Synthesis of new pyridazine derivatives as potential anti-HIV-1 agents

Article abstract of DOI:10.1002/jhet.230

(Chemical Equation Presented) We herein report conventional and microwave-assisted methods for the synthesis of a series of compounds containing some bivalent ion chelating requirements useful to exert potential HIV-1 integrase (IN) enzyme inhibition acti

Full text of DOI:10.1002/jhet.230

1420
Synthesis of New Pyridazine Derivatives as Potential
Anti-HIV-1 Agents
Vol 46
Stefania Ferro,a* Stefano Agnello,a Maria Letizia Barreca,b Laura De Luca,a
Frauke Christ,c and Rosaria Gittoa
aDepartment of Medicinal Chemistry, University of Messina, Viale Annunziata,
I-98168 Messina, Italy
bDepartment of Pharmaceutical Chemistry and Technology, University of Perugia,
Via del Liceo 1, I-06123 Perugia, Italy
cMolecular Medicine, Katholieke Universiteit Leuven and IRC KULAK,
Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
*E-mail: sferro@pharma.unime.it
Received April 15, 2009
DOI 10.1002/jhet.230
Published online 10 November 2009 in Wiley InterScience (www.interscience.wiley.com).
We herein report conventional and microwave-assisted methods for the synthesis of a series of com-
pounds containing some bivalent ion chelating requirements useful to exert potential HIV-1 integrase
(IN) enzyme inhibition activity. The biological screening highlighted that only derivative 5 proved to be
a strand-transfer step inhibitor of the virus integration process at micromolar concentration.
J. Heterocyclic Chem., 46, 1420 (2009).
INTRODUCTION
Food and Drug Administration (FDA), belongs to the
DKAs analogs class, in which the diketo acid moiety, as
‘‘ketoenole,’’ has been included in a rigid system.
Taking into account these findings and considering
the results of our previously reported studies [7–10], we
planned the synthesis of new derivatives containing a
pyridazine core as well as the chemical features able to
inhibit IN activity as chelating agents for bivalent metal
ions. The obtained compounds were characterized and
tested to evaluate their antiviral activity and enzymatic
inhibition.
Human immunodeficiency virus (HIV), the etiological
agent of acquired immunodeficiency syndrome (AIDS)
continues to be a major health threat worldwide. The
main steps in the viral life cycle include viral entry, rep-
lication of the viral genome, and proteolytic processing
of viral polyproteins. In addition, HIV replication
depends on the integration of the proviral DNA into the
genome of infected cells, which is catalyzed by HIV
integrase (IN). All these steps are potential targets for
antiviral drugs.
The IN-catalyzed insertion of viral DNA into the host
cell chromosome is a multistep process that includes
two catalytic reactions: 30-Processing (30P) and strand-
transfer (ST) [1]. Moreover, it has been suggested that
the integration mechanism involves bivalent metal ions
(Mg2þ or Mn2þ) in the enzyme catalytic site [2,3].
After many years of research, several molecules were
identified as IN inhibitors and among them b-diketo
acids (DKAs) [4] and their analogs [5] represent one of
the major leads in the identification of new antiviral
agents. In particular, it has been reported that the DKA
pharmacophoric motif could be able to sequester one or
both metal ions necessary for IN-enzymatic activity [2].
In fact, the only one integrase strand-transfer inhibitor
(INSTI) (Raltegravir) (Fig. 1) [6] so far approved by
RESULTS AND DISCUSSION
The synthesis of the first designed compound 4 was
carried out, as reported in Scheme 1, starting from the
commercially available nitrophenylhydrazine 1.
At first it was converted into 3-ethoxy-3-[(2-nitrophe-
nyl)hydrazono]propanoic acid ethyl ester (2) by reaction
with 3-amino-3-ethoxy-acrylic acid ethyl ester. This is a
novel synthetic approach with respect to a previously
reported procedure [11] thus improving the yields
and reducing reaction time. The obtained mixture of Z-
and E-isomers (2), without further separation, was succes-
sively used for the cyclocondensation with oxalyl chloride
to give the ethyl 5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxylate (3). The pyridazine
C
V
2009 HeteroCorporation

November 2009
Synthesis of New Pyridazine Derivatives as Potential Anti-HIV-1 Agents
1421
Scheme 2. Reagents and conditions: a) ArCH2NH2, CH2CI2, r.t. 15 h;
b) ArCH2NH2, two steps in the same conditions: 280 W, 50ꢀC,
30 min; and c) oxalyl chloride, toluene, 0ꢀC, 2 h.
