Synthesis and structure elucidation of novel fused 1,2,4-triazine derivatives as potent inhibitors targeting CYP1A1 activity

Article abstract of DOI:10.1016/j.bmc.2012.02.041

Synthesis and structure elucidation of new series of novel fused 1,2,4-triazine derivatives 3a-3f, 4a-4i and 6a-6b and their inhibitory activities are presented. Molecular structures of the synthesized compounds were confirmed by ¹H NMR, ¹³C NMR, MS spectra and elemental analyses. X-ray crystallographic analysis was performed on 2-acetyl-8-(N,N- diacetylamino)-6-(4-methoxybenzyl)-3-(4-methoxy-phenyl)-7-oxo-2,3-dihydro-7H-[1, 2,4]triazolo[4,3-b][1,2,4]triazine 3d and 2-acetyl-8-(N-acetylamino)-6-benzyl-3- (4-chlorophenyl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4] triazine 4e to secure their structures. The inhibitory effect of these compounds toward the CPY1A1 activity was screened to determine their potential as promising anticancer drugs. Our data showed that compounds 4e, 5a, 5b and 6b possess the highest inhibitory effects among all tested compounds. Furthermore, analysis of triazolotriazine derivatives docking showed that these compounds bind only at the interface of substrate recognition site 2 (SRS2) and (SRS6) at the outer surface of the protein. Amino-acids ASN214, SER216 and ILE462 participate in the binding of these compounds through H-bonds.

Full text of DOI:10.1016/j.bmc.2012.02.041

Bioorganic & Medicinal Chemistry 20 (2012) 2624–2637
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure elucidation of novel fused 1,2,4-triazine derivatives
as potent inhibitors targeting CYP1A1 activity q
Abdel Moneim EL Massry a,c, , Ahmed Mosaad Asal b, Sherine Nabil Khattab c, , Nesreen Saied Haiba b,
,
Hala A. Awney d, , Mohamed Helmy e,f, Vratislav Langer g, Adel Amer c, ,
⇑
⇑
a Department of Chemistry, Rabigh College of Science and Art, King Abdulaziz University, Saudi Arabia
b Department of Chemistry and Physics, Faculty of Education, Alexandria Univeristy, Alexandria, Egypt
c Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt
d Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt
e Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt
f Department of Biology, Faculty of sciences, Hail University, Saudi Arabia
g Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and structure elucidation of new series of novel fused 1,2,4-triazine derivatives 3a–3f, 4a–4i and
6a–6b and their inhibitory activities are presented. Molecular structures of the synthesized compounds
were confirmed by 1H NMR, 13C NMR, MS spectra and elemental analyses. X-ray crystallographic analysis
was performed on 2-acetyl-8-(N,N-diacetylamino)-6-(4-methoxybenzyl)-3-(4-methoxy-phenyl)-7-oxo-
2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine 3d and 2-acetyl-8-(N-acetyl- amino)-6-benzyl-3-
(4-chlorophenyl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine 4e to secure their
structures. The inhibitory effect of these compounds toward the CPY1A1 activity was screened to deter-
mine their potential as promising anticancer drugs. Our data showed that compounds 4e, 5a, 5b and 6b
possess the highest inhibitory effects among all tested compounds. Furthermore, analysis of triazolotri-
azine derivatives docking showed that these compounds bind only at the interface of substrate recognition
site 2 (SRS2) and (SRS6) at the outer surface of the protein. Amino-acids ASN214, SER216 and ILE462
participate in the binding of these compounds through H-bonds.
Received 12 December 2011
Revised 6 February 2012
Accepted 15 February 2012
Available online 25 February 2012
Keywords:
Triazine
Triazolotriazine
Triazinotetrazine
NMR
X-ray
Aryl hydrocarbons hydroxylase
CYP1A1
Docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
endogenous substances. These isoenzymes catalyze a variety of
metabolic pathways and thus play an important role primarily in
In recent years, the screening of natural products and chemical
compounds has led to the development of the anticancer drugs
currently utilized clinically.2–8 However, cancer still carries a high
mortality rate globally regardless of therapeutic advances. Among
the treatment choices available, chemotherapy is one of the most
widely used treatment options in spite of its numerous side effects,
such as suppression of bone marrow and immune system as well
as its development of drug resistance.9,10 Therefore, the search
for new chemo-preventive anticancer agents that are able to not
only target chemical carcinogens, but also display increased effi-
cacy and overall decreased systemic toxicity continues to be a
great challenge for medical science.
phase I metabolism of xenobiotics facilitating their excretion, as
well as in the activation of endogenous compounds to reactive con-
version products involved in the regulation of physiological and
cellular processes.11
CYPs family 1 in particular, are involved in the metabolic activa-
tion of numerous chemical carcinogens such as polycyclic aromatic
hydrocarbons (PAHs) which are ubiquitous pollutants in human
environment. This is the case for benzo[a]pyrene (BP), an infamous
environmental PAH considered as a pro-carcinogen for various
organs, including breast, lung and skin.2 The metabolic activation
of BP via drug-metabolizing enzymes, such as cytochromes
CYP1A1 (aryl hydrocarbon hydroxylase), can produce genotoxic
electrophile intermediates that can covalently bind DNA and form
mutagenic DNA adducts that might be involved in the initial events
of carcinogenesis.3
Cytochrome P450s (CYPs) are heme containing isoenzymes
that participate in the metabolism of various xenobiotics and
CYPs inhibitors are being considered by many pharmaceutical
companies as possible therapeutic targets. Over-expressed CYPs,
with altered substrate/inhibitor specificity, offer great advantage
for the preparation/formulation of safe anticancer molecules, as
q
See Ref. 1.
⇑
Corresponding authors. Tel.: +20 122219463; fax: +20 33911794.
E-mail address: aamer@sci.alex.edu.eg (A. Amer).
Authors participated equally in the study.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.041

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2625
levels of these CYPs are either undetectable or miniscule in normal
cells. Inhibition of these endogenous CYPs in cancerous cells thus
might offer a novel target for designing new anticancer agents.
Accordingly, various natural and synthetic compounds that are
capable of reducing or inhibiting CYPs-mediated bioactivation of
PAHs have been reported as potential chemopreventive anticancer
agents.5,6 Among wide variety of natural compounds, flavonoids
have traditionally been described as CYP1 inhibitors due to their
inhibition of carcinogenic product formation as well as their conse-
quent blockage of the initiation stage of carcinogenesis. One of
their most significant characteristics, responsible for their cancer
preventive properties, is their selective interaction with cyto-
chrome CYP1 enzymes.12,13 Moreover, several synthetic com-
pounds have been reviewed for their potency and selectivity as
chemical inhibitors for the major human hepatic CYP isoforms.
For example, furafylline was found to be the most selective inhib-
itor available for CYP1A2; 2-phenyl-2-(1-piperidinyl) propane
(PPP) for CYP2B6; montelukast for CYP2C8; sulfaphenazole for
CYP2C9; (ꢀ)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine
for CYP2D6.14 Recently, potent CYP17 inhibitors which provide
effective treatment of prostate cancer patients were reported.15
However, more-selective inhibitors are clearly still needed for phe-
notyping of some CYP isoforms.14
Among a wide variety of synthetic compounds being screened
for anticancer activities various condensed 1,2,4-triazines have
been reported by independent groups to be extremely potent.
Sztanke et al. reported that several heterobicyclic systems contain-
ing the 1,2,4-triazine moiety have shown significant biological
activities. For example the imidazo[2,1-c][1,2,4]triazin-4(6H)-one
core system showed distinctly lower cytotoxicity towards normal
cells and several-times higher against cancer cell lines.16–20 Ab-
del-Rahman et al. demonstrated that several heterobicyclic nitro-
gen systems bearing the 1,2,4-triazine moiety possess Leukemia,
Lung, Breast and CNS anticancer activities.21–23 Ciciani et al. syn-
thesized a series of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives
showing selective cytotoxicity on human colorectal adenocarci-
noma cell line HCT-8 in hypoxic as well as normoxic condi-
tions.24,25 Nevertheless, the role of 1,2,4-triazines derivatives as
selective synthetic inhibitors of the major hepatic CYP isoenzymes
is yet to be studied.
In our effort to identify new anticancer agents, we synthesized
various heterocyclic derivatives bearing the 1,2,4-triazine moiety.
Herein, we report syntheses and characterization of the novel com-
pounds as well as their inhibitory activities on CYP1A1.
2. Chemistry
The multi nucleophilic centered molecules, namely, 4-amino-6-
substituted benzyl-3-hydrazinyl-1,2,4-triazin-5(4H)-one 1a–b
were selected as building block units for the preparation of the tar-
get compounds (Fig. 1). A simple synthetic strategy was used to
prepare the fused triazines required for the present study. Thus,
the reaction of 1 with a variety of one carbon donors, such as
substituted benzaldehydes and acetophenones afforded the corre-
sponding Schiff’s bases 2a–2t in good yields (Scheme 1).
Cyclocondensation of 2a–2t followed pathway A. Treatment
of compound 2a–2t with acetic anhydride to give either
corresponding triacetylated heterobicyclic derivatives 2-acetyl-8-
(N,N-(diacetylamino)-6-substituted
benzyl-7-oxo-3-substituted
phenyl-2,3-dihydro-7H-[1,2,4]-triazolo[4,3-b][1,2,4] triazine 3 or
diacetylated derivatives 4 (Scheme 2). The structures of these com-
pounds were established by IR, 1H NMR, 13C NMR, HMQC, and ele-
mental analysis. Even though their structures can be inferred from
their spectroscopic and analytical data, the structures of 3d and 4e
were secured by X-ray diffraction analysis as prototypes (Tables 1
and 2; Figs. 2 and 3). The structure of 4e is centrosymmetric, and
hence racemic. There are two crystallographically independent
molecules A (R on C3) and B (S on C3) in the asymmetric unit
(Fig. 3).
Another attempt for cyclocondensation was performed by
allowing 4-amino-6-substituted benzyl-3-hydrazinyl-1,2,4-tria-
zin-5(4H)-one 1 to react with isatin as an example for 1,2-dicar-
bonyl analogue. In this case, condensation occurred only between
the isatin ketonic carbonyl group and the NH2 group of the hydra-
zine moiety of 1 to form the hydrazone 5 (Scheme 3). The struc-
tures of compounds 5a and 5b were confirmed by their IR, 1H
N
N
Ar
N
Ar
NH
NH2
NH2
O
N
NH
O
N
N
NH2
NH2
1
monocarbon donar
pathway A
pathway B
N
N
N
Ar
N
Ar
N
N
O
N
NH
N
N
O
HN
NH2
Figure 1. Pathways for the annelation reaction of 1 with monocarbon donor.

