Antimycobacterial activity of pyrimido[4,5-b]diazepine derivatives

Article abstract of DOI:10.1002/ardp.201100433

Three series of novel 8,9-dihydro-7H-pyrimido[5,4-b][1,4]diazepines, 4a-d, 5a-d, and 7a-d, were efficiently obtained in good yields using simple reaction methodologies. These pyrimidodiazepines were evaluated against 15 Mycobacterium spp. strains. Moderat

Full text of DOI:10.1002/ardp.201100433

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
1
Full Paper
Antimycobacterial Activity of Pyrimido[4,5-b]diazepine
Derivatives
1
1
2
1
1
Braulio Insuasty , Angelica Garcıa , Juan Bueno , Jairo Quiroga , Rodrigo Abonia , and
´
´
Alejandro Ortiz1
1 Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, Cali, Colombia
2
´
´
Grupo Micobacterias, Subdireccion Red Nacional de Laboratorios, Instituto Nacional de Salud, Bogota,
´
Centro Colombiano de Investigacion en Tuberculosis CCITB, Bogota, D.C., Colombia
´
Three series of novel 8,9-dihydro-7H-pyrimido[5,4-b][1,4]diazepines, 4a–d, 5a–d, and 7a–d, were
efficiently obtained in good yields using simple reaction methodologies. These pyrimido-
diazepines were evaluated against 15 Mycobacterium spp. strains. Moderate activity in the
inhibition of 13 microorganisms was obtained for the four compounds 4a, 5a, 5c, and 5d.
Keywords: 4,5-Diaminopyrimidines / Methylenedioxychalcones / Pyrimido[4,5-b]diazepine / Tuberculosis
Received: November 29, 2011; Revised: May 2, 2012; Accepted: May 11, 2012
DOI 10.1002/ardp.201100433
Introduction
recent years, some examples of a heterocyclic ring fused to
the seven-membered diazepine ring system have appeared in
the literature [4].
Human tuberculosis (TB) is a contagious–infectious disease
mainly caused by Mycobacterium tuberculosis, which is an aero-
bic pathogenic bacterium that establishes its infection
usually in the lungs [1]. About one-third of the world’s popu-
lation is currently infected with M. tuberculosis; 10% of those
infected will develop clinical disease, particularly those who
are also infected with the human immunodeficiency virus
(HIV) [1]. Although medical regimens exist for treating TB,
they are far from ideal [2]. In addition, new drugs are needed
to combat the increasing number of multidrug-resistant
strains (MDR-TB) and extensively drug-resistant (XDR-TB)
strains and HIV epidemics, leading to an increased need to
understand the molecular mechanisms of drug action and
drug resistance, which should provide significant insights
into the development of new compounds [2]. In this effort,
diazepine derivatives are scaffolds that have shown a broad
spectrum of biological activities as anticancer, antibacterial,
psychotropic, anticonvulsant, antiviral, and herbicidal
agents; these facts present them as a promising source of
new compound leaders [3]. Benzodiazepines are an important
class of psychotherapeutic compounds. Although a benzene
ring is usually needed for pharmacological activity, in
In previous papers [5], we reported on the synthesis of
pyrimidodiazepines from the reaction of 4,5-diaminopyrimi-
dines with chalcones. Because of the biological importance of
these heterocycles, the aim of this study was the synthesis of
the new pyrimidodiazepines 4, 5, and 7 by the reaction of 4,5-
diaminopyrimidines 1 and 6 with chalcones 2 and 3 contain-
ing the dioxymethylene moiety, and the determination of
their activity against 15 Mycobacterium spp., in comparison
between strains and clinical isolates.
Results and discussion
Chemistry
The synthesis of pyrimidodiazepines 4, 5, and 7 was achieved
by two different methodologies (Scheme 1): Compounds 4
and 5 were obtained by a mixture of the dihydrochloride of
2,4,5,6-tetraaminopyrimidine 1 (0.100 g, 0.47 mmol) and
methylenedioxychalcone 2 or 3 (0.94 mmol) in methanol
(30 mL) under reflux for 30 min. Compound 7 was obtained
using microwave irradiation (1208C, 75 W; CEM Discovery)
during 4–14 min, in the reaction of 2-methylthio-4,5,6-tri-
aminopyrimidine 6 (0.60 mmol) and chalcone 3 (0.30 mmol)
in dimethylformamide (DMF) (0.5 mL) and a catalytic amount
of BF3ꢀOEt2. The structures of all new pyrimidodiazepines
were appropriately established by the usual spectroscopic
methods (see Experimental section).
Correspondence: Braulio Insuasty, Heterocyclic Compounds Research
Group, Department of Chemistry, Universidad del Valle, A. A. 25360, Cali,
Colombia.
