Synthesis, characterization and anticancer screening of some novel piperonyl-tetrazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.11.008

A series of new 1,2-substituted tetrazole derivatives were synthesized and evaluated on MCF-7 (ER positive), MDA-MB-231 and ZR-75 (ER negative) breast cancer cell lines. Out of the fourteen compounds, three compounds 10, 12 and 14 showed higher inhibitory effects on MCF-7 cells. Whereas, compound 8 exhibited higher inhibition in MDA-MB-231 and ZR-75 cells at 10^-5 M concentration. Total RNA was extracted and effect of compounds on different marker genes was studied. The gene expression of CD44, BRAC and BAX were significantly affected. The compounds were screened against the HepG2 cell line, to know if they are selectively targeting specific cancers and only 1-10 percent inhibition was found at 10^-5 M concentration.

Full text of DOI:10.1016/j.ejmech.2013.11.008

Accepted Manuscript
Synthesis, Characterization and Anticancer Screening of Some Novel Piperonyl-
Tetrazole Derivatives
Mohammad Arshad, Abdul Roouf Bhat, Smritee Pokharel, Ji-Eun Kim, Eun Ju Lee,
Fareeda Athar, Inho Choi
PII:
S0223-5234(13)00742-3
10.1016/j.ejmech.2013.11.008
EJMECH 6554
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 September 2012
Revised Date: 31 October 2013
Accepted Date: 7 November 2013
Please cite this article as: M. Arshad, A.R. Bhat, S. Pokharel, J.-E. Kim, E.J. Lee, F. Athar, I. Choi,
Synthesis, Characterization and Anticancer Screening of Some Novel Piperonyl-Tetrazole Derivatives,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.11.008.
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ACCEPTED MANUSCRIPT
Synthesis, Characterization and Anticancer Screening of Some Novel Piperonyl-Tetrazole Derivatives
$1
$2
2
2
1
2
Mohammad Arshad , Abdul Roouf Bhat , Smritee Pokharel , Eun Ju Lee , Fareeda Athar *, Inho Choi *.
1
Centre for Interdisciplinary research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025
2
School of Biotechnology, Yeungnam University, Gyeongsan 712-749, South Korea
$
Authors contributed Equally
*
*
Corresponding author. fathar@jmi.ac.in (Fareeda Athar).
Corresponding author. inhochoi@ynu.ac.kr (Inho Choi)
1,2 substitued tetrazole derivatives were synthesized and screened in ER positive and negative Breast cancer cell lines. Derivatives which showed 10-30
percent negative growth were studied unto marker gene expression.
1

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Research Highlights
•
•
•
•
•
Synthesis of new series of 1,2-substituted tetrazoles.
The compounds were screened for ER+/- breast cancer cell lines.
Three cell lines MCF-7, MDA-MB-231 and ZR-75 cells were used.
HepG2 cells were used to find out the target specific character of compounds.
The compounds with 10-30% retarding effects were screened unto gene level.

ACCEPTED MANUSCRIPT
Synthesis, Characterization and Anticancer Screening of Some Novel
Piperonyl-Tetrazole Derivatives
$
1
$2,3
2
2
Mohammad Arshad , Abdul Roouf Bhat , Smritee Pokharel , Ji-Eun Kim, Eun Ju Lee ,
1
2
Fareeda Athar *, Inho Choi *.
1
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India.
2
School of Biotechnology, Yeungnam University, Gyeongsan 712-749, South Korea.
3
Present address; P.G. Department of Chemistry, Sri Pratap College, Srinagar (J&K)- 190001, India .
ABSTRACT
A series of new 1,2-substituted tetrazole derivatives were synthesized and evaluated on MCF-7
(ER positive), MDA-MB-231 and ZR-75 (ER negative) breast cancer cell lines. Out of the
fourteen compounds, three compounds 10, 12 and 14 showed higher inhibitory effects on MCF-7
-5
cells. Whereas, compound 8 exhibited higher inhibition in MDA-MB-231 and ZR-75 cells at 10
M concentration. Total RNA was extracted and effect of compounds on different marker genes
was studied. The gene expression of CD44, BRAC and BAX were significantly affected. The
compounds were screened against the HepG2 cell line, to know if they are selectively targeting
-5
specific cancers and only 1-10 percent inhibition was found at 10 M concentration.
Key Words: Tetrazole derivatives, Anticancer, MCF-7, MDA-MB-231, ZR-75
Corresponding authors:
Fareeda Athar, Email- fathar@jmi.ac.in, fareeda_athar@yahoo.co.in, Phone+91-11-26981717 Ext.
4
492 Fax: +91-11-26980164
Inho Choi, Email: inhochoi@ynu.ac.kr, Phone: +82-53-810-3024, Fax: +82-53-810-4769
$
Authors contributed equally
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1
. Introduction
Breast cancer is one of the leading causes of cancer related deaths and illnesses in the world.
Based on the responsiveness towards hormone treatment, breast cancers are grouped as ER-
negative and ER-positive. ER-positive, which show response to estrogen or progesterone
treatment are responsible for two third deaths and ER-negative accounts for one third of deaths
[
1-2]. ER is considered as a marker to determine the ability of endocrine therapy against breast
cancer. Despite having available relatively good prognosis, surgery, chemo and radiotherapy,
breast cancer remain the leading cause of cancer mortality in women [3]. Several therapeutic
agents such as tamoxifen; a selective estrogen receptor modulator (SERM), femara, zoladex and
taxol are widely used for breast cancer treatment [4-6]. However, their use involves the risk of
life threatening host toxicity. Cancer chemotherapy is moving through an era of targeted
therapeutics, where specific molecules are used, which are able to selectively recognize targeting
sites through different DNA sequences and are not associated with the serious toxicities like
conventional cytotoxic drugs [7]. Heterocycles form a fundamental core of many
pharmacophores, like substituted acridine (DACA), benzimidazoles, benzothiazoles, pacitexol
derivatives, isatin, phenanthrene etc., which are used for the treatment of many cancers [8].
Tetrazoles are well known compounds with a number of biological activities such as
antibacterial, antifungal and analgesic [9-11]. Recently, several tetrazoles have been invented as
promising candidates for anticancer activity [12-14]. Losartan with a biphenyl tetrazolyl moiety
is being studied for use in the treatment of breast cancer tumors positive for AGTR1 [15-17].
Hydrazones owing to their physiological activity and co-ordination capability yielded a number
of pharmacophores with antiplatelet, antiulcer, antitumor, antiprotozoal, antibacterial and
antifungal activities [18-23]. Hydrazone derivatives of thiadiazole demonstrated markable effects
2