Figure 1. Chemical structure of Raltegravir.
derivative 3 was finally treated with platinum dioxide
under hydrogen atmosphere to give the ethyl 4-hydroxy-
2-oxo-1,2-dihydropyridazino[1,6-a]benzimidazole-3-car-
boxylate (4) resulting from the nitro-group reduction and
the consequent ring closure.
With the aim to prepare the amide derivatives 5–22,
we used the synthetic route described in Scheme 2. The
(Z) isomer of intermediate 2, obtained as red crystals
from ethanol, was used as starting material, and was
converted into the corresponding (Z)N-benzyl-3-ethoxy-
3-[(2-nitrophenyl)hydrazono]propanamides 5–10, by
reaction with appropriate benzylamine. With the aim to
optimize the synthetic procedure, we performed this step
both at room temperature and under reflux (50ꢀC). We
found that even elevating the reaction temperature no
significant reduction in reaction time occurred. Alterna-
tively, when we used the microwave-assisted organic
synthesis (MAOS) approach we observed a remarkable
reduction of reaction time (1 h rather than 15 h). More-
over, this approach was used in free-solvent conditions
(see experimental section). Then, the condensation with
oxalyl chloride gave a mixture of two different series
(11–16 and 17–22 in a ratio of 2:1) of pyridazine deriv-
atives depending on the tautomeric form of the amide
precursors 5–10 as well as the hydrolytic processes
occurred on their ethoxy functionality.
strand-transfer step. The biological results showed that
compound 5 was able to inhibit the enzymatic activity
at micromolar concentration (Table 1).
Moreover, the HIV-induced cytopathic effect (CPE)
in human lymphocyte MT-4 cell culture was determined
by the MT-4/MTT-assay. The screening data put in evi-
dence that only the N-benzyl-3-ethoxy-3-[(2-nitrophe-
nyl)hydrazono]propanamides (5–10) showed antiviral
activity in cells test, but also toxicity at same
concentration.
EXPERIMENTAL
The N-benzyl-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxamides 11–16 were
directly recovered as crude precipitate from reaction
mixture, while the evaporation of the resulting solution
furnished the N-benzyl-3-ethoxy-1-(2-nitrophenyl)-5,6-
dioxo-1,2,5,6-tetrahydropyridazine-4-carboximidic acids
17–22.
All the synthesized compounds were tested for their
IN enzymatic inhibitory effect both against the overall
integration reaction and more specifically against the
Chemistry. All microwave-assisted reactions were carried
out in a CEM Focused Microwave Synthesis System, Model
Discover working at the potency necessary for refluxing under
atmospheric conditions. Melting points were determined on a
BUCHI Melting Point B-545 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
60.4% of the theoretical values. Merck silica gel 60 F254
plates were used for analytical tlc. 1H NMR spectra were
recorded in DMSO-d6 on a Varian Gemini-300 spectrometer.
Chemical shifts were expressed in d (ppm).
Scheme 1. Reagents and conditions: a) 3-amino-3-ethoxy-acrylic acid ethyl ester, dry EtOH, N2, rt, 2h; b) oxalyl chloride, toluene, 0ꢀC, 5 h; and
c) PtO2, H2, EtOH, rt, 5 h.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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S. Ferro, S. Agnello, M. L. Barreca, L. De Luca, F. Christ, and R. Gitto
Vol 46
Table 1
Inhibition of HIV-1 integrase enzymatic activity, replication of HIV-1 (IIIB), and cytotoxicity in MT-4 cells.