E-mail: braulio.insuasty@correounivalle.edu.co
Fax: þ57 2 3339-2440
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B. Insuasty et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
NH2
N
NH2
NH2
N
X
1
6
or
X = NH2 or SCH3
O
O
O
O
a)
O
b)
a)
R
N
2
O
3
R
R
O
O
O
O
NH2
N
NH2
N
NH2
N
N
N
N
N
X
N
H
X
X
N
N
H
N
H
O
O
R
R
4
5
7
R = Cl, F, H, CF3
X = NH2
R = Cl, H, OCH3, CF3
X = SCH3
R = Cl, F, H, OCH3
X = NH2
a) MeOH reflux, 30 minutes. b) BF3.OEt2 (cat.), DMF (5 mL), MW close vessel, 120ºC, 75 W, 4−14 minutes.
Compd 4a 4b 4c 4d 5a 5b 5c 5d 7a 7b 7c 7d
NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 SCH3 SCH3 SCH3 SCH3
Cl OCH3 Cl CF3 Cl OCH3 CF3
X
R
F
H
F
H
H
Scheme 1. Synthesis of pyrimidodiazepines 4a–d, 5a–d, and 7a–d.
1
In the H NMR spectra of the compounds, one proton on
The formation of the pyrimidodiazepines 4, 5, and 7 pro-
ceeded regioselectively, despite the existence of two different
amino groups in the starting 1,2-diaminopyrimidines 1 and
6, which can also be explained as a matter of the high
nucleophilicity of the amino groups on C-5 of the 1,2-diami-
nopyrimidines mentioned above, enhanced by the electronic
effect of the pyrimidine ring and the other amino groups
attached to it [7, 8]. Therefore, condensation of the amino
group in C-5 with the carbonyl group in chalcones 2 and 3 can
be followed by Michael’s reaction of the amino group at C-4 of
the diastereotopic center C-7 of the 1,4-diazepine ring
appears, for compounds 4a–d, as a doublet of doublets at
d ¼ 3.07–3.13 ppm, with a coupling constant of Jgem ¼ 14.4–
14.8 Hz, Jtrans ¼ 7.2–7.6 Hz, and the other diastereotopic pro-
ton in C-7 appears as a doublet at d ¼ 3.29–3.38 ppm with a
coupling constant of Jgem ¼ 14.4–14.8 Hz. For compounds 5
and 7, one proton on the diastereotopic center C-7 appears as
a doublet at d ¼ 2.76–3.21 ppm with a coupling constant of
Jgem ¼ 14.4–14.5 Hz, and the other diastereotopic proton
appears as a doublet of doublets at d ¼ 3.58–3.66 ppm with
a coupling constant of Jgem ¼ 14.4–14.5 Hz; proton H-8 is
usually observed as a broad signal at d ¼ 4.66–5.22 ppm.
Finally, 13C NMR, DEPT-135, and two-dimensional heteronu-
clear NMR spectra provided the final structural elucidation of
the derivatives. The mass spectra of all compounds exhibit
well-defined molecular ions with particular fragmentation
–
the C C double bond.
–
Antimycobacterial screening
Mycobacterium species were obtained from the Laboratorio
´
Micobacterias, Instituto Nacional de Salud, Bogota,
Colombia. M. tuberculosis H37Rv (ATCC 27294), the resistant
variants (ATCC 35837 ethambutol resistant, ATCC 35838
rifampicin resistant, ATCC 35822 isoniazid resistant, and
ATCC 35820 streptomycin resistant), five strains of
M. tuberculosis Beijing genotype belonging to the Colombia
National Study of Drug Resistance, and five clinical isolates
–
patterns involving the loss of Ar–CH CH . This fragmenta-
2
–
tion occurs by scission on the C–C bond in b position to the
diazepine NH, followed by a C–N cleavage generating a stable
cation radical with high relative intensity [6].
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antimycobacterial Activity of Pyrimido[4,5-b]diazepine Derivatives
3
from humans with mesotherapy-associated outbreak caused
by nontuberculous mycobacteria (NTM; M. chelonae,
M. fortuitum, M. intracellulare, M. scrofulaceum, and M. szulgai)
were used. The antimycobacterial activity of the derivates
dissolved in dimethylsulfoxide (DMSO; Sigma, NJ, USA) was
evaluated following the MTT colorimetric microdilution pro-
tocol described by Caviedes et al. [9], with modifications. For
standard tests, the minimum inhibitory concentration (MIC)
Table 1. In vitro activity (MIC in mg/mL) of 12 diazepine derivates against 15 Mycobacterium spp.
Compd LogP
Dipole (D)
MIC (mg/mL)
Mycobacterium spp.