2626
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
N
N
N
Ar
N
Ar
N
Ar '-CRO , EtOH
NH2
N
Ar'
N
O
N
H
ACOH
O
NH
NH2
NH2
R
2
1
R
Ar
C6H5
Ar'
Ar
2a
2b
2c
2d
2e
2f
H
C6H5
C6H5
H
H
H
H
H
H
H
H
H
4-OCH3-C6H4
C6H5
1a
1b
C6H5
4-OCH3-C6H4
4-OCH3-C6H4
4-OCH3-C6H4
4-OCH3-C6H4
C6H5
4-OCH3-C6H4
C6H5
3,4,5-(OCH3)3-C6H2
3,4,5-(OCH3)3-C6H2
4-OH-C6H4
4-OH-C6H4
4-NO2-C6H
4-NO2-C6H
C6H5
2g
2h
2i
4-OCH3-C6H4
C6H5
2j
4-OCH3-C6H4
2k CH3 C6H5
2l CH3 4-OCH3-C6H4
2m CH3 C6H5
2n CH3 4-OCH3-C6H4
C6H5
4-Cl-C6H4
4-Cl-C6H4
4-CH3-C6H4
4-CH3-C6H4
2-pyridyl
2o
CH3 C6H5
2p CH3 4-OCH3-C6H4
2q CH3 C6H5
2r CH3 4-OCH3-C6H4
2s CH3 C6H5
2-pyridyl
2-furyl
2t
CH3 4-OCH3-C6H4
2-furyl
Scheme 1. General synthesis of compound 2.
NMR, 13C NMR and elemental analysis. In turn, these compounds
have undergone acylation with acetic anhydride to afford the cor-
responding spiro compound 6a and 6b as evident from their IR, 1H
NMR, 13C NMR and elemental analysis (Scheme 3). In 1H NMR
spectrum of 6a four methyl groups beside the benzylic and aro-
matic protons were observed (see Section 5). Furthermore, the
structure was supported by 13C NMR which showed the spiro qua-
ternary carbon at d 83.06. Similarly, the structure of 6b was con-
firmed. Based on the number of NMR signals for methyl groups,
the structural possibilities like 7 and 8 were ruled out.
The condensation reaction between 1 and triethyl orthoformate
as the monocarbon donor in methanol and the presence of catalytic
amount of acetic acid furnished [1,2,4]triazolo[4,3-b][1,2,4]triazin-
7(8H)-one 9 (Scheme 4). The assigned structures of the reaction
products such as 9 rather than 10 were based on their spectroscop-
ical data. Thus, the IR spectra of 9a and 9b were quite distinct
allowing the observation of the characteristic peaks for the NH2
groups at 3321, 3220 and 3292, 3241 cmꢀ1, respectively, and sup-
ported by 1H NMR that revealed the exchangeable protons at d 5.94
and 6.03 with two protons intensity each.
isolate the diacetylated analogue 4j and 2-methyl-1H-
benzo[d]imidazole.
3. Biology
3.1. Microsomal aryl hydrocarbon hydroxylase activity
Microsomal aryl hydrocarbon hydroxylase (AHH) activity was
determined with or without triazolotriazine derivatives and per-
centage of changes in AHH activities were calculated according
to the methods reported earlier.26–28 Data presented in Table 3
showed that triazolotriazine derivatives affect the aryl hydrocar-
bon hydroxylase (AHH) activity in different manner; marked inhib-
itory effects were seen with 5a (92.23% 2.07), 5b (90.45% 2.25),
4e (87.90% 2.30) and 6b (81.57% 1.9) whereas moderated
inhibitory effects were detected with 4f (75.58% 0.64), 6a
(64.56% 4.54), 4d (59.81% 4.26), 3b (57.59% 2.65) and 3c
(51.47% 0.64). Nevertheless, low inhibition effect were detected
with 3a (20.63 3.32), 4c (37.92 3.41) and 3f (48.16 3.55).
Table 3 also showed that compound 5a exhibits the lowest IC50
value. In the present study, we report for the first time to the best
of our knowledge that 1,2,4-triazine derivatives, constitute potent
inhibitors of drug-metabolizing CYP 450 enzymes. These data
showed that compounds with 5a, 5b, 4e and 6b may be identified
as a constituting new chemical class of CYP1A1 inhibitors.
However, the reaction of 1 with acetic anhydride led to the for-
mation of the 1H-[1,2,4]triazino[4,3-b][1,2,4,5]tetrazin-6(4H)-one
11 via pathway B. The structures of 11a and 11b were confirmed
using NMR experiments including NOE together with their ele-
mental analyses. For example, in the NOE spectrum of 11a a corre-
lation was observed between the two methyl groups of 91.86%
indicating the short distance between them and thus eliminating
structure 12 for the reaction product.
3.2. Acute toxicity test
In order to test the capability of the multiacetylated com-
pounds 3 and 6 as acetyl donors, compounds 3c, 3d and 6a as
prototype were allowed to react with o-phenylenediamine
(o-PDA) in ethanol and the reaction was monitored by TLC which
detected the formation of 2-methyl-1H-benzo[d]imidazole. In
case of compound 3c the reaction mixture of was worked up to
Oral and intraperitoneal acute toxicity test results indicated
that compounds 5a, 5b 4e and 6b proved to be non-toxic and well
tolerated by the experimental animals up to 350 and 150 mg/kg in
oral and intraperitoneal acute toxicity test, respectively. Based on
these results, the single acute oral LD50 of the tested compounds
is greater than 350 mg/kg of body weight.

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2627
N
Ar
N
N
Ar'
N
O
NH
NH2
R
2
Ac2O
R
Ar'
R
N
Ar'
N
Ar
N
O
Ar
N
O
N
N
N
N
N
O
N
N
O
O
O
O
NH
3
4
Ar
R
Ar'
4-NO2-C6H4
4-OCH3-C6H4 4-NO2-C6H4
C6H5
R
H
H
H
H
Ar
C6H5
Ar'
C6H5
C6H5
4-OCH3-C6H4
4-OCH3-C6H4
4-Cl-C6H4
3a
3b
3c
3d
4a,
4b,
4c,
H
H
C6H5
4-OCH3-C6H4
C6H5
CH3 C6H5
4d, CH3 4-OCH3-C6H4 C6H5
4e, CH3 C6H5
4f, CH3 C6H5
4g, CH3 C6H5
4-OCH3-C6H4
3e CH3 4-OCH3-C6H4
3f CH3 4-OCH3-C6H4
4-Cl-C6H4
4-CH3-C6H4
4-CH3-C6H4
2-pyridyl
2-furyl
4h,
CH3 C6H5
4i, CH3 4-OCH3-C6H4 2-furyl
Scheme 2. General synthesis of compounds 3 and 4.
substrates and/or inhibitors. To answer this question, binding
mode simulation was carried out with ANF which is a natural sub-
strate for CYP1A1, a natural inhibitor of CYP1A232–34 and has al-
ready been used in studying CYP1A2 3D structure (PDB: 2hi4).32
It was found that, besides binding the active site, ANF binds the
SRS2/SRS6 site as for triazolotriazine derivatives (Fig. 4F). Further-
more, the docking fullfitness energy difference between the active
site and the SRS2/SRS6 site was not significant (Table 4) since it
was less than 5 kcal/mol. This indicates that ANF binds freely to
the active site, the SRS2/SRS6 site or both of them. If this informa-
tion is true, it can be assumed that at the molecular level more
than one triazolotriazine derivative will bind to the enzyme. Con-
sequently the obtained data confirm the assumption of the pres-
ence of two or more substrate binding sites and the allosterism
of CYP1A1.35 This information also indicates that certain sites out-
side the active site play a role in the activity of CYP1A1. One of
these probable sites is SRS2/SRS6 site. The probable role may be
through the modification of the enzyme 3D structure due to bind-
ing to the SRS2/SRS6 site, leading to reduction or prevention of
binding of the substrate to the active site. This can, in part, explains
the mechanism by which triazolotriazine derivatives inhibit
CYP1A1.
4. Structural modeling and inhibitors docking
CYP1A1 3D structure model was generated using sequence-se-
quence comparative modeling. Through PSI-BLAST, CYP1A2 (PDB:
2hi4) was determined as the most suitable template for predicting
CYP1A1 structure based on a sequence identity of 73.5%. The valid-
ity of the obtained structure was confirmed by Raman plot which
showed only 0.5% of the amino acids in the disallowed region (data
not shown). The predicted CYP1A1 structure was then used
through EAdock29 and SwissDock30 to predict binding modes of al-
pha-naphthoflavone (ANF) and the triazolotriazine derivatives (i.e.,
4e, 5a, 5b, 6b). Binding modes were analyzed using UCSF chi-
mera.31 According to the obtained data (Fig. 4A–E) none of triazol-
otriazine derivatives gave a binding mode in the active site. This is
not surprising. Based on the structure of CYP1A2 active site32 and
the sequence identity between CYP1A2 and CYP1A1, the active site
structure of CYP1A1 will be highly adapted for positioning and oxi-
dation of relatively large, planar substrates. Furthermore, CYP1A1
substrates will be bulkier than those of CYP1A2.32 However; this
is not the case of our studied compounds. The lowest fullfitness en-
ergy indicates that the most probable binding mode for all the
studied triazolotriazine derivatives is at the interface between
SRS2 (Fig. 4, purple) and SRS6 (Fig. 4, blue) at the outer surface
of the protein. This finding raised the question about the possibility
that this docking site (i.e., SRS2/SRS6) is accessible in nature by
However, the difference in the inhibitory potential of the stud-
ied triazolotriazine derivatives remains to be explained. Therefore,
further analysis was carried out to determine the regions that par-