IN enzymatic activity
Activity in MT-4 cells
Cytotoxicityd CC50 (lM)
Compound
Over-alla IC50 (lM)
STb IC50 (lM)
HIV-1c EC50 (lM)
SIe
5
6
7
8
9
10
15.32
>250
>250
>250
>250
>250
0.08
37.39
>250
>250
>250
>250
>250
0.14
0.82
53.28
15.91
5.17
>30.68
18.16
0.59
0.82
53.28
15.91
5.17
>30.68
18.16
41.1
1
1
1
1
1
1
70
CHI 10439
a Concentration required to inhibit the in vitro overall integrase activity by 50%.
b Concentration required to inhibit the in vitro strand transfer step by 50%.
c Effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells.
d Cytotoxic concentration to reduce MT-4 cell viability by 50%.
e Selectivity index: ratio CC50 /EC50
.
resulting solution was stirred for 15 h. Precipitate was filtered
and crystallized from ethanol.
Synthesis of (Z-E)3-ethoxy-3-[(2-nitrophenyl)hydrazono]-
propanoic acid ethyl ester (2). 2-Nitrophenylhydrazine (0.001
mol, 137.14 mg) (1) and 3-amino-3-ethoxy-acrylic acid ethyl
ester hydrochloride (0.001 mol, 195.65 mg) were suspended in
ethanol (3 mL), under N2 atmosphere, and stirred at room tem-
perature for 2 h. The precipitate was filtered off and the solu-
tion concentrated in vacuo. Yield 69%. Spectral measumerents
(1H NMR) and experimental data were in accordance with
reported data in the literature [11].
Synthesis of ethyl 5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxylate (3). Oxalyl chlo-
ride (0.001 mol, 126.93 mg) was added dropwise at 0ꢀC to a
solution of (Z-E)3-ethoxy-3-[(2-nitrophenyl)hydrazono]propa-
noic acid ethyl ester (2) (0.001 mol, 295.30 mg) in toluene (6
mL). The mixture was stirred for 5 h and allowed to reach
room temperature. Yellow crystals were formed, the precipitate
was filtered off, and crystallized from diethyl ether. Mp 260–
262ꢀC yield 81%; 1H NMR: d 1.15 (t, 3H, J ¼ 7.1, CH3),
3.40 (q, 2H, J ¼ 7.1, CH2), 6.78–8.12 (m, 4H, ArH), 9.40 (bs,
1H, NH). Anal. Calcd. for C13H11N3O7: C, 48.61; H, 3.45; N,
13.08. Found: C, 48.54; H, 3.38; N, 13.24.
Microwave-assisted synthesis. The appropriate benzylamine
(0.010 mol) was added to the (Z)3-ethoxy-3-[(2-nitrophenyl)-
hydrazono]propanoic acid ethyl ester (2) (0.001 mol, 295.30
mg), under N2 atmosphere, and the resulting mixture was
placed in a cylindrical quartz tube (Ø 2 cm). The reaction mix-
ture was then stirred and irradiated in a microwave oven for
two subsequent periods in the same conditions: 280 W, 30
min, and 50ꢀC. After cooling the work-up was carried out as
described for the conventional method.
(Z)N-Benzyl-3-ethoxy-3-[(2-nitrophenyl)hydrazono]propa-
1
namide (5). Mp 172–174ꢀC, yield 100%; H NMR: d 1.30 (t,
3H, J ¼ 7.1, CH3), 3.55 (s, 2H, CH2), 4.23 (q, 2H, J ¼ 7.1,
CH2), 4.31 (d, 2H, J ¼ 5.99, CH2), 6.76–8.08 (m, 9H, ArH),
8.68 (t, 1H, J ¼ 5.99, NH), 10.93 (bs, 1H, NH). Anal. Calcd.
for C18H20N4O4: C, 60.67; H, 5.66; N, 15.72. Found: C,
60.60; H, 5.59; N, 15.78.
(Z)N-(4-Bromobenzyl)-3-ethoxy-3-[(2-nitrophenyl)hydra-
zono]propanamide (6). Mp 175–177ꢀ C, yield 99%; 1H
NMR: d 1.30 (t, 3H, J ¼ 6.9, CH3), 3.54 (s, 2H, CH2), 4.22
(q, 2H, J ¼ 6.9, CH2), 4.27 (d, 2H, J ¼ 5.8, CH2), 6.76–8.08
(m, 8H, ArH), 8.71 (t, 1H, J ¼ 5.8, NH), 10.92 (bs, 1H, NH).
Anal. Calcd. for C18H19BrN4O4: C, 49.67; H, 4.40; N, 12.87.