35838
H37Rv
35837
35822
35820
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
7c
7d
Iso
Rif
3.28
2.88
2.72
2.60
3.28
2.88
2.72
3.64
4.52
3.97
3.84
4.89
–
1.751
1.759
1.785
1.678
1.119
1.611
1.576
1.430
3.463
4.424
4.754
3.005
–
32
>32
>32
>32
32
>32
32
32
>32
>32
>32
>32
0.25
0.125
32
>32
>32
>32
32
>32
32
32
>32
>32
>32
>32
0.25
0.125
32
>32
>32
>32
32
>32
32
32
>32
>32
>32
>32
0.25
>0.5
32
>32
32
>32
32
32
32
32
>32
>32
>32
>32
>0.5
0.125
32
>32
32
>32
32
32
32
32
>32
>32
>32
>32
0.25
0.125
–
–
Compd LogP
Dipole (D)
MTB411
MTB985
MNT1407
MNT1073
MNT1100
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
7c
7d
Iso
Rif
3.28
2.88
2.72
2.60
3.28
2.88
2.72
3.64
4.52
3.97
3.84
4.89
–
1.751
1.759
1.785
1.678
1.119
1.611
1.576
1.430
3.463
4.424
4.754
3.005
–
16
>32
32
>32
32
>32
32
32
>32
>32
>32
>32
>0.5
>0.5
16
>32
32
>32
32
>32
32
32
>32
>32
>32
>32
>0.5
>0.5
32
>32
>32
>32
32
>32
32
32
>32
>32
>32
>32
>0.5
0.125
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>0.5
>0.5
16
>32
>32
32
32
32
32
32
>32
>32
>32
>32
>0.5
>0.5
–
–
Compd LogP
Dipole (D)
MTB2556
MTB4000
UT544
MNT1193
MNT1408
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
7c
7d
Iso
Rif
3.28
2.88
2.72
2.60
3.28
2.88
2.72
3.64
4.52
3.97
3.84
4.89
–
1.751
1.759
1.785
1.678
1.119
1.611
1.576
1.430
3.463
4.424
4.754
3.005
–
16
>32
32
>32
32
32
32
32
>32
>32
>32
>32
>0.5
>0.5
16
>32
32
>32
32
32
32
32
>32
>32
>32
>32
>0.5
>0.5
16
>32
32
>32
32
32
16
16
>32
>32
>32
>32
>0.5
>0.5
32
>32
>32
>32
32
32
32
32
>32
>32
>32
>32
>0.5
>0.5
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>0.5
>0.5
–
–
M. tuberculosis H37Rv (27294) susceptible to all five first-line antituberculosis drugs (streptomycin, isoniazid, rifampicin, ethambutol,
and pirazinamide), H37Rv (35837) ethambutol resistant, H37Rv (35838) rifampicin resistant, H37Rv (35822) isoniazid resistant, and
H37Rv (35820) streptomycin resistant; MTB2556, MTB4000, UT544, MTB411, MTB985 (M. tuberculosis clinical isolates Beijing genotype,
isoniazid and rifampicin resistant); MNT 1407 (M. chelonae clinical isolate), MNT1073 (M. fortuitum clinical isolate), MNT1100 (M. szulgai
clinical isolate), MNT1193 (M. scrofulaceum clinical isolate), and MNT1408 (M. intracellulare clinical isolate).
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4
B. Insuasty et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
values of rifampicin and isoniazid (Sigma) were determined
each time. The MIC of each molecule corresponds to the
lowest concentration at which the bacteria tested do not
species. Four diazepine derivates (4a, 5a, 5c, and 5d) were
active against 13 microorganisms.
show any growth. Susceptibility testing was performed three Experimental
times. The results consigned in Table 1 were expressed as
Commercially available starting materials, reagents, and sol-
geometric mean (GM) of the MIC.
vents were used as supplied. TLC analyses were performed on
Merck silica gel 60 F254 aluminum plates. Melting points were
determined in a Buechi melting point apparatus and are uncor-
rected. The 1H and 13C NMR spectra were run on a Bruker
AVANCE 400 spectrometer operating at 400 and 100 MHz,
respectively, using DMSO-d6 and CDCl3 as solvents and tetrame-
thylsilane as internal reference. The mass spectra were scanned
on a Shimadzu GCMS-QP 2010 spectrometer (equipped with a
direct inlet probe) operating at 70 eV. The elemental analyses
were obtained using a Thermo Finnigan Flash EA1112 CHN
(STIUJA) elemental analyzer. Microwave experiments were car-
ried out on a focused microwave reactor (300W CEM DiscoverTM).