2628
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
Table 1
Crystal data and structure refinement for 3d
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C25H26N6O6
506.52
173(2) K
0.71073 Å
Triclinic
ꢀ
P1
Unit cell dimensions
a = 9.4281(5) Å; b = 10.2953(5) Å;
c = 12.6745(7) Å
91.896(1)°, 103.660(1)°, 90.882(1)°
1194.46(11) Å3
Angles a, b, c
Volume
Z
2
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data
collection
1.408 Mg/m3
0.103 mmꢀ1
532
0.32 ꢁ 0.30 ꢁ 0.20 mm3
2.44 to 25.10°
Index ranges
ꢀ11<=h<=11, ꢀ12<=k<=12, ꢀ15<=l<=15
Reflections collected
Independent reflections
Completeness to
13170
4247 [R(int) = 0.0735]
99.9%
theta = 25.10°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Multi-scan
0.9797 and 0.9677
Full-matrix least-squares on F2
4247/0/339
Figure 2. Numbering scheme with atomic displacement ellipsoids drawn at 50%
probability level of compound 3d.
0.999
Final R indices [I >2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.0548, wR2 = 0.1427
R1 = 0.0790, wR2 = 0.1585
0.234 and ꢀ0.313 e.Åꢀ3
olotriazine derivatives will change. It was found that certain amino
acids are involved in the triazolotriazine derivatives binding modes
by H-bond formation. These amino acids are ASN214, SER216 and
ILE462. The possible binding modes are determined by the fullfit-
ness energy range in Table 4. According to these findings, one
can assume that the position of amino acids and the extent of their
implication in fixing the triazolotriazine derivatives will determine
the degree of change in enzyme structure and hence its function.
The obtained data can partially explain the difference in the inhibi-
tion potential of the triazolotriazine derivatives (Table 4). Further
studies are now being carried out to determine the role of the as-
sumed amino acids in CYP1A1 activity.
Table 2
Crystal data and structure refinement for 4e
Empirical formula
Formula weight
Temperature
C22H21ClN6O3
452.90
153(2) K
Wavelength
0.71073 Å
Crystal system
Space group
Monoclinic
P21/n
Unit cell dimensions
a = 10.3866(5) Å; b = 15.8554(8) Å;
c = 26.7122(14) Å
90°, 93.763(1)°, 90°
4389.6(4) Å3
8
In summary, our results suggest that compounds such 4e, 5a, 5b
and 6b may be considered as leads for chemoprevention anticancer
studies.
Angles a, b, c
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data
collection
1.371 Mg/m3
0.211 mmꢀ1
1888
5. Experimental protocols
5.1. Chemistry
0.42 ꢁ 0.24 ꢁ 0.11 mm3
2.00 to 25.39°
Index ranges
ꢀ12<=h<=12, ꢀ19<=k<=19, ꢀ32<=l<=32
Melting points were determined with a Mel-Temp apparatus
and are uncorrected. Magnetic resonance spectra (1H NMR and
13C NMR spectra) were recorded using a JEOL 500 MHz spectrom-
eter with the chemical shift values reported in d units (part per
million). Infrared data were obtained using a Perkin–Elmer 1600
series Fourier transform instrument as KBr pellets. The compounds
were named using Chem. Draw Ultra version 12, Cambridge soft
Corporation. Follow up of the reactions and checking the purity
of the compounds were made by TLC on silica gel-protected alumi-
num sheets (Type 60 GF254, Merck) and the spots were detected
by exposure to UV-lamp at k 254 nm for few seconds. X-ray
diffraction data were collected using a Siemens SMART CCD dif-
Reflections collected
Independent reflections
Completeness to theta =
25.39°
Absorption correction
Max. and min. transmission 0.9771 and 0.9165
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I
>2sigma(I)]
R indices (all data)
Largest diff. peak and hole
48135
8063 [R(int) = 0.1024]
99.9%
Multi-scan
Full-matrix least-squares on F2
8063/0/583
1.022
R1 = 0.0637, wR2 = 0.1584
R1 = 0.1104, wR2 = 0.1836
0.533 and ꢀ0.512 e.Åꢀ3
fractometer with Mo–Ka radiation (k = 0.71073 Å, graphite mono-
chromator). The crystals were cooled to 173(2)K for 3d and
ticipate in the interaction between the SRS2/SRS6 site and triazol-
otriazine derivatives (Fig. 5). These regions were found to be ILE18-
LEU30, ASN191-PHE220 and ILE462-LEU465. According to the
binding mode of the triazolotriazine derivatives, the amino acids
in the vicinity of the SRS/SRS6 site that can interact with the triaz-
153(2)K for 4e by a flow of nitrogen gas using the LT-2A device.
A full sphere of reciprocal space was scanned by 0.3 steps in
x with
a crystal-to-detector distance of 3.97 cm. Elemental analyses were
performed on Perkin–Elmer 2400 elemental analyzer, and the ob-
tained values were within 0.3% of the theoretical values.

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2629
Figure 3. A perspective drawing of molecules A and B of compound 4e, showing the atom-numbering scheme. Displacement ellipsoids are drawn at 50% probability level.
O
N
Ar
N
O
N
O
N
N
O
N
H
H
1
MeOH
NH2
N
H
5
Ac2O
O
N
O
N
O
Ar
N
N
Ar
N
N
N
O
a
b
C6H5
4-OCH3-C6H4
O
O
6
Scheme 3. Synthesis of compounds 5 and 6.
5.2. General procedure for preparation of 4-amino-6-substi
tuted benzyl-3-hydrazinyl-1,2,4-triazin-5(4H)-one (1)
5.2.1. 4-Amino-6-benzyl-3-hydrazinyl-1,2,4-triazin-5(4H)-one
(1a)
Compound 1a was obtained as pale yellow needles, 0.21 g
(53.8%) yield, mp—(°C) 194–195 (Lit. mp (°C)—254–255).36 1H
NMR (500 MHz: CDCl3): d: 3.89 (s, 2H, CH2), 4.38 (s. 2H, NH2,
D2O exchangeable), 5.51 (s, 2H, NH2, D2O exchangeable), 7.13-
7.16 (m, 1H, Ar-H), 7.22 (d, 4H, J = 3.8 Hz, Ar-H), 8.18 (s, 1H, NH,
D2O exchangeable).
To a solution of 4-amino-6-aryl-3-(methylthio)-1,2,4-triazin-
5(4H)-one (1.6 mmol) in 5 mL isopropyl alcohol, 1 mL hydrazine
hydrate (80%) was added. The reaction mixture was refluxed for
about 3 h then cooled to room temperature. The product that pre-
cipitated out was filtered off and washed well with ethanol.36

2630
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
N
Ar
N
O
N
N
O
N
N
N
N
Ar
N
N
O
N
N
N
N
O
O
O
7
8
N
N
Ar
N
N
N
O
EtO
EtO
N
N
OEt
Ar
N
NH2
NH2
9
EtOH, AcOH
O
N
H
NH2
N
N
Ar
N
Ar
N
Ar
O
N
NH
N
O
N
N
NH
NH
a
b
C6H5
4-OCH3-C6H4
HN
10
Scheme 4. Synthesis of compound 9.
Table 3
ethanol, the corresponding aldehyde (2.5 mmol) or acetophenone
(2.5 mmol) was added and heated under reflux for 2 h. The product
obtained on cooling was filtered off and recrystallized from
ethanol.
The effect of triazolotriazine derivatives on the aryl hydrocarbon hydroxylase activity
(AHH)
Code
Ar
Ar0
% Inhibition
IC50
nd
M
2d
3a
3b
3c
3f
4c
4d
4e
4f
5a
5b
6a
6b
4-OCH3-C6H4
C6H5
4-OCH3-C6H4
C6H5-
4-OCH3-C6H4
C6H5
4-OCH3-C6H4
C6H5
C6H5
C6H5
4-OCH3-C6H4
C6H5
4-OCH3-C6H4
4-OCH3-C6H4
C6H5
C6H5
4-OCH3-C6H4
4-CH3-C6H4
C6H5
C6H5
4-Cl-C6H4
4-CH3-C6H4
nd
20.63 3.32
57.59 2.65
51.47 0.64
48.16 3.55
37.92 3.41
59.81 4.26
87.90 2.30
75.58 0.64
92.23 2.07
90.45 2.25
64.56 4.54
81.54 1.9
2.42 ꢁ 10ꢀ8
8.67 ꢁ 10ꢀ8
9.71 ꢁ 10ꢀ9
1.04 ꢁ 10ꢀ8
1.32 ꢁ 10ꢀ8
8.36 ꢁ 10ꢀ9
5.69 ꢁ 10ꢀ9
6.62 ꢁ 10ꢀ9
5.42 ꢁ 10ꢀ9
5.53 ꢁ 10ꢀ9
7.74 ꢁ 10ꢀ9
6.14 ꢁ 10ꢀ8
5.3.1. 4-Amino-6-benzyl-3-(2-benzylidenehydrazinyl)-1,2,4-
triazin-5(4H)-one (2a)
Compound 2a was obtained as yellow crystals, 0.69 g (95.8%)
yield, mp (°C)—198–199. IR (KBr): 3302, 3232 (NH2+NH),
1650(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.84 (s, 2H,
.
CH2), 5.76 (s, 2H, NH2, D2O exchangeable), 7.17–7.36 (m, 8H, Ar-
H), 7.90 (d, 2H, J = 6.1 Hz, Ar-H), 8.27 (s, 1H, CH), 12.26 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C17H16N6O: C, 63.74; H,
5.03; N, 26.23. Found: C, 63.55; H, 5.27; N, 26.01.
The results were expressed as mean S.E.M. Data were analyzed by one way of
variance. Student’s t test for unpaired observations was used. p value <0.001 and
was significant. Number of experiments was 3.
5.3.2. 4-Amino-3-(2-benzylidenehydrazinyl)-6-(4-
methoxybenzyl)-1,2,4-triazin-5(4H)-one (2b)
Compound 2b was collected as yellow crystals, 0.52 g (81.1%)
yield, mp (°C)—166–161. IR (KBr): 3288, 3229 (NH2+NH), 1666
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.70 (s, 3H, OCH3),
.
5.2.2. 4-Amino-6-(4-methoxybenzyl)-3-hydrazinyl-1,2,4-
triazin-5(4H)-one (1b)
3.76 (s, 2H, CH2), 5.75 (s, 2H, NH2, D2O exchangeable), 6.82 (d,
2H, J = 8.4 Hz, Ar-H), 7.15 (d, 2H, J = 9.2 Hz, Ar-H), 7.37–7.38 (m,
3H, Ar-H), 7.90 (d, 2H, J = 6.1 Hz, Ar-H), 8.27 (s, 1H, CH), 12.31 (s,
1H, NH, D2O exchangeable). Anal. Calcd for C18H18N6O2: C, 61.70;
H, 5.18; N, 23.99. Found: C, 61.14; H, 5.41; N, 23.78.
Compound 1b was obtained as yellow needles, 0.35 g (87.5%)
yield, mp (°C)—258–259 °C (Lit. mp (°C)—243–244).36
5.3. General procedure for the reaction of 4-amino-6-aryl-3-
hydrazinyl-1,2,4-triazin-5(4H)-one (1) with aldehydes and
acetophenones
5.3.3. 4-Amino-6-benzyl-3-(2-(4-methoxybenzylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2c)
Compound 2c was obtained as yellow crystals, 0.69 g (87.9%)
yield, mp (°C)—150–151. IR (KBr): 3308, 3219 (NH2+NH), 1650
To a solution of 4-amino-6-benzyl-3-hydrazinyl-1,2,4-triazin-
5(4H)-one 1a (2.5 mmol) or 4-amino-6-(4-methoxybenzyl)-3-
hydrazinyl-1,2,4-triazin-5(4H)-one 1b (2.5 mmol) in 10 mL
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.77 (s, 3H, OCH3),
.
3.91 (s, 2H, CH2), 6.96 (d, 2H, J = 8.4 Hz, Ar-H), 7.14–7.22 (m, 2H,

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2631
Figure 4. Binding mode of triazolotriazine derivatives to CYP1A1 in comparison to ANF. CYP1A1 is shown in gray. The hetero-compounds are the triazolotriazine derivatives.
3a derivative, (A) 4e derivative, (B) 5a derivative, (C) 5b derivative, (D) 6b derivative, (E) and ANF, (F) Substrate recognition sites (SRS) are colored as follows: SRS1, orange;
SRS2, purple; SRS3, yellow; SRS4, green; SRS5, cyan; SRS6, blue.
NH2, D2O exchangeable), 7.28–7.30 (m, 5H, Ar-H), 7.87 (d, 2H,
J = 8.4 Hz, Ar-H), 8.41 (s, 1H, CH), 12.20 (s, 1H, NH, D2O exchange-
able). Anal. Calcd for C18H18N6O2: C, 61.70; H, 5.18; N, 23.99.
Found: C, 61.42.; H, 5.46; N, 23.74.
5.3.5. 4-Amino-6-benzyl-3-(2-(3,4,5-trimethoxybenzylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2e)
Compound 2e was obtained as pale yellow crystals, 0.93 g
(98.9%) yield, mp (°C)—157–158. IR (KBr): 3310, 3224 (NH2+NH),
1682 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO): d 3.35 (s, 3H,
.
5.3.4. 4-Amino-6-(4-methoxybenzyl)-3-(2-(4-methoxybenzyl
idene)hydrazinyl)-1,2,4-triazin-5(4H)-one (2d)
OCH3), 3.80 (s, 6H, OCH3), 3.85 (s, 2H, CH2), 5.76 (s, 2H, NH2, D2O
exchangeable), 7.28–7.25 (m, 7H, Ar-H), 8.18 (s, 1H, CH), 12.35
(s, 1H, NH, D2O exchangeable). Anal. Calcd for C20H22N6O4: C,
58.53; H, 5.40; N, 20.48. Found: C, 58.97; H, 5.14; N, 20.32.
Compound 2d was obtained as yellow crystals, 0.77 g (89.6%)
yield, mp (°C)—150–151. IR (KBr): 3331, 3244 (NH2+NH), 1650
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.67 (s, 2H, CH2),
.
3.76 (s, 6H, OCH3), 5.72 (s, 2H, NH2, D2O exchangeable), 6.81 (d,
2H, J = 8.4 Hz, Ar-H), 6.92 (d, 2H, J = 8.4 Hz, Ar-H.), 7.15(d, 2H,
J = 8.4 Hz, Ar-H), 7.84 (d, 2H, J = 8.4 Hz, Ar-H), 8.21 (s, 1H, CH),
12.24 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C19H20N6O3:
C, 59.99; H, 5.30; N, 22.09. Found: C, 75.2; H, 5.58; N, 22.124.
5.3.6. 4-Amino-6-(4-methoxybenzyl)-3-(2-(3,4,5-trimethoxy-
benzylidene)hydrazinyl)-1,2,4-triazin-5(4H)-one (2f)
Compound 2f was obtained as pale yellow crystals, 0.81 g (92%)
yield, mp (°C)—100–101. IR (KBr): 3308, 3229 (NH2+NH), 1676
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO): d 3.64–3.65 (m, 12H,
.

