assays. These assays use an oligonucleotide sub-
strate of which one oligonucleotide (59- ACTGCTAGAGATTTTC-
CACACTGACTAAAAGGGTC -39) is labeled with biotin at the 39-
end and the other oligonucleotide is labeled with digoxigenin at
the 59-end. For the overall integration assay the second 59-digoxi-
genin labeled oligonucleotide is (59- GACCCTTTTAGTCAGTGTG-
GAAAATCTCTAGCAGT -39). For the strand-transfer assay, the sec-
ond oligonucleotide lacks GT at the 39-end. The integrase enzyme
was diluted in 750mM NaCl, 10 mM Tris, pH 7.6, 10% glycerol,
and 1 mM b-mercapto ethanol. To perform the reaction, 4 lL
diluted integrase (corresponding to a concentration of 1.6 lM)
and 4 lL of annealed oligonucleotides (7 nM) was added in a
final reaction volume of 40 lL containing 10 mM MgCl2, 5 mM
DTT, 20 mM HEPES, pH 7.5, 5% PEG, and 15% DMSO. The reac-
tion was carried out for 1 h at 378C. Reaction products were
denatured with 30 mM NaOH and detected by an immunosorb-
ent assay on avidin-coated plates [11].
1
0.001 mol). Mp. 172–1748C, yield 52%; H-NMR (d) 3.76 (s, 3H,
CH3) 5.13 (s, 2H, CH2), 5.48 (s, 2H, CH2), 6.86–8.57 (m, 8ArH).
Anal. Calcd for C21H16FNO5: C, 66.14; H, 4.23; N, 3.67. Found: C,
66.25; H, 4.32; N, 3.52.
4-[5-Methoxy-1-(4-(trifluoromethyl)benzyl)-1H-indol-3-
oyl]-3-hydroxyfuran-2(5H)-one 37
Accordingly, 37 was obtained starting from 27 (447 mg,
1
0.001 mol). Mp. 189–1918C, yield 67%; H-NMR (d) 3.76 (s, 3H,
CH3) 5.14 (s, 2H, CH2), 5.63 (s, 2H, CH2), 6.86–8.58 (m, 8H, ArH).
Anal. Calcd for C22H16F3NO5: C, 61.26; H, 3.74; N, 3.25. Found: C,
61.06; H, 3.82; N, 3.18.
4-[1-(4-Cyanobenzyl)-5-methoxy-1H-indol-3-oyl]-3-
hydroxyfuran-2(5H)-one 38
Accordingly, 38 was obtained starting from 28 (404 mg,
0.001 mol). Mp. 168–708C, yield 72%; 1H-NMR (d) 3.77 (s, 3H,
CH3) 5.13 (s, 2H, CH2), 5.62 (s, 2H, CH2), 6.86–7.81 (m, 8H, ArH).
Anal. Calcd for C22H16N2O5: C, 68.04; H, 4.15; N, 7.21. Found: C,
68.11; H, 4.30; N, 7.09.
In-vitro anti-HIV and drug susceptibility assays
The inhibitory effect of antiviral drugs on the HIV-induced cyto-
pathic effect (CPE) in human lymphocyte MT-4 cell culture was
determined by the MT-4/MTT-assay [12]. This assay is based on
the reduction of the yellow coloured 3-(4,5-dimethylthiazol-2yl)-
2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehy-
drogenase of metabolically active cells to a blue formazan deriv-
ative, which can be measured spectrophotometrically. The 50%
cell culture infective dose (CCID50) of the HIV (IIIB) strain was
determined by titration of the virus stock using MT-4 cells. For
the drug-susceptibility assays, MT-4 cells were infected with
100–300 CCID50 of the virus stock in the presence of five-fold
serial dilutions of the antiviral drugs. The concentration of vari-
ous compounds achieving 50% protection against the CPE of the
different HIV strains, which is defined as the EC50, was deter-
mined. In parallel, the 50% cytotoxic concentration (IC50) was
determined.
4-[1-(2,6-Difluorobenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid 43
According to the procedure reported in reference [1] derivative
43 was obtained starting from 23 (420 mg, 0.001 mol). Mp. 136–
1388C, yield 48%; 1H-NMR (d) 5.41 (s, 2H, CH2), 6.89 (s, 1H, CH),
6.96–8.30 (m, 7H, ArH). Anal. Calcd for C19H12ClF2NO4: C, 58.25;
H, 3.09; N, 3.58. Found: C, 58.03; H, 3.28; N, 3.44.
4-[1-(2,6-Difluorobenzyl)-6-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid 44
Accordingly, 44 was obtained starting from 24 (420 mg,
1
0.001 mol). Mp. 180–1828C, yield 45%; H-NMR (d) 5.40 (s, 2H,
CH2), 6.91 (s, 1H, CH), 7.01–8.22 (m, 7H, ArH). Anal. Calcd for
C19H12ClF2NO4: C, 58.25; H, 3.09; N, 3.58. Found: C, 58.41; H, 3.01;
N, 3.47.
References
[1] M. L. Barreca, S. Ferro, A. Rao, L. De Luca, et al., J. Med.
Chem. 2005, 48, 7084–7088.
4-[1-(4-Trifluoromethylbenzyl)-5-Methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid 47
Accordingly, 47 was obtained starting from 27 (447 mg,
[2] T. Imamichi, Curr. Pharm. Des. 2004, 10, 4039–4053.
[3] N. J. Anthony, Curr. Top. Med. Chem. 2004, 4, 979–990.
1
0.001 mol). Mp. 183–1848C dec., yield 39%; H-NMR (d) 3.92 (s,
3H, OCH3), 5.42 (s, 2H, CH2), 6.89 (s, 1H, CH), 7.15–7.94 (m, 8H,
ArH). Anal. Calcd for C21H16F3NO5: C, 64.15; H, 3.85; N, 3.39.
Found: C, 64.33; H, 4.02; N, 3.17.
[4] A. A. Johnson, C. Marchand, Y. Pommier, Curr. Top. Med.
Chem. 2004, 4, 1059–1077.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

298
S. Ferro et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 292–298
[5] D. J. Hazuda, P. Felock, M. Witmer, A. Wolfe, et al., Science
2000, 287, 646–650.
[9] M. L. Barreca, A. Rao, L. De Luca, M. Zappalꢀ, et al., J.
Chem. Inf. Comput. Sci. 2004, 44, 1450–1455.
[6] J. A. Grobler, K. Stillmock, B. Hu, M. Witmer, et al., Proc.
Natl. Acad. Sci. USA 2002, 99, 6661–6666.
[10] D. J. Hazuda, P. Felock, M. Witmer, A. Wolfe, et al., Science
2000, 287, 646–650.
[7] L. Zhuang, J. S. Wai, M. W. Embrey, T. E Fisher, et al., J.
Med. Chem. 2003, 46, 453–456.
[11] Y. Hwang, D. Rhodes, F. Bushman, Nucleic Acids Res. 2000,
28, 4884–4892.
[8] M. Melek, J. M. Jones, M. H. O'Dea, G. Pais, et al., Proc. Natl.
Acad. Sci. USA 2002, 99, 134–137.
[12] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, et al., J. Virol.
Methods 1988, 20, 309–321.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com