Four diazepine derivates (4a, 5a, 5c, and 5d) were moder-
ately active (good activity MIC below 10 mg/mL) [10] against
13 microorganisms; the rest inhibited the growth of
M. tuberculosis strains. Animal studies have shown that the
globally emerging Beijing genotype strains of M. tuberculosis
are more virulent than other strains [11]. Diazepine derivates
have been shown to be fatty acid biosynthesis inhibitors in
other microorganisms [12]. This is an important molecular
target in mycobacteria [13]. In addition, the antimicrobial
activity against Mycobacterium species depends on how effi-
ciently solutes cross the cell wall, which constitutes an effi-
cient permeability barrier. Mycobacteria have a lipid-rich,
hydrophobic cell wall, principally in the form of very-long-
chain fatty acids (mycolic acids), which account for 30–60% of
the weight of the cell wall. This cell wall obviously acts as a
barrier to the penetration of hydrophilic solutes, causing
that mycobacteria are often more susceptible to less polar
compounds [14, 15]. Furthermore, we performed molecular
modeling studies, using the CS Chem-Office Software version
9.0 (Cambridge software) [16]. All molecules were constructed
using Chem Draw Ultra 9.0 and saved as template structures.
For every compound, the template structure was suitably
changed considering its structural features and copied
General procedure for the synthesis of 8-(benzo[d][1,3]-
dioxol-5-yl)-6-aryl-8,9-dihydro-7H-pyrimido[5,4-b][1,4]-
diazepine-2,4-diamines (4a–d) and 6-(benzo[d][1,3]dioxol-
5-yl)-8-aryl-8,9-dihydro-7H-pyrimido[5,4-b][1,4]diazepine-
2,4-diamines (5a–d)
Compounds 4 and 5 were obtained by a mixture of the dihydro-
chloride of 2,4,5,6-tetraaminopyrimidine 1 (0.100 g, 0.47 mmol)
and methylenedioxychalcone 2 or 3 (0.94 mmol) in methanol
(30 mL) under reflux for 30 min. Reaction progress was con-
trolled by TLC. The precipitate formed was filtrated and purified
by column chromatography on silica gel by using a mixture of
CH2Cl2/MeOH (20:1) as eluent.
8-(Benzo[d][1,3]dioxol-5-yl)-6-(4-chlorophenyl)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-diamine
(4a)
to Chem 3D 9.0 to create
a three-dimensional (3D)
model. Finally, the model was cleaned up and subjected
to energy minimization using MOPAC, with the Austin
Model method. The minimization was executed until the
root mean square (RMS) gradient value reached a value
smaller than 0.0001 kcal/mol. The lowest energy structure
was used for each molecule to calculate LogP and dipole
values (Table 1), as lipophilicity measures [17]. The four dia-
zepine derivates, 4a, 5a, 5c, and 5d, showed appropriate
values for LogP and dipole, in accordance with their antimi-
crobial activity. In contrast, the derivatives 7a, 7b, 7c, and 7d
showed the lowest lipophilicity in comparison with the other
diazepines reported, and therefore less antimycobacterial
activity.
This compound was obtained as a yellow solid in 93% yield. M.p.
1868C; 1H NMR (CDCl3, 400 MHz): d ¼ 3.13 (dd, Jgem ¼ 14.4 Hz,
Jtrans ¼ 7.6 Hz, 1H, CHgem), 3.31 (d, J ¼ 14.4, 1H, CHgem), 4.66
(s, 1H), 4.69 (s, 2H, NH2), 5.51 (s, 1H, NH), 5.57 (s, 2H, NH2),
5.93 (s, 2H), 6.74–6.75 (m, 3H), 7.29 (d, J ¼ 8.4, 2H), 7.59
(d, J ¼ 8.4, 2H) ppm; 13C NMR (CDCl3, 100 MHz): d ¼ 41.5,
58.5, 101.2, 102.6, 106.6, 108.4, 119.5, 127.6, 128.4, 128.5,
135.0, 136.9, 139.2, 139.3, 147.3, 148.2, 153.9, 155.7, 160.2,
and 163.9 ppm; FTIR (KBr) n ¼ 3477, 3383, 3161, 3071, 2988,
1587, 1567, and 1550 cmꢁ1; MS (EI, 70 eV), m/z (%) 410 [Mþ2]
(29), 408 [Mþ] (86), 393 (43), 260 (100). Anal. Calcd.
for C20H17ClN6O2: C, 58.75; H, 4.19; N, 20.56. Found: C, 58.83;
H, 4.06; N, 20.68.
8-(Benzo[d][1,3]dioxol-5-yl)-6-(4-fluorophenyl)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-
diamine (4b)
Conclusion
We described herein the synthesis and antimycobacterial
activity of three novel series of pyrimido[4,5-b]diazepines,
which have a methylenedioxyaryl moiety. The new com-
pounds were obtained in a straightforward manner and were
fully characterized. The pyrimido[4,5-b]diazepines synthe-
sized were tested against a wide range of Mycobacterium
This compound was obtained as a yellow solid in 87% yield.
M.p. 1468C; 1H NMR (CDCl3, 400 MHz): d ¼ 3.13 (dd,
Jgem ¼ 14.6 Hz, Jtrans ¼ 7.2 Hz, 1H, CHgem), 3.29 (d, J ¼ 14.6,
1H, CHgem), 4.67 (d, Jtrans ¼ 7.2 Hz, 1H), 4.78 (s, 2H, NH2), 5.63
(s, 1H, NH), 5.69 (s, 2H, NH2), 5.91 (s, 2H), 6.71–6.74 (m, 3H), 7.00
(d, J ¼ 8.4, 2H), 7.63 (d, J ¼ 8.4, 2H) ppm; 13C NMR (CDCl3,
100 MHz): d ¼ 41.5, 58.6, 101.2, 102.5, 106.6, 108.4, 115.1,
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antimycobacterial Activity of Pyrimido[4,5-b]diazepine Derivatives
5
119.4, 128.2, 128.3, 128.5, 137.0, 147.3, 148.1, 153.8, 156.0, 160.2,
162.1, 163.9, and 164.6 ppm; FTIR (KBr) n ¼ 3488, 3387, 3162,
3071, 2990, 1599, 1584, and 1566 cmꢁ1; MS (EI, 70 eV), m/z (%)
392 [Mþ] (91), 377 (46), 244 (100). Anal. Calcd. for C20H17FN6O2: C,
61.22; H, 4.37; N, 21.42. Found: C, 61.18; H, 4.36; N, 21.48.
107.8, 115.1, 120.9, 128.3, 128.4, 136.3, 140.7, 140.8, 147.9, 148.1,
154.8, 155.0, 155.1, 160.9, and 164.1 ppm; FTIR (KBr) n ¼ 3543,
3477, 3427, 3244, 3125, 1606, and 1549 cmꢁ1; MS (EI, 70 eV), m/z
(%) 392 [Mþ] (100), 377 (53), 270 (48). Anal. Calcd. for C20H17FN6O2:
C, 61.22; H, 4.37; N, 21.42. Found: C, 61.18; H, 4.36; N, 21.48.
8-(Benzo[d][1,3]dioxol-5-yl)-6-phenyl-8,9-dihydro-7H-
pyrimido[5,4-b][1,4]diazepine-2,4-diamine (4c)
6-(Benzo[d][1,3]dioxol-5-yl)-8-phenyl-8,9-dihydro-7H-
pyrimido[5,4-b][1,4]diazepine-2,4-diamine (5c)
This compound was obtained as a yellow solid in 72% yield. M.p.
1428C; 1H NMR (CDCl3, 400 MHz): d ¼ 3.13 (dd, Jgem ¼ 14.8 Hz,
Jtrans ¼ 7.6 Hz, 1H, CHgem), 3.38 (d, J ¼ 14.8, 1H, CHgem), 4.66
(d, Jtrans ¼ 7.6 Hz, 1H), 4.72 (s, 2H, NH2), 5.58 (s, 1H, NH), 5.64
(s, 2H, NH2), 5.92 (s, 2H), 6.74–6.77 (m, 3H), 7.32 (s, 1H), 7.33
(d, J ¼ 8.4, 2H), 7.66 (d, J ¼ 8.4, 2H) ppm; 13C NMR (CDCl3,
100 MHz): d ¼ 41.6, 58.4, 101.1, 102.4, 106.6, 108.3, 119.4,
126.3, 128.2, 128.3, 128.8, 137.1, 140.8, 147.2, 148.0, 153.7,
157.0, 159.9, 160.0, and 163.8 ppm; FTIR (KBr) n ¼ 3470, 3385,
2968, 1608, 1583, and 1577 cmꢁ1; MS (EI, 70 eV), m/z (%) 374 [Mþ]
(100), 359 (46), 226 (75). Anal. Calcd. for C20H18N6O2: C, 64.16; H,
4.85; N, 22.45. Found: C, 64.20; H, 4.86; N, 22.40.
This compound was obtained as a yellow solid in 70% yield. M.p.
1
1158C; H NMR (400 MHz, DMSO-d6): d ¼ 2.49 (m, 1H, H-7), 3.52
(dd, Jgem ¼ 14.8 Hz, J1 ¼ 6.0 Hz, 1H, H-7), 4.97 (m, 1H, H-8), 5.28
(s, 2H, NH2), 5.89 (s, 2H, NH2), 5.94 (s, 2H), 6.41 (m, 1H, NH), 6.72–
7.21 (m, 8H) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 41.0, 58.6,
101.5, 102.3, 106.8, 107.8, 121.0, 126.4, 127.2, 128.5, 128.6, 136.4,
144.5, 147.8, 148.1, 155.0, 155.1, 160.6, 160.7, and 164.0 ppm;
FTIR (KBr) n ¼ 3553, 3489, 3435, 3239, 3112, 1600, and
1552 cmꢁ1; MS (EI, 70 eV), m/z (%) 374 [Mþ] (100), 357 (42), 270
(23). Anal. Calcd. for C20H18N6O2: C, 64.16; H, 4.85; N, 22.45.
Found: C, 64.12; H, 4.90; N, 22.47.
6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-trifluoromethylphenyl)-
8,9-dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-
diamine (5d)
8-(Benzo[d][1,3]dioxol-5-yl)-6-(4-methoxyphenyl)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-
diamine (4d)
This compound was obtained as a yellow solid in 84% yield.
M.p. 1488C; 1H NMR (400 MHz, DMSO-d6): d ¼ 2.86 (dd, Jgem
¼
This compound was obtained as a yellow crystal solid in
86% yield. M.p. 1438C; 1H NMR (CDCl3, 400 MHz): d ¼ 3.07
14.4 Hz, J1 ¼ 3.6 Hz, 1H, H-7), 3.66 (dd, Jgem ¼ 14.5 Hz,
J2 ¼ 6.4 Hz, 1H, H-7), 5.12 (m, 1H, H-8), 5.38 (s, 2H, NH2), 5.93
(s, 2H, NH2), 5.94 (s, 2H), 6.70 (d, J ¼ 3.6, 1H, NH), 6.72–7.54
(m, 7H) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 41.0, 58.3,
101.5, 102.4, 106.7, 107.8, 120.9, 125.3, 125.3, 127.3, 127.4,
136.2, 147.9, 148.1, 149.0, 154.6, 155.1, 155.2, 160.9, 164.1,
and 171.3 ppm; FTIR (KBr) n ¼ 3474, 3387, 1587, and
1569 cmꢁ1; MS (EI, 70 eV), m/z (%) 442 [Mþ] (100), 427 (46), 270
(35). Anal. Calcd. for C21H17F3N6O2: C, 57.01; H, 3.87; N, 19.00.
Found: C, 56.98; H, 4.00; N, 19.97.
(dd, Jgem ¼ 14.4 Hz, Jtrans ¼ 7.2 Hz, 1H, CHgem), 3.33 (d, Jgem
¼
14.4, 1H, CHgem), 3.81 (s, 3H, OCH3), 4.65 (d, Jtrans ¼ 7.2 Hz, 1H),
4.72 (s, 2H, NH2), 5.56 (s, 1H, NH), 5.62 (s, 2H, NH2), 5.91 (s, 2H),
6.73–6.77 (m, 3H), 6.84 (d, J ¼ 8.8, 2H), 7.63 (d, J ¼ 8.8, 2H) ppm;
13C NMR (CDCl3, 100 MHz): d ¼ 41.3, 55.3, 58.9, 101.2, 102.7,
106.7, 108.4, 113.6, 119.5, 127.9, 128.0, 133.4, 137.3, 147.2,
148.1, 153.6, 157.1, 159.7, 159.9, 160.5, and 163.7 ppm; FTIR
(KBr) n ¼ 3475, 3385, 3178, 2961, 1599, 1584, and 1577 cmꢁ1
;
MS (EI, 70 eV), m/z (%) 404 [Mþ] (100), 389 (64), 256 (50). Anal.
Calcd. for C21H20N6O3: C, 62.37; H, 4.98; N, 20.78. Found: C, 62.43;
H, 5.04; N, 20.64.
General procedure for the synthesis of 6-
(benzo[d][1,3]dioxol-5-yl)-8-aryl-2-(methylthio)-8,9-
6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-chlorophenyl)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepin-4-amines (7a–d)
Compound 7 was obtained using microwave irradiation (1208C,
75 W, CEM Discovery) during 4–14 min, in the reaction of 2-
methylthio-4,5,6-triaminopyrimidine 6 (0.60 mmol) and chal-
cone 3 (0.30 mmol) in DMF (0.5 mL) and a catalytic amount of
BF3ꢀOEt2. After the reaction mixture was cooled to room tempera-
ture, ethanol (5.0 mL) was added and the precipitate formed was
filtrated and purified by column chromatography on silica gel by
using a mixture of CH2Cl2/MeOH (20:1) as eluent.
dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-diamine (5a)
This compound was obtained as a yellow solid in 95% yield. M.p.
1738C; 1H NMR (400 MHz, DMSO-d6): d ¼ 3.21 (m, 2H), 4.74 (m, 3H,
H-8, NH2), 5.30 (s, 1H, NH), 5.60 (s, 2H, NH2), 5.96 (s, 2H), 6.69–7.26
(m, 7H) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 41.0, 58.5, 101.3,
102.7, 106.6, 107.6, 120.7, 127.5, 127.6, 129.0, 133.7, 135.3, 141.4,
148.0, 148.6, 153.8, 156.5, 159.9, 160.0, and 163.7 ppm; FTIR (KBr)
n ¼ 3485, 3381, 2900, 1586, and 1568 cmꢁ1; MS (EI, 70 eV), m/z
(%) 410 [Mþ2] (30), 408 [Mþ] (100), 395 (14), 393 (43), 270 (67). Anal.
Calcd. for C20H17ClN6O2: C, 58.75; H, 4.19; N, 20.56. Found: C,
58.78; H, 4.16; N, 20.62.
6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-chlorophenyl)-2-
(methylthio)-8,9-dihydro-7H-pyrimido[5,4-b][1,4]-
diazepin-4-amine (7a)
6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-fluorophenyl)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepine-2,4-
diamine (5b)
This compound was obtained as a yellow solid in 95% yield. M.p.
2588C; 1H NMR (DMSO-d6, 400 MHz): d ¼ 2.39 (s, 3H), 2.76 (d,
Jgem ¼ 14.4, 1H, H-7), 3.76 (dd, Jgem ¼ 14.4, J ¼ 5.6, 1H, H-7), 5.11
(m, 1H, H-8), 5.96 (s, 2H), 6.47 (s, 2H, NH2), 6.71–7.33 (m, 7H), 7.47
(d, J ¼ 5.2, 1H, NH) ppm; 13C NMR (DMSO-d6, 100 MHz): d ¼ 13.7,
39.3, 58.5, 101.6, 104.8, 107.1, 107.8, 121.7, 121.8, 128.3, 128.4,
131.7, 135.2, 143.1, 147.8, 148.6, 153.5, 159.2, 159.3, 162.5, and
166.2 ppm; FTIR (KBr) n ¼ 3513, 3432, 3118, 2978, and
1568 cmꢁ1; MS (EI, 70 eV), m/z (%) 441 [Mþ2] (41), 439 [Mþ]
This compound was obtained as a yellow solid in 92% yield.
M.p. 2608C; 1H NMR (400 MHz, DMSO-d6): d ¼ 2.85 (dd, Jgem
¼
14.4 Hz, J1 ¼ 3.6 Hz, 1H, H-7), 3.58 (dd, Jgem ¼ 14.4 Hz,
J2 ¼ 6.4 Hz, 1H, H-7), 5.01 (m, 1H, H-8), 5.38 (s, 2H, NH2), 5.95
(s, 2H, NH2), 5.96 (s, 2H), 6.60 (d, 1H, NH), 6.72–7.21 (m, 7H) ppm;
13C NMR (100 MHz, DMSO-d6): d ¼ 39.8, 58.0, 101.5, 102.3, 106.7,
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
B. Insuasty et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
(100), 426 (18), 424 (48), 301 (57). Anal. Calcd. for
C21H18ClN5O2S: C, 57.33; H, 4.12; N, 15.92. Found: C, 57.95; H,
3.98; N, 16.04.
References
[1] A. Wright, M. Zignol, WHO/IUATLD Global Project on Anti-
tuberculosis Drug Resistance Surveillance, in Anti-
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The World Health Organization/International Union Against
Tuberculosis and Lung Disease (WHO/UNION) Global Project on
Anti-Tuberculosis Drug Resistance Surveillance, 2002–2007,
World Health Organization, Geneva, Switzerland 2008,
p. 151.
6-(Benzo[d][1,3]dioxol-5-yl)-8-phenyl-2-(methylthio)-8,9-
dihydro-7H-pyrimido[5,4-b][1,4]diazepin-4-amine (7b)
This compound was obtained as a yellow solid in 78% yield. M.p.
2608C; 1H NMR (DMSO-d6, 400 MHz): d ¼ 2.40 (s, 3H), 2.78
(d, Jgem ¼ 14.4, 1H, H-7), 3.72 (dd, Jgem ¼ 14.4, J ¼ 6.0, 1H, H-7),
5.10 (m, 1H, H-8), 5.95 (s, 2H), 6.44 (s, 2H, NH2), 6.70–7.27 (m, 8H),
7.41 (d, J ¼ 5.2, 1H, NH) ppm; 13C NMR (DMSO-d6, 100 MHz):
d ¼ 13.7, 39.6, 59.1, 101.6, 104.7, 107.1, 107.8, 121.7, 126.4,
127.3, 128.5, 128.6, 135.4, 144.1, 147.7, 148.5, 153.6, 159.4,
162.4, and 166.1 ppm; FTIR (KBr) n ¼ 3549, 3402, 3183, 2956,
and 1518 cmꢁ1; MS (EI, 70 eV), m/z (%) 405 [Mþ] (100), 390 (50),
301 (50). Anal. Calcd. for C21H19N5O2S: C, 62.21; H, 4.72; N, 17.27.
Found: C, 62.23; H, 4.76; N, 17.28.
[2] Y. L. Janin, Bioorg. Med. Chem. 2007, 15, 2479–2513.