Found: C, 49.62; H, 4.43; N, 12.83.
(Z)N-(4-Chlorobenzyl)-3-ethoxy-3-[(2-nitrophenyl)hydra-
zono]propanamide (7). Mp 171–173ꢀC, yield 100%; 1H
NMR: d 1.30 (t, 3H, J ¼ 6.9, CH3), 3.69 (s, 2H, CH2), 4.22
(q, 2H, J ¼ 6.9, CH2), 4.29 (d, 2H, J ¼ 5.8, CH2), 6.76–8.08
(m, 8H, ArH), 8.72 (t, 1H, J ¼ 5.8, NH), 10.93 (bs, 1H, NH).
C12H12O4: C, 65.45; H, 5.49; C, 65.31; H, 5.63. Anal. Calcd.
for C18H19ClN4O4: C, 55.32; H, 4.90; N, 14.34. Found: C,
55.28; H, 4.95; N, 14.31.
Synthesis of ethyl 4-hydroxy-2-oxo-1,2-dihydropyrida-
zino[1,6-a]benzimidazole-3-carboxylate (4). Compound
3
(0.001 mol, 321.25 mg) was dissolved in dry ethanol (50 mL)
in a flask suitable for a normal pressure hydrogenation. Plati-
num dioxide (0.002 mol, 32 mg) was added and the compound
3 was hydrogenated under ambient pressure. The mixture was
stirred for 5 h. The catalyst was filtered off on diatomaceous
earth and the solvent removed under reduced pressure. The
residue was powdered by treatment with diethyl ether and
crystallized from ethanol. Mp 230–232ꢀC, yield 26%; 1H
NMR: d 1.22 (t, 3H, J ¼ 7.1, CH3), 4.16 (q, 2H, J ¼ 7.1,
CH2), 6.55–7.97 (m, 4H, ArH), 10.43 (bs, 1H, NH). Anal.
Calcd. for C13H11N3O4: C, 57.14; H, 4.06; N, 15.38. Found:
C, 57.20; H, 4.12; N, 15.30.
(Z)N-(4-Fluorobenzyl)-3-ethoxy-3-[(2-nitrophenyl)hydra-
1
zono]propanamide (8). Mp 180–182ꢀC, yield 93%; H NMR:
d 1.30 (t, 3H, J ¼ 7.1, CH3), 3.69 (s, 2H, CH2), 4.22 (q, 2H, J
¼ 7.1, CH2), 4.29 (d, 2H, J ¼ 5.8, CH2), 6.78–8.08 (m, 8H,
ArH), 8.68 (t, 1H, J ¼ 5.8, NH), 10.93 (bs, 1H, NH). Anal.
Calcd. for C18H19FN4O4: C, 57.75; H, 5.12; N, 14.97. Found:
C, 57.71; H, 5.08; N, 14.93.
General procedure for the synthesis of (Z)N-benzyl-3-
ethoxy-3-[(2-nitrophenyl)hydrazono]propanamides (5–10)
Conventional method. (Z)3-Ethoxy-3-[(2-nitrophenyl)hydra-
zono]propanoic acid ethyl ester (2) (0.001 mol, 295.30 mg)
was dissolved in methylene chloride (2 mL), under N2 atmos-
phere, and the solution stirred at room temperature for 10 min.
The appropriate benzylamine (0.010 mol) was added and the
(Z)N-(4-Methoxybenzyl)-3-ethoxy-3-[(2-nitrophenyl)hydra-
1
zono]propanamide (9). Mp 171–173ꢀC, yield 84%; H NMR:
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DOI 10.1002/jhet

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Synthesis of New Pyridazine Derivatives as Potential Anti-HIV-1 Agents
1423
d 1.30 (t, 3H, J ¼ 7.3, CH3), 3.52 (s, 2H, CH2), 3.72 (s, 3H,
CH3O), 4.21 (q, 2H, J ¼ 7.3, CH2), 4.23 (d, 2H, J ¼ 5.7,
CH2), 6.75–8.07 (m, 8H, ArH), 8.58 (t, 1H, J ¼ 5.7, NH),
10.92 (bs, 1H, NH). Anal. Calcd. for C19H22N4O5: C, 59.06;
H, 5.74; N, 14.50. Found: C, 59.02; H, 5.69; N, 14.55.