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on an ovarian cancer cell line [24]. Some quinazolinone hydrazide derivatives showed most
favorable growth inhibition against a renal cancer cell line [25]. 5-chloro-3-phenylindole-2-
carboxylic acid(4-nitrobenzylidene)hydrazide was found to be highly active in a standard growth
inhibition assay in breast cancer cells [26] and 6-amino-4-aryl-2-oxo-1-(1-pyrid-3-yl- or 4-yl-
ethylidene-amino)-1,2-dihydropyridine-3,5-dicarbo-nitrile series exhibited a high percentage of
tumor growth inhibition in all cancer cell lines [27]. Likewise, Piperonyl ring found in a number
of naturally occurring compounds such as Podophyllotoxin (an aryltetralin, lignin isolated from
the genus podophyllum) and Diphyllin (an arylnaphthalene lignin lactone isolated from many
traditional plants) have been reported to possess anticancer and antiviral activities [28]. In
addition, synthetic compounds containing piperonyl ring possess enormous biological activity
such as anti-cancer [29, 30], anti-convulsant [31, 32], anti-amoebic [33], anti-proliferative [34],
antiviral [35], anti-tumor [36], anti-plasmodial [37], COX-2 inhibitor [38]. Considering the
importance of tetrazole, Piperonyl and hydrazone molecules in pharmaceutical field and the
unmet demand of target specific chemotherapeutic agents, we have developed a series of
tetrazolo-hydrazones and screened them against the ER +ve and ER -ve breast cancer cell lines.
Besides, the effect of these compounds on different marker genes was studied.
2
. Discussion
2
.1. Chemistry
The 5-(1,3-benzodioxol-5-yl)-1-tetrazole molecules were synthesized from 1,3-benzodioxazole-
-carbaldehyde using multistep reaction system. The aldehyde was converted into an oxime
5
which when treated with excess of acetic anhydride yielded piperonylonitrile. The nitrile
functionality was converted to corresponding tetrazoles with addition of azide and in situ
3

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cyclization using zinc bromide. This tetrazole core molecule was used to synthesize a series of
substituted derivatives using the reaction sequence describes in figure 1A and 1B. All the
compounds were characterized by various spectroscopic techniques.
Ethyl[5-(1,3-benzodioxol-5-yl)1-tetrazol-1H-yl]acetate C was prepared from a simple chemical
entity 3’4’(methylenedioxyl)acetophenone, by converting later first into an oxime “A” and then
into a nitrile “B” compound. The conversions were strictly established using TLC and IR
1
spectroscopy and structure established by H NMR. The structure of compound “C” was
1
established using H NMR. When compared with compound “B” an additional singlet at 11.01
ppm was observed, which depicted the conversion and cyclization of nitrile group into a
tetrazole molecule. The compound C, when reflected with dry acetone in presence of
ethylchloroacetate yielded ethyl[5-(1,3-benzodioxol-5-yl)tetrazol-1-yl]acetate. TLC confirmed
1
the conversion and H NMR helped in establishing the structure. The absence of the singlet that
arises due to tetrazole proton was sufficient to establish the conversion. In addition the signals at
1
.11-1.26 ppm, a triplet for CH and 4.10-4.26 ppm, a quartet for CH proton confirmed the
3 2
condensation reaction. The compound “D” was condensed with hydrazine hydrate to get the core
molecule tetrazole-acetohydrazide. A singlet at around 9.59 ppm confirmed this conversion. The
core compound was further condensed with different aldehyde groups to produce a series of
tetrazolohydrazones (1-14) molecules which were screened for anticancer activity. The reaction
was monitored by TLC and structures were elucidated using various spectroscopic techniques.
The purity of compounds was established from sharp melting points and elemental analysis data.
Assignment of selected characteristic IR bands provides significant indications for the formation
-1
of the tetrazolohydrazones (1-14). The band around 1617-1501, 1703-1639 cm represent the
4

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stretching frequency of C=N and C=O groups, respectively. Similarly the band around 3220-
-1
3
068 and 1043-1031 cm were assigned to N-H and C-N, respectively. The structures of
1
13
1
compounds were further established on the basis of H and C NMR. In H NMR, besides the
aromatic region, four prominent singles were observed. A singlet around (12.257-11.309) ppm
was assigned to NH proton. The downfield shift of the NH signal is attributed to presence of π-
electron cloud around the NH proton. Similarly downfield shift was observed in CH protons
2
adjacent to carbonyl group and a singlet around 5.67-5.34 ppm were assigned to them. The
singlet of methylenedioxyl protons was found at around 6.91-5.97 ppm. The presence of
delocalised π-electron cloud on one hand and the π-electron cloud on other hand shifted the CH
proton to 8.50-7.97 ppm. Besides these various proton signals aromatic regions were also present.
1
3
The C-NMR data were in accordance with the proposed structures. Besides the common
signals which appear for the phenyl rings, the signals of the core compounds were found slightly
down fielded. The chemical shift may be due to the π-electron cloud and the inductive effect. A
signal at 57.06-53.09 ppm was assigned to the α-methylene group attached to the carbonyl group,
while the carbonyl carbon signal appeared at 172.31-166.41 ppm. The epoxy carbon signal was
found downshifted to 101.11-101.80 ppm and the nitrile carbon signals for N-C=N and ph-C=N
were found around 168.16-161.23 ppm and 165.55-159.41ppm respectively. In addition to NMR,
the compounds were subjected to ESI-MS analysis for detecting the molecular mass of the
compounds.
2
.2. Pharmacology
The mechanism of breast cancer progression and metastasis is not fully understood. However,
breast cancer frequency tends to increase. Moreover the ability of these cancers to metastasize
5