[3] R. Kumar, Y. Joshi, J. Chem. Sci. 2009, 121, 497–502.
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[5] B. Insuasty, F. Orozco, A. Garcıa, J. Quiroga, R. Abonia,
M. Nogueras, J. Cobo, J. Heterocyclic Chem. 2008, 45, 1659–
6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-methoxyphenyl)-2-
(methylthio)-8,9-dihydro-7H-pyrimido[5,4-b][1,4]-
diazepin-4-amine (7c)
´
1663; B. Insuasty, F. Orozco, J. Quiroga, R. Abonıa,
M. Nogueras, J. Cobo, Eur. J. Med. Chem. 2008, 43, 1955–
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Abonia, M. Hursthouse, M. Nogueras, J. Cobo, Bioorg. Med.
Chem. 2008, 16, 8492–8500.
This compound was obtained as a yellow solid in 83% yield. M.p.
2018C; 1H NMR (DMSO-d6, 400 MHz): d ¼ 2.41 (s, 3H), 2.80
(d, Jgem ¼ 14.6, 1H, H-7), 3.68 (dd, Jgem ¼ 14.6, J ¼ 6.4, 1H, H-7),
3.73 (s, 3H, OCH3), 5.03 (m, 1H, H-8), 5.88 (s, 2H), 6.48 (s, 2H, NH2),
6.61–7.67 (m, 7H), 7.37 (d, J ¼ 5.2, 1H, NH) ppm; 13C NMR (DMSO-
d6, 100 MHz): d ¼ 13.7, 39.5, 58.8, 101.2, 104.8, 107.1, 108.2,
113.8, 119.6, 128.7, 128.8, 133.4, 138.4, 146.3, 147.5, 151.6,
153.4, 159.7, 159.8, 160.5, 162.5, and 166.0 ppm; FTIR (KBr)
n ¼ 3441, 3385, 3126, 2904, and 1535 cmꢁ1; MS (EI, 70 eV),
m/z (%) 436 [Mþ] (18), 435 (64), 420 (46), 301 (11), 287 (100).
Anal. Calcd. for C22H21N5O3S: C, 60.67; H, 4.86; N, 16.08.
Found: C, 60.63; H, 4.80; N, 16.02.
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M. R. Mohammadizadeh, J. Org. Chem. 2005, 70, 1471–
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6-(Benzo[d][1,3]dioxol-5-yl)-8-(4-trifluorophenyl)-2-
(methylthio)-8,9-dihydro-7H-pyrimido[5,4-b][1,4]-
diazepin-4-amine (7d)
[11] B. Parwati, L. Alisjahbana, R. D. Apriani, T. H. Soetikno,
A. G. Ottenhoff, J. van der Zanden, D. van der Meer,
R. van Soolingen, R. van Crevel, J. Infect Dis. 2010, 201 (4),
553–557.
This compound was obtained as a yellow solid in 81% yield. M.p.
2158C; 1H NMR (DMSO-d6, 400 MHz): d ¼ 2.41 (s, 3H), 2.81
(d, Jgem ¼ 14.4, 1H, H-7), 3.81 (dd, Jgem ¼ 14.4, J ¼ 6.0, 1H, H-7),
5.22 (m, 1H, H-8), 5.95 (s, 2H), 6.47 (s, 2H, NH2), 6.69–7.54 (m, 7H),
7.50 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 100 MHz): d ¼ 13.7,
39.3, 58.9, 101.6, 104.9, 107.1, 107.7, 121.7, 125.3, 125.4, 127.3,
127.4, 135.1, 147.8, 148.5, 148.6, 151.6, 153.5, 159.2, 159.3, 162.5,
and 166.3 ppm; FTIR (KBr) n ¼ 3500, 3489, 3109, 2976, and
1513 cmꢁ1; MS (EI, 70 eV), m/z (%) 473 [Mþ] (100), 458 (41), 301
(37). Anal. Calcd. for C22H18F3N5O2S: C, 55.81; H, 3.83; N, 14.79.
Found: C, 55.83; H, 3.86; N, 14.78.
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447–453.
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S. G. Franzblau, Life Sci. 2005, 78 (5), 485–494.
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[16] CS. Chem. Office, Version 9.0, Cambridge Soft Corporation,
100 Cambridge Park Drive, Cambridge, MA, 2005.
The authors are grateful to the Instituto Nacional de Salud, INS,
Universidad del Valle and COLCIENCIAS for the support of this project.
´
[17] M. Sortino, P. Delgado, S. Juarez, J. Quiroga, R. Abonia,
B. Insuasty, M. Nogueras, L. Rodero, F. M. Garibotto,
R. D. Henriz, S. A. Zacchino, Bioorg. Med. Chem. 2007, 15,
484–494.
The authors have declared no conflict of interest.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com