(Z)N-(4-Methylbenzyl)-3-ethoxy-3-[(2-nitrophenyl)hydra-
zono]propanamide (10). Mp 179–181ꢀC, yield 90%; 1H
NMR: d 1.30 (t, 3H, J ¼ 7.1, CH3), 2.27 (s, 3H, CH3), 3.53
(s, 2H, CH2), 4.22 (q, 2H, J ¼ 7.1, CH2), 4.26 (d, 2H, J ¼
5.7, CH2), 6.76–8.08 (m, 8H, ArH), 8.63 (t, 1H, J ¼ 5.7, NH),
10.93 (bs, 1H, NH). Anal. Calcd. for C19H22N4O4: C, 61.61;
H, 5.99; N, 15.13. Found: C, 61.58; H, 5.95; N, 15.16.
yield 21%; 1H NMR: d 1.33 (t, 3H, J ¼ 6.8, CH3), 4.20 (q,
2H, J ¼ 6.8, CH2), 4.54 (s, 2H, CH2), 6.90–8.16 (m, 9H,
ArH), 11.19 (bs, 1H, NH). Anal. Calcd. for C20H18N4O6: C,
58.54; H, 4.42; N, 13.65. Found: C, 58.51; H, 4.47; N, 13.61.
N-(4-Bromobenzyl)-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-1,
2,5,6-tetrahydropyridazine-4-carboximidic acid (18). Mp
184–186ꢀC, yield 23%; 1H NMR: d 1.35 (t, 3H, J ¼ 6.8,
CH3), 4.24 (q, 2H, J ¼ 6.8, CH2), 4.52 (s, 2H, CH2), 6.96–
8.19 (m, 8H, ArH), 11.23 (bs, 1H, NH). Anal. Calcd. for
C20H17BrN4O6: C, 49.10; H, 3.50; N, 11.45. Found: C, 49.16;
H, 3.57; N, 11.50.
N-(4-Chlorobenzyl)-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-
1,2,5,6-tetrahydropyridazine-4-carboximidic acid (19). Mp
184–186ꢀC, yield 25%; 1H NMR: d 1.34 (t, 3H, J ¼ 6.8,
CH3), 4.19 (q, 2H, J ¼ 6.8, CH2), 4.19 (s, 2H, CH2), 6.99–
8.16 (m, 8H, ArH), 11.26 (bs, 1H, NH). Anal. Calcd. for
C20H17ClN4O6 : C, 54.00; H, 3.85; N, 12.59. Found: C, 54.05;
H, 3.89; N, 12.53.
General procedures for the synthesis of N-benzyl-5-
hydroxy-1-(2-nitrophenyl)-3,6-dioxo-1,2,3,6-tetrahydropyri-
dazine-4-carboxamides (11–16) and N-benzyl-3-ethoxy-
1-(2-nitrophenyl)-5,6-dioxo-1,2,5,6-tetrahydropyridazine-4-
carboximidic acids (17–22). The appropriate (Z)N-benzyl-3-
ethoxy-3-[(2-nitrophenyl)hydrazono]propanamide
(5–10)
N-(4-Fluorobenzyl)-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-
1,2,5,6-tetrahydropyridazine-4-carboximidic acid (20). Mp
186–188ꢀC, yield 23%; 1H NMR: d 1.34 (t, 3H, J ¼ 6.8,
CH3), 4.19 (q, 2H, J ¼ 6.8, CH2), 4.67 (s, 2H, CH2), 6.97–
8.17 (m, 8H, ArH), 11.27 (bs, 1H, NH). Anal. Calcd. for
C20H17FN4O6: C, 56.08; H, 4.00; N, 13.08. Found: C, 56.03;
H, 4.05; N, 13.03.
(0.001 mol) was dissolved in dry toluene (6.5 mL). Oxalyl
chloride (0.001 mol, 126.93 mg) was added dropwise at 0ꢀC
and the reaction mixture stirred for 2 h. The obtained precipi-
tate was filtered and crystallized from ethanol/methanol (1:1,
v/v) to give derivatives 11–16. The solution was concentrated
under reduced pressure and the residue treated with diethyl
ether to afford derivatives 17–22.
