EurJMC 2015

Accepted Manuscript
A facile synthesis and microtubule-destabilizing properties of 4-(1H-
benzo[d]imidazol-2-yl)-furazan-3-amines
Andrei I. Stepanov, Alexander A. Astrat’ev, Aleksei B. Sheremetev, Nataliya K.
Lagutina, Nadezhda V. Palysaeva, Aleksei Yu. Tyurin, Nataly S. Aleksandrova,
Nataliya P. Sadchikova, Kyrill Yu. Suponitsky, Olga P. Atamanenko, Leonid D.
Konyushkin, Roman V. Semenov, Sergei I. Firgang, Alex S. Kiselyov, Marina N.
Semenova, Victor V. Semenov
PII: S0223-5234(15)00147-6
DOI: 10.1016/j.ejmech.2015.02.051
Reference: EJMECH 7733
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2014
Revised Date: 18 January 2015
Accepted Date: 27 February 2015
Please cite this article as: A.I. Stepanov, A.A Astrat’ev, A.B Sheremetev, N.K. Lagutina, N.V. Palysaeva,
A.Y. Tyurin, N.S. Aleksandrova, N.P. Sadchikova, K.Y. Suponitsky, O.P. Atamanenko, L.D. Konyushkin,
R.V. Semenov, S.I. Firgang, A.S. Kiselyov M.N. Semenova, V.V. Semenov, A facile synthesis and
microtubule-destabilizing properties of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.051.
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A facile synthesis and microtubule-destabilizing properties of 4-(1H-
benzo[d]imidazol-2-yl)-furazan-3-amines
Andrei I. Stepanov,a
Alexander A. Astrat’ev,a
Aleksei B. Sheremetev,b
Nataliya K. Lagutina,c
Nadezhda V. Palysaeva,b
Aleksei Yu. Tyurin,b
Nataly S. Aleksandrova,b
Nataliya P. Sadchikova,c
Kyrill Yu. Suponitsky,d
Olga P. Atamanenko,b
Leonid D. Konyushkin,b
Roman V. Semenov,b
Sergei I. Firgang,b
Alex S. Kiselyov,e
Marina N. Semenova,f
Victor V. Semenovb,*
a
Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint
Petersburg, 192076, Russian Federation
b
N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow,
Russian Federation
c
I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow,
Russian Federation
d
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28
Vavilov Str., 119991 Moscow, Russian Federation
e
Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology,
Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation
f
N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow,
Russian Federation
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru

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E-mail addresses:
Andrei I. Stepanov stepanoff@pisem.net
Alexander A. Astrat’ev astrchim@yandex.ru
Aleksei B. Sheremetev sab@ioc.ac.ru
Nataliya K. Lagutina mpcpr@yandex.ru
Nadezhda V. Palysaeva naduasha.85@mail.ru
Aleksei Yu. Tyurin tyurin@ioc.ac.ru
Nataly S. Aleksandrova natali.aleksandrova.50@mail.ru
Nataliya P. Sadchikova cska76@gmail.com
Kyrill Yu. Suponitsky kirshik@yahoo.com
Olga P. Atamanenko info@chemblock.com
Leonid D. Konyushkin LeonidK@chemical-block.com
Roman V. Semenov rs@chemical-block.com
Sergei I. Firgang sfirgang@yandex.ru
Alex S. Kiselyov akiselyov@chemdiv.com
Marina N. Semenova ms@chemical-block.com
Victor V. Semenov vs@zelinsky.ru

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ABSTRACT
A series of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFAs) were prepared in good yields
(60–90% for each reaction step) via a novel procedure from aminofurazanyl hydroximoyl chlorides
and o-diaminobenzenes. The synthetic sequence was run under mild reaction conditions, it was
robust and did not require extensive purification of intermediates or final products. Furthermore,
there was no need for protection of reactive moieties allowing for the parallel synthesis of diverse
BIFA derivatives. Subsequent biological evaluation of the resulting compounds revealed their anti-
proliferative effects in the sea urchin embryo model and in cultured human cancer cell lines. The
most active compounds showed 0.2–2 µM activities in both assay systems. The unsubstituted
benzene ring of the benzoimidazole template as well as the unsubstituted amino group in the
furazan ring were essential prerequisites for the antimitotic activity of BIFAs. Compound 57
bearing the 2-chlorophenyl acetamide substituent at the nitrogen atom of the imidazole ring was the
most active molecule in the examined set.
Keywords:
Benzoimidazolfurazanamines
Inhibitors of tubulin polymerization
Sea urchin embryo
Cytotoxicity
Abbreviations:
BIFA, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine;
SAR, structure-activity relationship.

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1. Introduction
Molecules exhibiting 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine (BIFA) scaffold have
attracted considerable attention of medicinal chemists in the past decade. This core is found in
multiple inhibitors of protein kinases, enzymes that represent an important class of cellular drug
targets in the treatment of hypertension, neoplastic, autoimmune, neurodegenerative, and
inflammatory diseases [1]. BIFA derivatives blocking glycogen synthase kinase GSK-3 signaling
were introduced as agents for the treatment of diabetes, Alzheimer’s disease, and as
immunomodulators [2]. BIFA-based inhibitors of p60 ribosomal S6 kinase 1 (RSK1) involved in
the cell cycle regulation [3] were suggested as promising antitumor agents [4]. BIFAs were reported
to work as potent selective modulators of ribosomal p70S6 kinase that control cell growth [5]. They
were also shown to suppress the activity of mitogen and stress-activated rho-kinase (MSK-1,
ROCK 1 and 2) [6] involved in apoptosis, cell proliferation and migration. Basilea team completed
synthesis and screening of a series of BIFAs (Fig. 1, I) [7]. Based on their cytotoxicity and
proapoptotic properties, BIFAs were proposed as agents for the treatment of various malignancies
and autoimmune disorders [7–9]. BAL27862 (Fig. 1) exhibiting low nM cytotoxicity across
multiple cancer cell lines [7,10–12] was selected for further optimization to yield a water-soluble
prodrug BAL101553 (Fig. 1) [8]. This compound is currently undergoing phase II clinical trials as
both an antimitotic and vascular targeting agent [13].
Insert Fig. 1.
N
N
N
O
N
NH
O
R1
R2
N
N
N
O
N
NH
O
NH2
CN
N
N
N
O
N
NH
O
NH
CN
NH2
O
NH2
NH
N
OH
N
H3CO
O
H3CO N
OH
O
O
O OCH3
H
Vinblastine
H3CO
H3CO
H3CO
OCH3
O
NH
O
Colchicine
I
BAL27862
BAL101553
Fig. 1. Structures of reported BIFAs and reference compounds colchicine and vinblastine.
Mechanism of BIFAs anti-proliferative activity has been tied to microtubule impairment.
For example, BAL27862 caused unique alterations of interphase and mitotic spindle microtubules
in cultured cancer cells [10,15]. The compound was shown to inhibit purified tubulin
polymerization and to bind tubulin dimers at the colchicine site [16]. Despite of this molecular
interaction, the specific effect of BAL27862 on microtubule dynamics differed from those of
colchicine and vinblastine suggesting its novel microtubule destabilizing mode of action [16]. It
should be noted that of the reported BIFAs the anti-tubulin mechanism was proved only for

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BAL27862. The indirect anti-tubulin effect of BIFAs could be also mediated by GSK-3 known to
affect microtubule stability, mitotic spindle formation and orientation [14].
Due to the diverse biological activity of BIFAs, we developed a robust protocol yielding a
library of respective derivatives and evaluated their microtubule destabilizing activity using the in
vivo sea urchin embryo model. Selected compounds were also studied in vitro using tubulin
polymerization assay, cell cycle distribution analysis, and further screened against a panel of human
cancer cell lines to assess their cytotoxicity.
BIFA scaffold 9 was first reported by Tselinskii et al. in 2001 [17]. To date, there are three
main routes towards 9 described in the literature (Scheme 1). Route A [17] involves treatment of
(un)substituted o-phenylenediamines 3 with carbimidate 2 [18] easily accessible from the
amidoxime 1. Route B [6] is based on recyclization of 5 upon heating with o-phenylenediamines 3
in acetic acid. Route C [2,8] employs condensation of 3 with ethylcyanoacetate at high temperature
to yield 6 [19] followed by its sequential conversion to cyano oxime 7, amidoxime 8 and finally, the
targeted aminofurazan derivative 9 [20–22].
Insert Scheme 1.
CH2(CN)2
NC CN
NOH NN
O
NH2
NH2
NOH
NN
O
NH2 CN
NN
O
NH2
MeO
NH
N
NMe
OH
NH2
N
NMe
OH
NH2
NO
N
N N
O
N
Me
OH
NH2
NHR
e
g
1
3 8
2
5
3
4
6 7
9
Route A
Route B
Route C
h
a b c d
N
N CN
R2
N
N CN
R2
NOH N
N
R2
NOH
NOH
NH2
3
NH2
NHR
R1
N
N
R2
N
O
N
NH2
fa
a b
e
R1
R1R1
R1R1
Scheme 1. Reported syntheses of BIFA scaffold. Reagents and conditions: (a) NaNO2, H+
; (b)
NaOH, NH2OH⋅HCl, H2O, reflux [18]; (c) Pb2O3, AcOH; (d) MeOH, HCl or MeOH, MeONa; (e)
o-phenylenediamine 3, EtOH, reflux, 20 h; (f) Pb(OAc)4, AcOH, r.t.; (g) NCCH2CO2Et, 100–190
°C; (h) NaOH, H2O, reflux.
All three strategies summarized in Scheme 1 afford poor yields of the targeted compounds
9. They require elevated temperatures, extensive purification of intermediates, convoluted synthetic
manipulations and toxic or costly reagents. In this study, we developed a robust and rapid access to

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a library of diverse BIFAs. The described protocol does not employ anhydrous solvents, inert
atmosphere, toxic chemicals and chromatographic purification of the targeted molecules.
2. Results and discussion
2.1. Chemistry
Benzimidazoles are an important class of biologically active compounds [23–25]. A plethora
of methods exist for their syntheses, including condensation of hydroximoyl chloride and
unsubstituted o-diaminobenzene. This procedure was originally described by Sasaki et al. for aryl
hydroximoyl chloride [26] and further expanded by Paton et al. onto carbohydrate-derived
hydroximoyl chloride [27,28]. Considering these results, we turned our attention to optimize the
reaction of o-diaminobenzenes 3 and 4-aminofurazan-3-carbohydroximoyl chloride 10 [29,30]
(Scheme 2), easily available from amidoxime 1 and sodium nitrite in HCl. We found that the
reaction of 10 with o-diaminobenzene (1:2 molar ratio) in ethanol at 60 °C for 0.5 h afforded the
benzoimidazole 9a in 81% yield after work-up and recrystallization. Using these conditions, we
evaluated the reaction of hydroximoyl chloride 10 with a variety of o-diaminobenzenes 3a–n
(Scheme 2). The desired BIFA derivatives 9a–n were obtained in moderate to good yields for both
electron-rich and electron-deficient o-diaminobenzenes 3a–n. Condensation of 10 with o-Me-
substituted o-diaminobenzenes 3c,d furnished 9c,d as a mixture of two isomers exhibiting a defined
proton position(s) at different N-atoms in the imidazole ring. The existence of isomers was further
confirmed by a doubling of signals in 1
H and 13
C NMR spectra.
N-substituted o-diamines 3o,p were also effective reagents in condensation with
hydroximoyl chloride 10 (Scheme 2) to afford 26 and 29. BIFAs 9a,h,i were condensed with 2,5-
dimethoxytetrahydrofuran to afford pyrrole derivatives 11a,h,i (Scheme 2).
Insert Scheme 2.