2632
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
Table 4
5.3.8. 4-Amino-3-(2-(4-hydroxybenzylidene)hydrazinyl)-6-(4-
methoxybenzyl)-1,2,4-triazin-5(4H)-one (2h)
Docking fullfitness energy for the triazolotriazine derivatives and alpha-naphthof-
lavone (ANF) on both the susbtrate recognition sites (SRS) and the active site of
CYP1A1. The fullfitness energy range is related to all the possible binding modes that
have no significant difference in the energy level and can take place at the SRS2/SRS6
site
Compound 2h was obtained as pale yellow crystals, 0.42 g
(49.7%) yield, mp (°C)—210–211. IR (KBr): 3276, 3136 (NH2+NH),
.
3276–2943 (br, OH), 1686 (NCO) cmꢀ1 1H NMR (500 MHz:
DMSO-d6): d 3.67 (s, 3H, OCH3); 3.75 (s, 2H, CH2), 4.04 (s, 1H,
OH, D2O exchangeable), 5.71 (s, 2H, NH2, D2O exchangeable),
6.73 (d, 2H, J = 8.4 Hz, Ar-H), 6.81 (d, 2H, J = 8.4 Hz, Ar-H), 7.15
(d, 2H, J = 8.4 Hz, Ar-H), 7.73 (d, 2H, J = 8.4 Hz, Ar-H), 8.10 (s, 1H,
CH), 12.17(s, 1H, NH, D2O exchangeable). Anal. Calcd for
Code
Fullfitness energy (kcal/mol)
SRS2/SRS6 siteb
Active site
% Inhibition
3a
6b
4e
5b
ꢀ1436 to ꢀ1431
ꢀ1417 to ꢀ1412
ꢀ1420 to ꢀ1415
ꢀ1384 to ꢀ1376
ꢀ1383 to ꢀ1378
ꢀ1463 to ꢀ1458
NAc
NA
NA
NA
NA
20.63 3.32
81.54 1.90
87.90 2.30
90.45 2.25
92.23 2.07
NA
C18H18N6O3: C, 59.01; H, 4.95; N, 22.94. Found: C, 59.27; H, 4.76;
N, 22.77.
5a
ANFa
ꢀ1461
a
5.3.9. 4-Amino-6-benzyl-3-(2-(4-nitrobenzylidene)hydrazinyl)-
1,2,4-triazin-5(4H)-one (2i)
Alpha-naphthoflavone.
SRS = Substrate recognition sites. The energy range is due to the possible
b
docking models of the hetero-compound to CYP1A1.
Compound 2i was obtained as yellow crystals, 0.84 g (96.1%)
c
NA = not applicable.
yield, mp (°C)—180–181. UV/VIS (CH3CN) kmax = 234 nm,
235,500; kmax = 289 nm, = 62,300. IR (KBr): 3331, 3223
(NH2+NH), 1691 (NCO), 1522, 1321 (NO2) cmꢀ1 1H NMR
e =
e
;
OCH3), 3.66 (s, 2H, CH2), 5.75 (s, 2H, NH2, D2O exchangeable), 6.77,
6.81 (2d, 2H, J = 8.4H, Ar-H), 7.09, 7.13 (2d, 2H, J = 8.4 Hz, Ar-H),
7.23 (s, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 8.25 (s, 1H, CH), 12.34 (s,
1H, NH, D2O exchangeable). Anal. Calcd for C21H24N6O5: C, 57.26;
H, 5.49; N, 19.08. Found: C, 57.02; H, 5.62; N, 19.33.
(500 MHz: DMSO-d6): d 3.86 (s, 2H, CH2), 5.81 (s, 2H, NH2,D2O
exchangeable), 7.18–7.29 (m, 5H, Ar-H), 8.17–8.2 (m, 4H, Ar-H),
8.37 (s, 1H, CH), 12.58 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C17H15N7O3: C, 55.89; H, 4.14; N, 26.84. Found: C, 55.60; H,
4.42; N, 26.63.
5.3.7. 4-Amino-6-benzyl-3-(2-(4-Hydroxybenzylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2g)
5.3.10. 4-Amino-6-(4-methoxybenzyl)-3-(2-(4-
nitrobenzylidene)hydrazinyl)-1,2,4-triazin-5(4H)-one(2j)
Compound 2j was obtained as orange yellow crystals, 0.8 g
(87.2%) yield, mp (°C)—195–196. IR (KBr): 3385, 3306 (NH2+NH),
Compound 2g was obtained as pale yellow crystals, 0.73 g
(96.1%) yield, mp (°C)—228–229. IR (KBr): 3425, 3292, 3180
(NH2+NH), 3425–3180 (br, OH), 1683 (NCO) cmꢀ1
.
1H NMR
1641 (NCO), 1524, 1323 (NO2) cmꢀ1 1H NMR (500 MHz: DMSO-
.
(500 MHz: DMSO-d6): d 3.82 (s, 2H, CH2), 4.04 (s, 1H, OH, D2O
exchangeable), 5.71 (s, 2H, NH2, D2O exchangeable), 6.73 (d, 2H,
J = 8.4 Hz, Ar-H), 7.18–7.28 (m, 5H, Ar-H), 7.71 (d, 2H, J = 8.4 Hz,
Ar-H), 8.15 (s, 1H, CH), 13.14 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C17H16N6O2: C, 60.71; H, 4.79; N, 24.99. Found: C, 60.43.;
H, 5.04; N, 24.70.
d6): d 3.68 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 5.81 (s, 2H, NH2, D2O
exchangeable), 6.82 (d, 2H, J = 8.4 Hz, Ar-H), 7.16 (d, 2H,
J = 8.4 Hz, Ar-H), 8.17–8.24 (m, 4H, Ar-H), 8.38 (s, 1H, CH), 12.56
(s, 1H, NH, D2O exchangeable). Anal. Calcd for C18H17N7O4: C,
54.68; H, 4.33; N, 24.80. Found: C, 54.42; H, 4.62; N, 24.90.
Figure 5. The amino acid structure of the SRS2/SRS6 site. SRS2, purple; SRS6, blue; asn214, red; ser216, dark green; ile462, magenta and other amino acids are shown in gray.
H-bond between HD21 of asn214 (red) and oxygen from triazolotriazine derivative (orange) is shown in light green.

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2633
5.3.11. 4-Amino-6-benzyl-3-(2-(1-phenylethylidene)-
5.3.17. 4-Amino-6-benzyl-3-(2-(1-(pyridin-2-yl)ethylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2k)
hydrazinyl)-1,2,4-triazin-5(4H)-one (2q)
Compound 2k was obtained as yellow crystals, 0.55 g (72.3%)
yield, mp (°C)—138–139. IR (KBr): 3462, 3302, 3222 (NH2+NH),
Compound 2q was obtained as yellow crystals, 0.79 g (95.1%)
yield, mp (°C)—133–134. IR (KBr): 3420, 3300, 3215 (NH2+NH),
1650 (NCO) cmꢀ1
.
1H NMR (500 Mz: DMSO-d6): d 2.36 (s, 3H,
1677 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.40 (s, 3H,
.
CH3), 3.84 (s, 2H, CH2), 5.84 (s, 2H, NH2, D2O exchangeable),
7.18–7.32 (m, 10H, Ar-H), 12.16 (s, 1H, NH, D2O exchangeable).
Anal. Calcd for C18H18N6O: C, 64.66; H, 5.43; N, 25.13. Found: C,
64.55; H, 5.64; N, 25.35.
CH3), 3.86 (s, 2H, CH2), 5,88 (s, 2H, NH2, D2O exchangeable),
7.18–7.26 (m, 6H, Ar-H), 7.73 (br s, 1H, Py-H), 8.52 (br s, 1H, Py-
H), 8.63 (br s, 1H, Py-H), 12.33 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C17H17N7O: C, 60.88; H, 5.11; N, 29.24. Found: C, 60.69; H,
5.38; N, 29.51.
5.3.12. 4-Amino-6-(4-methoxybenzyl)-3-(2-(1-phenyl-
ethylidene)hydrazinyl)-1,2,4-triazin-5(4H)-one (2l)
5.3.18. 4-Amino-6-(4-methoxybenzyl)-3-(2-(1-(pyridin-2-
yl)ethylidene)hydrazinyl)-1,2,4-triazin-5(4H)-one (2r)
Compound 2r was obtained as yellow crystals, 0.87 g (95.5%)
yield, mp (°C)—246–247. IR (KBr): 3322, 3187 (NH2+NH), 1685
Compound 2l was obtained as yellow crystals, 0.62 g (71.2%)
yield, mp (°C)—88–89. IR (KBr): 3406, 3304, 3218 (NH2+NH),
1659 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.36 (s, 3H,
.
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.45 (s, 3H, CH3),
.
CH3), 3.67 (s, 3H, OCH3), 3.76 (s, 2H, CH2), 5.85 (s, 2H, NH2, D2O
exchangeable), 6.78–6.80 (m, 2H, Ar-H), 7.17 (d, 2H, J = 8.4 Hz,
Ar-H), 7.36 (m, 3H, Ar-H), 7.75 (d, 2H, J = 6.1 Hz, Ar-H), 12.16 (s,
1H, NH, D2O exchangeable). Anal. Calcd for C19H20N6O2: C, 62.62;
H, 5.53; N, 23.06. Found: C, 62.50; H, 5.75; N, 22.87.
3.68 (s, 3H, OCH3), 3.81 (s, 2H, CH2), 5.76 (s, 2H, NH2, D2O
exchangeable), 6.80 (d, 2H, J = 8.4 Hz, Ar-H), 7.13 (d, 2H,
J = 8.4 Hz, Ar-H), 7.84 (m, 1H, Py-H), 8.26 (d, 1H, J = 6.1 Hz, Py-H),
8.43 (m, 1H, Py-H), 8.79–8.82 (m, 1H, Py-H), 13.14 (s, 1H, NH,
D2O exchangeable). Anal. Calcd for C18H19N7O2: C, 59.17; H, 5.24;
N, 26.83. Found: C, 58.92; H, 5.47; N, 26.73.
5.3.13. 4-Amino-6-benzyl-3-(2-(1-(4-chlorophenyl)ethylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2m)
5.3.19. 4-Amino-6-benzyl-3-(2-(1-(furan-2-yl)ethylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2s)
Compound 2m was obtained as yellow crystals, 0.66 g (78.5%)
yield, mp (°C)—140–141. IR (KBr): 3462, 3328, 3241 (NH2+NH),
1650 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.34 (s, 3H,
.
Compound 2s was obtained as brown crystals, 0.78 g (96.2%)
yield, mp (°C)—69–70. IR (KBr): 3402, 3310, 3128 (NH2+NH),
CH3), 3.84 (s, 2H, CH2), 5.85 (s, 2H, NH2, D2O exchangeable),
7.18–7.25 (m, 5H, Ar-H), 7.38 (d, 2H, J = 8.4 Hz, Ar-H), 8.08 (d,
2H, J = 8.4 Hz, Ar-H), 12.23 (s, 1H, NH, exchangeable). Anal. Calcd
for C18H17ClN6O: C, 58.62; H, 4.65; N, 22.79. Found: C, 58.86; H,
4.59; N, 22.53.
1684 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.26 (s, 3H,
.
CH3), 3.83 (s, 2H, CH2), 5.83 (s, 2H, NH2, D2O exchangeable), 6.55
(m, 1H, Fur-H4), 7.20–7.26 (m, 6H, Ar-H), 7.72 (br s, 1H, Fur-H5),
12.06 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C16H16N6O2:
C, 59.25; H, 4.97; N, 25.91. Found: C, 59.13; H, 5.10; N, 26.10.
5.3.14. 4-Amino-3-(2-(1-(4-chlorophenyl)ethylidene)-
5.3.20. 4-Amino-3-(2-(1-(furan-2-yl)ethylidene)hydrazinyl)-6-
(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one (2t)
hydrazinyl)-6-(4-methoxybenzyl)-1,2,4-triazin-5(4H)-one (2n)
Compound 2n was obtained as yellow crystals, 0.68 g (70.8%)
yield, mp (°C)—168–169. IR (KBr): 3267, 3227, 3190 (NH2+NH),
Compound 2t was obtained as brown crystals, 0.81 g (92.0%)
yield, mp (°C)—84–85. IR (KBr): 3399, 3309, 3200 (NH2+NH),
1675 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.34 (s, 3H,
.
1638 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6) d 2.25 (s, 3H,
.
CH3), 3.67 (s, 3H, OCH3), 3.80 (s, 2H, CH2), 5.55 (s, 2H, NH2, D2O
exchangeable), 6.79 (d, 2H, J = 8.4 Hz, Ar-H), 6.81 (d, 2H,
J = 8.4 Hz, Ar-H), 7.12(d, 2H, J = 8.4 Hz, Ar-H), 7.15 (d, 2H,
J = 8.4 Hz, Ar-H), 12.51 (br s, 1H, NH, D2O exchangeable). Anal.
Calcd for C19H19ClN6O2: C, 57.22; H, 4.80; N, 21.07. Found: C,
57.05; H, 4.98; N, 21.30.
CH3), 3.67 (s, 3H, CH3), 3.75 (s, 2H, CH2), 5.82 (s, 2H, NH2, D2O
exchangeable), 6.54 (m, 1H, Fur-H4), 6.81 (d, 2H, J = 7.7 Hz, Ar-H),
7.14 (d, 2H, J = 7.7 Hz, Ar-H), 7.20 (d, 1H, J = 3.1 Hz, Fur-H3), 7.72
(br s, 1H, Fur-H5) 12.01 (s, 1H, NH, D2O exchangeable. Anal. Calcd
for C17H18N6O3: C, 57.62; H, 5.12; N, 23.72. Found: C, 57.85; H,
4.86; N, 23.50.
5.3.15. 4-Amino-6-zbenzyl-3-(2-(1-p-tolylethylidene)-
hydrazinyl)-1,2,4-triazin-5(4H)-one (2o)
5.4. General procedure for the reaction of Schiff’s bases with
acetic anhydride
Compound 2o was obtained as yellow crystals, 0.65 g (83.3%)
yield, mp (°C)—146–147. IR (KBr): 3460, 3326, 3240 (NH2+NH),
1650 (NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 2.30 (s, 3H,
.
A solution of the corresponding Schiff’s base (2.5 mmol) in 6 mL
acetic anhydride was heated under reflux for 10 h. The reaction
mixture was then cooled and poured into crushed ice. The crude
product was obtained by filtration, washed well with water, and
recrystallized from ethanol.
CH3), 2.33 (s, 3H, CH3), 3.83 (s, 2H, CH2), 5.83 (s, 2H, NH2, D2O
exchangeable), 7.13–7.26 (m, 7H, Ar-H), 7.94 (d, 2H, J = 7.6 Hz, Ar-
H), 12.11 (br s, 1H, NH, exchangeable). Anal. Calcd for C19H20N6O:
C, 65.50; H, 5.79; N, 24.12. Found: C, 65.63; H, 5.91; N, 23.82.
5.3.16. 4-Amino-6-(4-methoxybenzyl)-3-(2-(1-p-
5.4.1. 2-Acetyl-8-(N,N-diacetylamino)-6-benzyl-7-oxo-3-
phenyl-2,3-dihydro-7H-[1,2,4] triazolo[4,3-b][1,2,4]triazine (3a)
Compound 3a was obtained as brown powder, 0.95 g (94.0%)
tolylethylidene)hydrazinyl)-1,2,4-triazin-5(4H)-one (2p)
Compound 2p was obtained as yellow crystals, 0.70 g (73.6%)
yield, mp (°C)—110–111. IR (KBr): 3462, 3334, 3235 (NH2+NH),
yield, mp (°C)—60–61. IR (KBr): 1755, 1708, 1699 (NCO) cmꢀ1 1H
.
1674 (NCO) cmꢀ1
.
1H NMR (500 MHz: DMSO-d6): d 2.21 (s, 3H,
NMR (500 MHz: DMSO-d6): d 1.87 (s, 3H, CH3), 2.02 (s, 3H, CH3),
2.07 (s, 3H, CH3), 4.01 (s, 2H, CH2), 7.33–7.40 (m, 9H, Ar-H+CH),
7.45–7.46 (m, 1H, Ar-H). 13C NMR (125 MHz: DMSO-d6): d 21.01,
21.24, 21.59, 37.32, 89.61, 128.83, 129.28, 129.32, 129.81, 146.67,
CH3), 2.31 (s, 3H, CH3), 3.67 (s, 3H, OCH3), 3.76 (s, 2H, CH2), 5.82
(s, 2H, NH2, D2O exchangeable), 6.81 (d, 2H, J = 8.4 Hz, Ar-H),
7.15–7.27 (m, 2H, Ar-H), 7.76 (d, 2H, J = 7.6 Hz, Ar-H), 7.93 (d, 2H,
J = 7.6 Hz, Ar-H), 12.10 (s, 1H, NH). Anal. Calcd for C20H22N6O2: C,
63.48; H, 5.86; N, 22.21. Found: C, 63.20; H, 5.54; N, 22.04.
149.89, 154.73, 167.74, 169.27, 170.02. Anal. Calcd for C23H22
-
N6O4: C, 61.87; H, 4.97; N, 18.82. Found: C, 61.48; H, 5.23; N, 19.02.

2634
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
5.4.2. 2-Acetyl-8-(N,N-diacetylamino)-6-(4-methoxybenzyl)-7-
oxo-3-phenyl-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (3b)
J = 8.4 Hz, Ar-H). 13C NMR (125 MHz: DMSO-d6): .d 21.18, 22.04,
22.12, 24.27, 24.90, 35.38, 55.50, 87.00, 114.28, 126.52, 139.53,
130.05, 136.26, 139.01, 143.08, 143.41, 152.07, 158.53, 166.04,
168.90, 170.03. Anal. Calcd for C26H28N6O5: C, 61.89; H, 5.59; N,
16.66. Found: C, 62.06; H, 5. 32; N, 16.53.
Compound 3b was obtained as yellow crystals, 0.96 g (83.4%)
yield, mp (°C)—155–156. IR (KBr): 1738, 1645 (NCO) cmꢀ1 1H
.
NMR (500 MHz: DMSO-d6): d 2.01 (s, 3H, CH3), 2.36 (s, 3H, CH3),
2.46 (s, 3H, CH3), 3.66 (s, 5H, CH2+OCH3), 6.74 (d, 2H, J = 7.6 Hz,
Ar-H), 6.96 (d, 2H, J = 8.4 Hz, Ar-H), 7.00 (s, 1H, CH), 7.41–7.42
(m, 5H, Ar-H). 13C NMR (125 MHz: DMSO-d6), HMQC: d 21.04
(CH3), 24.10 (CH3), 25.03 (CH3), 40.00 (OCH3), 55.52 (CH2), 79.61
(CH), 114.29 (Ar-CH), 127.74 (Ar-CH), 128.29 (Ar-CH), 130.04
(Ar-CH), 130.21 (Ar-C), 136.80 (Ar-C), 143.26 (Ar-C), 151.93 (Ar-
C), 158.55 (C@O), 167.19 (C@O), 168.83 (C@O), 170.09 (C@O). Anal.
Calcd for C24H24N6O5: C, 60.50; H, 5.08; N, 17.64. Found: C, 60.24;
H, 5. 32; N, 17.45.
5.4.7. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-(4-nitrophenyl)-
7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine (4a)
Compound 4a was obtained as brown crystals, 0.96 g (85.7%)
yield, mp (°C)—242–243. IR (KBr): 3193 (NH), 1727, 1706
(C@O) cm ꢀ1. 1H NMR (500 MHz: DMSO-d6): (Isomer 1, 57%) d
2.00(s, 3H, CH3), d 2.02(s, 3H, CH3), 3.62–3.76 (m, 2H, CH2), 7.09–
7.20 (m, 6H, Ar-H+CH), 7.64 (d, 1H, J = 8.4 Hz), 7.75 (d, 1H,
J = 8.4 Hz, Ar-H), 8.24–8.28 (m, 2H, Ar-H), 11.14 (d, 1H, NH, D2O
exchangeable). (Isomer 2, 43%) d 2.00 (s, 3H, CH3), d 2.05(s, 3H,
CH3), 3.62–3.76 (m, 2H, CH2), 7.09–7.20 (m, 6H, Ar-H+CH), 7.64
(d, 1H, J = 8.4 Hz), 7.75 (d, 1H, J = 8.4 Hz, Ar-H), 8.24–8.28 (m, 2H,
Ar-H), 11.14 (d, 1H, NH, D2O exchangeable). 13C NMR (125 MHz:
DMSO-d6): d 20.73, 20.95, 39.66, 78.22, 129.13, 129.16, 136.59,
136.66, 143.22, 145.91, 152.25, 161.36, 167.28, 168.70. Anal. Calcd
for C21H19N7O5: C, 56.12; H, 4.26; N, 21.82. Found: C, 55.84; H,
4.52; N, 22.06.
5.4.3. 2-Acetyl-8-(N,N-diacetylamino)-6-benzyl-3-(4-
methoxyphenyl)-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (3c)
Compound 3c was obtained as yellow crystals, 0.95 g (87.9%)
yield, mp (°C)—140–141. IR (KBr): 1741, 1641 (NCO) cmꢀ1 1H
.
NMR (500 MHz: DMSO-d6): d 2.00 (s, 3H, CH3), 2.35 (s, 3H, CH3),
2.46 (s, 3H, CH3), 3.74–3.76 (m, 5H, CH2+OCH3), 6.95 (d, 2H,
J = 3.0 Hz, Ar-H), 6.97 (s, 1H, CH), 7.06 (d, 2H, J = 7.3 Hz, Ar-H),
7.14–7.22 (m, 3H, Ar-H), 7.31 (d, 2H, J = 8.4 Hz, Ar-H). 13C NMR
(125 MHz: DMSO-d6): d 21.08, 24.05, 25.06, 40.00, 55.80, 79.42,
114.60, 128.90, 129.21, 136.68, 142.85, 144.62, 152.00, 160.72,
167.07, 168.79, 170.14. Anal. Calcd for C24H24N6O5: C, 60.50; H,
5.08; N, 17.64; O. Found: C, 60.77; H, 4.87; N, 17.52.
5.4.8. 2-Acetyl-8-(N-acetylamino)-6-(4-methoxybenzyl)-3-(4-
nitrophenyl)-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (4b)
Compound 4b was obtained as dark brown crystals, 0.89 g
(68.4%) yield, mp (°C)—120–121. IR (KBr): 3465 (NH), 1744, 1656
(C@O) cmꢀ1 1H NMR (500 Hz: DMSO): d 1.99–2.04 (m, 6H, CH3),
.
3.36 (s, 3H, OCH3), 3.65 (s, 2H, CH2), 6.74 (d, 2H, J = 8.4 Hz, Ar-H),
6.97–7.01 (m, 2H, Ar-H), 7.15 (s, 1H, CH), 7.69 (d, 1H, J = 7.6 Hz,
Ar-H), 8.24 (d, 3H, J = 5.3 Hz, Ar-H), 11.03 (br s, 1H, NH, D2O
exchangeable). Anal. Calcd for C22H21N7O6: C, 55.11; H, 4.41; N,
20.45. Found: C, 55.38; H, 4.14; N, 20.72.
5.4.4. 2-Acetyl-8-(N,N-diacetylamino)-6-(4-methoxybenzyl)-3-
(4-methoxyphenyl)-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (3d)
Compound 3d was obtained as pale brown crystals, 0.92 g
(72.7%) yield, mp (°C)—170–171. UV/VIS (CH3CN) kmax = 201 nm,
e
= 11850; kmax = 229 nm,
e
= 13210; kmax = 258 nm,
.
e
= 9820. IR
5.4.9. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-methyl-7-oxo-3-
phenyl-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine (4c)
Compound 4c was obtained as brown crystals, 0.85 g (81.7%)
(KBr) 1738, 1644 (C@O) cmꢀ1
1H NMR (500 MHz: DMSO-d6): d
1.99 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.46 (s, 3H, CH3), 3.66 (s, 6H,
OCH3), 3.68 (s, 2H, CH2), 6.75 (d, 2H, J = 8.4 Hz, Ar-H), 6.95–6.98
(m, 5H, Ar-H+CH), 7.31 (d, 2H, J = 8.4 Hz, Ar-H). 13C NMR
(125 MHz: DMSO-d6): d 21.09, 24.07, 25.07, 35.40, 55.50, 55.79,
79.38, 114.29, 114.60, 128.97, 129.20, 130.03, 143.14, 144.64,
151.96, 158.53, 160.71, 167.04, 168.78, 170.16. Anal. Calcd for
yield, mp (°C)—58–59. IR (KBr): 3455 (NH), 1738, 1649 (C@O) cm
ꢀ1
.
1H NMR (500 MHz: DMSO-d6): (Isomer 1, 26%) d 2.01 (s, 3H,
CH3), 2.17 (s, 3H, CH3), 2.36 (s, 3H, CH3), 3.67 (s, 2H, CH2), 7.00–
7.04 (m, 2H, Ar-H), 7.15–7.21 (m, 3H, Ar-H), 7.37–7.41 (m, 5H,
Ar-H), 11.04 (s, 1H, NH, D2O exchangeable). (Isomer 2, 74%) d
1.99 (s, 3H, CH3), 2.22 (s, 3H, CH3), 2.39 (s, 3H, CH3), 3.73 (s, 2H,
CH2), 7.00–7.04 (m, 2H, Ar-H), 7.15–7.21 (m, 3H, Ar-H), 7.37–
7.41 (m, 5H, Ar-H), 11.04 (s, 1H, NH, D2O exchangeable). 13C
NMR (125 MHz: DMSO-d6): d 22.11, 24.26, 24.92, 39.822, 87.01,
128.89, 128.95, 129.02, 142.93, 143.47, 152.10, 168.42, 168.90,
170.07. Anal. Calcd for C22H22N6O3: C, 63.15; H, 5.30; N, 20.08.
Found: C, 63.02; H, 5.52; N, 20.18.
C
25H26N6O6: C, 59.28; H, 5.17; N, 16.59. Found: C, 59.56; H, 4.
95; N, 16.32.
5.4.5. 2-Acetyl-8-(N,N-diacetylamino)-3-(4-chlorophenyl)-6-(4-
methoxybenzyl)-3-methyl-7-oxo-2,3-dihydro-7H-
[1,2,4]triazolo[4,3-b][1,2,4]triazine (3e)
Compound 3e was obtained as pale yellow crystals, 0.78 g (65%)
yield, mp (°C)—135–136. IR (KBr): 3439 (NH), 1730 (C@O) cmꢀ1. 1H
NMR (500 MHz: DMSO-d6): d 2.36 (s, 6H, 2CH3), 2.50 (s, 3H, CH3),
3.68 (s, 3H, OCH3), 4.03 (s, 2H, CH2), 6.82 (s, 1H, NH, D2O exchange-
able), 6.84 (d, 4H, J = 8.4 Hz, Ar-H), 7.23 (d, 4H, J = 8.4 Hz, Ar-H).
Anal. Calcd for C25H25ClN6O5: C, 57.20; H, 4.80; N, 16.01. Found:
C, 57.02; H, 4.57; N, 15.79.
5.4.10. 2-Acetyl-8-(N-acetylamino)-6-(4-methoxybenzyl)-3-
methyl-7-oxo-3-phenyl-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (4d)
Compound 4d was obtained as orange crystals, 0.95 g (77.8%)
yield, mp (°C)—103–104. IR (KBr): 3449 (NH), 1707, 1638
(C@O) cmꢀ1 1H NMR (500 MHz: DMSO-d6): (Isomer 1, 47%) d
.
5.4.6. 2-Acetyl-8-(N,N-diacetylamino)-6-(4-methoxybenzyl)-3-
methyl-7-oxo-3-p-tolyl-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (3f)
1.93 (s, 3H, CH3), 2.01 (s, 3H, CH3), 2.16 (s, 3H, CH3), 3.57 (s, 2H,
CH2), 3.66 (s, 3H, OCH3), 6.74 (d, 2H, J = 8.4 Hz, Ar-H), 6.94 (t, 2H,
J = 7.6 Hz, Ar-H), 7.37–7.42 (m, 5H, Ar-H), 12.23 (s, 1H, NH, D2O,
exchangeable). (Isomer 2, 53%) d 1.94 (s, 3H, CH3), 2.00 (s, 3H,
CH3), 2.19 (s, 3H, CH3), 3.59 (s, 2H, CH2), 3.66 (s, 3H, OCH3), 6.74
(d, 2H, J = 8.4 Hz, Ar-H), 6.94 (t, 2H, J = 7.6 Hz, Ar-H), 7.37–7.42
(m, 5H, Ar-H), 12.23 (s, 1H, NH, D2O, exchangeable). 13C NMR
(125 MHz: DMSO-d6): d 21.95, 22.18, 22.22, 35.43, 55.53, 86.06,
114.25, 126.40, 128.98, 129.34, 130.21, 139.44, 143.14, 144.62,
Compound 3f was obtained as dark orange crystals, 0.97 g
(76.9%) yield, mp (°C)—140–141. IR (KBr): 1740, 1649 (C@O) cm
ꢀ1
.
1H NMR (500 MHz: DMSO-d6): d 1.99 (s, 3H, CH3), 2.19 (s, 3H,
CH3), 2.28 (s, 3H, CH3), 2.38 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.64
(d, 5H, J = 10.0 Hz, CH2+OCH3), 6.74 (d, 2H, J = 8.4 Hz, Ar-H), 6.92
(d, 2H, J = 8.4 Hz, Ar-H), 7.20 (d, 2H, J = 8.4 Hz, Ar-H), 7.24 (d, 2H,

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2635
152.09, 152.34, 158.52, 165.85, 168.75. Anal. Calcd for C23H24N6O4:
C, 61.60; H, 5.39; N, 18.74. Found: C, 61.63; H, 5.37; N, 18.76.
(s, 3H, CH3), 2.12 (s, 3H, CH3), 3.70 (s, 2H, CH2), 6.44 (d, 1H,
J = 8 Hz, Ar-H), 6.63–6.70 (m, 1H, Ar-H), 7.04–7.08 (m, 2H, Ar-H),
7.10–7.22 (m, 3H, Ar-H), 7.64 (s, 1H, Ar-H), 11.01 (s, 1H, NH, D2O
exchangeable). 13C NMR (125 Hz: DMSO): d 20.71, 20.89, 22.05,
31.32, 81.78, 110.89, 111.17, 128.83, 129.12, 129.17, 143.00,
5.4.11. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-(4-chlorophenyl)
-3-methyl-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]
triazine (4e)
144.24, 151.93, 152.26, 166.40, 168.41. Anal. Calcd for C20H20
-
Compound 4e was obtained as orange crystals, 1.00 g (81.3%)
yield, mp (°C)—210–211. IR (KBr): 3448 (NH), 1717, 1634
N6O4: C, 58.82; H, 4.94; N, 20.58. Found: C, 59.01; H, 4.69;
N, 20.71.
(C@O) cmꢀ1 1H NMR (500 MHz: DMSO-d6): (Isomer 1, 50%) d
.
1.98 (s, 6H, CH3), 2.13 (s, 3H, CH3), 3.66–3.72 (m, 2H, CH2), 7.06
(d, 2H, J = 7.6 Hz, Ar-H), 7.15–7.21 (m, 3H, Ar-H), 7.38 (d, 1H,
J = 6.8 Hz, Ar-H), 7.43–7.48 (m, 3H, Ar-H), 10.46 (br s, 1H, NH,
D2O exchangeable). (Isomer 2, 50%) d 2.01 (s, 6H, CH3), 2.18 (s,
3H, CH3), 3.66–3.72 (m, 2H, CH2), 7.06 (d, 2H, J = 7.6 Hz, Ar-H),
7.15–7.21 (m, 3H, Ar-H), 7.38 (d, 1H, J = 6.8 Hz, Ar-H), 7.43–7.48
(m, 3H, Ar-H), 10.46 (br s, 1H, NH, D2O exchangeable). 13C NMR
5.4.15. 2-Acetyl-8-(N-acetylamino)-3-(furan-2-yl)-6-(4-
methoxybenzyl)-3-methyl-7-oxo-2,3-7H-dihydro-
[1,2,4]triazolo[4,3-b][1,2,4]triazine (4i)
Compound 4i was obtained as pale yellow crystals, 0.83 g
(76.1%) yield, mp (°C)—218–219 °C. IR (KBr): 3202 (NH), 1745,
1656 (C@O) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 1.97 (s, 3H,
.
CH3), 2.14 (s, 3H, CH3), 2.40 (s, 3H, CH3), 3.66 (brs, 5H, CH2+OCH3),
6.47–6.48 (m, 1H, Ar-H), 6.67 (d, 1H, J = 3.8 Hz, Ar-H), 6.77–6.79
(m, 2H, Ar-H), 6.94–7.00 (m, 2H, Ar-H), 7.64 (d, 1H, J = 11.4 Hz,
Ar-H), 12.26 (s, 1H, NH, D2O exchangeable). Anal. Calcd for
(125 MHz: DMSO-d6):
d 20.79, 21.84, 22.124, 36.22, 85.85,
127.22, 128.43, 128.89, 129.09, 129.16, 136.69, 152.12, 152.33,
166.04, 166.07, 168.52. Anal. Calcd for C22H21ClN6O3: C, 58.34; H,
4.67; N, 18.56. Found: C, 58.58; H, 4.83; N, 18.25.
C21H22N6O5: C, 57.53; H, 5.06; N, 19.17. Found: C, 57.84; H, 4.86;
N, 18.99.
5.4.12. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-methyl-7-oxo-3-
p-tolyl-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine (4f)
Compound 4f was obtained as orange crystals, 0.95 g (80.5%)
5.4.16. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-(4-
methoxyphenyl)-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-
b][1,2,4]triazine (4j)
yield, mp (°C)—194–195. IR (KBr): 3409 (NH), 1702 (C@O) cmꢀ1
.
1H NMR (500 MHz: DMSO-d6): (Isomer 1, 54%) d 1.98 (s, 3H,
CH3), 2.00 (s, 3H, CH3), 2.01 (s, 3H, CH3), 2.27 (s, 3H, CH3), 3.65–
3.75 (m, 2H, CH2), 7.04–7.06 (m, 2H, Ar-H), 7.15–7.28 (m, 6H, Ar-
H), 7.30 (d, 1H, J = 7.6 Hz, Ar-H), 12.21 (s, 1H, NH, D2O exchange-
able). (Isomer 2, 46%) d 1.98 (s, 3H, CH3), 2.01 (s, 3H, CH3), 2.15
(s, 3H, CH3), 2.28 (s, 3H, CH3), 3.65–3.75 (m, 2H, CH2), 7.04–7.06
(m, 2H, Ar-H), 7.15–7.28 (m, 6H, Ar-H), 7.30 (d, 1H, J = 7.6 Hz, Ar-
H), 12.21 (s, 1H, NH, D2O exchangeable). 13C NMR (125 MHz:
DMSO-d6): d 20.79, 21.18, 22.00, 22.22, 36.27, 86.39, 126.34,
126.62, 127.15, 128.90, 136.22, 136.58, 138.86, 142.65, 144.54,
152.38, 165.82, 165.91, 168.43. Anal. Calcd for C23H24N6O3: C,
63.88; H, 5.59; N, 19.43. Found: C, 63.65; H, 5.71; N, 19.73.
To a solution of N-acetyl-N-(2-acetyl-6-benzyl-2,3-dihydro-3-
(4-methoxyphenyl)-7-oxo-[1,2,4]triazolo[4,3-b][1,2,4]triazin-
8(7H)-yl)acetamide 3c (0.48 g, 1 mmol) in 2 ml ethanol, a solution
of o-phenylene diamine (0.1 g, 1 mmol) in 1 ml ethanol was added.
The reaction mixture was then heated under reflux for about 3 h.
The product was collected from the cooled reaction mixture by fil-
tration and recrystallized from ethanol to afford compound 4j as
brown crystals, 0.39 g (92.8%) yield, mp (°C)—252–253. IR (KBr):
3146 (NH), 1730, 1701 (C@O) cmꢀ1 1H NMR (500 MHz: DMSO-
.
d6): d 1.99 (s, 3H, CH3), 2.02 (s, H, 3CH3), 3.61–3.74 (m, 5H,
CH2+OCH3), 6,88 (s, 1H, CH), 6.94–6.96 (m, 2H, Ar-H), 7.08 (d,
2H, J = 6.9 Hz, Ar-H), 7.15–7.20 (m, 3H, Ar-H), 7.27 (d, 1H,
J = 8.4 Hz, Ar-H), 7.35 (d, 1H, J = 8.4 Hz, Ar-H), 11.17 (s, 1H, NH,
5.4.13. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-methyl-7-oxo-3-
(pyridin-2-yl)-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]
triazine (4g)
D2O exchangeable). 13C NMR (125 MHz: DMSO-d6):
d 20.72,
20.87, 39.19, 55.86, 79.14, 114.71, 128.83, 129.16, 136.60, 143.08,
145.63, 152.16, 160.61, 167.19, 169.14. EIMS (m/z): 434 (44,
[M]+), 91 (100). Anal. Calcd for C22H22N6O4: C, 60.82; H, 5.10; N,
19.34. Found: C, 61.05; H, 4.98; N, 19.74.
Compound 4g was obtained as orange crystals, 0.94 g (91.3%)
yield, mp (°C)—280–281. IR (KBr): 3200 (NH), 1713, 1672
(C@O) cmꢀ1 1H NMR (500 MHz: DMSO-d6): (Isomer 1, 45%) d
.
1.96 (s, 3H, CH3), 2.21(s, 3H, CH3), 2.23 (d, 3H, J = 13 Hz, CH3),
3.59–3.72 (m, 2H, CH2), 6.94 (d, 1H, J = 7.6 Hz, Ar-H), 7.01 (d, 1H,
J = 7.6 Hz, Ar-H), 7.14–7.18 (m, 3H, Ar-H), 7.37–7.40 (m, 1H, Ar-
H), 7.65–7.66 (m, 1H, Ar-H), 7.82–7.87 (m, 1H, Ar-H), 8.53–8.56
(m, 1H, Ar-H), 11.10 (s, 1H, NH, D2O exchangeable). (Isomer2,
55%) d 1.97 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.23 (d, 3H, J = 13 Hz,
CH3), 3.59–3.72 (m, 2H, CH2), 6.94 (d, 1H, J = 7.6 Hz, Ar-H), 7.01
(d, 1H, J = 7.6 Hz, Ar-H), 7.14–7.18 (m, 3H, Ar-H), 7.37–7.40 (m,
1H, Ar-H), 7.65–7.66 (m, 1H, Ar-H), 7.82–7.87 (m, 1H, Ar-H),
8.53–8.56 (m, 1H, Ar-H), 11.10 (s, 1H, NH, D2O exchangeable).
13C NMR (125 MHz: DMSO-d6): d 20.88, 21.02, 21.33, 39.96,
86.62, 122.16, 124.94, 128.78, 129.10, 136.70, 136.82, 142.80,
144.81, 149.50, 152.00, 156.79, 166.19, 168.36, 168.49. Anal. Calcd
for C21H21N7O3: C, 60.13; H, 5.05; N, 23.38. Found: C, 60.18; H,
5.30; N, 23.26.
5.5. General procedure for the reaction of 4-amino-6-aryl-3-
hydrazinyl-1,2,4-triazin-5(4H)-one (1) with isatine (5)
A mixture of 4-amino-6-aryl-3-hydrazinyl-1,2,4-triazin-5(4H)-
one (1) (0.5 mmol) and isatine (0.07 g, 0.5 mmol) in 5 mL methanol
was heated under refluxed for 2 h then cooled to room tempera-
ture. The product that separated out was then collected by filtra-
tion and recrystallized from ethanol.
5.5.1. 3-(2-(4-Amino-6-benzyl-5-oxo-4,5-dihydro-1,2,4-triazin-
3-yl)hydrazono)indolin-2-one (5a)
Compound 5a was obtained as yellow powder, 0.12 g (79.8%)
yield, mp (°C)—250–251. IR (KBr): 3408, 3308 (NH2+NH), 1700
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.90 (s, 2H, CH2),
.
6.02 (s, 2H, NH2, D2O exchangeable), 6.83–6.86 (m, 1H, Ar-H),
6.97–7.00 (m, 1H, Ar-H), 7.18–7.31 (m, 6H, Ar-H), 8.33–8.37 (m,
2H, Ar-H+NH, D2O exchangeable), 10.63 (s, 1H, NH, D2O exchange-
able). 13C NMR (125 MHz: DMSO-d6): 36.35, 110.39, 127.07,
128.19, 129.46, 131.84, 137.37, 143.37, 145.08, 150.88, 151.11,
166.18. Anal. Calcd for C18H15N7O2: C, 59.83; H, 4.18; N, 27.13.
Found: C, 59.55; H, 4.44; N, 27.33.
5.4.14. 2-Acetyl-8-(N-acetylamino)-6-benzyl-3-(furan-2-yl)-3-
methyl-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]
triazine (4h)
Compound 4h was obtained as yellow crystals, 0.89 g (87.2%)
yield, mp (°C)—220–221. IR (KBr): 3177 (NH), 1741, 1664
(C@O) cmꢀ1 1H NMR (500 MHz: DMSO): d 1.97 (s, 3H, CH3), 1.98
.

2636
A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
5.5.2. 3-(2-(4-Amino-6-(4-methoxybenzyl)-5-oxo-4,5-dihydro-
1,2,4-triazin-3-yl)hydrazono) indolin-2-one (5b)
5.7.1 8-Amino-6-benzyl-[1,2,4]triazolo[4,3-b][1,2,4]triazin-
7(8H)-one (9a)
Compound 5b was obtained as yellow crystals, 0.1 g (47.6%)
yield, mp (°C)—235–236. IR (KBr): 3431, 3302 (NH2+NH), 1701
Compound 9a was obtained as colorless crystals, 0.08 g (66.6%)
yield, mp (°C)—152–153. UV/VIS (CH3CN): kmax = 222 nm,
(NCO) cmꢀ1
.
1H NMR (500 MHz: DMSO-d6):
d
3.67 (s, 3H,
e
= 10870; kmax = 254 nm,
e
= 7000; kmax = 320 nm,
e = 2030. IR
OCH3), 3.81 (s, 2H, CH2), 6.00 (s, 2H, NH2, D2O exchangeable),
6.82 (d, 2H, J = 8.4 Hz, Ar-H), 6.94 (t, 1H, J = 7.6 Hz, Ar-H),
7.16–7.24 (m, 4H, Ar-H), 8.31–8.32 (m, 3H, Ar-H+NH, D2O
exchangeable), 10.56 (s, 1H, NH, D2O exchangeable). Anal. Calcd
for C19H17N7O3: C, 58.31; H, 4.38; N, 25.05. Found: C, 59.05; H,
4.68; N, 25.15.
(KBr): 3321, 3220 (NH2), 1689 (NCO) cmꢀ1
;
1H NMR (500 MHz:
DMSO-d6): d 4.02 (s, 2H, CH2), 5.94 (s, 2H, NH2, D2O exchangeable),
7.20–7.29 (m, 5H, Ar-H), 9.7 (s, 1H, CH). 13C NMR (125 MHz:
DMSO-d6):
d 37.05, 127.33, 128.92, 129.93, 135.93, 138.91,
145.28, 152.18, 153.73. Anal. Calcd for C11H10N6O: C, 54.54; H,
4.16; N, 34.69. Found: C, 54.32; H, 4.47; N, 34.39.
5.7.2. 8-Amino-6-(4-methoxybenzyl)-[1,2,4]triazolo[4,3-
b][1,2,4]triazin-7(8H)-one (9b)
5.6. General procedure for the preparation of 8-(N,N-
diacetylamino)-10,2-diacetyl-6-substituted benzyl-2H-
spiro[1,2,4]triazolo[4,3-b][1,2,4]triazine-3,30-indoline]-20,7(8H)-
dione (6)
Compound 9b was obtained as yellow crystals, 0.09 g (65.6%)
yield, mp (°C)—272–273. IR (KBr): 3292, 3241 (NH2), 1687
(NCO) cmꢀ1 1H NMR (500 MHz: DMSO-d6): d 3.67 (s, 3H, OCH3),
;
3.70 (s, 2H, CH2), 6.03 (s, 2H, NH2, D2O exchangeable), 6.80 (d,
2H, J = 7.6 Hz, Ar-H), 7.12 (d, 2H, J = 8.6 Hz, Ar-H), 11.91 (s, 1H,
CH). 13C NMR (125 MHz: DMSO-d6): d 39.84, 55.55, 114.22,
129.66, 130.58, 141.31, 143.09, 150.02, 158.41. Anal. Calcd for
A
solution of 3-(2-(4-amino-6-aryl-5-oxo-4,5-dihydro-1,2,4-
triazin-3-yl)hydrazono) indolin-2-one (5) (0.5 mmol) in 3 mL
acetic anhydride was heated under reflux for 10 h. The reaction
mixture was then cooled and poured into crushed ice. The result-
ing precipitate was filtered, washed with water, and recrystallized
from ethanol.
C12H12N6O2: C, 52.94; H, 4.44; N, 30.87. Found: C, 52.67; H, 4.40;
N, 30.93.
5.8. General procedure for the reaction of 4-amino-6-aryl-3-
hydrazinyl-1,2,4-triazin-5(4H)-one (1) with acetic anhydride
(11)
5.6.1. 8-(N,N-Diacetylamino)-10,2-diacetyl-6-benzyl-2H-
spiro[[1,2,4]triazolo[4,3-b][1,2,4]triazine-3,30-indoline]-
20,7(8H)-dione (6a)
Compound 6a was collected as yellow crystals, 0.21 g (89.3%)
A solution of 4-amino-6-aryl-3-hydrazinyl-1,2,4-triazin-5(4H)-
one 1 (2.5 mmol) in 6 mL acetic anhydride was heated under reflux
for 6 h. The reaction mixture was then cooled and poured into
crushed ice. The resulting precipitate was filtered, washed with
water, and recrystallized from ethanol.
yield, mp (°C)—165–166. IR (KBr): 1737, 1659 (NCO) cmꢀ1 1H
.
NMR (500 MHz: DMSO-d6): d 2.06 (s, 3H, CH3), 2.47 (s, 6H, 2CH3),
2.57 (s, 3H, CH3), 3.74 (s, 2H, CH2), 6.98 (d, 2H, J = 7.6 Hz, Ar-H),
7.13–7.20 (m, 3H, Ar-H), 7.34 (t, 1H, J = 16.0 Hz, Ar-H), 7.54–7.57
(m, 2H, Ar-H), 8.13 (d, 1H, J = 8.4 Hz, Ar-H).13C NMR (125 MHz:
DMSO-d6): d 20.51, 24.57, 24.68, 26.50, 36.13, 83.06, 122.45,
126.78, 127.30, 128.90, 133.09, 135.94, 141.07, 144.67, 146.04,
151.88, 167.14, 169.14, 170.27, 170.35. Anal. Calcd for
5.8.1. 4-Acetyl-7-benzyl-3-methyl-1H-[1,2,4]triazino[4,3-
b][1,2,4,5]tetrazin-6(4H)-one (11a)
Compound 11a was obtained as yellow crystals, 0.2 g (28.5%)
C
26H23N7O6: C, 58.98; H, 4.38; N, 18.52.Found: C, 59.75; H, 4.13;
N, 18.44.
yield, mp (°C)—80–81. UV/VIS (CH3CN): kmax = 210 nm,
e = 18500;
kmax = 256 nm,
e
= 10430. IR (KBr): 3166 (NH), 1711 (NCO) cmꢀ1
.
1H NMR (500 MHz: DMSO-d6), NOE: d 2.03 (s, 3H, CH3), 2.47 (s,
3H, CH3), 3.99–4.11 (m, 2H, CH2), 7.26–7.30 (m, 5H, Ar-H), 11.35
(s, 1H, NH, D2O exchangeable). 13C NMR (125 MHz: DMSO-d6): d
9.71, 20.79, 37.18, 127.47, 128.99, 129.73, 135.74, 145.06, 146.87,
151.36, 153.25, 168.56. Anal. Calcd for C14H14N6O2: C, 56.37; H,
4.73; N, 28.1. Found: C, 56.12; H, 4.99; N, 27.85.
5.6.2. 8-(N,N-Diacetylamino)-10,2-diacetyl-6-(4-
methoxybenzyl)-2H-spiro[[1,2,4]triazolo [4,3-b][1,2,4]triazine-
3,30-indoline]-20,7(8H)-dione (6b)
Compound 6b was collected as yellow crystals, 0.13 g (85.5%)
yield, mp (°C)—205–206. IR (KBr): 1746, 1655 (NCO) cmꢀ1 1H
.
NMR (500 MHz: DMSO-d6): d 2.06 (s, 3H, CH3), 2.45 (s, 3H, CH3),
2.46 (s, 3H, CH3), 2.57 (s, 3H, CH3), 3.65 (s, 5H, CH2+OCH3), 6.74
(d, 2H, J = 8.4 Hz, Ar-H), 6.89 (d, 2H, J = 8.4 Hz, Ar-H), 7.37 (t, 1H,
J = 7.6 Hz, Ar-H), 7.53–7.64 (m, 2H, Ar-H), 8.13 (d, 2H, J = 8.4 Hz,
Ar-H). 13C NMR (125 MHz: DMSO-d6): d 20.52, 24.55, 24.70,
26.51, 35.36, 55.55, 83.05, 114.31, 116.79, 125.33, 126.80, 127.58,
130.04, 133.09, 141.05, 144.66, 146.28, 151.84, 158.61, 167.14,
169.73, 170.27, 170.37. Anal. Calcd for C27H25N7O7: C, 57.96; H,
4.50; N, 17.52. Found: C, 57.77; H, 4.73; N, 17.65.
5.8.2. 4-Acetyl-7-(4-methoxybenzyl)-3-methyl-1H-
[1,2,4]triazino[4,3-b][1,2,4,5]tetrazin-6(4H)-one (11b)
Compound 11b was obtained as brown crystals, 0.25 g (36.2%)
yield, mp (°C)—65–66. IR (KBr): 3234 (NH), 1697 (NCO) cmꢀ1 1H
.
NMR (500 MHz: DMSO-d6): d 2.01 (s, 3H, CH3), 2.42 (s, 3H, CH3),
3.76 (s, 5H, OCH3+CH2), 6.83 (d, 2H, J = 8.4 Hz, Ar-H), 7.24 (d, 2H,
J = 8.4 Hz Ar-H), 11.34 (s, 1H, NH, D2O exchangeable). 13C NMR
(125 MHz: DMSO-d6):
d 11.32, 20.93, 36.36, 55.56, 114.40,
129.19, 130.86, 132.16, 151.32, 153.47, 158.78, 168.68, 168.75.
Anal. Calcd for C15H16N6O3: C, 54.87; H, 4.91; N, 25.60. Found: C,
55.15; H, 4.62; N, 25.46.
5.7. General procedure for the reaction of 4-amino-6-aryl-3-
hydrazinyl-1,2,4-triazin-5(4H)-one (1) with
triethylorhtoformate (9)
5.9. Biology
To
a solution of 4-amino-6-aryl-3-hydrazinyl-1,2,4-triazin-
5.9.1. Microsomal AHH activity
5(4H)-one 1 (0.5 mmol) in 5 mL ethanol, triethyl orthoformate
(0.1 mL, 0.5 mmol) and 2 drops of glacial acetic acid were added.
The reaction mixture was then heated under reflux for about
10 h. The product was obtained on cooling, filtered and washed
well with ethanol.
Microsomal aryl hydrocarbon hydroxylase (AHH) activity was
determined according to the method of Wiebel and Gelboin,
1975.26 Briefly, the volume of the incubation mixture was 1 mL
containing 50 mM Tris–HCl buffer, pH 7.4; 3 mmol MgCl2;
0.6 mmol NADPH; 100 nmol of each triazine derivatives; 0.1 mL

A. M. EL Massry et al. / Bioorg. Med. Chem. 20 (2012) 2624–2637
2637
of microsomal protein (10 mg:mL). The reaction was started by the
addition of 100 nmol benzo[ ]pyrene then incubated at 37 °C for
10 min. The reaction was terminated by the addition of 1 mL ace-
tone. The 3-hydroxy benzo[ ]pyrene was extracted in 2 mL hex-
CYP1A1 was determined using EAdock29 and Swissdock.30 The
analysis of the obtained binding modes was through UCSF
Chimera.31
a
a
ane. The fluorescence intensity was measured at excitation and
emission wavelengths of 396 and 522 nm, respectively. Hepatic
microsome was prepared from PAH induced rat liver tissues which
dissected into very small pieces before homogenization in 3 vol of
0.1 M of potassium phosphate buffer, pH 7.4. After centrifugation
at 12,000 g for 20 min at 4 °C, the supernatant fractions was recen-
trifuged at 105,000 g for 1 h at 4 °C to yield a microsomal pellet
which was then re-suspended in 0.1 M potassium phosphate buf-
fer, pH 7.4.27,28
Acknowledgments
The authors gratefully acknowledge Professor Mostafa H. Mos-
tafa, Professor of Biochemistry, Alexandria University and former
Beirut Arab University president for his excellent advices and sup-
port for the biology part of this study. The authors also thank Al-
shima’a A. Massoud for X-ray data collection of compound 3d.
References and notes
5.9.2. Oral and intraperitoneal acute toxicity tests
Oral and intraperitoneal acute toxicity tests were conducted
with male mice (24 1.9 g) obtained from Medical Research Insti-
tute, Alexandria University to determine the potential of test com-
pounds 4e, 5a, 5b and 6b to produce toxicity. Animals were divided
into groups of six mice each. Tested compounds were suspended in
corn oil as a drug vehicle and given orally by oral gavage in doses of
1, 10, 100, 200 or 350 mg/kg or intraperitoneal in doses of 1, 5, 10,
50 or 150 mg/kg. Control group received corn oil only. Bodyweight
were recorded prior to administration and again on day 7 and 14
(termination). The animals were observed for mortality, signs of
gross toxicity and behavioral changes at least once daily and the
percentage survival was recorded at termination.29 The animal
care local committee approved the design of the experiment, and
the protocol conformed to the guidelines of the National Institutes
of Health for animal care and handling.
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