N-(4-Methoxylbenzyl)-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-
1,2,5,6-tetrahydropyridazine-4-carboximidic acid (21). Mp
184–186ꢀC, yield 18%; 1H NMR: d 1.33 (t, 3H, J ¼ 6.8,
CH3), 3.72 (s, 3H, CH3O), 4.19 (q, 2H, J ¼ 6.8, CH2), 4.61 (s,
2H, CH2), 6.88–8.16 (m, 8H, ArH), 11.26 (bs, 1H, NH). Anal.
Calcd. for C21H20N4O7: C, 57.27; H, 4.58; N, 12.72. Found:
C, 57.22; H, 4.53; N, 12.66.
N-(4-Methylbenzyl)-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-
1,2,5,6-tetrahydropyridazine-4-carboximidic acid (22). Mp
185–187ꢀC, yield 17%; 1H NMR: d 1.33 (t, 3H, J ¼ 6.8,
CH3), 2.27 (s, 3H, CH3), 4.19 (q, 2H, J ¼ 6.8, CH2), 4.63 (s,
2H, CH2), 6.97–8.16 (m, 8H, ArH), 11.26 (bs, 1H, NH). Anal.
Calcd. for C21H20N4O6: C, 59.43; H, 4.75; N, 13.20. Found:
C, 59.47; H, 4.71; N, 13.25.
N-Benzyl-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-1,2,3,6-tet-
rahydropyridazine-4-carboxamide (11). Mp 164–166ꢀC, yield
56%; H NMR: d 4.41 (s, 2H, CH2), 6.87–8.12 (m, 9H, ArH),
1
9.44 (bs, 1H, NH). Anal. Calcd. for C18H14N4O6: C, 56.55; H,
3.69; N, 14.65. Found: C, 56.49; H, 3.65; N, 14.68.
N-(4-Bromobenzyl)-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxamide (12). Mp 171–
173ꢀC, yield 64%; 1H NMR: d 4.36 (s, 2H, CH2), 6.84–8.11
(m, 8H, ArH), 9.41 (bs, 1H, NH). Anal. Calcd. for
C18H13BrN4O6: C, 46.87; H, 2.84; N, 12.15. Found: C, 46.61;
H, 2.55; N, 12.82.
N-(4-Chlorobenzyl)-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxamide (13). Mp 182–
184ꢀC, yield 48%; 1H NMR: d 4.36 (s, 2H, CH2), 6.82–8.09
(m, 8H, ArH), 9.41 (bs, 1H, NH). Anal. Calcd. for
C18H13ClN4O6: C, 51.87; H, 3.14; N, 13.44. Found: C, 51.63;
H, 3.38; N, 13.21.
Bioassays
Overall integrase assay using an enzyme-
linked immunosorbent assay and strand-transfer inhibition. We
used enzyme-linked immunosorbent assays to determine the
susceptibility of the HIV-1 integrase enzyme towards different
compounds. These assays use an oligonucleotide substrate of
which one oligonucleotide (50-ACTGCTAGAGATTTTCCA-
CACT GACTAAAAGGGTC-30) is labeled with biotin at the
30 end and the other oligonucleotide is labeled with digoxige-
nin at the 50 end. For the overall integration assay the second
50-digoxigenin labeled oligonucleotide is (50-GACCCTTT-
TAGT CAGTGTGGAAAATCTCTAGCAGT-30). For the
strand transfer assay the second oligonucleotide lacks GT at
the 30 end. The integrase enzyme was diluted in 750 mM
NaCl, 10 mM Tris pH 7.6, 10% glycerol, and 1 mM b-mer-
capto 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) were added in a final reac-
tion 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 at 37ꢀC for 1 h. Reaction products were
denatured with 30 mM NaOH and detected by an immunosor-
bent assay on avidin-coated plates [12].
N-(4-Fluorobenzyl)-1-(2-nitrophenyl)-3,6-dioxo-1,2,3,6-
tetrahydropyridazine-4-carboxamide (14). Mp 172–174ꢀC,
1
yield 59%; H NMR: d 4.37 (s, 2H, CH2), 6.86–8.09 (m, 8H,
ArH), 9.42 (bs, 1H, NH). Anal. Calcd. for C18H13FN4O6: C,
54.01; H, 3.27; N, 14.00. Found: C, 54.06; H, 3.31; N, 14.05.