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and their heterogeneity, clearly require new therapies, targeted to specific genes and proteins
actively engaged in the physiopathology of breast cancer rather than the conventional cytotoxic
chemotherapy. The synthesized 5-(1,3-benzodioxol-5-yl)-1-tetrazole derivatives were evaluated,
to find out the effect on in vitro growth of ER positive (MCF-7) and ER negative (MDA-MB-
2
4
31 and ZR-75) cells. The cells were incubated with different concentrations of compounds for
8 hours. End point determination was made by MTT-a colorimetric assay. The results for each
compound are reported as percent growth (GP %) of treated cells compared to untreated controls
Fig. 2). The inhibitory effects were detectable in compounds 2, 8, 10, 12 and 14. These
(
compounds showed a negative growth of 10-30 % compared to the untreated cells. The percent
growth inhibition of compounds varied not only with the substituent groups but also with the
nature of the cell line. The variable effect on the cell lines was clear from the gene expression.
Compounds 10, 12 and 14 were found more effective in retarding the growth of MCF-7 cells
while compound 8 showed more growth retarding effects in ER negative MDA-MB-231 and ZR-
7
5. To understand the effects on different cell lines, we studied the gene expression level of
different breast cancer marker genes. BAX is a member of Bcl2 family, and several studies have
documented a strong correlation between estrogen receptor (ER) positivity and Bcl-2
immunostaining in primary adenocarcinomas of the breast [39]. In our experiments, BAX
showed more than two fold expression in treated cells compared to control (untreated) in MCF -7
cells (Fig. 4). A defect in the expression of BAX at mRNA and protein level has been found
proportional to apoptosis [40]. Furthermore, restoration of BAX expression in breast cancer cell
lines inhibits tumorigenicity and increased sensitivity to drug therapy. This demonstrated that
the compounds showed a better inhibitory effect at mRNA level. However the expression level
was little decreased in MDA-MB-231, an ER negative cell line. The CD44 gene expression is
6

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related to prognosis and metastasis. In vitro studies showed that the effect of CD44 engagement
with hyaluronan during cancer progression and metastatic spread is cell type specific. However,
increased expression of CD44 has been correlated with poor prognosis in diseases, such as
thyroid, prostate and breast cancer. Lopez et. al. [41] provided an in vivo evidence of CD44
antagonism to breast cancer metastasis. In our studies we found an increase in CD44 (Fig. 4) in
MCF-7, when treated with compounds 2, 8, 10, 12 and 14. The expression was about two folds
higher in compounds 8 and 10. This shows that these compounds have ability to decrease the
prognosis of the cell. The slight decrease in expression of CD44 in MDA-MB-231 cells may be
due to the highly aggressive and mesenchymal-type behavior. BRAC-1; a caretaker gene which
produces breast cancer type-1 susceptible protein, responsible for DNA repairing. However the
mutations in BRAC-1 gene lead to an increased risk of breast cancer and ovarian cancer [42].
The decrease in expression of BRAC-1 gene is directly proportional to prognosis of the disease.
Fig. 5 shows the effect of tetrazolohydrazones in expression of BRAC-1 in MDA-MB-231 cells.
From the gene expression, a minimum of two folds decrease was found upon treatment.
Compound 8 having p-nitrophenyl substituent had highly suppressed BRAC-1. This is consistent
with the idea that most BRAC-1 mutant tumors are ER negative and manifest a higher amount of
lymphocytic infiltrate [43]. In MCF-7 cells also, we can find a slight decrease in BRAC-1
expression, however the results are not significant. In similar cases the RNA expression of P53
and KRT-19 was found with low statistical significance.
To find out the cell specific effects of tetrazolohydrazones, we counted on cell proliferation test
using the HepG2 cell line. Only 0-10% (Fig. 3) negative growth was recorded in HepG2 cells
showing that these compounds are more specific to breast cancer cell lines.
7

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3
. Conclusion
A series of tetrazolohydrazones compounds (1-14) was synthesized starting from the simple
chemical molecules. All the compounds were screened against the ER +/- breast cancer cell lines.
Five compound 2, 8, 10, 12 and 14 were found to retard the growth of breast cancer cells. Based
on the gene study the compounds 10, 12 and 14 were found more effective in retarding the
growth of MCF-7 cells. While compound 8 showed more growth retarding effects in ER
negative MDA-MB-231 and ZR-75 cell lines. From the results it was observed that the
substituent groups have an important role in deciding the fate of the molecule. Further studies in
this direction will lead to develop an effective chemo-core, which can be used to cure the breast
cancer patients.
4
. Materials and Method:
Solvents and organic reagents purchased from Sigma Aldrich, Merck (Germany) was used
without purification. Melting points (mp) were performed using a Mel-temp instrument, and the
results are uncorrected. Precoated aluminum sheets (silica gel 60 F , Merck Germany) were
2
54
used for thin-layer chromatography (TLC) using solvent ethyl acetate and hexane, spots were
visualized under UV light. Elemental analysis was performed on Heraeus Vario EL III analyzer
and the results were within ± 0.3% of theoretical values. IR spectra were recorded on a Perkin-
1
13
Elmer model 1600 FT-IR RX1 spectrophotometer as KBr discs. H NMR and C NMR spectra
were recorded on Bruker Avance 300 spectrometer using DMSO-d as solvent with TMS as
6
internal standard. Splitting patterns are designated as follows; s, singlet; d, doublet; t, triplet; and
m, multiplet. Chemical shift values are given in ppm. ESI-MS was recorded on a Micromass
Quattro II triple quadrupole mass spectrometer.
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4
.1. Chemistry
4
.1.1. General Procedure for the synthesis of piperonyl oxime [A]
To a solution of aldehyde (10 mmol) and hydroxylamine hydrochloride (12.5 mmol) in water (5
mL), a solution of sodium bicarbonate (12.5 mmol) in water (10 mL) was added gradually with
stirring. The mixture was stirred for further 2-5 hours with the formation of a solid precipitate.
0
The compound was filtered using whatman no. 1 and air dried. Yield 95%; mp 100-102 C;
-1
1
white crystals; IRνmaxcm : 3225 (NO-H), 2921 (CH-Ar), 1660 (C=N), 1608, 937 (N-O); H
NMR DMSO/(ppm): 10.85 (broad s, 1H, N-OH), 7.90 (s, 1H, CH=N-OH), 7.30-6.50 (m, 3H,
Ar-H), 5.90 (s, -O-CH -O-).
2
4
.1.2 General procedure for the synthesis of piperonyl nitrile [B]
A mixture of piperonal oxime A (30 mmol) and acetic anhydride (20 mL) was refluxed for 24 h;
vigorous stirring is essential. The reaction was monitored by thin layer chromatography. After
completion, the reaction mixture was poured into ice cold water to obtain a solid precipitate. The
0
-1
precipitate was filtered, and air dried. Yield 92%; mp 95-98 C; white crystals; IR ν cm :
max
1
2
928 (CH-Ar), 2204 (CN); H NMR DMSO/(ppm): 7.18-6.90 (m, 3H, Ar-H), 6.03 (s, 2H, -O-
CH -O-)
2
4
.1.3. General Procedure for the preparation of 5-(1,3-benzodioxol-5-yl)-1-H-tetrazole [C]
To a 250 mL round-bottomed flask, a mixture of nitrile B (20 mmol), sodium azide (22 mmol),
zinc bromide (20 mmol), 40 ml of water and isopropylalcohol (10 mL) were added. The reaction
mixture was refluxed for 24 h with vigorous stirring. HCl (3 N, 30 mL) and ethyl acetate (100
mL) were added and reaction was continued until the solid material present in the flask
9

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disappeared and pH of aqueous layer reached to 1. If necessary, additional ethyl acetate was
added. The organic layer was isolated and aqueous layer was treated with 2×100 mL of ethyl
acetate. The combined organic layers were evaporated. 200 ml of 0.25 N NaOH was added, and
the mixture was stirred for 30 min, until the original precipitate was dissolved and a suspension
of zinc hydroxide was formed. The suspension was filtered, and the solid washed with 20 ml of 1
N NaOH. To the filtrate was added 40 ml of 3 N HCl with vigorous stirring causing the tetrazole
to precipitate. The tetrazole was filtered and washed with 2×20 mL of 3 N HCl and dried in a
drying oven to furnish the tetrazole as a yellow powder. HCl used in the process does not affect
the piperonal ring [44]. If necessary, the solid was triturated with hexane or 10% ethyl acetate in
0
-1
hexane. Yield: 85%; mp 170-172 C; yellow crystals; IRν (cm ): 3285 (NH) 2924 (CH-Ar),
max
1
1
608 (C=N); H NMR DMSO/(ppm): 6.156 (s, 2H, O-CH -O), 7.135 (J=8.1, d, 1H, Ar-H), 7.548
2
(s, 1H, Ar-H), 7.596 (J=7.8, d, 1H, Ar-H), 11.011 (s, 1H, NH).
4
.1.4. General procedure for the preparation of Ethyl[5-(1,3-benzodioxol-5-yl)-1-H-tetrazol-1-
yl]acetate [D]
A mixture of 5-(1,3-benzodioxol-5-yl)-1-H-tetrazole C (10 mmol), ethyl chloroacetate (10
mmol) and potassium carbonate (15 mmol) in dry acetone (100 mL) was refluxed for 24 h. The
reaction mixture was filtered hot and the solvent was distilled off from the filtrate. The crude
0
ester thus obtained was purified by recrystallization from ethanol. Yield: 95%; mp 188-190 C;
-1
1
white crystals; IRνmax(cm ): 1780 (C=O), 2920 (CH-Ar), 1605 (C=N), 1043 (C-N); H NMR
DMSO/(ppm): 1.117-1.263 (t, 3H, CH ), 4.101-4.265 (q, 2H, CH ), 5.851 (s, 2H, CH ), 6.141 (s,
3
2
2
2
H, O-CH O), 7.094 (J=8.1, d, 1H, Ar-H), 7.523 (s, 1H, Ar-H), 7.619 (J=9.6, d, 1H, Ar-H).
2-
1
0

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4
.1.5. General procedure for the preparation of 2-[5-(1,3-benzodioxol-5-yl)-1-H-tetrazol-1-
yl]acetohydrazide [E]
A mixture of Ethyl[5-(1,3-benzodioxol-5-yl)-1-H-tetrazol-1-yl]acetate D (10 mmol) and
hydrazine hydrate (99%, 10 mmol) in ethanol (50 mL) was refluxed for 8 h. The solution on
cooling gave a solid mass of hydrazide, which was collected by filtration, and recrystallized from
0
-1
ethanol. Yield: 90%; mp 180-182 C; white crystals; IRν (cm ): 1676 (C=O), 3001 (CH-Ar),
max
1
3
7
1
320 (NH ) 3210 (NH); H NMR DMSO/(ppm): 5.377 (s, 2H, CH ), 6.099 (s, 2H, O-CH -O),
2
2
2
.051 (J=8.1, d, 1H, Ar-H), 7.468 (s, 1H, Ar-H), 7.564 (J=8.1, d, 1H, Ar-H), 9.598 (s, 2H, NH2),
1.90 (s, 1H, NH).
4
.1.6. General procedure for the synthesis of compounds 1-14
A mixture of compound E (10 mmol), appropriate aldehyde (10 mmol) and few drops of glacial
acetic acid in ethanol (50 mL) was refluxed for 12 h. The product was precipitated, collected by
filtration and re-crystallized from ethanol.
4
.1.7. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[4-chlorophenyl)methylidene]acetohydr-
azide (1)
0
Yield: 92%; mp 206-208 C; white crystals; Anal. calc. for C H N O Cl: C 58.28, H 4.03, N
1
7
13
6
3
-
1
2
3
3.99, found C 58.29, H 4.02, N 23.98; IRνmax(cm ): 1031 (C-N), 1617 (C=N), 1693 (C=O),
1
001 (CH-Ar), 3210 (NH); H NMR DMSO-d /(ppm): 5.656 (s, 2H, CH ), 6.105 (s, 2H, O-
6
2
CH -O), 7.083 (J=8.1, d, 1H, Ar-H), 7.496 (J=8.7, d, 2H, Ar-H), 7.521 (s, 1H, Ar-H), 7.619
2
1
3
(
J=8.1, d, 2H, Ar-H), 7.739 (J=11.4, d, 1H, Ar-H), 8.059 (s, 1H, HC=N), 12.038 (s, 1H, NH); C
NMR DMSO-d /(ppm): 55.44 (CH ), 101.71 (O-C-O), 106.20, 109.07, 114.44, 120.55, 120.88,
6
2
1
1

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1
24.99, 130.54, 148.03, 149.21, 151.99 (Phenyl), 165.45, 168.11(CH=N), 170.63 (C=O); ESI-
+
MS (m/z): [M +1] 385.07 (385.78).
4
.1.8. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(2-chlorophenyl)methylidene]aceto-
hydrazide (2)
0
Yield 93%; mp 198-200 C; white crystals; Anal. calc. for C H N O Cl: C 58.28, H 4.03, N
1
7
13
6
3
-
1
2
2
3.99, found C 58.29, H 4.02, N 23.98; IRνmax(cm ): 1039 (C-N), 1605 (C=N), 1702 (C=O),
1
955 (CH-Ar), 3128 (NH); H NMR DMSO-d /(ppm): 5.638 (s, 2H, CH ), 6.109 (s, 2H, O-
6
2
CH -O), 7.066 (J=7.8, d, 1H, Ar-H), 7.392-7.527 (m, 3H, Ar-H), 7.593 (J=8.1, d, 1H, Ar-H),
2
13
8
.056 (J=7.5, d, 1H, Ar-H), 8.424 (s, 1H, HC=N), 12.118 (s, 1H, NH); C NMR DMSO-
d /(ppm): 55.41 (CH ), 101.69 (O-C-O), 106.19, 109.10, 114.50, 120.35, 120.43, 124.79, 130.44,
6
2
+
1
3
48.23, 149.23, 150.79 (Phenyl), 165.55, 168.13 (CH=N), 170.65 (C=O); ESI-MS (m/z): [M +1]
85.07 (385.78).
4
.1.9. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(4-ethoxyphenyl)methylidene]acetohy-
drazide (3)
0
Yield 93%; mp 208-210 C; white crystals; Anal. calc. for C H N O : C 57.86, H 4.60, N
1
9
18
6
4
-
1
2
2
2
1.31, found: C 57.87, H 4.59, N 21.30; IRνmax(cm ): 1043 (C-N), 1607 (C=N), 1688 (C=O),
1
959 (CH-Ar), 3142 (NH); H NMR DMSO-d /(ppm): 1.285-1.331 (t, 3H, CH ), 4.009-4.078 (q,
6
3
H, CH ), 5.587 (s, 2H, CH ), 6.034 (s, 2H, O-CH -O), 6.942 (J=8.4, d, 1H, Ar-H), 7.061 (J=8.1,
2
2
2
d, 1H, Ar-H), 7.495 (s, 1H, Ar-H), 7.591 (J=7.8, d, 2H, Ar-H), 7.688 (J=8.4, d, 2H, Ar-H), 8.162
1
3
(
s, 1H, HC=N), 11.811 (s, 1H, NH); C NMR DMSO-d /(ppm): 25.74 (CH ), 55.41 (CH ),
6 3 2
6
5.22 (O-CH ), 101.69 (O-C-O), 106.19, 109.10, 114.50, 120.35, 120.43, 124.79, 130.44, 148.2,
2
1
2

ACCEPTED MANUSCRIPT
+
1
49.23, 151.79 (Phenyl), 165.55, 168.13 (CH=N), 170.65 (C=O); ESI-MS (m/z): [M +1] 395.14
(395.38).
4
.1.10. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(4-methoxyphenyl)methylidene]aceto-
hydrazide (4)
0
Yield 95%; mp 198-200 C; white crystals; Anal. calc. for C H N O : C 56.84, H 4.24, N
1
8
16
6
4
-
1
2
3
6
2.10, found: C 56.85, H 4.25, N 22.11%; IRνmax(cm ): 1043 (C-N), 1606 (C=N), 1685 (C=O),
1
000 (CH-Ar), 3210 (NH); H NMR DMSO-d /(ppm): 3.809 (s, 3H, OCH ), 5.614 (s, 2H, CH ),
6
3
2
.134 (s, 2H, O-CH -O), 6.977 (J=13.5, d, 1H, Ar-H), 7.092 (J=7.8, d, 1H, Ar-H), 7.526 (s, 1H,
2
1
3
Ar-H), 7.623-7.714 (m, 4H, Ar-H), 8.015 (s, 1H, HC=N), 11.831 (s, 1H, NH); C NMR DMSO-
d /(ppm): 55.48 (OCH ), 57.06 (CH ), 101.70 (O-C-O), 106.19, 109.06, 114.48, 120.58, 120.87,
6
3
2
1
24.96, 130.54, 148.03, 149.21, 162.67, 163.98, 165.44, 168.16 (C=N), 170.61 (C=O); ESI-MS
+
(
m/z): [M +1] 381.12 (381.12).
4
.1.11. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[phenylmethylidene]acetohydrazide
5)
(
0
Yield 93%; mp 204-206 C; white crystals; Anal. calc. for C H N O : C 58.28, H 4.03, N
1
7
14
6
3
-
1
2
2
3.99, found C 58.29, H 4.02, N 24.01; IRνmax(cm ): 1037 (C-N), 1607 (C=N) 1687 (C=O),
1
909 (CH-Ar), 3068 (NH); H NMR DMSO-d /(ppm): 5.627 (s, 2H, CH ), 6.082 (s, 2H, O-CH -
6
2
2
O), 7.076 (J=8.1, d, 1H, Ar-H), 7.431 (J=3.6, d, 1H, Ar-H), 7.510 (s, 1H, Ar-H), 7.606 (J=8.1, d,
1
3
1
H, Ar-H), 7.735-7.758 (m, 4H, Ar-H), 8.056 (s, 1H, HC=N), 11.954 (s, 1H, NH); C NMR
DMSO-d /(ppm): 56.00 (CH ), 101.69 (O-C-O), 106.21, 109.06, 120.67, 120.85, 127.12, 127.27,
6
2
1
3

ACCEPTED MANUSCRIPT
1
28.77, 130.19, 133.73, 144.94, 148.03, 149.17 (Phenyl), 163.95 (C=N), 166.20, 168.65 (C=O);
+
ESI-MS (m/z): [M +1] 351.11 (351.11).
4
.1.12. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-
[
(4-methylphenyl)methylidene]acetohydr-azide (6)
0
Yield 95%; mp 203-205 C; white crystals; Anal. calc. for C H N O : C 58.28, H 4.03, N
1
8
16
6
3
-
1
2
2
6
3.99, found C 58.27, H 4.02, N 24.00; IRνmax(cm ): 1034 (C-N), 1607 (C=N), 1676 (C=O),
1
911 (CH-Ar), 3199 (NH); H NMR DMSO-d /(ppm): 2.317 (s, 3H, CH ), 5.609 (s, 2H, CH ),
6
3
2
.046 (s, 2H, O-CH -O), 7.064 (J=8.7, d, 1H, Ar-H), 7.227 (J=7.8, d, 1H, Ar-H), 7.498 (s, 1H,
2
Ar-H), 7.583 (J=6.6, d, 2H, Ar-H), 7.626 (J=1.5, d, 2H, Ar-H), 8.012 (s, 1H, HC=N), 11.861 (s,
1
3
1
1
H, NH); C NMR DMSO-d /(ppm): 25.69 (CH ), 56.10 (CH ), 101.68 (O-C-O), 106.22,
6 3 3
09.16, 120.77, 120.87, 127.22, 127.24, 128.75, 130.18, 133.59, 144.82, 148.13, 149.27
+
(
Phenyl), 162.21, 163.91 (C=N), 169.36 (C=O); ESI-MS (m/z): [M +1] 365.16 (365.13).
4
.1.13. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(2-ethoxyphenyl)methylidene]aceto-
hydrazide (7)
0
Yield 96%; mp 196-198 C; white crystals; Anal. calc. for C H N O : C 57.86, H 4.60, N
1
9
18
6
4
-
1
2
2
2
1.31, found C 57.87, H 4.59, N 21.30; IRνmax(cm ): 1035 (C-N), 1608 (C=N), 1685 (C=O),
1
905 (CH-Ar), 3211 (NH); H NMR DMSO-d /(ppm): 1.349-1.414 (t, 3H, CH ), 4.089-4.156 (q,
6
3
H, CH ), 5.612 (s, 2H, CH ), 6.074 (s, 2H, O-CH -O), 6.968-7.429 (m, 3H, Ar-H), 7.525 (s, 1H,
2
2
2
Ar-H), 7.617 (J=2.1, d, 1H, Ar-H), 7.644 (J=1.8, d, 1H, Ar-H), 7.898 (J=7.8, d, 1H, Ar-H), 8.440
1
3
(
s, 1H, HC=N), 11.898 (s, 1H, NH); C NMR DMSO-d /(ppm): 15.74 (CH ), 55.41 (CH ),
6 3 2
6
3.91 (O-CH ), 101.69 (O-C-O), 106.19, 109.10, 114.50, 120.35, 120.43, 124.79, 130.44,
2
1
4

ACCEPTED MANUSCRIPT
+
1
3
48.23, 149.23, 152.79 (Phenyl), 165.55, 168.13 (C=N), 170.65 (C=O); ESI-MS (m/z): [M +1]
95.13 (395.14).
4
.1.14. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(3,4-dimethoxyphenyl)methylidene]-
acetohydrazide (8)
0
Yield 94%; mp 200-202 C; white crystals; Anal. calc. for C H N O : C 55.61, H 4.42, N
1
9
18
6
5
-
1
2
2
0.48, found C 55.60, H 4.41, N 20.49; IRνmax(cm ): 1033 (C-N), 1604 (C=N), 1685 (C=O),
1
960 (CH-Ar), 3222 (NH); H NMR DMSO-d /(ppm): 3.790 (s, 3H, OCH ), 3.806 (s, 3H,
6
3
OCH ), 5.591 (s, 2H, CH ), 6.065 (s, 2H, O-CH -O), 6.974 (J=8.1, d, 1H, Ar-H), 7.023 (d, 1H,
3
2
2
Ar-H), 7.184 (J=8.4, d, 1H, Ar-H), 7.377 (s, 1H, Ar-H), 7.502 (s, 1H, Ar-H), 7.603 (J=8.1, d, 1H,
1
3
Ar-H), 7.970 (s, 1H, HC=N), 11.804 (s, 1H, NH); C NMR DMSO-d /(ppm): 55.48 (CH ),
6
2
5
1
7.06 (OCH ), 101.70 (O-C-O), 106.19, 109.06, 114.48, 120.58, 120.87, 124.96, 130.54, 148.03,
3
+
49.21, 165.44, 168.16 (C=N), 170.61 (C=O); ESI-MS (m/z): [M +1] 411.13 (411.13).
4
.1.15. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[thiophen-2-yl-methylidene]acetohydr-
azide (9)
0
Yield 95%; mp 200-202 C; white crystals; Anal. calc. for C H N O S: C 50.56, H 3.39, N
1
5
12
6
3
-
1
2
2
3.58; found C 50.57, H 3.40, N 23.57; IRνmax(cm ): 1038 (C-N), 1501 (C=N), 1692 (C=O),
1
971 (CH-Ar), 3139 (NH); H NMR DMSO-d /(ppm): 5.615 (s, 2H, CH ), 5.973 (s, 2H, O-CH -
6
2
2
O), 7.089-7.503 (m, 3H, Ar-H), 7.523 (s, 1H, Ar-H), 7.643 (J=1.2, d, 1H, Ar-H), 7.675 (J=4.8, d,
1
3
1
1
H, Ar-H), 8.255 (s, 1H, HC=N), 11.926 (s, 1H, NH); C NMR DMSO-d /(ppm): 53.6 (CH ),
6 2
01.20 (O-C-O), 112.3, 115.8, 120.8, 124.0, 125.1, 125.8, 127.1, 127.4, 144.2, 148.8, 149.3
+
(
Phenyl), 162.3, 163.5 (C=N), 173.2 (C=O); ESI-MS (m/z): [M +1] 357.06 (357.07).
1
5

ACCEPTED MANUSCRIPT
4
.1.16. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(2-nitrophenyl)methylidene]aceto-
hydrazide (10)
0
Yield 97%; mp 210-212 C; yellow crystals; Anal. calc. for C H N O : C 51.65, H 3.31, N
1
7
13
7
5
-
1
2
2
4.80, found C 51.66, H 3.30, N 24.81; IRνmax(cm ): 1039 (C-N), 1614 (C=N), 1693 (C=O),
1
967 (CH-Ar), 3148 (NH); H NMR DMSO-d /(ppm): 5.685 (s, 2H, CH ), 6.138 (s, 2H, O-CH -
6
2
2
O), 7.069 (J=16.2, d, 1H, Ar-H), 7.529 (s, 1H, Ar-H), 7.625-8.127 (m, 4H, Ar-H), 8.200 (J=7.8,
13
d, 1H, Ar-H), 8.473 (s, 1H, HC=N), 12.257 (s, 1H, NH); C NMR DMSO-d /(ppm): 53.80
6
(
CH ), 101.71 (O-C-O), 106.24, 109.09, 120.64, 123.94, 128.14, 140.03, 142.65, 147.96, 148.25,
2
+
1
49.21(Phenyl), 161.41, 162.32 (C=N), 166.72 (C=O), ESI-MS (m/z): [M +1] 396.09 (396.10).
4
.1.17. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(4-ethylphenyl)methylidene]aceto-
hydrazide (11)
0
Yield 93%; mp: 200-202 C; white crystals; Anal. calc. for C H N O : C 60.31, H 4.79, N
1
9
18
6
3
-
1
2
2
2
2.21, found C 60.30, H 4.80, N 22.22; IRνmax(cm ): 1034 (C-N), 1528 (C=N), 1680 (C=O),
1
959 (CH-Ar), 3198 (NH); H NMR DMSO-d /(ppm): 1.169-1.216 (t, 3H, CH ), 2.504-2.655 (q,
6
3
H , CH ), 5.629 (s, 2H, CH ), 6.076 (s, 2H, O-CH -O), 7.089 (J=7.8, d, 1H, Ar-H), 7.281
2
2
2
(J=6.9, d, 1H, Ar-H), 7.625 (J=5.1, d, 2H, Ar-H), 7.653 (J=7.5, d, 2H, Ar-H), 7.525 (s, 1H, Ar-
1
3
H), 8.042 (s, 1H, HC=N), 11.903 (s, 1H, NH); C NMR DMSO-d /(ppm): 14.81 (CH ), 32.80
6
3
(
CH ), 53.66 (CH , 101.11 (O-C-O), 115.12, 115.82, 119.98, 124.51, 129.40, 129.61, 130.56,
2 2)
1
31.93, 136.42, 142.99, 147.73, 149.21, 162.21, 163.42 (C=N), 172.31(C=O); ESI-MS (m/z):
+
[
M +1] 379.14 (379.14).
1
6

ACCEPTED MANUSCRIPT
4
.1.18. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(3-nitrophenyl)methylidene]aceto-
hydrazide (12)
0
Yield 93%; mp 208-210 C; Yellow crystals; Anal. calc. for C H N O : C 51.65, H 3.31, N
1
7
13
7
5
-
1
2
2
4.80, found C 51.66, H 3.30, N 24.81; IRνmax(cm ): 1035 (C-N), 1615 (C=N), 1703 (C=O),
1
964 (CH-Ar), 3102 (NH); H NMR DMSO-d /(ppm): 5.343 (s, 2H, CH ), 5.962 (s, 2H, O-CH -
6
2
2
O), 6.910 (J=7.8, d, 1H, Ar-H), 7.096 (J=8.1, d, 1H, Ar-H), 7.529 (s, IH, Ar-H), 7.566 (s, 1H,
Ar-H), 7.588-7.719 (m, 1H, Ar-H), 8.036 (J=8.1, d, 1H, Ar-H), 8.231 (J=8.1, d, 1H, Ar-H), 8.509
1
3
(
s, 1H, HC=N), 11.092 (s, 1H, NH); C NMR DMSO-d /(ppm): 53.79 (CH ), 101.80 (O-C-O),
6 2
1
1
06.26, 109.11, 120.54, 123.84, 128.40, 140.23, 142.39, 147.12, 148.23, 149.31 (Phenyl),
+
59.11, 161.31 (C=N), 166.66 (C=O); ESI-MS (m/z): [M +1] 396.09 (396.10).
4
.1.19. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[furan-2-yl-methylidene]acetohydrazide
(13)
0
Yield 95%; mp 212-214 C; white crystals; Anal. calc. for C H N O : C 52.94, H 3.55, N
1
5
12
6
4
-
1
2
2
4.70, found C 52.95, H 3.56, N 24.71; IRνmax(cm ): 1034 (C-N), 1612 (C=N), 1693 (C=O),
1
918 (CH-Ar), 3124 (NH); H NMR DMSO-d /(ppm): 5.601 (s, 2H, CH ), 5.963 (s, 2H, O-CH -
6
2
2
O), 6.619 (J=1.8, d, 1H, Ar-H), 7.058-7.091 (m, 3H, Ar-H), 7.492 (s, 1H, Ar-H), 7.587 (J=8.1, d,
1
3
1
1
1
H, Ar-H), 8.108 (s, 1H, HC=N), 11.898 (s, 1H, NH); C NMR DMSO-d /(ppm): 53.63 (CH );
6 2
01.23 (O-C-O), 105.91, 109.91, 112.33, 115.83, 120.82, 124.02, 134.72, 143.99, 148.83,
+
49.11, 149.31 (Phenyl), 161.32, 163.52 (C=N), 170.11 (C=O); ESI-MS(m/z): [M +1] 341.09
(341.09).
1
7

ACCEPTED MANUSCRIPT
4
.1.20. 2-[5-(1,3-benzodioxol-5-yl)-1H-tetrazol-1-yl]-N'-[(E)-(4-nitrophenyl)methylidene]aceto-
hydrazide (14)
Yield 97%; mp 204-206 C; white crystals; Anal. calc. for C H N O : C 51.65, H 3.31, N
0
1
7
13
7
5
-
1
2
2
4.80; found C 51.66, H 3.32, N 24.81; IRνmax(cm ): 1036 (C-N), 1599 (C=N), 1689 (C=O),
1
913 (CH-Ar), 3089 (NH); H NMR DMSO-d /(ppm): 5.672 (s, 2H, CH ), 6.103 (s, 2H, O-CH -
6
2
2
O), 7.066 (J=8.1, d, 1H, Ar-H), 7.498 (s, 1H, Ar-H), 7.420 (J=3.6, d, 1H, Ar-H), 7.997 (J=8.7, d,
1
3
2
H, Ar-H), 8.244 (J=8.7, d, 2H, Ar-H), 8.148 (s, 1H, HC=N), 11.091 (s, 1H, NH); C NMR
DMSO-d /(ppm): 53.09 (CH ), 101.67 (O-C-O), 106.44, 109.13, 120.59, 123.88, 128.14, 140.13,
6
2
1
42.75, 147.86, 148.31, 149.43 (Phenyl), 160.21 and 162.32 (CH=N), 166.41[C=O]; ESI-
+
MS(m/z): [M +1] 396.09 (396.10).
4
.2. Pharmacology
4
.2.1. In vitro Anti-Cancer activity
The human breast cancer cell lines MCF-7, HepG2 and MDA-MB-231 were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) (HyClone Laboratories, Logan, UT, USA) and 1% penicillin: streptomycin (Invitrogen,
Carlsbad, CA, USA) in 12 well plates. The test compounds were dissolved in DMSO and one
microliter of the stock solution was used per milliliter of the culture to make a working
-5
-6
-7
concentration 1x10 , 1x10 and 1x10 M. Each test includes control wells (culture media with
4
cell line). The cell suspension was diluted to 1x10 cells/mL by adding fresh medium, and 1 ml
4
of this suspension was added to the test and control wells. An inoculum of 1x10 cells/well was
chosen, so that confluent, but not excessive growth took place. The plates were incubated at 37
°
C under CO for 24 hours. After incubation, the culture media was changed and cells were
2
1
8

ACCEPTED MANUSCRIPT
treated with 1 µL/mL of stock solution. Cells were incubated at 37 °C under CO for 48 hours.
2
The growth of the cells was checked with a low power microscope and MTT assay was used to
confirm the viability. In the similar experiments, the ZR-75 cells were grown in RPMI 1640
(HyClone Laboratories, Logan, UT, USA) using similar culture conditions.
4
.2.2. Total RNA extraction and Real time RT-PCR

Total RNA was extracted from the cells using Trizol reagent (Invitrogen) according to the
manufacturer’s protocol, stored in diethylpyrocarbonate-treated water at -80°C until use. Total
2
µg RNA was used for the synthesis of cDNA. All the primers used were designed with Primer 3
software (http://frodo.wi.mit.edu) using sequence information listed at NCBI. Following
conditions were applied for the RT-PCR: pre-denaturation of the synthesized cDNA at 95°C for
1
0 min followed by 40 cycles of denaturation at 95°C for 33 s, annealing at each gene-specific
primer T (°C) (primer information is provided in Supplemental Table S1), and extension at
m
7
2°C for 33 s. 1.2% of the agarose gel electrophoresis were used to verify the purity of the
amplified genes.
4
.2.3. Statistical analysis
All values represent the mean ± SEM. The means were compared by Tukey’s Studentized Range
(
HSD) to detect significant differences at *p<0.05, **p<0.001, and ***p<.0001. All the
statistical analysis were performed with the SAS software package, ver. 9.0 (SAS, Cary, NC,
USA).
4
.2.4. HepG2 cell viability Test
1
9

ACCEPTED MANUSCRIPT
Human hepatocellular carcinoma cell line (HepG2) was cultured in Dulbecco’s Modified Eagle’s
0
medium with 10% fetal bovine serum and 1% penicillin: streptomycin at 37 C in a saturated
humidity atmosphere containing 95% air/5% CO . The cells when reached to the 70%
2
-5
confulence were treated with 10 M of the tetrazoles. The cells were grown under the infulence
of the compounds for 48 hours. The cells were subjected to MTT assay for confirming the
viability [45].
4
.2.5. MTT assay
The MTT assay is a standard colorimetric assay for measuring the activity of enzymes that
reduces the MTT to the formazan dyes. These reductions take place only when reductase
enzymes are active and their activity is based on number of viable cells. For viability testing,
The culture medium was aspirated and cells were washed twice with DMEM to remove the
residual activity of FBS. 500 µL of 10 % MTT ( 5mg, thiazolyl blue tetrazolium bromide,
M2128 from Sigma in 1 ml of PBS) in DMEM were added to each well of cells and incubated
for 3 hours at 37 °C under CO atmosphere. The MTT solution was aspirated and 500 µL of
2
DMSO were added to dissolve the purple colour. The plates were placed on rotator for 10
minutes and 100 µL of blue coloured solution was transferred to the 96 well plate. Optical
density was measured at 510 nm using a Versa Max microplate reader (Sunnyvale, CA, USA)
[
46].
Acknowledgement:
2
0

ACCEPTED MANUSCRIPT
The authors are thankful to Yeungnam University, Republic of Korea, for their kind support. The
authors are also thankful to UGC (F. No. 41-236/2012) and UGC MANF scheme wide letter no.
F.40-65(C/M)/2009(SA-III/MANF) for providing financial assistance.
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1

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[
[
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Captions:
Figure 1A: Schematic diagram of hydrazine-tetrazole derivatives (1-14)
Figure 1B: Different R substituent groups.
Figure 2: Graph showing the effect of different substituted hydrazine-tetrazole derivatives on
growth of MCF-7, MDA-MB-231 and ZR-75 compounds.
Figure 3: Effect of Tetrazole compounds on HepG2 cell proliferation.
Figure 4: Effect of effective hydrazine-tetrazole derivatives in expression of different breast
cancer marker genes in MCF-7 cells.
Figure 5: Effect of effective hydrazine-tetrazole derivatives in expression of different breast
cancer marker genes in MDA-MB-231 cells.
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Figure 1 A: Schematic diagram of hydrazine-tetrazole derivatives (1-14)
O
CHO
O
NOH
NH OH.HCl
2
NaHCO ,H O
3
2
reflux
O
O
A
Acetic anhydride
reflux
N
N
CN
O
N
NaN ,ZnBr
3
2
O
O
2
-propanol
N
H
H O, reflux
2
O
B
C
ECA
Dry acetone
reflux
N
N
N
N
N
N
N
O
O
O
O
N
OC H
NH2NH2.H2O
2
5
NHNH2
Ethanol
reflux
O
D
E
O
Acetic acid R-CHO
reflux
Ethanol
N
N
N
N
O
O
R
NHN
O
1-14
2
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Figure 1B: Different R substituent groups.
Cl
1
2
6
3
4
Cl
OC2H5
OCH3
OCH3
5
9
7
8
OC H
2
5
OCH3
NO2
O N
2
10
14
11
12
S
C H
2
5
1
3
O
NO2
2
7