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R1 R2 R4
63: H Et pMeO-C6
H4
-
64: Cl H C6H5-CH2-
c
d e
NN
O
NH2
Cl
NOH
10
NN
O
NH2
NH2
NOH
1
NN
O
NHR4
Cl
HON
62v,w
NN
O
NH2
R4HN
NOH
60v-x
NN
O
NHR4
NH2
HON
61v-x
59v-x
R4NH2 59-70%
78-82% 60-87%
N
H
N
N
O
N
NH2
9a-n
NH2
NHR2
3o,p
12'-57'
R2Hal
NH2
NH2
3a-n
54-92%
a
70-90%
b
a
R3COCl or (R3CO)2O
58r-u
N
N
R2
N
O
N
NH2
12-57
g
70-80%
N
N
R2
N
O
N
NH
R3
O
14r,16s,19r,20r,24r,
25r,26t,28r,32r,34s,
38r,39r,u,40r,51t
R1
N
N
R2
N
O
N
N
11a,h,i
O OMeMeO
g
R1
R1
R1
R1
R1
3f,q
NH2
NHR2
R1
N
N
R2
N
O
N
R4HN
R1
f
59-70%
o-Phenylenediamines (3a-q)
NH2
NH2
NH2
NH2
F
a e
NH2
NH2
Cl
f
NH2
NH2
MeONH2
NH2
Cl
hg
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
MeO
MeOb c d i
NH2
NH2
NC NH2
NH
F3C
OMe
NH2
NH2
NH2
NH2
NO
N
NH2
NH2
MeOOC
k l m n
NH2
NHMe
NH2
NHEt
p qo
NH2
NH2
HOOC
j
Acid anhydrates or chloroanhydrates R3COCl, (R3CO)2O (58r-u)
u
N
Cl
O
F
Cl
O
ts
O
EtOEt
O
r
Cl
O
Me
O
MeOMe
O
w
Amines R4NH2 (59v-x)
NH2
MeO MeO
NH2
x
NH2
v
Cl
O
Et
Scheme 2. Facile synthesis of BIFAs. Reagents and conditions: (a) hydroximoyl chloride 10, o-
diaminobenzene 3, EtOH, 60 °C, 0.5 h; 5 h for 9c; (b) R2Hal, K2CO3, DMF, 50−90 °C, 4–12 h; (c)
R4NH2, NEt3, EtOH, i-PrOH, r.t., 3 h; (d) KOH, (CH2OH)2, reflux, 4 h; (e) NaNO2, HCl, AcOH,
≤10 °C, 3 h, r.t., 1 h; (f) o-phenylenediamine 3f,q, EtOH, reflux, 0.5 h, r.t., 1 h; (g) R3COCl,
toluene, reflux, 8–20 h; (R3CO)2O, AlkCOONa, reflux, 3 h.
Alkylation of scaffold 9 proceeded regioselectively at a nitrogen atom of the benzimidazole
ring, whereas the amino group in the furazan ring was not affected. N-substituted benzimidazoles
12–57 (Table 1) were prepared from the unsubstituted precursors 9a–n and respective alkyl halides
12′′′′–57′′′′ (Scheme 2). BIFAs 12–57 exhibiting free amino group in the furazan ring were coupled
with the appropriate acid chlorides or anhydrides 58r–u to form amides 14r, 16s, 19r, 20r, 24r,
25r, 26t, 28r, 32r, 34s, 38r, 39r,u, 40r, and 51t (Scheme 2; Table.1).

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Aminofurazans 63 and 64 were synthesized via the recyclization route (Scheme 2).
Treatment of hydroximoyl chloride 10 with amines 59v–x in the presence of NEt3 yielded the
corresponding amidoximes 60v–x. These were converted to the isomeric amidoximes 61v–x via a
recyclization reaction [31–33] in refluxing ethylene glycol with KOH. The corresponding
hydroximoyl chlorides 62v,w were synthesized via deazotization of 61v,w with sodium nitrite in
HCl. Condensation of the hydroximoyl chloride 62v,w with 1,2-diaminobenzenes in ethanol at 60
°C for 1 h afforded the desired benzoimidazoles 63 and 64 in moderate yields. Cyclization
conditions were similar for substituted (3o–q) and unsubstituted (3a–n) o-phenylenediamines
(Scheme 2, a and f).
Treatment of compound 9a with chloroacetonitrile gave cyanomethyl derivative 40. It was
reacted with sodium azide in hot DMF to give tetrazole 65. Alternatively, the nitrile group of
compound 40 was coupled with thiosemicarbazide at reflux in trifluoroacetic acid to yield of 2-
amino-1,3,4-thiadiazole derivative 66 (Scheme 3).
Insert Scheme 3.
N
N
N
O
N
NH2
CN
N
N
N
O
N
NH2
N N
NH
N
N
N
N
O
N
NH2
N N
S
NH2
a b
63% 71%
4065 66
Scheme 3. Synthesis of N-(hetarylmethyl) benzoimidazol derivatives 65 and 66. Reagents and
conditions: (a) NaN3, NH4Cl, DMF, 100 °C, 8 h; (b) H2NC(S)NHNH2, CF3COOH, reflux, 8 h.
The structures of all synthesized products were confirmed by spectroscopy. Both 1
H and 13
C
NMR data were consistent with the presence of furazan and benzimidazole moieties. Specifically,
for the N-unsubstituted benzimidazole group, there were two distinct signals at ca. 138 ppm and ca.
124–130 ppm for C-2 and C-5/6 respectively, notably signals corresponding to other carbon atoms
were broadened and overlapped. This phenomenon has been described for benzimidazoles earlier
[34]. It was explained by the rapid proton exchange between N-1 and N-3 atoms. Signals at ca. 140
(C-C-NH2) and ca. 155 ppm (C-NH2) were attributed to carbons of the furazan ring [31,35].
Structures of benzimidazoles 27 and 29 were unequivocally established by X-ray crystallography
(Fig. 2). Experimental details are given in Supplementary data.
Insert Figure 2.

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Fig. 2. Molecular structure of compounds 27 and 29 showing the atom numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
The N–O bonds in the furazan ring of both structures showed different length (N1–O1 of
1.40–1.41 Å, N2–O1 of 1.37–1.38 Å) affected by substituents [36]. Crystal packing of 27 and 29
and the details of X-ray data collection are presented in Fig. S2 and Table S1, Supplementary data
[37].
2.2. Biological effects
2.2.1. Antiproliferative activity in the sea urchin embryo model
The synthesized BIFA analogs were evaluated for their antiproliferative activity using in
vivo phenotypic sea urchin embryo assay [38]. This assay has been extensively validated in our lab
to afford a reliable insight into specific antimitotic, cytotoxic, and microtubule destabilizing effects
of tested compounds. A typical experimental protocol includes (i) fertilized egg test for antimitotic
activity displayed by cleavage alteration/arrest, and (ii) swimming pattern observation of blastulae
treated by compounds after hatching. The lack of forward movement, settlement to the bottom of
the culture vessel, and rapid spinning of embryos around the animal–vegetal axis suggests a
microtubule destabilizing activity caused by a molecule (video illustrations are available at
http://www.chemblock.com). The attainment of specific tuberculate shape of arrested eggs, which is
typical for microtubule destabilizing agents, is considered an indirect evidence of targeting
tubulin/microtubules [38–40]. The test results are listed in Table 1. Colchicine and vinblastine
sulfate served as reference microtubule destabilizing compounds.
Insert Table 1.

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Table 1.
Structures of BIFAs and their effects on sea urchin embryos and human cancer cells.
Compd R1 R2 R3
Sea urchin embryo effects, EC (µM)a
NCI60 screen
Cleavage
alteration
Cleavage
arrest
Embryo
spinning
Mean GI50,
µMb
Mean cell
growth, %c
Colchicine 50 100 TEd
50 0.132e
Vinblastine 0.1 0.2 TEd
2 0.00137f
BAL 27862 0.0065–0.017g
9a H H – 2 >4 >4 102.97
11a H H R3C(O)NH=Pyrrole >4 >4 >4 104.11
9b 5-Me H – NDh
9c 4-Me H – NDh
9d 4,5-diMe H – NDh
9e 5-F H – 2 >4 >5 97.83
9f 5-Cl H – 2 >4 >5 97.13
R3COCl or (R3CO)2O
58r-u
9-57
N
N
R2
N
O
N
NH
R3
O
14r,16s,19r,20r,24r,
25r,26t,28r,32r,34s,
38r,39r,u,40r,51t
R1N
N
R
N
O
N
H2N
2
1
R
2
4
7
6
5
1
3O
OMeOMe
N
N
R2
N
O
N
N
11a,h,i
R1

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9g 4-Cl H – 1 >4 >4 99.8
9h 5-OMe H – 0.5 >4 >5 101.01
11h 5-OMe H R3C(O)NH=Pyrrole 2 >4 >4 104.43%
9i 4,7-diOMe H – 2 >4 >4 NDh
11i 4,7-diOMe H R3C(O)NH=Pyrrole NDh
9j 5-COOH H – NDh
9k 5-COOMe H – NDh
9l 5-CN H – >4 >4 >4 107.07%
9m 6
5
H – 0.5 4 >4 NDh
9n
NO
N
6
7
H – NDh
12 H OH – NDh 102.07
13 H CH – >4 >4 >4 99.56
14 H CH2 – NDh
14r H CH2 Me >4 >4 >4 96.53
15 H
F
– 1 >4 >4 3.09
16 H
F
– 0.5 >4 >4 NDh

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12
16s H
F
Et 4 >4 >4 7.76
17 H
F
– 2 >4 >4 8.91
18 H
Br
– 0.5 >4 2 0.603
19 H
F3C
– 2 >4 >4 0.676
19r H
F3C
Me >4 >4 >4 82.18
20 H
MeO
– 0.2 2 TEd
>5 0.457
20r H
MeO
Me 1 >4 >4 3.98
21 H
MeO
Me
– 0.2 2 TEd
>4 0.479
22 H
EtO
EtO
– >4 >4 >4 80.97
23 H
Me
– NDh
70.38
24 H
t-Bu
– >4 >4 >4 67.38

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13
24r H
t-Bu
Me NDh
73.48
25 H
NC
– 4 >4 >4 79.81
25r H
NC
Me >4 >4 >4 94.08
26 H Me – NDh
26t H Me
F
NDh
99.5
27 H Et – NDh
28 H
MeO
– NDh
28r H
MeO
Me >4 >4 >4 84.96
29 5-CF3
MeO
– >4 >4 >4 81.06
30 H
Cl
– NDh
3.39
31 H
Cl
– >4 >4 >4 84.97
32 H
Cl
Cl
– NDh

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32r H
Cl
Cl
Me NDh
76.68
33 H
F
Cl
– >4 >4 >4 91.9
34 H
F
Cl
– NDh
34s H
F
Cl
Et >4 >4 >4 64.55
35 H
Cl
O
O
– >4 >4 >4 88.34
36 H
Br
O
O
– >4 >4 >4 86.07
37 H – 0.2 2 TEd
2 NDh
38 H
N
– 2 >4 >4 89.31
38r H
N
Me >4 >4 >4 103.6

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39 H N
– NDh
96.96
39r H N
Me >4 >4 >5 101.38
39u H N N
>4 >4 >4 97.07
40 H NC – >4 >4 >4 102.45
40r H NC Me NDh
65 H
NN
NH
N – >4 >4 >4 102.74
66 H
NN
SNH2
– NDh
41 H N
N
N
NH2
NH2
– >4 >4 >4 101.61
42 H N
N
N
NH2
NMe2
– 2 >4 >4 80.77
43 H
N
N
N
NH2
N
– 4 >4 >4 87.71

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44 H
NH
O
Me
Me
– >4 >4 >4 108.54
45 H
NH
O
Me
Me
– >4 >4 >4 90.2
46 H
NH
O
MeO
OMe
MeO
– 1 4 TEd
>5 9.77
47 H
NH
O
F
F
– 1 >4 >4 3.98
48 H
NH
O
F
F
– 1 4 >4 3.55
49 H
NH
O
F
Cl
– 2 >4 >4 70.49
50 H
NH
O
Cl
MeO
– >4 >4 >4 100.44
51 H
NH
O
Me
F
– NDh
51t H
NH
O
Me
F
F
NDh
71.77
52 H
NH
O
O
F3C – 4 >4 >4 104.79

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53 H
NH
O
O
O
– 2 4 >4 80.78
54 H
NH
O
Me
NHS
NH
– >4 >4 >4 NDh
55 H NH
F
O
– 4 >4 >4 101.92
56 H
NH
O
Cl
– 4 >4 >4 NDh
57 H
NH
O
Cl
– 0.05 0.5 TEd
>5 0.24
57h 5-OMe
NH
O
Cl
– 2 >4 >4 NDh
57h′′′′ 6-OMe
NH
O
Cl
– >4 >4 >4 NDh
57i 4,7-diOMe
NH
O
Cl
– Not tested due to poor solubility
a
The sea urchin embryo assay was conducted as described previously [38]. Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values.
b
GI50: concentration required for 50% cell growth inhibition.
c
Cell growth percent at 10 µM concentration.

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d
TE: tuberculate eggs typical for microtubule destabilizing agents.
e
NCI60 screen data for colchicine NSC 757. For the structure see Fig. 1.
f
NCI60 screen data for vinblastine NSC 49842. For the structure see Fig. 1.
g
Ref. [11].
h
ND: Not determined.