N-(4-Methoxylbenzyl)-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxamide (15). Mp 184–
186ꢀC, yield 37%; 1H NMR: d 3.72 (s, 3H, CH3O), 4.33 (s,
2H, CH2), 6.87–8.13 (m, 8H, ArH), 9.43 (bs, 1H, NH). Anal.
Calcd. for C19H16N4O7: C, 55.34; H, 3.91; N, 13.59%. Found:
C, 55.31; H, 3.96; N, 13.52%.
N-(4-Methylbenzyl)-5-hydroxy-1-(2-nitrophenyl)-3,6-dioxo-
1,2,3,6-tetrahydropyridazine-4-carboxamide (16). Mp 180–
1
182ꢀC, yield 44%; H NMR: d 2.27 (s, 3H, CH3), 4.36 (s, 2H,
CH2), 6.87–8.12 (m, 8H, ArH), 9.43 (bs, 1H, NH). Anal.
Calcd. for C19H16N4O6: C, 57.58; H, 4.07; N, 14.14. Found:
C, 57.53; H, 4.02; N, 14.19.
N-Benzyl-3-ethoxy-1-(2-nitrophenyl)-5,6-dioxo-1,2,5,6-tetra-
hydropyridazine-4-carboximidic acid (17). Mp 190–192ꢀC,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1424
S. Ferro, S. Agnello, M. L. Barreca, L. De Luca, F. Christ, and R. Gitto
Vol 46
[3] Yang, W.; Steitz, T. A. Structure 1995, 3, 131.
In vitro anti-HIV and drug susceptibility assays. The inhib-
[4] Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Still-
mock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif, W.;
Blau, C.; Miller, M. D. Science 2000, 287, 646.
itory effect of antiviral drugs on the HIV-induced cytopathic
effect (CPE) in human lymphocyte MT-4 cell culture was
determined by the MT-4/MTT-assay [13]. This assay is based
on the reduction of the yellow colored 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial
dehydrogenase of metabolically active cells to a blue formazan
derivative, 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 pres-
ence of fivefold serial dilutions of the antiviral drugs. The con-
centration of various compounds achieving 50% protection
against the CPE of the different HIV strains, which is defined
as the EC50, was determined. In parallel the 50% cytotoxic
concentration (CC50) was determined.
[5] Johnson, A. A.; Marchand, C.; Pommier, Y. Curr Top Med
Chem 2004, 4, 1059.
[6] www.fda.gov/bbs/topics/NEWS/2007/NEW01726.html.
[7] Barreca, M. L.; Ferro, S.; Rao, A.; De Luca, L.; Zappala,
M.; Monforte, A. M.; Debyser, Z.; Witvrouw, M.; Chimirri, A. J Med
Chem 2005, 48, 7084.
[8] Ferro, S.; Barreca, M. L.; De Luca, L.; Rao, A.; Monforte,
A. M.; Debyser, Z.; Witvrouw, M.; Chimirri, A. Arch Pharm (Wein-
heim) 2007, 340, 292.
[9] De Luca, L.; Barreca, M. L.; Ferro, S.; Iraci, N.; Michiels,
M.; Christ, F.; Debyser, Z.; Witvrouw, M.; Chimirri, A. Bioorg Med
Chem Lett 2008, 18, 2891.
[10] Hombrouck, A.; Van Remoortel, B.; Michiels, M.; Noppe,
W.; Christ, F.; Eneroth, A.; Sahlberg, B. L.; Benkestock, K.; Vrang,
L.; Johansson, N. G.; Barreca, M. L.; De Luca, L.; Ferro, S.; Chimirri,
A.; Debyser, Z.; Witvrouw, M. Antimicrob Agents Chemother 2008,
52, 2861.
Acknowledgments. This work was supported by the European
Commission (HEALTH-F3-2008-201032) (THINC project).
[11] Wilde, H. H. S.; Kanitz, A.; Franzheld, M.; Mann, G. J fuer
Praktische Chemie 1985, 327, 297.
[12] Hwang, Y.; Rhodes, D.; Bushman, F. Nucleic Acids Res
2000, 28, 4884.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet