3-Aryl/Heteroaryl-5-amino-1-(3′,4′,5′-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization

Article abstract of DOI:10.1016/j.bioorg.2018.06.037

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3′,4′,5′-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC₅₀ < 1 μM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC₅₀ values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC₅₀ value of 0.66 μM, half that obtained in simultaneous experiments with CA-4 (IC₅₀ = 1.3 μM).

Full text of DOI:10.1016/j.bioorg.2018.06.037

Accepted Manuscript
3
-Aryl/Heteroaryl-5-Amino-1-(3’,4’,5’-Trimethoxybenzoyl)-1,2,4-Triazoles
as Antimicrotubule Agents. Design, Synthesis, Antiproliferative Activity and
Inhibition of Tubulin Polymerization
Romeo Romagnoli, Filippo Prencipe, Paola Oliva, Stefania Baraldi, Pier
Giovanni Baraldi, Andrea Brancale, Salvatore Ferla, Ernest Hamel, Roberta
Bortolozzi, Giampietro Viola
PII:
S0045-2068(18)30441-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.037
YBIOO 2417
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 May 2018
13 June 2018
29 June 2018
Please cite this article as: R. Romagnoli, F. Prencipe, P. Oliva, S. Baraldi, P. Giovanni Baraldi, A. Brancale, S. Ferla,
E. Hamel, R. Bortolozzi, G. Viola, 3-Aryl/Heteroaryl-5-Amino-1-(3’,4’,5’-Trimethoxybenzoyl)-1,2,4-Triazoles as
Antimicrotubule Agents. Design, Synthesis, Antiproliferative Activity and Inhibition of Tubulin Polymerization,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.037
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3
-Aryl/Heteroaryl-5-Amino-1-(3’,4’,5’-Trimethoxybenzoyl)-1,2,4-
Triazoles as Antimicrotubule Agents. Design, Synthesis,
Antiproliferative Activity and Inhibition of Tubulin Polymerization
a
a
a
a
Romeo Romagnoli, * Filippo Prencipe, Paola Oliva, Stefania Baraldi, Pier Giovanni
a
b
b
c
d
Baraldi, Andrea Brancale, Salvatore Ferla, Ernest Hamel, Roberta Bortolozzi and
d
Giampietro Viola *
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, 44121
Ferrara, Italy;
b
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII
Avenue, Cardiff, CF10 3NB, UK;
c
Screening Technologies Branch, Developmental Therapeutics Program, Division of
Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer Research,
National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,
USA;
d
Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia,
Università di Padova, 35131 Padova, Italy
*
To whom correspondence should be addressed. E-mail:rmr@unife.it; Phone: 39-
(
0)532-455303. Fax: 39-(0)532-455953. (R.R.); E-mail:giampietro.viola.1@unipd.it
Phone: 39-(0)49-8215485. Fax: 39-(0)49-8211462. (G.V.).
1

Abstract: Many natural and synthetic substances are known to interfere with the
dynamic assembly of tubulin, preventing the formation of microtubules. In our search
for potent and selective antitumor agents, a novel series of 1-(3’,4’,5’-
trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had
different heterocycles, including thiophene, furan or the three isomeric pyridines, and
they possessed a phenyl ring bearing electron-releasing or electron-withdrawing
substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-
two tested compounds showed moderate to potent antiproliferative activities against a
panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing
strong antiproliferative activity (IC50<1 M) against selected cancer cells. Among them,
several molecules preferentially inhibited the proliferation of leukemic cell lines, showing
IC50 values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines
derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly
inhibited tubulin assembly, with an IC value of 0.66 M, half that obtained in
5
0
simultaneous experiments with CA-4 (IC =1.3 M).
5
0
Keywords. Microtubule targeting-agent, structure-activity relationship, tubulin
polymerization, antiproliferative activity, molecular docking.
1
. Introduction
The microtubule system of eukaryotic cells, a protein system composed of α-tubulin
heterodimers in a dynamic equilibrium with microtubules, is a critical element in a variety
of fundamental cellular processes, including mitosis and cell division, determination and
maintenance of cell shape, organization of intracellular architecture, secretion,
intracellular transport and regulation of motility [1-3]. Given their significant role in the
2

cellular functions that are critical to the life cycle of the cell, microtubules are a proven
molecular target for cancer chemotherapeutic agents and continue to receive substantial
attention for development of new and improved drugs [4-6].
Among the distinct classes of tubulin-binding agents that have been identified on the
basis of their different mechanisms of action and binding sites on tubulin, two groups are
of particular importance in the clinical treatment of cancer [7, 8]. First are the vinca
alkaloid microtubule-destabilizers, typified by vinblastine, vincristine, vinorelbine and
vinflunine. Second are the taxoids, paclitaxel (Taxol), docetaxel (Taxotere) and
cabazitaxel (Javetana). These compounds act with an opposite effect, interfering with
microtubule disassembly and stabilizing the microtubule structure. These drugs are used
to treat a broad range of leukemias and lymphomas as well as many types of solid tumors
[
9-11]. Nevertheless, the clinical success of these two classes of compounds has
stimulated intensive research aimed at discovering additional microtubule-targeting drugs
with clinical potential [12].
A third class of note, due to the discovery of numerous natural products and synthetic
compounds that target this site, are agents that bind in the colchicine site [13, 14]. Such
compounds inhibit the polymerization of tubulin into microtubules and display wide
structural heterogeneity [15, 16]. One of the most notable of these compounds, both for
its potency and structural simplicity is the naturally occurring cis-stilbene derivative
combretastatin A-4 (CA-4, 1, Figure 1) [17, 18]. Moreover, several studies have
documented that compounds targeting the colchicine site also act as powerful vascular
disrupting agents [19], particularly for the neovasculature of tumors.
While the vinca alkaloids and the taxoids have well-established roles in the treatment of
human cancers, no colchicine site inhibitor is currently approved for cancer therapy,
despite considerable synthetic efforts [20-22], including the preparation of many water-
3

soluble prodrugs of CA-4, such as the phosphorylated derivative of combretastatin A-4
CA-4P, 2) in phase III clinical trials [23, 24] or the serine amino acid prodrug of 3’-
(
amino-deoxycombretastatin A-4 (Ombrabulin or AVE8062, 3) [25], which is in phase I
clinical studies for patients with solid tumors [26].
These observations emphasize the need to discover novel scaffolds active at the
colchicine site and amenable to rapid derivatization because such compounds could be
readily prepared in a cost-effective way by rapid and concise synthetic procedures [27].
In addition, several colchicine site binders have been shown to inhibit p53 mutant cell
lines, and this further emphasizes the importance of novel inhibitors of tubulin assembly
that have activity against resistant tumors [28].
Among the synthetic inhibitors of tubulin polymerization, in a previous article we have
reported the synthesis and biological evaluation of a series of compounds with general
structure 4 based on the 1-(3’,4’,5’-trimethoxybenzoyl)-5-amino-1,2,4-triazole
molecular skeleton, that showed strong antiproliferative activity against a panel of cell
lines and act as inhibitors of microtubule polymerization by interfering with the
colchicine site of tubulin [29]. Structure-activity relationships were elucidated with
various substituents on the phenyl ring of the anilino moiety at the C-3 position of the
1
,2,4-triazole ring, with the p-toluidino derivative 4a exhibiting the best cell growth
inhibitory activity among the tested compounds, with IC50 values of 0.21-3.2 nM. This
derivative was also a potent inhibitor of tubulin polymerization, with an IC50 value of
0
.75 M for tubulin assembly.
A molecular docking study of compound 4a in the colchicine site of tubulin showed that
the trimethoxyphenyl unit of this derivative is placed in proximity of Cys241, while the
phenyl ring of the aniline moiety at the 3-position of 1,2,4-triazole ring occupies a
4

relatively small hydrophobic region deep in the binding site, establishing a series of non-
polar interactions with amino acids Met259, Thr314 and Lys352 in this subpocket,
which stabilizes the binding of the molecule in the colchicine site. The same docking
simulation on compound 4a did not show any hydrogen bond interactions of the amino
bridge function of the aniline moiety with the amino acid residues located in this small
pocket of the colchicine site. X-ray crystallographic analysis on compound 4a showed
the formation of an intramolecular hydrogen bond between the carbonyl oxygen of the
3
’,4’,5’-trimethoxybenzoyl function and the hydrogen of the amino moiety at the 1- and
5
-position, respectively, of the 1,2,4-triazole ring.
Inspired by these observations and encouraged by the activity obtained with compound
a, here we report a new series of 1-(3’,4’,5’-trimethoxybenzoyl)-3-aryl/heteroaryl-5-
4
amino-1,2,4-triazole analogues with general structure 5, obtained by removing the amino
function spacer of the anilino group of compounds with general structure 4, connecting
the aryl/heteroaryl moiety at the 3-position of 1,2,4-triazole ring and keeping constant
the 3’,4’,5’-trimethoxybenzoyl function and the amino group at the 1- and 5-positions,
respectively, of the triazole nucleus.
Thus, once the 1-(3’,4’,5’-trimethoxybenzoyl)-5-amino-1,2,4-triazole motif was
identified as the minimum structural requirement for activity, modifications were focused
on varying the substituent at the 3-position of the 1,2,4-triazole ring to develop new
potent antiproliferative agents and to understand the minimum structural requirements
for this class of compounds to exhibit potent and selective activity against cancer cell
lines. The substituents examined included heterocycles such as thien-2-yl (5b), furan-2-yl
(
5c) and the three isomeric pyridines (5d-f) as well as the phenyl ring with various
5

electron-withdrawing (F, Cl, Br, CN and NO
EtO) groups [30].
2
) and electron-releasing (Me, Et, MeO and
Figure 1. Structures of CA-4 (1), CA-4P (2) and Ombrabulin (3). General structure of 1-(3’,4’,5’)-3-
anilino-5-amino-1,2,4-triazole 4. Molecular formulas of synthesized 1-aroyl-3-substituted-5-amino-
1
,2,4-triazoles 5a-v.
While for compounds 5a-t, the 3’,4’,5’-trimethoxyphenyl of the benzoyl moiety at the N-
position of the 1,2,4-triazole ring was kept unchanged because this ring is the essential
1
structural requirement to maximize potency in a large series of inhibitors of tubulin
polymerization, such as colchicine, steganacin, CA-4 and podophyllotoxin [31, 32], in an
effort to further confirm whether the 3,4,5-trimethoxybenzoyl moiety of compound 5k
6

played an essential role for antiproliferative activity, the corresponding 3,4-
dimethoxybenzoyl and 4-methoxybenzoyl derivatives 5u and 5v, respectively, were
prepared.
2
. Chemistry
Synthesis of 1-aroyl-3-aryl/heteroaryl-5-amino-1,2,4-triazoles 5a-v was accomplished
using a three-step procedure described in Scheme 1.
Scheme 1. Reagents. a: aminoguanidine hydrogen carbonate pyridine, 0 °C for 30 min. then 12 h rt; b:
water, microwave irradiation, 100 W; c: 3',4',5'-(OMe) C H COCl for the preparation of 5a-t, 4',5'-
3
6
2
(
OMe) C H COCl or 4'-OMe-C H COCl for the synthesis of 5u and 5v, respectively, pyridine, 0 °C.
2
6
3
6
4
The required 3-aryl/heteroaryl-5-amino-1,2,4-triazoles 8a-t were synthesized by reaction
of aroyl/heteroaroyl chloride 6a-t with aminoguanidine hydrogen carbonate using
7

pyridine as solvent to furnish the corresponding aroyl/heteroaryl amidoguanidines 7a-t,
followed by subsequent ring closure of the intermediate in water under microwave
irradiation. Treatment of 8a-t with an equimolar quantity of 3’,4’,5’-trimethoxybenzoyl
chloride resulted in the formation of compounds 5a-t. Using the same synthetic
methodology, compounds 5u and 5v were prepared by the reaction of 3-(p-tolyl)-5-
amino-1,2,4-triazole 8k with 4’,5’-dimethoxybenzoyl chloride and 4’-methoxybenzoyl
chloride, respectively.
3
. Biological Results and Discussion
3
.1. In vitro antiproliferative activities.
The synthesized compounds 5a-v were tested for their antiproliferative activity against a
panel of five human cancer cell lines, T-leukemia (Jurkat), acute lymphoblastic leukemia
(
RS4;11), cervix carcinoma (HeLa), colon adenocarcinoma (HT29) and breast
adenocarcinoma (MCF-7), by the MTT assay for 48 h. The results are summarized in
Table 1, using CA-4 (1) and 4a as positive controls. In general, the antiproliferative
activities of compounds 5a, 5h, 5k-r and 5u were greater against the leukemia cells as
compared with the solid tumor cells.
The unsubstituted phenyl derivative 5a showed modest antiproliferative activity (IC50>10

M) against HT29 and MCF-7 cells, while it was more active, with IC50 values of 1.4,
1
.7 and 6.6 M, against Jurkat, RS4;11 and HeLa cells, respectively.
8

Table 1. In vitro inhibitory effects of compounds 4a, 5a-v and CA-4
a
IC50 (M)
Compd
Jurkat
RS4;11
HeLa
HT29
MCF-7 Average
5
5
a
1.4±0.3
1.7±0.1
6.6±1.3 27.3±9. 19.1±2.
11.2
21.5
nc
6
0
b
43.3±7.3 17.7±7.7 10.5±2. 15.9±1. 19.9±3.
1
3
1
5
c
>100
>100
>100
44.3±16. 21.3±1. 18.5±2. 19.1±1.
8
6
2
1
5
d
10.8±3.2 17.7±1. 23.9±2. 21.9±3.
nc
3
6
1
5
e
68.3±10.
9
>100
>100
>100
nc
5
f
>100
49.0±4.6
>100
>100
>100
nc
5.9
5
5
g
8.1±1.3
1.4±0.8
>100
4.0±0.5
1.3±0.5
>100
5.4±1.6 4.7±1.5 7.2±3.2
4.0±0.5 3.8±1.4 2.7±0.7
h
2.6
5
i
>100
>100
>100
>100
2.5
5
j
2.6±0.6
0.5±0.2
3.9±0.1
0.8±0.2
2.8±0.9 0.5±0.1 5.6±1.6
5
k
1.8±0.06 5.5±1.2 4.9±0.8 3.6±1.0
4.2±0.9 10.1±2.6 13.2±1.8 11.5±1.4
3.3
5
l
8.6
5
m
34.0±0.6 19.3±0.9 19.8±3.1 25.2±2.5 23.6±3.6
24.4
12.0
8.2
5
n
o
p
q
2.6±0.09
1.2±0.5
4.0±1.3
4.3±0.8
4.0±0.1
>100
2.2±0.1 15.6±1.9 21.1±2.9 18.6±2.1
0.3±0.1 6.6±1.3 23.4±2.1 9.6±1.5
5
5
5
0.9±0.02 9.8±1.2 11.2±1.1 10.3±2.0
8.5±1.5 11.2±2.3 19.9±1.6 18.8±2.6
7.2
12.5
7.9
5
r
s
3.6±1.1
9.8±1.1 12.1±1.5 10.2±0.9
>100 >100 >100
5
99.3±22.8
nc
5
t
59.3±17.6 47.7±9.3 75.6±3.5 88.9±5.2 82.3±3.9
70.8
11.3
5
u
v
9.3±1.1
>100
1.8±0.6
15.1±1. 17.2±2. 13.2±1.
9
5
6
5
38.9±7.2 45.5±3. 65.3±4. 39.3±4.
nc
6
2
1
4
a (nM)
0.8±0.3
nd
3.2±1.3 0.8±0.1
1±0.6
nc
CA-4 (nM) 0.8±0.2
370±100
4±1 180±30
5±0.6
112
a
IC = compound concentration required to inhibit tumor cell proliferation by 50%.
5
0
nc= not calculated
nd=not determined
9

In contrast, if the phenyl at position 3 was replaced by the biososteric 2’-thienyl (5b), 2’-
furanyl (5c) or the three isomeric pyridines (compounds 5d-f), significantly reduced
activity was observed against the leukemia cell lines, while derivatives 5b-d, but not 5e-f,
were comparable to 5a against the solid tumor cells. Comparing the activities of p-tolyl
and p-toluidino derivatives 5k and 4a, respectively, the presence of the amino (NH)
spacer between the p-tolyl group and the 3-position of 1,2,4-triazole ring played a
crucial role in affecting antiproliferative activity, with the p-tolyl derivative 5k almost
three orders of magnitude less potent than the p-toluidino derivative 4a.
The data presented in Table 1 for compounds 5g-t examine the effects of different
substituents on the phenyl ring on antiproliferative activity. Considering the average IC50
against the five cell lines, compounds 5h, 5j and 5k were the most active in the series,
with mean IC50’s, respectively, of 2.6, 2.5 and 3.3 µM. In addition, these data showed
that the nature and the location of the substituent on the phenyl at the 3-position of the
1
,2,4-triazole core played a critical role in antiproliferative activities, and only a few
compounds showed any IC50 values lower than 1 M against any of the cancer cell lines.
RS4;11 and HT29 cells were the most sensitive to the p-bromophenyl derivative 5j, with
IC50 values of 0.8 and 0.5 M, respectively. The p-tolyl compound 5k showed an IC50 of
0
.5 M against the Jurkat cells, and the p-methoxyphenyl derivative 5o and its isomeric
m-methoxy analogue 5p had IC50 values of 0.3 and 0.9 M, respectively, against RS4;11
cells.
Substituents clearly had effects on antiproliferative activity. For example,
antiproliferative activity was almost eliminated with a m-Cl (5i) or a m-CN (5s).
Compounds with other meta-substituents, such as methyl (5l) or methoxy (5p), had
reasonable activity relative to 5a (in terms of mean IC50, 8.6, 7.2 and 11.2 µM,
10

respectively). With para-substituents, the best activities, in terms of mean IC50 values
2.5-3.3 µM) occurred with a p-Cl (5h), a p-Br (5j) and a p-CH (5k). A second group
of para-substituents had mean IC50 values in the 5.9-12.5 µM range (p-F, 5g; p-ethyl,
n; p-methoxy, 5o; p-ethoxy, 5q; p-CN, 5r). Compound 5t, p-NO , was an outlier with
(
3
5
2
a mean IC50 of 70.8 µM. With this large group of para-substituents, there was no clear
pattern in terms of electron-withdrawing or electron-donating properties. The only
compound with an ortho-substituent (5m, o-CH
IC50 of 24 µM, as compared with 3.3 and 8.6 µM for its para and meta analogs 5k and
l, respectively. In terms of position, para seems best, ortho worst, based on the methyl
3
) was relatively inactive with a mean
5
series 5k-m. The superiority of the para-substituent over the meta held in every case
except for the methoxy pair, where the mean IC50 for 5p (meta) was 7.2 while that for
5
o (para) was 8.2.
Turning to individual cell lines, comparing the antiproliferative activities of compounds
g (p-F), 5h (p-Cl) and 5j (p-Br), revealed that on RS4;11, HeLa and HT-29 cells, the
5
antiproliferative activity increased with increasing size of the halogen atom at the para-
position of the phenyl ring. In contrast, for Jurkat and MCF7 cells, the effect of the
halogen atom on activity was chlorine>bromine>fluorine. Thus, replacement of the
fluorine at the para-position of the phenyl ring with a chlorine (compounds 5g and 5h,
respectively) led to 6-, 3- and 2.5-fold increase of potency against Jurkat, RS4;11 and
MCF-7 cells, while the activity was unchanged against HeLa and HT29 cells. Moving the
chlorine from the para- to the meta-position on the phenyl ring (compounds 5h and 5i,
respectively) eliminated antiproliferative activity. A similar loss of potency was observed
for the para-cyano derivative 5r versus the isomeric meta-cyano analogue 5s. Replacing
para-chlorine with bromine (compounds 5h and 5j, respectively) had a contrasting
11

effect. Derivative 5j was 2-fold less potent than chlorine agent 5h against Jurkat and
MCF-7 cells, while it retained activity against HeLa cells. Nevertheless, the p-
bromophenyl derivative 5j showed strong antiproliferative activities in the
submicromolar range against RS4;11 and HT29 cells, with IC50 values of 0.8 and 0.5

M, respectively, and was thus 2- and 7-fold more potent than its chlorine counterpart
5
h.
It should be noted that replacement of the weak electron-withdrawing fluorine with the
weak electron-releasing methyl group, to furnish derivative 5k, significantly improved
potency from 2- to 16- fold against Jurkat, RS4;11 and MCF-7 cells, while the two
derivatives showed similar activity toward HeLa and HT29 cells. However, the
introduction of the methyl at the para-position of the phenyl ring (compound 5k)
enhanced activity from 3- to 5-fold against Jurkat, HT-29 and MCF-7 cells in
comparison with the unsubstituted phenyl analogue 5a, while the two compounds are
equipotent against RS4;11 and HeLa cells.
To compare the effects of ortho-, meta- and para-methyl substitution on the phenyl ring,
the m-tolyl and o-tolyl derivatives 5l and 5m, respectively, were also synthesized and
showed different activities, indicating that the position of the methyl group is crucial for
antiproliferative activity. The o-tolyl derivative 5m was 3-70-fold less potent the p-tolyl
analogue 5k, whereas the m-tolyl derivative 5l had intermediate activity, being 2-7-fold
less active than the p-tolyl isomer 5k against all cancer cell lines.
As noted above, our findings indicate that a methoxy group located at the para-position
of the phenyl ring (compound 5o) resulted in strong antiproliferative activity against
RS4;11 cells (IC50=0.3 M), while shifting it to the meta-position, to furnish derivative
5
p, decreased activity 3-fold. A similar effect was observed against Jurkat and HeLa
12

cells. Additionally, our findings indicate that moving substituents from the para- to the
meta- or ortho-position on the phenyl ring was generally not favorable for maintaining
antiproliferative activity within the series of analogues reported here (5h vs. 5i, 5k vs. 5l
and 5m, 5o vs. 5p and 5r vs. 5s).
Replacement of p-methoxy with p-ethoxy (compounds 5o and 5q, respectively) reduced
activity from 2- to 28-fold against Jurkat, RS4;11, Hela and MCF-7 cells, although
activity was retained against HT29 cells.
Compound 5u, with a 3’,4’-dimethoxybenzoyl group at the N
1
-position of the 3-(p-toly)-
5
-amino-1,2,4-triazole system, had 3-18-fold reduced cell growth inhibitory activity as
compared with the 3’,4’,5’-trimethoxybenzoyl derivative 5k against four of the five cell
lines, but the two derivatives were equipotent against RS4;11 cells. The 4’-
methoxybenzoyl analogue 5v showed substantially reduced activity (IC50>30 M)
compared to 5k in all cell lines.
Among the electron-withdrawing groups, introduction of the nitro substituent, resulted
in compound (5t) with reduced antiproliferative activity compared with those bearing
halogen or cyano substituents.
In summary, small structural modifications are responsible for great variations of the IC50
values obtained against the cancer cell lines studied, with the methyl and methoxy
moieties (compounds 5k and 5o, respectively) being optimal at the para-position of the
phenyl at the 3-position of the 1,2,4-triazole ring.
3
.2. In vitro inhibition of tubulin polymerization and colchicine binding.
To investigate whether the antiproliferative activities of selected substituted phenyl
derivatives 5j (p-Br), 5k (p-Me), 5o (p-OMe) and 5p (m-OMe) derived from an
interaction with tubulin, these agents were evaluated for their ability to inhibit tubulin
13

3
polymerization and for effects on the binding of [ H]colchicine to tubulin (Table 2). For
comparison, CA-4 (1) and 4a were examined in contemporaneous experiments as
reference compounds.
Table 2. Inhibition of tubulin polymerization and colchicine binding by compounds
CA-4, 4a, 5j-k and 5o-p.
a
b
Tubulin assembly Colchicine binding
Compound
IC50±SD (M)
>20
% ±SD
5
j
17±6
61±4
5
k
o
0.66±0.09
0.97±0.1
>20
5
51±3
5
p
3.8±0.2
92±2
4
a
0.75±0.1
1.2±0.1
CA-4
98±0.5
a
Inhibition of tubulin polymerization. Tubulin was at 10 M.
b
3
Inhibition of [ H]colchicine binding. Tubulin, colchicine and tested compound were at 1, 5 and 5 M,
respectively.
The p-tolyl derivative 5k, the most potent compound of the series against Jurkat (T-
leukemia) cells, was found to be also a strong inhibitor of tubulin polymerization, with an
IC50 value of 0.66 M, nearly twice as potent as CA-4 (IC50:1.2 M). Note that 4a had
much greater antiproliferative activity than 5k, despite the similar effects on tubulin
assembly. Such discrepancies in antitubulin versus antiproliferative activity are not
infrequently observed, but the reasons are usually uncertain, as is the case here. Among
possible explanations is that we are using bovine brain tubulin in the former studies, and
its composition in terms of tubulin isotypes differs significantly from that of different
human cancer cell lines [33].
Compound 5o, as the most potent derivative of the series against RS4;11 (acute
lymphoblastic leukemia) cells, strongly inhibited tubulin polymerization (IC50:0.97 M)
14

with activity comparable to that of CA-4. The results obtained demonstrate that the
antiproliferative activities of compounds 5k and 5o are related to inhibition of tubulin
polymerization. It is intriguing to note that compounds 5j and 5p were inactive as
inhibitors of tubulin polymerization, with minimal activity (IC50>20 M), although these
derivatives demonstrated significant antiproliferative activity against RS4;11 cells, with
IC50 values of 0.8 and 0.9 M, respectively, only 3-fold less potent as antiproliferative
agents relative to 5o against this cancer cell line. The weak activity against tubulin
suggests the possibility that compounds 5j and 5p may possess an additional mechanism
of inhibition of cancer cell growth beyond that attributable to the tubulin-based
mechanism.
Comparing the IC50 values of the inhibition of tubulin assembly for compounds 5o and
5
p, corresponding to 0.97 and >20 M, respectively, it appears that a small structural
modification, such as moving the methoxy group from the para- to the meta-position of
the phenyl ring, is responsible for great variation in antitubulin activity, suggesting that
there is a space-limited pocket surrounding the phenyl at the 3-position of 1,2,4-triazole
ring.
In the colchicine binding studies, compounds 5k and 5o displayed 61% and 51%,
3
respectively, inhibition of [ H]colchicine binding at 5 M, with 1 µM tubulin. Both
compounds were significantly less potent than CA-4 in this assay, despite their greater
potency as inhibitors of tubulin assembly. Such differences are commonly observed, and
3
CA-4 is a particularly potent inhibitor of [ H]colchicine binding [17, 18]. The data
suggested that compounds 5k and 5o bind to the colchicine site and inhibit the
polymerization of tubulin. In this assay, derivative 4a potently inhibited the binding of
15

3
[
H]colchicine to tubulin, and with 92% inhibition was 1.5-fold more active than 5k,
which in this experiment inhibited colchicine binding by 61%.
3
In general, in these experiments, inhibition of [ H]colchicine binding correlated more
closely with inhibition of tubulin assembly than with antiproliferative activity. Thus,
compound 5o was as active as CA-4 as an inhibitor of tubulin polymerization, although
this derivative was less active in its effects on cell growth against the whole panel of
cancer cell lines.
3
.3. Molecular modeling studies.
In order to further understand the different activity profiles of the tested compounds,
several molecular modeling studies were performed. According to a recent publication,
the tubulin colchicine domain is formed by a main site, where colchicine binds (zone 1),
and two additional neighboring pockets (zones 2 and 3) [34]. After studying the tubulin
crystal structures in complex with different ligands, these authors concluded that
globular or butterfly like shaped molecules bind to zones 1 and 2, mimicking colchicine
binding, whereas planar compounds tend to bind in zones 2 and 3 [34]. In order to
identify the most likely binding area of the new derivatives, a series of docking studies
were performed with compounds 5j, 5k, 5o and 5p. Two different tubulin crystal
structures were used: one co-crystallized with colchicine (PDB ID: 4O2B), representing
the colchicine-like binding site area (zones 1 and 2), and one co-crystallized with the
inhibitor G2N (PDB ID: 3N2G), representing the planar compounds binding in zones 2
and 3 [35, 36]. The 3N2G crystal structure was chosen because the ligand included a
free amino group in the central portion of G2N, which is a feature also present in the
compounds we examined. Docking studies were performed using the Glide SP method,
and the results were then refined using Glide XP scoring [37]. The proposed binding
16

modes of the two active derivatives 5k and 5o in the colchicine binding site are
consistent with that previously reported for similar derivatives, and these binding modes
are very similar to that found for the co-crystallized colchicine. In these models, the
trimethoxyphenyl ring is in proximity of βCys241, with the 4’-methylphenyl or the 4’-
methoxyphenyl ring occupying the area close to βMet259 [29] (Figure 2A).
Figure 2. Proposed binding for compound 5k (A, carbon atoms in purple) and 5p (B, carbon atoms in
orange) in the colchicine binding site (4O2B). Co-crystallized colchicine is shown in green. The
hydrophobic subpocket nearby, including Met259, is highlighted with a red surface. Note how the 3’-
methoxyphenyl ring of 5p is pointing in the opposite direction of the co-crystallised colchicine, as
compared with the analogous structural feature of 5k as shown in Panel A.
We found no plausible binding poses for the two inactive compounds, 5p and 5j, by the
docking program, but there was a binding pose for 5p (shown in Figure 2B), where the
3
-methoxyphenyl ring pointed in the opposite direction relative to the co-crystallized
17

colchicine. We then examined the potential binding mode of the new compounds into the
G2N site. Even though the compounds occupy the binding site in a very similar manner
to the co-crystallized inhibitor, it was not possible to rationalize the differences seen in
the tubulin polymerization inhibition assay, which appeared to be linked to the
substituent on the phenyl ring (Figure 3A and 3B).
Figure 3. Proposed binding for compound 5k (A, carbon atoms in purple) and 5p (B, carbon atoms in
orange) in the G2N binding site (3N2G). Co-crystallized G2N is shown in yellow. The different
location of this binding site in comparison with the colchicine binding site, can be easily seen by noting
the spatial orientation of Cys241. No substantial differences in the binding modes were found between
the active (5k) and inactive (5p) derivatives.
Based on the docking results, a binding mode similar to that of colchicine might be more
relevant for these compounds (zone 1). However, the planar conformation of the
molecules and the relatively low colchicine binding competition values found in the
18

biological assay, would suggest that the binding site for these new compounds only
partially overlaps the one for colchicine and could involve only zones 2 and 3 (3N2G).
Therefore, in order to help select the potential binding site from the two choices
presented in Figures 2 and 3, a series of short 20 ns molecular dynamic (MD) simulations
on selected compounds (5k, 5o, 5p) were performed using the Desmond software
package [38]. The relative binding free energies (∆Gbinding) of the compounds were then
calculated using the Prime/MM-GBSA based calculation method [39]. All the protein-
ligand systems reached stability after an initial 6 ns of equilibration, as shown by the C-
alpha RMSD variation (see Figure S1 in the Supporting Information data), so therefore
only the remaining 14 ns of the simulation were considered in our analysis.
Figure 4. Ligand-protein interaction diagram for compound 5k in the two different binding sites, 4O2B
(a) and 3N2G (b). The most persistent interactions formed during the MD simulation are reported
together with the interaction strengths. The interaction between the methoxy groups and Cys241,
mediated by a water bridge, is only present in the colchicine binding site simulation. The first 6 ns of
MD, in which the protein-ligand system reached stability, were excluded from the diagram.
The position of the trimethoxyphenyl ring and the disposition of the substituted phenyl
ring near the area of βMet259 were maintained during the entire simulation of 5k and 5o,
confirming the reliability of the docking in predicting the binding mode in the colchicine
binding site (4O2B). In particular, water bridge formation between the methoxy groups
and βCys241 and the interaction between βLys352 and the central triazole ring were
seen for both derivatives during the entire MD, potentially contributing to protein-ligand
19

stability (Figure 4A). Instead, the proposed binding for 5p was very variable, with the
compound not able to occupy in a stable manner the binding area during the whole
simulation. In the case of the G2N binding site, for all the derivatives studied, the
trimethoxyphenyl ring was no longer able to interact with Cys241, moving away from
this residue and potentially losing a fundamental interaction point for the tubulin
inhibition function (Figure 4B). Table 3 reports the calculated ligand-interaction energies
with the two different binding sites for the compounds analysed by MD. Overall, the
∆Gbinding calculated for 5k and 5o in the colchicine binding site (4O2B) is lower than the
one estimated for each corresponding compound in the G2N binding area, potentially
meaning that the colchicine binding site ligands system is more stable in comparison with
the G2N-binding site ligands system, and therefore the former is the more likely binding
area for the these new derivatives.
Table 3. Calculated ligand-interaction energies for the compounds analyzed by MD
simulation.
a
a
Compound
∆Gbinding (kJ/mol) ±SD
∆Gbinding (kJ/mol) ±SD
4
O2B
3N2G
-58.685 ± 3.338
-69.161 ± 3.379
-65.809 ± 2.318
5
k
o
-69.512 ± 1.769
-74.471 ± 0.400
-59.613 ± 1.964
5
5
p
a
Average values calculated as mean of three ΔGbinding obtained from three independent MD simulations
triplicate) for each compound. For each replicate, the average ΔGbinding value was calculated excluding
the first 6 ns of MD in which the protein-ligand system reached stability. Standard deviation (SD) is
reported.
(
Water bridge formation between the methoxy groups and βCys241, the interaction
between βLys352 and the central 1,2,4-triazole ring and the accommodation of the
substituted phenyl ring in the hydrophobic sub-pocket near βMet259 could confer
20

stability on the protein-ligand system leading to lower energy values, reflecting binding
of the ligand resulting in inhibition of tubulin polymerization. The insertion of the
methoxy group in position 3’, as in compound 5p, abolishes inhibition of tubulin
polymerization, and a completely different binding pose with a higher calculated ∆Gbinding
value was found. The higher ∆Gbinding values obtained for the G2N-binding site is an
indication that the protein-ligand system could be less stable if the compounds bind to
this site. Moreover, the higher standard deviation calculated for the three average
∆Gbinding values may indicate that the ligand binding in this area was very variable in all
the performed simulations, giving a further indication that it is very unlikely that the
G2N-binding site is the potential binding site for these new derivatives.
3
.4 Analysis of cell cycle effects.
The effects of 24 h treatments with different concentrations of 5k and 5p on cell cycle
progression in HeLa cells were determined by flow cytometry (Figure 5). Compound 5k
caused a significant G2/M arrest in a concentration-dependent manner in HeLa cells,
with an increase in G2/M cells occurring at 5 µM. The cell cycle arrest in G2/M phase
was accompanied by a slight reduction of both G1 and S phase cells. In contrast,
treatment with compound 5p even at high concentrations (20 µM) had no effect on cell
cycle distribution, and, in particular, it did not cause the increase in G -M cells always
2
observed with antitubulin agents, in good agreement with the negative result observed in
the tubulin polymerization assay. It is worthwhile noting that similar results wereb also
obtained after 48 h treatments (data not shown).
21

Figure 5. Percentage of cells in each phase of the cell cycle in HeLa cells treated with compounds 5k
and 5p at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by
flow cytometry as described in the Experimental Section. Data are expressed as mean ± SEM.
3
.5 Compounds 5k and 5p both induce apoptosis.
To evaluate the mode of cell death induced by 5k and 5p, a biparametric cytofluorimetric
analysis was performed using propidium iodide (PI), which stains DNA and enters only
dead cells, and fluorescent immunolabeling of the protein annexin-V, which binds to
phosphatidyl serine in a highly selective manner. Dual staining for annexin-V and with PI
-
-
permits discrimination between live cells (annexin-V /PI ), early apoptotic cells (annexin-
+
-
+
+
-
+
V /PI ), late apoptotic cells (annexin-V /PI ) and necrotic cells (annexin-V /PI ). As
shown in Figure 6, HeLa cells treated with 5k or 5p showed an accumulation of annexin-
V positive cells in comparison with the control, in a concentration- and time-dependent
manner.
22

2
4 h
48 h
A -/P I+
A + /P I+
A + /P I-
A -/P I-
A -/P I+
A + /P I+
A + /P I-
A -/P I-
1
0 0
1 0 0
8 0
6 0
4 0
2 0
0
8
6
4
2
0
0
0
0
0
tr
tr
M
M
M
M
M
M
C



C



0
5
0
0
5
0
1
2
1
2
5
k
5k
2
4
h
48 h
A -/P I+
A + /P I+
A + /P I-
A -/P I-
A -/P I+
A + /P I+
A + /P I-
A -/P I-
1
0 0
1 0 0
8 0
6 0
4 0
2 0
0
8
6
4
2
0
0
0
0
0
tr
t
r
M
M
M
M
M
M
C



C



0
5
0
0
5
0
1
2
1
2
5
p
5p
Figure 6. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with compounds 5k
or 5p at the indicated concentrations after incubation for 24 or 48 h. The cells were harvested and
labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are represented as
mean±SEM of three independent experiments.
4
. Conclusions
We have discovered a new class of simple synthetic inhibitors of tubulin polymerization
based on the 1-(3’,4’,5’-trimethoxybenzoyl)-3-aryl/heteroaryl-5-amino-1,2,4-triazole
molecular skeleton. The introduction of different electron-withdrawing (F, Cl and Br)
and electron-releasing (Me and OMe) groups at the para-position of the phenyl ring had
variable effects on cell growth inhibitory activity against different cancer cell lines,
revealing an avenue for future optimization. An enhancement of antiproliferative activity
was observed against leukemic RS4;11 cells by the introduction of a bromine or a
methoxy group at the para-position of the phenyl ring or moving the methoxy group
from the para- to the meta-position (compounds 5j, 5o and 5p, respectively).
23

Only compounds 5j (p-Br), 5k (p-Me), 5o (p-OMe) and 5p (m-OMe) showed cell
growth inhibitory activities in the submicromolar range (IC50<1 M) against selected
cancer cell lines, with 5j, 5k and 5o as the most potent antiproliferative agents against
HT29, Jurkat and RS4;11 cells, with IC50 values of 0.5, 0.5 and 0.3 M.
Compounds 5k and 5o, the most potent antiproliferative agents against Jurkat and
RS4;11 leukemic cells, respectively, proved to be strong inhibitors of tubulin
polymerization (IC50:0.66 M for 5k, IC50:0.97 M for 5o). These findings indicate that
the p-tolyl as well as the p-methoxyphenyl at the 3-position of 5-amino-1,2,4-triazole
core are crucial moieties for inhibition of tubulin polymerization. Analogues 5j (p-Br)
and 5p (m-OMe), with antiproliferative activity at submicromolar concentrations against
RS4;11 cells, showed IC50>20 M in the tubulin polymerization assay. This, together
with the minimal cell cycle effect of 5p, implies that these derivatives act against another
target. The complete loss of antitubulin activity of the m-OMe analogue 5p as compared
with the p-OMe counterpart 5o was presumably due primarily to steric limitations
around the phenyl ring at the C-3 position of the 1,2,4-triazole core. Although
derivatives 5j and 5p were 3-fold less active as antiproliferative agents as compared with
compound 5o against RS4;11 cells, the weak tubulin polymerization inhibition of 5j and
5
p suggested that the mechanism of antiproliferative activity of these two molecules was
different from inhibition of tubulin polymerization. These results were also confirmed by
analysis of cell cycle data in which only compound 5k arrested cells in the G2/M phase,
in good agreement with the tubulin polymerization data. Removing the amino spacer
between the p-tolyl moiety and the 1,2,4-triazole ring of 4a, to furnish derivative 5k,
resulted in a substantial reduction in antiproliferative activity, while the two compounds
were equipotent as inhibitors of tubulin polymerization. Assuming similar tubulin
24

content in the different cell lines, the most reasonable explanation for these differences
is that the tubulin assay is not a good predictor of antiproliferative activity in cells.
Finally, several compounds, such as 5j, 5k, 5o and 5p, retain antiproliferative activity at
submicromolar levels toward leukemic cells, and this should permit the future design of
compounds specifically directed against leukemia.
5
. Experimental Protocols.
5
.1. Chemistry.
5
.1.1. Materials and Methods.
1
H NMR spectra were recorded on either a Bruker AC 200 or a Varian 400 Mercury
1
3
Plus spectrometer, while C NMR spectra were recorded on Varian 400 Mercury Plus
spectrometer. Chemical shifts () are given in ppm upfield from tetramethylsilane as
internal standard, and the spectra were recorded in appropriate deuterated solvents, as
indicated. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a
double-focusing Finnigan MAT 95 instrument with BE geometry. Melting points (mp)
were determined on a Buchi-Tottoli apparatus and are uncorrected. Microwave-assisted
reactions were performed on a CEM Discover SP single-mode reactor (2450 MHz).
Closed vessel experiments were carried out in capped microwave-dedicated vials (10
mL). The temperature of the reaction was monitored by an external fiber optic
temperature sensor. After completion of the reaction, the mixture was cooled to 25 °C
1
13
via air-jet cooling. All products reported showed H and C NMR spectra in agreement
with the assigned structures. The purity of tested compounds was determined by
combustion elemental analyses conducted by the Microanalytical Laboratory of the
Chemistry Department of the University of Ferrara with a Yanagimoto MT-5 CHN
25

recorder elemental analyzer. All tested compounds yielded data consistent with a purity
of at least 95% as compared with the theoretical values. All reactions were carried out
under an inert atmosphere of dry nitrogen. Standard syringe techniques were used for
transferring dry solvents. Reaction courses and product mixtures were routinely
monitored by TLC on silica gel (precoated F
Merck plates), and compounds were
54
2
visualized with aqueous KMnO . Flash chromatography was performed using 230-400
4
mesh silica gel and the indicated solvent system. Organic solutions were dried over
anhydrous Na
2
SO .
4
5
.1.2. General method A for the synthesis of compounds 7a-t.
To a stirred solution of aminoguanidine hydrogen carbonate (2.8 g, 20 mmol) in dry
pyridine (30 mL) cooled at 0 °C was added the appropriate hetero/aroyl chloride 6a-t
(
20 mol, 1 equiv.) in small portions. The reaction mixture was stirred for 30 min at 0 °C
and then overnight at room temperature. Pyridine was removed by evaporation under
reduced pressure, and the residue was dissolved with water (20 mL). The stirred solution
was cooled with an ice bath, and an aqueous solution of NaOH (2 N) was slowly added
(
pH=10-11). The resultant solid was collected by filtration, washed with ethyl ether (15
mL) and dried overnight under vacuum on P to afford compounds 7a-t. Compounds
a and 7f showed spectroscopic and analytical data in agreement with those previously
2
O
5
7
published [40]. Compounds 7e, 7f, 7h, 7i and 7o were previously published, although
neither their spectroscopic and analytical data were reported [30].
5
.1.2.1. 2-Benzoylhydrazinecarboximidamide (7a). Synthesized according to method A,
1
compound 7a was obtained as a white solid (yield 61%); mp 184-185 °C. H-NMR (d
6
-
26

DMSO) : 6.84 (bs, 2H), 6.98 (bs, 2H), 7.28 (m, 3H), 7.86 (m, 2H), 10.5 (bs, 1H). MS
+
(
ESI): [M+1] =179.25.
5
.1.2.2. 2-(Thiophene-2-carbonyl)hydrazinecarboximidamide (7b). Synthesized
according to method A, derivative 7b was obtained as a yellow solid (yield 53%); mp
1
2
7
10-212 °C. H-NMR (d
6
-DMSO) : 6.77 (bs, 4H), 6.94 (dd, J=4.8 and 3.6 Hz, 1H),
+
.33 (m, 2H), 10.4 (bs, 1H). MS (ESI): [M+1] =185.07.
5
.1.2.3. 2-(Furan-2-carbonyl)hydrazinecarboximidamide (7c). Synthesized according to
1
method A, derivative 7c was obtained as a white solid (yield 44%); mp 214-215 °C. H-
NMR (d
6
-DMSO) : 6.39 (dd, J=3.2 and 1.6 Hz, 1H), 6.57 (d, J=3.2 Hz, 1H), 6.67 (bs,
+
4
H), 7.51 (d, J=1,6 Hz, 1H), 10.4 (bs, 1H). MS (ESI): [M+1] =169.22.
5
.1.2.4. 2-Picolinoylhydrazinecarboximidamide (7d). Synthesized according to method
1
A, derivative 7d was obtained as a yellow solid (yield 54%); mp 210-211 °C. H-NMR
(
d
6
-DMSO) : 6.78 (bs, 4H), 7.62 (dd, J=6.8 and 7.0 Hz, 1H), 8.06 (m, 1H), 8.72 (d,
+
J=6.8 Hz, 1H), 9.10 (d, J=2.0 Hz, 1H), 10.3 (bs, 1H). MS (ESI): [M+1] =180.56.
5
.1.2.5. 2-Nicotinoylhydrazinecarboximidamide (7e). Synthesized according to method
1
A, derivative 7e was obtained as a yellow solid (yield 62%); mp 203-205 °C. H-NMR
(d -DMSO) : 6.86 (bs, 4H), 7.26 (m, 1H), 8.19 (dd, J=5.8 and 2.0 Hz, 1H), 8.43 (dd,
6
J=4.8 and 1.6 Hz Hz, 1H), 9.10 (d, J=2.0 Hz, 1H), 10.2 (bs, 1H). MS (ESI):
+
[
M+1] =180.24.
5
.1.2.6. 2-Isonicotinoylhydrazinecarboximidamide (7f). Synthesized according to
1
method A, derivative 7f was obtained as a yellow solid (yield 56%); mp 187-189 °C. H-
27

NMR (d
6
-DMSO) : 6.94 (bs, 4H), 7.82 (dd, J=4.4 and 1.2 Hz, 2H), 8.47 (dd, J=4.4
+
and 1.2 Hz, 2H), 10.2 (bs, 1H). MS (ESI): [M+1] =180.36.
5
.1.2.7. 2-(4-Fluorobenzoyl)hydrazinecarboximidamide (7g). Synthesized according to
1
method A, compound 7g was obtained as a white solid (yield 50%); mp 187-188 °C. H-
NMR (d
6
-DMSO) : 6.74 (bs, 2H), 6.86 (bs, 2H), 7.02 (t, J=9.0 Hz, 2H), 7.93 (dd,
+
J=9.0 and 6.2 Hz, 2H), 10.4 (bs, 1H). MS (ESI): [M+1] =197.21.
5
.1.2.8. 2-(4-Chlorobenzoyl)hydrazinecarboximidamide (7h). Synthesized according to
method A, compound 7h was obtained as a yellow solid (yield 43%); mp 188-189 °C.
1
H-NMR (d
6
-DMSO) : 6.80 (bs, 2H), 6.95 (bs, 2H), 7.30 (d, J=8.6 Hz, 2H), 7.92 (d,
+
J=8.6 Hz, 2H), 10.4 (bs, 1H). MS (ESI): [M+1] =213.19.
5
.1.2.9. 2-(3-Chlorobenzoyl)hydrazinecarboximidamide (7i). Synthesized according to
method A, compound 7i was obtained as a yellow solid (yield 38%); mp 163-165 °C.
1
H-NMR (d
6
-DMSO) : 6.77 (bs, 2H), 6.97 (bs, 2H), 7.29 (m, 2H), 7.86 (t, J=7.2 Hz,
+
1
H), 7.97 (s, 1H), 10.4 (s, 1H). MS (ESI): [M+1] =213.31.
5
.1.2.10. 2-(4-Bromobenzoyl)hydrazinecarboximidamide (7j). Synthesized according to
method A, compound 7j was obtained as a yellow solid (yield 41%); mp 199-201 °C.
1
H-NMR (d
6
-DMSO) : 6.76 (bs, 2H), 6.91 (bs, 2H), 7.43 (d, J=7.8 Hz, 2H), 7.86 (d,
+
J=7.8 Hz, 2H), 10.4 (bs, 1H). MS (ESI): [M] =257.16 and 259.26.
5
.1.2.11. 2-(4-Methylbenzoyl)hydrazinecarboximidamide (7k). Synthesized according to
1
method A, compound 7k was obtained as a white solid (yield 64%); mp 179-181 °C. H-
NMR (d
6
-DMSO) : 2.30 (s, 3H), 6.97 (bs, 4H), 7.10 (d, J=8.0 Hz, 2H), 7.79 (s, 2H),
+
1
0,.3 (bs, 1H). MS (ESI): [M+1] =193.25.
28

5
.1.2.12. 2-(3-Methylbenzoyl)hydrazinecarboximidamide (7l). Synthesized according to
1
method A, compound 7l was obtained as a pink solid (yield 67%); mp 161-162 °C. H-
NMR (d
6
-DMSO) : 2.31 (s, 3H), 7.08 (bs, 4H), 7.15 (m, 2H), 7.63 (t, J=7.6 Hz, 1H),
+
7
.77 (s, 1H), 10.1 (bs, 1H), 7.84 (bs, 2H). MS (ESI): [M+1] =193.25.
5
.1.2.13. 2-(2-Methylbenzoyl)hydrazinecarboximidamide (7m). Synthesized according
to method A, compound 7m was obtained as a white solid (yield 52%); mp 105-107 °C.
1
H-NMR (d
6
-DMSO) : 6.57 (bs, 2H), 6.72 (bs, 2H), 7.11 (d, J=7.4 Hz, 1H), 7.26 (m,
+
2
H), 7.42 (d, J=7.4 Hz, 1H), 10.1 (bs, 1H). MS (ESI): [M+1] =193.25.
5
.1.2.14. 2-(4-Ethylbenzoyl)hydrazinecarboximidamide (7n). Synthesized according to
method A, compound 7n was obtained as a yellow solid (yield 43%); mp 170-171 °C.
1
H-NMR (d -DMSO) : 1.14 (t, J=7.4 Hz, 3H), 2.58 (q, J=7.4 Hz, 2H), 6.92 (bs, 4H),
6
7
.10 (d, J=8.0 Hz, 2H), 7.81 (d, J=8.0 Hz, 2H), 10.4 (bs, 1H). MS (ESI):
+
[
M+1] =207.24.
5
.1.2.15. 2-(4-Methoxybenzoyl)hydrazinecarboximidamide (7o). Synthesized according
to method A, compound 7o was obtained as a yellow solid (yield 45%); mp 183-185 °C.
1
H-NMR (d
6
-DMSO) : 3.73 (s, 3H), 6.78 (bs, 4H), 6.84 (d, J=8.8 Hz, 2H), 7.81 (d,
+
J=8.8 Hz, 2H), 10.3 (bs, 1H). MS (ESI): [M+1] =209.22.
5
.1.2.16. 2-(3-Methoxybenzoyl)hydrazinecarboximidamide (7p). Synthesized according
to method A, compound 7p was obtained as a white solid (yield 45%); mp 169-171 °C.
1
H-NMR (d
6
-DMSO) : 3.76 (s, 3H), 6.80 (bs, 3H), 6.84 (bs, 2H), 7.17 (t, J=8.0 Hz,
+
1
H), 7.49 (s, 1H), 7.54 (d, J=8.0 Hz, 1H), 10.4 (bs, 1H). MS (ESI): [M+1] =209.10.
29