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As evidenced from the data, compounds 18 and 37 exhibited antimitotic activity at ca. 0.2–
0.5 µM concentrations. This effect was likely related to their microtubule destabilizing activity, as
suggested by embryo spinning. Compounds 20, 21, 46, and 57 were also considered to target
tubulin/microtubules. This conclusion was based on the observation that the arrested sea urchin
eggs acquired the tuberculate shape typical of the microtubule destabilizers [38–40]. The additional
24 molecules, including 16, 9h, and 9n, that induced cleavage abnormalities at 0.5–4 µM
concentration but failed to cause both cleavage arrest and embryo spinning, were classified as
tubulin independent antiproliferative agents.
It is worth noting that the most active BIFAs were endowed with the unsubstituted phenyl
ring in benzimidazole fragment and aminofurazan moiety (R1 = R3 = H). For the R1 substituents, the
maximal activity was observed for R1 = 5-MeO. For derivatives with R2 = R3 = H, the activity order
was as follows: 5-MeO (9h) = naphtyl (9m)> 4-Cl (9g) > H (9a) = 5-F (9e) = 5-Cl (9f). For the
analogs 57, the activity decreased in the order: R1-unsubstituted 57 >6-MeO (57h′)>5-MeO (57h),
where 57h was inactive up to 4 µM. For the R2 substituents the order of activity was as follows: R1
= R3 = H; R2 = benzyl, m-MeO (20) = m-MeO-p-Me (21) = o-naphtyl (37) > m-F (16) = m-Br (18) >
o-F (15) > p-F (17) > p-CN (25). Compounds substituted with m,p-diEtO (22), p-t-Bu (24), p-Cl
(31), o-Cl-methylenedioxy (35), o-Br-methylenedioxy (36) in R2 = benzyl were inactive in the
assay. Notably, meta-substitution in R2 with MeO or Hal group was favorable to compound activity.
In contrast, presence of bulky groups especially in para-position was deteriorating. Similarly,
compounds with R2 = alkyl showed no activity, as evidenced by 13, 14r, 40, and 63. Heterocyclic
derivatives with R1 = H; R2 = -CH2-pyridin were inactive up to 4 µM (38r, 39r, and 39u) except for
38 that exhibited antiproliferative effect at 2 µM. For arylacetamide derivatives 44–57 (R2 = -CH2-
CO-NH-Ar) compound 57 substituted with o-Cl-phenyl group was the most active microtubule
destabilizer. There was a significant reduction in activity for the related p-Cl-phenyl analog 56. In
evaluating the effect of R3 substitution (aminofurazane fragment), we found that the best activity
was exhibited by the unsubstituted compounds. Specifically, the NH2 derivatives were more active
than the respective pyrrole analogs (9a vs 11a; 9h vs 11h). It is worth noting that as opposed to
BIFA, their regioisomers 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans I (Fig. 3) were generally
inactive in the sea urchin embryo assay. However, the respective 3-pyrrole-substituted furazans II
were reported to be potent antimitotic microtubule destabilizing agents [41].
Insert Figure 3.
N N
N
N O
N
NH2
N N
N
N O
N
N
(AlkO)n
I
(AlkO)n
II

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Fig. 3. Structures of 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans [41].
Furthermore, unsubstituted NH2 derivatives were generally more active than the respective
N-substituted analogs including NHAc (19 vs 19r; 20 vs 20r; 25 vs 25r; 38 vs 38r). Similarly, NH2
derivative was more potent that the COEt compound (16 vs 16s). Thus, the replacement of NH2 in
furazan ring with pyrrole, acetyl- or propionamide groups resulted in a marked decrease or loss of
the antiproliferative effect. In summary, our assay data suggested that for the synthesized series of
BIFAs both the unsubstituted phenyl ring in benzimidazole and the unsubstituted amino group of
aminofurazan were essential for the antimitotic activity. In contrast, the nature of R2 was critical to
the microtubule destabilizing mode of action. For example, 9h and 9m with R2=H altered cell
division in the sea urchin embryos at submicromolar concentrations likely via a tubulin-independent
manner (Table 1).
2.2.2. In vitro cytotoxicity
The sea urchin embryo test results for the derivatives of 9 correlated well with their
cytotoxicity against a panel of human cancer cell lines (NCI60 anticancer drug screen) (Table 1).
Compound 57 was the most active in both assays. All potent molecules that exhibited GI50 less than
1 µM (18, 19, 20, 21, and 57) featured the unsubstituted phenyl ring in the benzimidazole fragment
and the unsubstituted amino group in the aminofurazan ring. The NCI60 screen mean graphs for
these compounds are presented in Supplementary data, Figures S3–S7. These SAR results are in
agreement with the cytotoxicity and apoptosis induction data reported previously for the series of
related furazanobenzimidazoles [7]. Four human cancer cell lines, namely melanoma MDA-MB-
435, CNS cancer SF 539, renal cancer RXF 393, and ovarian cancer OVCAR-3 cells were the most
sensitive to 57 (Table 2). Notably, in melanoma MDA-MB-435 cells, this compound caused total
cell growth inhibition and 50% reduction of cell number at concentrations of 0.088 µM and 0.84
µM, respectively. In addition, 57 displayed higher cytotoxicity against NCI/ADR-RES multidrug
resistant ovarian cancer cells over expressing P-glycoprotein than against the parent OVCAR-8 cell
line (Table 2).
Insert Table 2

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Table 2.
Effects of compound 57 on human cancer cells
Panel Cell line
57 Vinblastinea
GI50, µMb
TGI, µMc
LC50, µMd
GI50, µMb
TGI, µMc
LC50, µMd
Melanoma MDA-MB-435 0.028 0.088 0.84 0.00025 0.00025 0.001
Renal cancer RXF 393 0.087 0.366 19.2 0.001 0.05 2.51
CNS cancer SF 539 0.152 0.511 16.9 0.0006 0.0025 0.79
Ovarian cancer OVCAR-3 0.122 0.427 8.05 0.0003 0.0016 0.5
OVCAR-8 0.371 >100 >100 0.0016 0.32 2
NCI/ADR-RESe
0.184 16.0 >100 0.1 0.79 1.58
a
NCI60 screen data for vinblastine NSC 49842.
b
GI50: concentration required for 50% cell growth inhibition.
c
TGI: concentration required for total (100%) cell growth inhibition.
d
LC50: concentration required for 50% reduction in cell number.
e
NCI/ADR-RES: P-glycoprotein-overexpressing multi-drug resistant cell line derived from OVCAR-8.

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2.2.3. Inhibition of tubulin polymerization and cell cycle analysis
Compounds 16, 18, 19, 37, and 57 that caused pronounced cleavage alteration/arrest in the
sea urchin embryos were evaluated for their anti-tubulin properties using in vitro inhibition of
purified tubulin polymerization assay [42] (Table 3). As indicated in Table 1, compounds 18 and 37
induced sea urchin embryo spinning suggestive of their direct microtubule destabilizing activity.
These molecules also inhibited tubulin polymerization (Table 3). Compound 57 that caused
formation of tuberculate eggs typical for microtubule destabilizers showed IC50 of 7.36 µM in in
vitro tubulin polymerization assay. Derivative 16 altered sea urchin embryo cleavage at 0.5 µM
(Table 1) whereas the inhibition of tubulin polymerization was observed with the IC50 value of 7.9
µM. Compound 19 was a less potent in vitro tubulin inhibitor. Correspondingly, it exhibited only
moderate antiproliferative activity in the sea urchin embryo assay.
Insert Table 3.
Table 3. Tubulin polymerization inhibition of selected BIFAs.
Compd ITP IC50, µMa
16 7.9
18 2.04
19 13.27
37 7.03
57 7.36
Vinblastineb
0.6
a
ITP IC50: concentration required for 50% inhibition of in vitro tubulin polymerization.
b
Data from [43].
Active BIFA derivatives 16, 18, 19, 37, and 57 were further tested for their effects on cell
cycle distribution in the mouse fibroblast 3T3 cell line at 1 µM concentration. Molecules 19 and 57
showed G2/M arrest confirming their anti-tubulin mode of action. These compounds were found to
induce G2/M block in human epidermoid carcinoma A431 cell line. Namely, A431 cells were
treated with 1 µM of 19 and 57 for 24 hrs followed by flow cytometry analysis to display induction
of cell cycle arrest of ca. 55% and 60% (percent of cells in G2/M phase, average of 2 experiments
with SD < 15%). A subsequent dose-response studies for 19 and 57 yielded EC50 values (the
compound concentration that causes 50% cells to arrest, average of 3 experiments with SD < 15%)
of ca. 1 µM and 0.5 µM, respectively. Once again, these data correlated well with the sea urchin
embryo data and NCI60 GI50 values (0.767 µM and 0.24 µM, respectively, Table 1). In our hands,
BIFAs were consistently less cytotoxic than BAL 27862. The sea urchin embryo assay data

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provided insight into the structure-activity relationship for these new series and further confirmed
the micritubule-destabilizing mechanism of their activity.
3. Conclusions
In summary, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFA) were prepared in good
yields using a novel robust procedure from aminofurazanyl hydroximoyl chlorides and o-
diaminobenzenes (Scheme 2). As opposed to the reported sequences, the developed protocol used
mild reaction conditions and accommodated a wide variety of functional groups to afford a diverse
array of targeted compounds. Furthermore, our approach to BIFAs requires neither protection of the
reactive moieties nor chromatographic purification of the respective intermediates. A subsequent
biological evaluation of the resulting library using the sea urchin embryo model and human cancer
cell lines revealed the antiproliferative effect of several derivatives. The activity of BIFAs in our
assay systems could be attributed to both direct microtubule destabilization and tubulin independent
mechanisms. The unsubstituted phenyl ring of benzoimidazole moiety as well as the unsubstituted
amino group in the furazan ring were essential prerequisites for the antimitotic activity of BIFAs.
The most active compound 57 was substituted with the 2-chlorophenyl acetamide moiety at the N
atom of the imidazole fragment. The potent synthetically feasible tubulin-targeting BIFA series will
be further evaluated as lead candidates for in vivo experiments.
4. Experimental protocols
4.1. Chemistry and chemical methods
Elemental microanalyses were obtained on an Perkin-Elmer 2400 CHN analyzer. Mass
spectra were collected on the Varian MAT-CH-6 spectrometer with direct sample injection at an
ionization voltage of 70 eV. IR spectra were recorded on IFS-113v Bruker in KBr pellets (1:200);
the frequencies were expressed in cm−1
. The 1
H NMR spectra were recorded on Bruker DRX-500
(500 MHz) and Bruker AM-300 (300 MHz) using internal standard with DMSO-D6 as the solvent;
the chemical shifts were reported in ppm (δ) and coupling constants (J) values were given in Hertz
(Hz).
The 13
C, and 15
N NMR spectra were recorded on Bruker AM-300 at 75.47 and 50.7 MHz,
respectively. Melting points were measured on a Kofler bench. Completion of the reactions and
purity of the obtained products were monitored by thin layer chromatography on the Silufol UV-
254 plates using hexane-acetone mixture (5:3) as an eluent and iodine vapor as a stain.
4.1.1. General procedure for synthesis of benzimidazoles 9a–n from hydroximoyl chloride 10
Hydroximoyl chloride 10 (0.16 g, 1 mmol) was added slowly by small portions to a solution
of the appropriate 1,2-diaminobenzene 3 (0.3 g, 1.5 mmol) in ethanol (5–10 mL) at room

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temperature. The mixture was stirred at reflux for 0.5 h followed by an additional hour at room
temperature, diluted with water (10 mL) and 0.1 M aq HCl (5 mL). The heterogeneous mixture was
stirred for 1 h. The precipitate was filtered, washed with water, and recrystallized from iPrOH/H2O.
4.1.1.1. 3-Amino-4-(1H-benzimidazol-2-yl)-furazan (9a). White solid; yield 0.16 g (80%); mp 268–
269 °C (lit. [17] 264–265 °C); 1
H NMR (DMSO-d6, 500 MHz): 6.84 (s, 2H, NH2), 7.31 (t, J = 7.3
Hz, 1H, H-5), 7.37 (t, J = 7.8 Hz, 1H, H-6), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.81 (d, J = 7.3 Hz, 1H,
H-4), 13.69 (s, 1H, NH); 13
C NMR (DMSO-d6): 112.6 (br), 120.2 (br), 123.8 (br), 135.0 (br), 139.1,
140.8, 143.2 (br), 156.1; EIMS m/z 201 [M]+
(20), 144 (100), 143 (29), 118 (42), 116 (11), 92 (18),
90 (20), 77 (6), 63 (35); Anal. Calcd for C9H7N5O: C 53.73; H 3.51; N 34.81. Found: C 53.61; H
3.47; N 34.93; IR (KBr): ν max 3406, 3303, 1635, 1621, 1604, 1561, 1495, 1459, 1423, 1322, 1278,
1125, 1012, 1000, 955, 900, 864, 748, 732, 697, 611.
4.1.1.2. 3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c).
Ratio of isomers 3:2.
White solid; yield 0.16g (73%); mp 213–216 °С; 1
H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H,
Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7),
7.38, 7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13
C NMR (DMSO-d6): 16.3,
17.1, 109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2,
142.3, 142.6, 155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18;
N 32.47; IR (KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268,
1239, 1157, 1138, 1005, 949, 899, 874, 748, 671, 561.
4.1.1.3. 3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d).
White solid; yield 0.211g (92%); mp 242–243 °C; 1
H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H,
Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43
(br. s, 2H, NH); 13
C NMR (DMSO-d6): 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2,
127.5, 129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for

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C11H11N5O: C 57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425,
3284, 3202, 1634, 1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741,
717, 661, 631, 561, 501.
4.1.2. General procedure for the synthesis of pyrroles 11a, 11h, and 11i
2,5-Dimethoxytetrahydrofuran (1.7 mmol) was added to a slurry of respective BIFAs (9a,
9h, 9i) (1.7 mmol) in AcOH (3 mL) at room temperature. The mixture was refluxed for 15 min,
cooled to room temperature, and the resulting suspension was stirred for 1 h. The precipitate was
ﬁltered, washed with ice water (2×5 mL) followed by 70% iPrOH (2×5 mL) and dried.
Crystallization of the crude product from 70% iPrOH afforded pure pyrroles 11a, 11h, and 11i
(white solids, 70–90% yield).
4.1.3. General procedure for alkylation of 9a–n
4.1.3.1. Alkylation by benzylhalides 12′′′′–39′′′′ and 41′′′′–43′′′′
A mixture of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a–n (0.01 mol), benzylhalide
12′′′′–43′′′′ (0.011 mol), and K2CO3 (1.52 g, 0.011 mol), in dry DMF (30 mL) was stirred at 80–90 °C
for 4–5 h (reflux condenser was used for volatile benzylhalides). The mixture was cooled to room
temperature and diluted with water (100 mL). The precipitate was filtered and recrystallized from
acetic acid. Yields of 12–39 and 41–43 were 70–90%.
4.1.3.2. Synthesis of [2-(4-amino-furazan-3-yl)-benzoimidazol-1-yl]-acetonitrile 40
BIFA 9a (2g, 0.01 mol) was alkylated by ClCH2CN (1.27 g, 0.02 mol) as described in the
following procedure 4.1.3.3 at ≤50 °C. The product was isolated and recrystallized from
EtOH:AcOH 3:1 v/v to yield 1.56 g (65%) of the targeted nitrile 40 as white solid.
4.1.3.3. Alkylation by N-aryl-acetamides 44′′′′–57′′′′
Alkylation by N-Aryl-acetamides 44′′′′–57′′′′ and separation of products was conducted as
alkylation by benzylhalides as described in 4.1.2.1., however at lower temperature (60 °C) and
increased reaction time (8–12 h). The precipitate was crystallized from acetic acid or mixture of
acetic acid–DMF (10–30% by volume) to give 44–57 (white solid, 70–90% yield).
4.1.3.4. Synthesis of isomeric 2-[2-(4-aminofurazan-3-yl)-5(6)-methoxy-1H-benzimidazol-1-yl]-N-
(2-chlorophenyl)acetamides 57h and 57h′′′′

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A mixture of 3-amino-4-(6-methoxy-1H-benzimidazol-2-yl)-furazan 9h (0.23 g, 0.001 mol),
2-chloro-N-(2-chlorophenyl)acetamide 57′′′′ (0.222 g, 0.0011 mol), K2CO3 (0.152 g, 0.0011 mol),
KBr (0.03 g), and glym (5 mL) was stirred at reflux for 9 h. The mixture was cooled to room
temperature and diluted with water (50 mL). The precipitate was filtered and washed with water (30
mL) to give a mixture of the respective isomers 57h and 57h′′′′. White solids, 91% yield (0.364 g);
mp 252–253 °C. Pure isomers were isolated by fractional crystallization from MeCN. Specifically,
isomer 57h′′′′ exhibited lower solubility in MeCN and was obtained in 38% yield (0.14 g). More
soluble isomer 57h was isolated in 46% yield (0.17 g).
4.1.4. Acylation of 4-(1-R-1H-Benzoimidazol-2-yl)-furazan-3-yl-amines
4.1.4.1. Acylation by anhydrates of aliphatic carbonic acids 58r,s
Corresponding BIFA (0.01 mol) was added to the neat acetic or propionic anhydrate (20
mL) and refluxed for 3 h in presence of 5 mmol of dry MeCO2Na or EtCO2Na, respectively. The
mixture was cooled to room temperature and diluted with water (100 mL). After a day at room
temperature, the precipitate was filtered and recrystallized from DMF–EtOH to yield 80% of the
desired products as white solids.
4.1.4.2. Acylation by chloroanhydrates of aromatic carbonic acids (aroylchloroanhydrates) 58t,u
i) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015
mol) in toluene (50 mL) was refluxed for 8–20 h until the evolution of HCl stopped. Toluene was
evaporated in vacuo, and the residue was recrystallized from DMF–EtOH to yield 80% of the
desired products as white solids.
ii) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015
mol) in freshly distilled pyridine (30 mL) was refluxed for 3 h, cooled, and diluted with water (100
mL). After 24 h the residue was filtered and recrystallized from DMF–EtOH to afford targeted
products as white solids (70% yield).
Crude alkylating products 14, 28, 32, 34, and 51 were filtered and without purification
acylated to afford 14r, 28r, 32r, 34s, and 51t.

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4.1.5. 4-Aminofurazan-3-carbox-N-(p-methoxyphenyl)amidoxime (60v). A solution of 4-
aminofurazan-3-carbohydroxymoyl chloride 10 (8.1 g, 50 mmol) in EtOH (100 mL) was added
drop wise to a solution of p-anizidine 59v (11.9 g, 97 mmol) and NEt3 (6 g, 60 mmol) in iPrOH (50
mL) at 0 °C over 10 min. The mixture was stirred at room temperature for 3 h, the solvent was
removed in vacuo and the residue was treated with 80 mL of water. The precipitate was filtered and
washed with H2O (2×50 mL) followed by benzene (30 mL). The solid residue (14 g, 86 mmol) was
further recrystallized to give 60v. Gray solid, yield 13.3 g, (62%); mp 197–198 °C (from
benzene/iPrOH); 1
H NMR (DMSO-d6) δ 3.69 (s, 3H, MeO), 6.18 (s, 2H, NH2), 6.80 (s, 4H, Ar), 8.4
(s, 1H, NH), 11.02 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 55.1, 113.6, 123.7, 133.2, 140.3, 140.7,
155.3, 155.4; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N, 28.10. Found (%): C, 48.23;
H, 4.51; N, 28.05. IR (KBr, cm-1
) 3472, 3372, 3272, 2964, 2840, 1652, 1612, 1568, 1536, 1516,
1440, 1400, 1304, 1252, 1156, 1108, 1044, 952.
4.1.6. 4-Aminofurazan-3-carbox-(N-benzylamid)oxime (60w). Gray solid; 7.93 g, 68% yield; mp
124–125 °C (from benzene/iPrOH); 1
H NMR (DMSO-d6) δ 4.63 (d, J = 7.3 Hz, 2H, CH2), 6.27 (s,
2H, NH2), 6.89 (t, J = 7.3 Hz, 1H, NH), 7.21 (m, 5H, Ph), 10.80 (s, 1H, NOH); 13
C NMR (DMSO-
d6) δ 46.3 (C4), 126.6 and 128.3 (C6, C7, C8), 139.7 (C5), 140.9 (C2), 144.8 (C3), 155.2 (C1);
Anal. Calcd for C10H11N5O2 (%): C, 51.50; H, 4.75; N, 30.03. Found (%): C, 51.55; H, 4.68; N,
28.75.
4.1.7. 4-Aminofurazan-3-carbox-N-(p-methoxybenzylamid)oxime (60x). Light-brown solid; 7.77 g,
59% yield; mp 141–142 °C (from benzene) (lit. [44] 140–142 °C); 1
H NMR (DMSO-d6) δ 3.69 (s,
3H, MeO), 4.54 (d, J = 7.1 Hz, 2H, CH2), 6.26 (s, 2H, NH2), 6.83 (m, 3H, NH, H-3',5'), 7.12 (d, J =
8.4 Hz, 2H, H-2',6'), 10.79 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 45.8, 55.0, 113.7, 128.1, 132.8,
139.7, 144.8, 155.3, 158.2; EIMS m/z 263 (M+), 233 [M+
–NO]; Anal. Calcd for C11H13N5O3 (%):
C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.27; H, 5.01; N, 26.54.
4.1.8. 4-(p-Methoxyphenylamino)furazan-3-carboxamidoxime (61v). A solution of 60v (13.3 g, 53
mmol) and KOH (2.92 g, 53 mmol) in ethylene glycol (50 mL) was refluxed for 4 h. The reaction
mixture was cooled, diluted with water (30 mL), and neutralized with 36% aqueous HCl. The
residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from
benzene–iPrOH to afford 61v. Light-brown solid; yield 10.84 g (82%); mp 191 °C (from
benzene/iPrOH) (lit. [44] 190–191 °C); 1
H NMR (DMSO-d6) δ 3.74 (s, 3H, MeO), 6.39 (s, 2H,
NH2), 6.97 (d, 2H, J = 8.6 Hz, 2H, ArH-3',5'), 7.38 (d, 2H, J = 8.6 Hz, 2H, ArH-2',6')), 8.79 (s, 1H,
NH), 10.71 (s, 1H, NOH); 13
C NMR (DMSO-d6) δ 55.2, 114.5, 118.4, 132.5, 139.7, 143.9, 151.0,

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154.5; EIMS m/z 249 (M+), 219 [M+
–NO]; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N,
28.10. Found (%): C, 48.22; H, 4.49; N, 28.03.
4.1.9. 4-(Benzylamino)furazan-3-carboxamidoxime (61w). White solid; yield 9.76 g (79%); mp 104
°C; 1
H NMR (DMSO-d6) δ 4.44 (d, J = 7.0 Hz, 2H, CH2), 6.25 (s, 2H, NH2)), 6.51 (t, J = 7.0 Hz, 1H,
NH), 7.31 (m, 5H, Ph), 10.50 (s, 1H, NOH)); 13
C NMR (DMSO-d6) δ 47.6 (C4), 127.3, 127.6,
128.5 (C6, C7, C8), 138.4 (C2), 139.7 (C5), 144.0 (C1), 154.9 (C3).
4.1.10. 4-(p-Methoxybenzylamino)furazan-3-carboxamidoxime (61x). A solution of 60x (5.2 g, 20
mmol) and KOH (1.1 g, 20 mmol) in ethylene glycol (15 mL) was refluxed for 4 h. The reaction
mixture was cooled, diluted with water (30 mL) and neutralized with 36% aqueous HCl. The
residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from
benzene–iPrOH to furnish 61x. Gray solid; yield 4 g (78%); mp 113–117 °C; 1
H NMR (DMSO-d6)
δ 3.72 (s, 3H, MeO), 4.36 (d, J = 6.0 Hz, 2H, CH2), 6.22 (s, 2H, NH2), 6.40 (br. s, 1H, NH), 6.89 (d,
J = 8.2 Hz, 2H ArH-3',5'), 7.33 (d, J = 8.2 Hz, 2H, ArH-2',6'), 10.45 (s, 1H, NOH); 13
C NMR
(DMSO-d6) δ 47.1, 55.1, 113.9, 129.2, 130.3, 139.7, 144.1, 154.9, 158.7; EIMS m/z 263 (M+);
Anal. calcd for C11H13N5O3 (%): C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.25; H, 5.00; N,
26.52
4.1.11. General procedure for synthesis of 4-(R-amino)furazan-3-carbohydroxymoyl chlorides
62v,w
A solution of NaNO2 (0.14 g, 2 mmol) in H2O (2 mL) was added drop wise to a stirred
solution of amidoxime 61v or 61w (2 mmol) in a mixture of conc. HCl (3.6 mL), AcOH (6 mL),
and H2O (3.2 mL) at < 10 °C. The reaction was allowed to stir for 3 h at 10 °C and for 1 h at room
temperature. The solid residue was filtered and washed with H2O (4×5 mL) to give 62v,w (60%–
87% yield) as white crystals.
4.1.11.1. N-hydroxy-4-((4-methoxyphenyl)amino)-1,2,5-oxadiazole-3-carbimidoyl chloride (62v).
White solid; yield 0.4 g (60%); mp 202–203 °C; 1
H NMR (DMSO-d6) δ 3.72 (s, 3H, OMe), 6.94 (d,
2H, J = 8.6, ArH-3'',5'' ), 7.38 (d, 2H, J = 8.6, Ar2H, H-2'',6'' ), 8.21 (s, 1H, NOH), 13.62 (s, 1H,
NH); 13
C NMR (DMSO-d6) δ 55.1, 114.3, 119.0, 126.1, 132.2, 141.8, 150.6, 154.7; Anal. Calcd for
C10H9N4O3: C 44.71; H 3.38; N 20.85. Found (%): C 44.75; H 3.34; N 20.24; IR (KBr): ν max
3363, 3253, 1617, 1602, 1566, 1512, 1466, 1444, 1270, 1231, 1183, 1025, 945, 876, 824, 792, 754,
701, 564, 516.

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4.1.11.2. 4-(Benzylamino)-N-hydroxy-1,2,5-oxadiazole-3-carbimidoyl chloride (62w). White solid;
yield 0.44 g (87%); mp 164–165 °C; 1
H NMR (DMSO-d6) δ 4.45 (d, J=5.8 Hz, 2H, CH2), 6.35 (s,
1H, NOH), 7.34 (m, 5H, Ph), 13.41 (s, 1H, NH); 13
C NMR (DMSO-d6) δ 47.8, 126.8, 127.4, 127.7,
128.6, 138.2, 141.6, 154.4; Anal. Calcd for C10H9ClN4O2: C 47.54; H 3.59; N 22.18. Found (%): C
47.58; H 3.52; N 22.01; IR (KBr): ν max 3392, 3263, 1619, 1601, 1562, 1440, 1354, 1233, 1031,
967, 946, 909, 856, 754, 696, 610, 557, 508.
4.1.12. 3-(1-Ethyl-1H-benzimidazol-2-yl)-4-(4-methoxyphenyl)amino-furazan (63). Hydroximoyl
chloride 62v (0.27 g, 1 mmol) was added in several portions to a vigorously stirred solution of 1,2-
diaminobenzene 3q (0.2 g, 1.5 mmol) in ethanol (5–10 mL) at room temperature. The mixture was
stirred at reflux for 0.5 h followed by stirring for 1 h at room temperature, dilution with water (10
mL) and 0.1 M aqueous HCl (5 mL). The heterogeneous mixture was stirred for 1 h, the resulting
precipitate was filtered, washed with water, and recrystallized from iPrOH–H2O to afford 63. White
solid; yield 0.22 g (65%); mp 135–136 °C; 1
H NMR (DMSO-d6): δ 1.43 (t, J = 7.1 Hz, 3H, CH3),
3.77 (s, 3H, OMe-4''), 4.76 (q, J = 7.1 Hz, 2H, CH2), 7.03 (d, J = 9.0 Hz, 2H, ArH-3'',5''), 7.43 (t, J
= 8.0 Hz, 1H, H-5), 7.49 (t, J = 8.2 Hz, 1H, H-6), 7.60 (d, J = 9.0 Hz, 2H, ArH-2'',6''), 7.86 (d, J =
8.2 Hz, 1H, H-7), 7.97 (d, J = 8.0 Hz, 1H, H-4), 9.97 (s, 1H, NH) 13
C NMR (DMSO-d6): δ 15.0,
40.6, 55.6, 110.1, 114.5 (C2), 119.0 (C2), 120.6, 123.5, 124.9, 133.2, 134.9, 137.9, 140.4, 142.1,
152.9, 155.0; 15
N NMR (DMSO-d6): δ 21.3, -20.9, -137.0, -221.7, -297.0; EIMS m/z 335 [M]+
, 295
[M+
- NO]; Anal. Calcd for C18H17N5O2: C 64.47; H 5.11; N 20.88. Found (%): C 64.54; H 5.14; N
20.77; IR (KBr): ν max 3249, 2977, 1623, 1577, 1513, 1439, 1334, 1245, 1180, 1115, 1977, 1035,
1008, 908, 872, 825, 753, 742, 717, 608, 557, 524.
4.1.13. 3-Benzylamino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (64). White solid; yield 0.19 g
(59%); mp 196 °C; 1
H NMR (DMSO-d6): δ 4.56 (d, 2H, J = 5.89 Hz, CH2), 7.34 (m, 6H, Ph, H-6),
7.68 (s, 2H, CH) 13.85 (br, s, 2H, NH); 13
C NMR (DMSO-d6): δ 47.5, 116.0 (br, s), 123.9, 127.1,
127.4, 127.5, 128.3, 128.4, 128.5, 128.4, 137.9, 138.5, 141.3, 155.7; EIMS m/z 325 [M]+
, 295 [M+
-
NO], 220, 178, 106, 91; Anal. Calcd for C16H12ClN5O: C 58.99; H 3.71; N 21.50. Found (%): C
59.02; H 3.69; N 21.42; IR (KBr): ν max 3358, 3259, 1630, 1594, 1519, 1496, 1432, 1371, 1332,
1299, 1262, 1236, 1141, 1063, 1029, 986, 955, 927, 855, 815, 741, 705, 640.
4.1.14. Synthesis of 3-amino-4-[1-((1H-tetrazol-5-yl)methyl)-1H-benzimidazol-2-yl]-furazan 65
A solution of nitrile 40 (2.4 g, 0.01 mol), NaN3 (1 g, 0.015 mol), and NH4Cl (0.8g, 0.015
mol) in dry DMF (30 mL) was stirred at 100 °C for 8 h, cooled, and diluted with water (100 mL).
The reaction mixture was stirred with activated charcoal (100 mg) for 15 min, filtered, and the pH

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was adjusted to ca. 1 with 36% aqueous HCl. The resulting precipitate of 65 was recrystallized from
EtOH to furnish the pure product (white solid, 1.78 g, 63% yield).
4.1.15. Synthesis of 3-Amino-4-[1-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-1H-benzimidazol-2-yl]-
furazan 66
A solution of nitrile 40 (2.4 g, 0.01 mol) and thiosemicarbazide (1.4 g, 0.015 mol) in neat
CF3COOH (30 mL) was refluxed for 8 h at stirring. The reaction mixture was cooled, diluted with
water (100 mL), and stirred for an additional 15 min with activated charcoal (100 mg). The mixture
was filtered and neutralized with aqueous NH4OH (25%) to afford the residue of crude 66. It was
further recrystallized from EtOH to afford the targeted pure thiadiazol (white solid, 2.23 g, 71%
yield).
Synthetic and analytical data for the other compounds are presented in Supplementary data.
4.2. Biology. Materials and methods
4.2.1. Phenotypic sea urchin embryo assay [38]
Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the
Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were
obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and
fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under
gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were
observed with a Biolam light microscope (LOMO, St. Petersburg, Russia). For treatment with the
test compounds, 5 mL aliquots of embryo suspension were transferred to six-well plates and
incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of compounds
were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 96% EtOH.
This procedure enhanced the solubility of the test compounds in the salt-containing medium
(seawater), as evidenced by microscopic examination of the samples. The maximal tolerated
concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.05% and 1%,
respectively. Higher concentrations of either DMSO (≥0.1%) or EtOH (>1%) caused nonspecific
alteration and retardation of the sea urchin embryo development independent of the treatment stage.
The compound solubility in the seawater was estimated by microscopic examination of sample
wells. Colchicine and vinblastine sulfate (Sigma-Aldrich) were applied as reference compounds,
using 20 mM and 5 mM stock solutions in distilled water, respectively.
The antiproliferative activity was assessed by exposing fertilized eggs (8–20 min after
fertilization, 45–55 min before the first mitotic cycle completion) to 2-fold decreasing
concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5–5.5 h

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after fertilization. The effects were estimated quantitatively as an effective threshold concentration,
resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these
concentrations all tested microtubule destabilizers caused 100% cleavage alteration and embryo
death before hatching, whereas at 2-fold lower concentrations the compounds failed to produce any
effect. For microtubule-destabilizing activity, the compounds were tested on free-swimming
blastulae just after hatching (8–10 h after fertilization), which originated from the same embryo
culture. Embryo spinning was observed after 15 min to 20 h of treatment, depending on the
structure and concentration of the compound. Both spinning and lack of forward movement were
interpreted to be the result of the microtubule-destabilizing activity of a molecule. Video
illustrations are available at http://www.chemblock.com. Both sea urchin embryo assay and DTP
NCI60 cell line activity data are available free of charge via the Internet at http://www.zelinsky.ru.
4.2.2. In vitro tubulin polymerization assay [42]
In vitro tubulin polymerization was determined using modified turbidity assay, developed
by Cytoskeleton Inc. (Cytodynamix 12), for maximized throughput and maintained sensitivity.
Lyophilized bovine tubulin (HTS02, Cytoskeleton Inc.) was re-suspended in G-PEM buffer (80
mM PIPES pH 7, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 5% glycerol) to a final concentration of
3 mg/mL and kept at 4 °C. Compounds in 100× stock solutions in DMSO were dotted into pre-
warmed 96-well plates (Corning Costar 3696), with the plates immediately transferred to a 37 °C
plate reader (SPECTRAmax Plus, Molecular Devices). Cold tubulin was added to the wells, plates
were mixed by shaking, and absorbance at 340 nm was read every minute for 30 min. Kinetic
curves with 30 points each were collected for tested compound, with a dynamic range between 0
and 0.4 OD units. Percentage inhibition values were calculated using the 30 minute data point,
based on control samples (1% DMSO). IC50 values were determined by sigmoidal curve fitting
using Excel-based software.
4.2.3. Cell cycle analysis [42]
Cell cycle analysis was assessed by flow cytometry. 3T3 mouse fibroblasts were cultured in
DMEM supplemented with 10% fetal bovine serum, 1 mg/mL L-glutamate, 100 units/mL penicillin
G, and 0.2 mg/mL streptomycin sulfate. Cells were plated onto 6-well plates at a final density of
500,000 cells/well at the time of treatment, treated with compounds at a final concentration of 1 µM
(0.1% final concentration of DMSO) for 24 h, then trypsinized, collected, rinsed in phosphate
buffer saline (PBS), and fixed in 70% cold ethanol overnight at 4 °C. Cells were rinsed in PBS, re-
suspended in PBS with 0.2% Tween, incubated with RNAse (final concentration of 1 µg/mL) at 37
°C for 15 min, followed by addition of propidium iodide (final concentration of 50 µg/mL) and 30

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min incubation at room temperature. Cell cycle distribution was determined by flow cytometry
using cell sorter Guava PCA-96. A compound was reported as a mitotic arrest inducer when the
amount of cells in G2/M phase exposed to 1 µM concentration of an agent was twice or more than
in control (DMSO).
Acknowledgments
The authors acknowledge the compounds screening at the National Cancer Institute (NCI)
(Bethesda, MD, USA) by the Developmental Therapeutics Program NCI/NIH
(http://dtp.cancer.gov). KYS is thankful to the Russian Scientific Foundation for financial support
(project no. 14-13-00884).
Supplementary data
Supplementary data associated with this article can be found in the online version, at
........................ These data include MOL files, experimental details regarding syntheses, analytical
data, and X-ray analysis.

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Supplementary data
A facile synthesis and antiproliferative properties of 4-(1H-benzo[d]imidazol-2-yl)-
furazan-3-amines
Andrei I. Stepanov,a
Alexander A. Astrat’ev,a
Aleksei B. Sheremetev,b
Nataliya K. Lagutina,c
Nadezhda
V. Palysaeva,b
Aleksei Yu. Tyurin,b
Nataly S. Aleksandrova,b
Nataliya P. Sadchikova,c
Kyrill Yu.
Suponitsky,d
Olga P. Atamanenko,b
Leonid D. Konyushkin,b
Roman V. Semenov,b
Sergei I. Firgang,b
Alex S. Kiselyov,e
Marina N. Semenova,f
Victor V. Semenovb,*
a
Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint Petersburg,
192076, Russian Federation
b
N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian
Federation
c
I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow,
Russian Federation
d
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov
Str., 119991 Moscow, Russian Federation
e
Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology,
Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation
f
N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow, Russian
Federation
Corresponding author: Victor V. Semenov
Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991,
Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966.
E-mail: vs@zelinsky.ru

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Table of contents
1) Single crystal X-ray crystallography of benzimidazoles 27 and 29
and Fig. S1. Molecular structure of compounds 27 and 29 showing the atom
numbering scheme. Page S3
2) Table S1. Crystallographic data for 27 and 29. Page S5
3) Figure S2. Fragment of the crystal packing of 27 and 29. Page S6
4) Figure S3. NCI60 5 dose screen. GI50 Mean Graph for 18. Page S7
9) The Mean Graphs interpretation. Page S12
9) Chemistry. General experimental procedures and synthetic and analytical
data for 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines. Pages S13–S45
10) References. Page S46

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Single crystal X-ray crystallography of benzimidazoles 27 and 29.
X-ray experiments were carried out using SMART APEX2 CCD (λ(Mo-Kα)=0.71073 Å,
graphite monochromator, ω-scans) at 120K. Collected data were analyzed by the SAINT and SADABS
software incorporated into APEX2 package (APEX2 and SAINT; Bruker AXS Inc., Madison,
Wisconsin, USA, 2009). All structures were solved by the direct methods and refined by the full-matrix
least-squares procedure against F2
in anisotropic approximation. The hydrogen atoms of the NH2 groups
were found in the difference Fourier synthesis. The H(C) positions were calculated. All the hydrogen
atoms were included in the refinement within isotropic approximation by the riding model with the
Uiso(H) = 1.5Ueq(Ci) for methyl groups and 1.2Ueq(Ci) for other carbon atoms, where Ueq(C) are
equivalent thermal parameters of the parent atoms. The refinement was carried out with the SHELXTL
software [i]. The details of data collection and crystal structures refinement are summarized in Table S1.
Fig. S1. Molecular structure of compounds 27 and 29 showing the atom numbering scheme.
In the molecular structure of 27 (Fig. S1), the aminofurazan moiety is nearly coplanar to the
benzimidazole ring (torsion angle C1–C2–C3–N4 is -1.0(7)°) suggesting conjugation between these
functionalities and stabilization by the intramolecular hydrogen bond N3–H3A…N4 (H…N 2.35Å,
N…N 2.883(5)Å, <NHN 118°). The N–O bonds in the furazan ring showed different length (N1–O1 of
1.402(5)Å, N2–O1 of 1.376(5)Å) affected by substituents [ii].
In the structure of 29 featuring anisole and CF3 substituents, the furazan and benzimidazole rings
are also coplanar (torsion angle C1–C2–C3–N4 is -1.0(4)°). For this groups, the conjugation is more
pronounced presumably due to the influence of the electron-withdrawing CF3 group as evidenced by the
greater difference in the N–O bonds of the furazan ring (N1–O1 of 1.413(3)Å, N2–O1 of 1.368(3)Å).
Further stabilization of the planar structure is likely attained by the intramolecular hydrogen bond N3–
H3A…N4 (H…N 2.23Å, N…N 2.859(4)Å, <NHN 127°), which is stronger than that in 27. It was

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further determined that the torsion angle C9–N5–C11–C12 of 92.2(3)° is defined by the intramolecular
steric effects, while the rotation of the anisole moiety about the C11–C12 bond is most probably
induced by crystal packing.
In addition to intramolecular H-bond, in the crystal structure of 27 the second hydrogen of the
amino group formed H-bond with the nitrogen atom of the furazan ring [N3–H3B…N1(1-x, -0.5+y, 1-z)
(H…N 2.14Å, N…N 3.025(5)Å, <NHN 169°)]. However, instead of formation of H-bonded dimers,
this resulted in a formation of H-bonded chains along the axis b. In these chains molecules were related
by the two-fold axis (Fig. S2).
In the crystal structure of 29 the second hydrogen atom of the amino group was bonded to the
oxygen atom of the methoxy group [N3-H3B…O2(x, 1+y, z) (H…O 2.23Å, N…O 3.056(3)Å, <NHO
152°)] causing a formation of chains along axis b (Fig. S2). Obviously this H-bond was weaker than the
N–H…N bond observed in 27 suggesting better nucleophilic properties of the furazan ring N3 atom as
compared to the O-atom of the methoxygroup [ii]. However the N–H…N bond was not observed in 29
that could be a consequence of the cumulative effect of the numerous weak intermolecular interactions
contributing to a stabilization of the 3-D crystal structure. Probably, for the same reason the H-bonded
chains, other than the H-bonded dimers, were formed in the crystal structure of 27.

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Table S1.
Crystallographic data for 27 and 29.
Parameter 27 29
Empirical formula
Fw
Crystal system
Space group
a, Å
b, Å
c, Å
α, deg
β, deg
γ, deg
V, Å3
Z
dcalc, g·cm-3
µ, mm-1
F(000)
θ range, deg.
Reflections collected
Independent reflections
Rint
Refined parameters
Completeness to theta θ, %
GOF (F2
)
Reflections with I>2σ(I)
R1(F) (I>2σ(I))a
wR2(F2
) (all data)b
Largest diff. peak/hole, e⋅Å-3
C11H11N5O
229.25
Monoclinic
P21
11.180(2)
4.1948(8)
11.823(2)
90.00
107.621(3)
90.00
528.44(17)
2
1.441
0.100
240
1.81 – 29.03
6268
1591
0.0403
155
99.6
1.021
1304
0.0667
0.1792
0.337 / -0.383
C18H18F3N5O2
393.37
Triclinic
P-1
5.0273(6)
11.0378(13)
16.3137(19)
107.435(3)
91.072(3)
97.262(3)
855.20(17)
2
1.528
0.125
408
1.95 – 27.00
9133
3715
0.0531
263
99.2
1.020
2229
0.0609
0.1648
0.735 / -0.443
a
R1 = ∑|Fo – |Fc||/∑(Fo).
b
wR2 = (∑[w(Fo
2
– Fc
2
)2
]/∑[w(Fo
2
)2
]1/2
.

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Fig. S2. Fragment of the crystal packing of 27 (top) and 29 (bottom). H-bonded chains are shown.

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Fig. S3. NCI60 5 dose screen. GI50 Mean Graph for 18.

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The Mean Graphs interpretation.
From: Methodology of the in vitro cancer screen (http://dtp.nci.nih.gov/branches/btb/ivclsp.html
Mean graphs facilitate visual scanning of data for potential patterns of selectivity for particular
cell lines or for particular subpanels with respect to a selected response parameter. Bars extending to the
right represent sensitivity of cell line to the test agent in excess of the average sensitivity of all tested
cell lines. Since the bar scale is logarithmic a bar 2 units to the right implies the compound achieved the
response parameter (e.g. GI50) for the cell line at a concentration one-hundredth the mean concentration
required over all cell lines, and thus the cell line is usually sensitive to that compound. Bars extending
to the left correspondingly imply sensitivity less than the mean.

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Alternative representative scale-up procedure for 3-amino-4-(1H-benzimidazol-2-yl)-furazan
9a. Step 1. Synthesis of 1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7.
7
N
H
N CN
NOH
6
N
H
N CN
A solution of NaNO2 (69 g, 1 mol) in H2O (100 mL) was added dropwise to a vigorously stirred
solution of 2-cyanomethylbenzimidazol 6 [iii] (152g, 1 mol) in AcOH (200 mL) at 5–10 °C. The
reaction mixture was allowed to stir for additional 0.5 h at 10–15 °C and diluted with water (200 mL).
The residue was filtered, washed with cold water, and dried to give crude acetonitrile 7 (150–160 g, 80–
86% yield), which was further used without purification.
Step 2. Synthesis of 2-(1H-benzimidazol-2-yl)-N'-hydroxy-2-(hydroxyimino)-ethanimidamide
and 2-(1H-benzoimidazol-2-yl)-N-hydroxy-2-hydroxyimino-acetamidine (mixture of isomers of 8).
87
N
H
N CN
NOH N
H
N
NOH
NOH
NH2
N
H
N
NOH
NH
NH
OH
+
A solution of NH2OH•HCl (77 g, 1.2 mol) in water (150 mL) was added to a solution of
1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7 (150 g, 0.8 mol) in EtOH or iPrOH (500 mL)
followed by pouring of K2CO3 (60 g, 0.6 mol) portionwise of 5–10 g. The reaction was stirred for 1 h at
room temperature, 1 h at 30 °C, 1 h at 40 °C, and 2 h at 50 °C, and cooled to room temperature. Most of
EtOH was evaporated in vacuo from the reaction mixture; the residue was diluted with hot water (70–80
°C) and cooled to room temperature. The precipitate was filtered, washed with cold water, and dried in
air to give crude glyoxime 8 (120–125 g, 68–71% yield), which was further used without purification.
Step 3. Synthesis of N,N'-bis(acetyloxy)-2-[(acetyloxy)imino]-2-(1H-benzimidazol-2-yl)-
ethanimidamide and 2-(1H-Benzoimidazol-2-yl)-N-acetoxy-2-acetoxyimino-N'-acetylacetamidine
(mixture of isomers of 8-triacetate).
8
isomers
N
H
N
NOH
NOH
NH2
N
H
N
NOAc
NOAc
AcNH
8-triacetate
isomers

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Acetic anhydrate (181 mL, 196 g, 1.92 mol) was added dropwise to a solution of glyoxime 8 (120 g,
0.55 mol) and dry AcONa (10 g 0.12 mol) in acetic acid at 25–30 °C. The reaction mixture was heated
to 90–95 °C during 1 h and stirred for 1 h. Acetic acid (ca. 250 mL) was evaporated in vacuo, the
residue was diluted with H2O (300 mL) and cooled to room temperature. The precipitate was filtered,
washed with cold water, and dried in air to afford crude 8-triacetate (155–165 g, 82–87% yield), which
was further used without purification.
Step 4. Synthesis of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a.
N
H
N
NOAc
NOAc
AcNH
8-triacetate 9{1,1,1}
N
H
N
N
O
N
NH2
8-triacetate (150 g, 0.43 mol) was added to a solution of NaOH (86 g, 2.15 mol) in hot H2O (250 mL,
50 °С). The reaction mixture was refluxed for 1 h and cooled to room temperature. The insoluble
impurities were removed by filtration, and the pH was adjusted to ca. 6 with acetic acid. The resulting
precipitate was filtered, washed with water, and recrystallized from acetic acid to furnish the pure
product 9a. White solid; 58–60 g; 67–70% yield; mp 269 °С (lit. [iv] 264–265 °С).
3-Amino-4-(5-methyl-1H-benzimidazol-2-yl)-furazan (9b).
N
H
N
N O
N
NH2
Me
White solid; yield 183 mg (85%); mp 268–269 °С; 1
H NMR (DMSO-d6): δ 2.41 (s, 3H, Me-5), 6.84 (s,
2H, NH2), 7.12 (s, 1H, (H-4), 7.50 (m, 2H, ArH-6,7, 13.53 (br. s, 1H, NH); 13
C NMR (DMSO-d6): δ
21.3, 111.6, 119.1, 124.4, 126.0, 134.1, 138.6, 139.8, 140.9, 155.5; Anal. Calcd for C10H9N5O: C 55.81;
H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR (KBr): ν max 3430, 3332, 3171, 1635, 1597,
1457, 1422, 1317, 1274, 1237, 1134, 1005, 952, 897, 877, 798, 761, 732, 668, 597, 562.
3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c).
N
N
H
N
O
N
H2NMe
N
N
H
N
O
N
H2N
Me Ratio of isomers 3:2
White solid; yield 157 mg (73%); mp 213–216 °С; 1
H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H,
Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7), 7.38,

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7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13
C NMR (DMSO-d6): δ 16.3, 17.1,
109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2, 142.3, 142.6,
155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR
(KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268, 1239, 1157, 1138,
1005, 949, 899, 874, 748, 671, 561.
3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d).
N
N
H
N
O
N
H2N
Me
Me
N
N
H
N
O
N
H2N
Me
Me
White solid; yield 211 mg (92%); mp 242–243 °C; 1
H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H,
Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43 (br. s,
2H, NH); 13
C NMR (DMSO-d6): δ 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2, 127.5,
129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for C11H11N5O: C
57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425, 3284, 3202, 1634,
1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741, 717, 661, 631, 561, 501.
3-Amino-4-(5-fluoro-1H-benzimidazol-2-yl)-furazan (9e).
N
H
N
N O
N
NH2
F
H NMR (DMSO-d6): δ 6.79 (s, 2H, NH2), 7.16 (t, J
= 8.2 Hz, 1H, H-6), 7.42 (d, J = 8.2 Hz, 1H, H-7), 7.67 (s, 1H, H-4), 13.76 (s, 1H, NH); 13
C NMR
(DMSO-d6): δ 101.3 (br), 111.83, 112.2, 117.5 (br), 138.4, 141.5, 155.5, 157.8, 161.0; 19
F NMR
(DMSO-d6): -118.10, -120.20; Anal. Calcd for C9H6FN5O: C 49.32; H 2.76; N 31.95. Found: C 49.38;
H 2.80; N 31.86; IR (KBr): ν max 3453, 3418, 3298, 1624, 1601, 1562, 1505, 1491, 1456, 1432, 1415,
1321, 1265, 1224, 1142, 1112, 1005, 954, 904, 861, 808, 771, 732, 699, 633, 614, 513.
3-Amino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (9f).
N
NH2
ON
H
N
NCl
H NMR (DMSO-d6): δ 6.80 (s, 2H, NH2), 7.30 (s,
1H, H-6), 7.59 (s, 1H, H-7), 7.74 (s, 1H, H-4), 13.82 (br. s, 1H, NH); 13
C NMR (DMSO-d6): δ 112.7

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(br. d), 120.1 (br. d), 123.9 (br. d), 128.1 (br. d), 134.1 (br. d), 138.4, 141.6, 143.6, 155.7; Anal. Calcd
for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.94; H 2.63; N 29.63; IR (KBr): ν max 3439,
3336, 3167, 3113, 632, 1623, 1597, 1454, 1414, 1309, 1271, 1228, 1134, 1057, 1005, 950, 926, 901,
875, 860, 803, 734, 676, 596, 563.
3-Amino-4-(7-chloro-1H-benzimidazol-2-yl)-furazan (9g).
N
NN
H
O
N
NH2
Cl
H NMR (DMSO-d6): δ 6.85 (s, 2H, NH2), 7.32,
7.36 (2t, J = 7.8 Hz, 1H, H-5), 7.41, 7.45 (2d, J = 7.8 Hz, 1H, H-6), 7.57, 7.79 (2d, J = 7.8 Hz, 1H, H-
4), 14.07, 14.24 (2s, 1H, NH); 13
C NMR (DMSO-d6): δ 111.2, 122.3, 123.4, 125.1, 135.4, 138.2, 139.7,
140.9, 155.5; Anal. Calcd for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.93; H 2.62; N 29.61;
IR (KBr): ν max 3452, 3339, 3278, 1616, 1589, 1499, 1453, 1453, 1419, 1318, 1257, 1200, 1113, 1004,
975, 948, 897, 778, 739,631, 606, 569.
3-Amino-4-(5-methoxy-1H-benzimidazol-2-yl)-furazan (9h).
N
NN
H
O
N
NH2
MeO
White solid; yield 143 mg (62%); mp 210–211°C; 1
H NMR (DMSO-d6): δ 3.79 (s, 3H, OMe-5), 6.81
(s, 2H, NH2), 6.91 (d, J = 8.0, 1H, H-6), 7.04 (s, 1H, H-7), 7.60 (s, 1H, H-4), 13.49 (s, 1H, NH); 13
C
NMR (DMSO-d6): δ 56.0 (OMe), 94.7 (br), 113.5 (br), 120.7 (br), 139.0, 155.9; Anal. Calcd for
C10H9N5O2: C 51.95; H 3.92; N 30.29. Found: C 52.01; H 3.90; N 30.21; IR (KBr): ν max 3434, 3287,
1624, 1591, 1510, 1462, 1435, 1411, 1270, 1204, 1164, 1119, 1025, 1001, 953, 896, 865, 819, 627.
3-Amino-4-(4,7-dimethoxy-1H-benzimidazol-2-yl)-furazan (9i).
N
NN
H
O
N
NH2
OMe
OMe
H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7, 6.70
(s, 2H, NH2), 6.72 (s, 2H, H-5,6), 13.95 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 55.9 (OMe), 103.2 (br),
105.2 (br), 138.3, 139.0, 155.5; Anal. Calcd for C11H11N5O3: C 50.57; H 4.24; N 26.81. Found: C 52.63;
H 4.26; N 26.71; IR (KBr): ν max 3419, 3335, 3208, 1630,1615, 1529,1464, 1449, 1420, 1349, 1275,
1256, 1213, 1176, 1108, 1097, 1004, 983, 951, 895, 855, 778, 748, 721, 681, 567,516, 486.

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2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid (9j).
1H, H-7), 7.91 (d, J = 8.3, 1H, H-6), 8.26 (br. s, 1H, H-4), 12.44 (br. s, 1H, OH), 13.77 (br. s, 1H, NH);
13
C NMR (DMSO-d6): δ 20.9, 113.9 (br. s), 119.0 (br. s), 124.5, 125.8, 138.3, 142.3, 155.5, 167.5,
171.9; Anal. Calcd for C10H7N5O3: C 48.98; H 2.88; N 28.56. Found: C 49.03; H 2.85; N 28.47; IR
(KBr): ν max 3473, 3352, 3102, 2629, 2553, 1686, 1635, 1599, 1493, 1455, 1411, 1326, 1287, 1233,
1150,1137, 1088, 1013, 957, 910, 869, 837, 771, 757, 689, 670, 570, 515.
2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid mehtyl ester (9k).
N
N
H
N
O
N
H2N
MeOOC
H NMR (DMSO-d6): δ 3.82 (s, 3H, OMe), 6,77 (s,
2H, NH2), 7.61 (s, 1H, H-7), 7.78 (s, 1H, H-6), 8.12 (s, 1H, H-4), 13.85 (br. s, 1H, NH); Anal. Calcd for
C11H9N5O3: C 50.97; H 3.50; N 27.02. Found: C 51.01; H 3.49; N 26.85; IR (KBr): ν max 3542, 3436,
3330, 1692, 1638, 1602, 1435, 1332, 1294, 1248, 1233, 1132, 1092, 1007, 979,952, 900, 863, 826, 772,
751, 567, 501.
2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carbonitrile (9l).
N
NN
H
O
N
NH2N
White solid; yield 195 mg (86%); mp 288 °C; 1
H NMR (DMSO-d6): δ 6.75 (s, 2H, NH2), 7.62 (d, J =
8.1 Hz, 1H, H-7), 7.74 (d, J = 8.1 Hz, 1H, H-6), 8.13 (s, 1H, H-4), 14.01 (br. .s, 1H, NH); 13
C NMR
(DMSO-d6): δ 105.8, 116.1, 120.0, 122.7, 123.5, 127.3, 138.7, 139.7, 143.7, 156.0; Anal. Calcd for
C10H6N6O: C 53.10; H 2.67; N 37.15. Found: C 53.14; H 2.64; N 37.07; IR (KBr): ν max 3421, 3311,
2234, 1644, 1624, 1603, 1445, 1407, 1326, 1286, 1236, 1142, 1096, 1012, 956, 907, 872, 826, 754, 628,
571.
3-Amino-4-(1H-naphtho[2,3-d]imidazol-2-yl)-furazan (9m).

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N
N
H
N
O
N
H2N
2H, H-4,9), 8.18 (m, 4H, H-5,6,7,8), 13.70 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 107.6, 116.6, 123.7,
1469, 1415, 1310, 1267, 1175, 1135, 1006, 954, 906, 874, 861, 735, 614, 477.
3-Amino-4-(6(8)H-imidazo[4',5':3,4]benzo[1,2-c][1,2,5]furazan-7-yl)-furazan (9n).
N
N
H
N
O
N
H2N
N
O N
H NMR (DMSO-d6): δ 6.74 (s, 2H, NH2), 7.84 (d, J
= 9.0 Hz, H-4,5), 14.77 (br..s, 1H, NH); 13
C NMR (DMSO-d6): δ 117.6, 127.4 (br), 140.1 (br), 143.2,
144.6, 154.1, 160.3; Anal. Calcd for C9H5N7O2: C 44.45; H 2.07; N 40.32. Found: C 44.49; H 2.01; N
40.25; IR (KBr): ν max 3450, 3354, 3148, 1614, 1627, 1605, 1562, 1525, 1486, 1443, 1414, 1376,
1331, 1261, 1195, 1121, 1081, 1005, 954, 884, 803, 782, 749, 695, 672, 606, 571, 508.
3-(1H-Benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11a).
N
N
ON
H
N
N
White solid; yield 333 mg (78%); mp 214 °С; 1
H NMR (DMSO-d6): δ 6.44 (t, J = 2.2 Hz, 2H, H-3'',4''),
7.36 (br. s, 2H, H-5,6), 7.66 (br. s, 1H, H-7), 7.79 (br. s, 1H, H-4), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''),
13.69 (s, 1H, NH); 13
C NMR (DMSO-d6): δ 111.9, 120.0 (br. s), 122.4, 134.5 (br. s), 138.1, 141.7,
143.0, 151.3; EIMS m/z 251 [M]+
; Anal. Calcd for C13H9N5O: C 62.15; H 3.61; N 27.87. Found: C
62.19; H 3.63; N 27.81; IR (KBr): ν max 2158-2586, 1565, 1504, 1480, 1435, 1399, 1390, 1374, 1324,
1281, 1232, 1195, 1149, 1116, 1065, 1034, 1003, 967, 919, 905, 886, 737, 604, 583, 484, 456.
3-(5-Methoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11h).

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N
NN
H
O
N
MeO
N
H NMR (DMSO-d6): δ 3.84 (s, 3H, OMe -5), 6.44
(t, J = 2.2 Hz, 2H, H-3'',4''), 6.94, 7.02 (2dd, J = 2.4 Hz, J = 8.9 Hz, 1H, H-6), 7.04, 7.33 (2d, J = 2.2
Hz, 1H, H-4), 7.50, 7.71 (2d, J = 8.9 Hz, 1H, H-7), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''), 13.50, 13.56 (2s,
1H, NH); EIMS m/z 281 [M]+
; Anal. Calcd for C14H11N5O2: C 59.78; H 3.94; N 24.90. Found: C 59.85;
H 3.96; N 24.81; IR (KBr): ν max 3144-2672, 1631, 1594, 1566, 1483, 1461, 1430, 1388, 1334, 1276,
1202, 1159, 1113, 1065, 1032, 971, 891, 820, 732, 603.
3-(4,7-Dimethoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11i).
N
NN
H
O
N
NOMe
OMe
White solid; yield 402 mg (76%); mp 188 °C; 1
H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7), 6.40 (br.
s, 2H, H-3'',4''), 6.65 (d, J = 7.8 Hz, 1H, CH), 6.76 (d, J = 7.8 Hz, 1H, CH), 7.75 (m, 2H, H-2'',5''),
13.94 (s, 1H, NH). 13
C NMR (DMSO-d6): δ 55.8, 55.9, 103.4, 105.0, 112.0, 121.9, 126.4, 135.0, 136.2,
140.6, 141.4, 145.7, 151.2; Anal. Calcd for C15H13N5O3: C 57.87; H 4.21; N 22.50. Found: C 57.95; H
4.16; N 22.32; IR (KBr): ν max 3244, 3111, 1582, 1561, 1528, 1484, 1461, 1388, 1342, 1270, 1174,
1107, 1095, 1063, 1037, 1005, 986, 970, 911, 892, 858, 787, 741, 722, 666, 598.
Alkylation by benzylhalides 12′′′′–43′′′′ (yields 75–90%).
2-[2-(4-Aminofurazan-3-yl)-1H-benzimidazol-1-yl]ethanol (12).
N
N
N O
N
NH2
OH
H NMR (DMSO-d6): δ 3.80 (q, J = 4.7 Hz, J = 5.5
Hz, 2H, CH2O), 4.74 (t, J = 5.5Hz, 2H, CH2N), 4.92 (t, J = 4.7 Hz, 1H, OH), 7.00 (s, 2H, NH2), 7.35 (t,
J = 8.1 Hz, 1H, H-5), 7.42 (t, J = 8.1 Hz, 1H, H-6), 7.77 (d, J = 8.1 Hz, 1H, H-7), 7.82 (d, J = 8.1 Hz,
1H, H-4); EIMS m/z 245 [M]+
(37), 228 (1), 215 (12), 206 (6), 188 (100), 172 (9), 157 (22), 156 (22),

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144 (96), 131 (5), 118 (12), 102 (6), 77 (10); Anal. Calcd for C11H11N5O2: C 53.87; H 4.52; N 28.56.
Found: C 53.82; H 4.50; N 28.61.
3-Amino-4-(1-propargyl-1H-benzimidazol-2-yl)-furazan (13).
N
N N
N
O
NH2
White solid; yield 1.72 g (72%); mp 210 °C; 1
H NMR (DMSO-d6): 3.33 (br. s, 1H, CH), 5.57, 5.58 (2s,
2H, CH2), 6.88 (br. s, 2H, NH2), 7.40 (t, J = 7.6 Hz, 1H, H-6), 7.49 (t, J = 7.6 Hz, 1H, H-5), 7.82 (d, J =
7.6 Hz, 1H, H-7), 7.86 (d, J = 7.6 Hz, 1H, H-4); EIMS m/z 239 [M]+
(21), 209 (31), 182 (38), 144
(100), 118 (37), 77 (8); Anal. Calcd for C12H9N5O: C 60.25; H 3.79; N 29.27. Found: C 60.29; H 3.82;
N 29.18.
N-[4-(1-Allyl-1H-benzimidazol-2-yl)-furazan-3-yl]acetamide (14r).
N
N N
N
O
NH
O
H NMR (DMSO-d6): δ 2.31 (s, 3H, CH3CO), 5.05
(d, J = 17.0 Hz, 1H, CH2=), 5.20 (d, J = 10.0 Hz, 1H, CH2=), 5.30 (s, 2H, CH2), 6.07 (m, 1H, CH=),
7.42 (t, J = 8.0 Hz, 1H, H-6), 7.47 (t, J = 8.0 Hz, 1H, H-5), 7.75 (d, J = 8.0 Hz, 1H, H-7), 7.90 (d, J =
8.0 Hz, 1H, H-4), 10.91 (s, 1H, NH); EIMS m/z 283 [M]+
(3), 268 (5), 253 (1), 241 (1), 227 (1), 211
(10), 200 (7), 194 (4), 184 (45), 182 (5), 169 (5), 156 (5), 144 (6), 129 (2), 116 (3), 102 (5), 90 (4), 77
(9), 43 (99), 41 (100); Anal. Calcd for C14H13N5O2: C 59.36; H 4.63; N 24.72. Found: C 59.43; H 4.66;
N 24.60.
3-Amino-4-[1-(2-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (15).
N
N
N O
N
NH2
F

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H NMR (DMSO-d6): δ 6.03 (s, 2H, CH2), 6.81 (t, J =
7.6 Hz, 1H, H-6''), 6.95 (s, 2H, NH2), 7.06 (t, J = 7.6 Hz, 1H, H-5''), 7.23 (t, J = 8.7 Hz, 1H, H-3''), 7.32
(m, 1H, H-4''), 7.38 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.7 Hz, 1H, H-6), 7.67 (d, J = 7.7 Hz, 1H, H-7),
7.88 (d, J = 7.7 Hz, 1H, H-4; EIMS m/z 309 [M]+
(5), 279 (4), 252 (6), 143 (8), 110 (5), 109 (100), 83
(18); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.19; H 3.94; N 22.57.
N
N
N O
N
NH2
F
H NMR (DMSO-d6): δ 6.00 (s, 2H, CH2), 6.89 (br. s,
2H, NH2), 6.98 (m, 3H, H-4'',5'',6''), 7.30 (t, J = 7.3 Hz, 1H, H-5), 7.36 (m, 2H, H-6,2''), 7.62 (d, J = 7.3
Hz, 1H, H-7), 7.82 (d, J = 7.3 Hz, 1H, H-4); EIMS m/z 309 [M]+
(7), 279 (6), 252 (9), 237 (6), 236 (7),
170 (1), 143 (13), 109 (100), 83 (25); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C
62.18; H 3.93; N 22.60.
N-(4-[1-(3-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan 3-yl)propionamide (16s).
N
N
N O
N
NH
F
O
H NMR (DMSO-d6): 1.20 (t, J = 7.4 Hz, 3H,
CH3CH2), 2.62 (q, J = 7.4 Hz, 2H, CH2CO), 5.96 (s, 2H, CH2), 7.03 (d, J = 7.7 Hz, 1H, H-6''), 7.08 (m,
2H, H-4'',5''), 7.35 (br. k, 1H, J = 6.8 Hz, J =7.7 Hz, H-2''), 7.41 (t, J = 7.7 Hz, 1H, H-5), 7.44 (t, J =
7.7 Hz, 1H, H-6), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.90 (d, J = 7.7 Hz, 1H, H-4), 11.00 (s, 1H, NH); EIMS
m/z 365 [M]+
(6), 335 (7), 309 (11), 308 (13), 237 (8), 170 (2), 143 (15), 109 (100), 83 (23); Anal.
Calcd for C19H16FN5O2: C 62.46; H 4.41; N 19.17. Found: C 62.52; H 4.45; N 19.05.

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N
N
N
N
O
NH2
F
H NMR (DMSO-d6): δ 5.95 (s, 2H, CH2), 6.94 (s, 2H,
NH2), 7.12 (t, J = 8.7 Hz, 2H, H-3'',5''), 7.24 (dd, J = 5.5 Hz, J = 8.7 Hz, 2H, H-2'',6''), 7.37 (t, J = 7.6
Hz, 1H, H-5), 7.41 (t, J =7.6 Hz, 1H, H-6), 7.71 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 8.1 Hz, 1H, H-4);
EIMS m/z 309 [M]+
(6), 379 (7), 263 (1), 143 (6), 110 (6), 109 (100), 83 (16), 63 (6); Anal. Calcd for
C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.05; H 3.88; N 22.69.
3-Amino-4-[1-(3-bromobenzyl)-1H-benzimidazol-2-yl]-furazan (18).
N
N
N O
N
NH2
Br
H NMR (DMSO-d6): δ 5.99 (s, 2H, CH2), 6.90 (br. s,
2H, NH2), 7.07 (d, J = 7.7 Hz, 1H, H-6''), 7.22 (t, J = 7.7 Hz, 1H, H-5''), 7.37 (m, 2H, H-5,6)), 7.40 (br.
d, J = 7.7 Hz, 1H, H-4''), 7.44 (br. s, 1H, H-2''), 7.62 (br. d, J = 7.3 Hz, 1H, H-7), 7.82 (br. d, J = 7.3 Hz,
1H, H-4); EIMS m/z 371 [M+1]+
(5), 369 [M-1]+
(5), 341 (6), 339 (6), 314 (9), 312 (9), 260 (4), 234 (5),
217 (7), 169 (100), 143 (38), 116 (10), 102 (9), 90 (84), 89 (54), 77 (12), 63 (27); Anal. Calcd for
C16H12BrN5O: C 51.91; H 3.27; N 18.92. Found: C 51.87; H 3.24; N 18.89.
3-Amino-4-[1-(3-trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan (19).
N
N
N O
N
NH2
CF3
H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.92 (s,
2H, NH2), 7.34 (d, J = 7.7 Hz, 1H, H-6''), 7.39 (m, 2H, H-5,6), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.60 (d, J
= 7.7 Hz, 1H, H-4''), 7.64 (s, 1H, H-2''), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4);
EIMS m/z 359 [M]+
(2), 329 (2), 302 (6), 287 (4), 286 (4), 159 (100), 143 (15), 119 (8), 109 (29), 90

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(7), 89 (6), 63 (7); Anal. Calcd for C17H12F3N5O: C 56.83; H 3.37; N 19.49. Found: C 56.77; H 3.35; N
19.55.
N-(4-[1-(3-Trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (19r).
N
N
N O
N
NH
CF3
O
H NMR (DMSO-d6): δ 2.32 (s, 3H, CH3CO), 6.03 (s,
2H, CH2), 7.41 (m, 2H, H-5,6,6''), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.61 (t, J = 7.7 Hz, 1H, H-4''), 7.68 (br.
s, 1H, H-2''), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.95 (s, 1H, NH); EIMS m/z
401 [M]+
(0.1), 386 (1), 329 (3), 302 (5), 159 (84), 109 (21), 43 (100); Anal. Calcd for C19H14F3N5O2: C
56.36; H 3.52; N 17.45. Found: C 56.22; H 3.48; N 17.57.
3-Amino-4-[1-(3-methoxybenzyl)-1H-benzimidazol-2-yl]-furazan (20).
N
N
N O
N
NH2
OMe
H NMR (DMSO-d6): δ 3.68 (s, 3H, OMe -3''), 5.94
(s, 2H, CH2), 6.66 (br. d, J = 7.3 Hz, 1H, H-4''), 6.76 ( br. s, 1H, H-2''), 6.82 (br. d, J = 8.2 Hz, 1H, H-
6''), 6.94 (s, 2H, NH2), 7.20 (t, J = 7.8 Hz, 1H, H-5''), 7.37 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.3 Hz,
1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4); EIMS m/z 321 [M]+
(6), 291
(17), 276 (3), 143 (5), 121 (100), 91 (37), 78 (19), 77 (25); Anal. Calcd for C17H15N5O2: C 63.54; H
4.71; N 21.79. Found: C 63.61; H 4.73; N 21.75.
N-(4-[1-(3-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (20r).

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N
N
N O
N
NH
CF3
O
H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3CO), 3.69 (s, 3H,
OMe-3''), 5.89 (s, 2H, CH2), 6.72 (br. d, J = 7.5 Hz, 1H, H-4''), 6.81 (br. s, 1H, H-2''), 6.83 (dd, J = 2.1
Hz, J = 8.2 Hz, 1H, H-6''), 7.21 (t, J = 7.9 Hz, 1H, H-5''), 7.40 (t, J = 7.2 Hz, 1H, H-5), 7.43 (t, J = 7.2
Hz, 1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.94 (s, 1H, NH); EIMS m/z
363 [M]+
(32), 348 (16), 333 (2), 321 (21), 291 (59), 264 (25), 143 (10), 121 (100), 91 (25), 78 (13), 77
(14), 43 (90); Anal. Calcd for C19H17N5O3: C 62.80; H 4.72; N 19.27. Found: C 62.72; H 4.70; N 19.34.
3-Amino-4-[1-(3-methoxy-4-methylbenzyl)-1H-benzimidazol-2-yl)-furazan (21).
N
N
N O
N
NH2
MeO
Me
H NMR (DMSO-d6): δ 2.11 (s, 3H, Me-4''), 3.84 (s,
3H, OMe-3''), 5.90 (s, 2H, CH2), 6.44 (br. s, 1H, H-2''), 6.79 (s, 2H, NH2), 6.84 (d, J = 8.3 Hz, 1H, H-
5''), 6.99 ( br. d, J = 8.3 Hz, 1H, H-6''), 7.29 (m, 2H, H-5,6), 7.40 (m, 1H, H-7), 7.78 (m, 1H, H-4');
EIMS m/z 335 [M]+
(31), 318 (3), 305 (19), 290 (1), 143 (13), 135 (100), 105 (40), 103 (9), 91 (13), 79
3-Amino-4-[1-(3,4-diethoxybenzyl)-1H-benzimidazol-2-yl]-furazan (22).
N
N
N O
N
NH2
EtO
OEt
H NMR (DMSO-d6): δ 1.34 (t, J = 7.0 Hz, 3H,
CH3CH2), 1.35 (t, J = 7.0 Hz, 3H, CH3CH2), 3.95 (q, J = 7.0 Hz, 4H, 2OCH2), 5.87 (s, 2H, CH2), 6.63

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(dd, J = 1.6 Hz, J = 8.2 Hz, 1H, H-6''), 6.73 (d, J = 8.2 Hz, 1H, H-5''), 6.81 (s, 2H, NH2), 6.86 (d, J = 1.6
Hz, 1H, H-2''), 7.33 (m, 2H, H-5',6'), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.78 (d, J = 7.6 Hz, 1H, H-4); EIMS
m/z 379 [M]+
(28), 349 (10), 179 (100), 151 (40), 144 (11), 143 (8), 133 (6), 123 (65), 105 (5), 94 (7),
77 (12); Anal. Calcd for C20H21N5O3: C 63.31; H 5.58; N 18.46. Found: C 63.40; H 5.61; N 18.56.
3-Amino-4-[1-(4-methylbenzyl)-1H-benzimidazol-2-yl]-furazan (23).
N
N
N O
N
NH2
Me
H NMR (DMSO-d6): 2.23 (s, 3H, Me-4''), 5.93 (s,
2H, CH2), 6.95 (s, 2H, NH2), 7.05 (d, J = 8.0 Hz, 2H, H-3'',5''), 7.10 (d, J = 8.0 Hz, 2H, H-2'',6''), 7.36
(t, J = 7.8 Hz, 1H, H-6'), 7.40 (t, J = 7.8 Hz, 1H, H-5), 7.68 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 7.8
Hz, 1H, H-4); EIMS m/z 305 [M]+
(18), 275 (16), 259 (1), 248 (5), 232 (7), 106 (7), 105 (100), 103 (8),
77 (10); Anal. Calcd for C17H15N5O: C 66.87; H 4.95; N 22.94. Found: C 66.94; H 4.98; N 22.80.
3-Amino-4-[1-(4-t
butylbenzyl)-1H-benzimidazol-2-yl]-furazan (24).
N
N
N O
N
NH2
t-Bu
H NMR (DMSO-d6): δ 1.24 (s, 9H, C(CH3)3-4''), 5.94 (s,
2H, CH2), 6.94 (s, 2H, NH2), 7.10 (d, J = 8.1 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.1 Hz, 2H, H-2'',6''), 7.35
(t, J = 7.8 Hz, 1H, H-6), 7.39 (t, J = 7.8 Hz, 1H, H-5), 7.67 (d, J = 7.8 Hz, 1H, H-7), 7.83 (d, J =7.8 Hz,
1H, H-4); EIMS m/z 347 [M]+
(15), 317 (11), 261 (11), 148 (11), 147 (100), 138 (6), 132 (18), 124 (22),
117 (23), 91 (12); Anal. Calcd for C20H21N5O: C 69.14; H 6.09; N 20.16. Found: C 69.25; H 6.14; N
20.02.
N-(4-[1-(4-t
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N
N
N O
N
NH
t-Bu
O
H NMR (DMSO-d6): 1.24 (s, 9H, C(CH3)3-4''), 2.33
(s, 3H, CH3CO), 5.91 (s, 2H, CH2), 7.14 (d, J = 8.2 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.2 Hz, 2H, H-2'',6''),
7.39 (t, J = 7.6 Hz, 1H, H-6), 7.42 (t, J = 7.6 Hz, 1H, H-5), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.89 (d, J =
(3), 374 (1), 347 (2), 317 (14), 261 (6), 147
(100), 132 (19), 124 (6), 119 (7), 117 (21), 105 (9), 91 (11); Anal. Calcd for C22H23N5O2: C 67.85; H
5.95; N 17.98. Found: C 67.95; H 6.00; N 17.87.
3-Amino-4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan (25).
N
N
N O
N
NH2
N
H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.91 (s,
2H, NH2), 7.32 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.39 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2
Hz, 2H, H-3'',5''), 7.86 (m, 1H, H-4); EIMS m/z 316 [M]+
(5), 286 (4), 259 (8), 143 (8), 117 (9), 116
(100), 89 (24); Anal. Calcd for C17H12N6O: C 64.55; H 3.82; N 26.57. Found: C 64.61; H 3.84; N 26.50.
N-(4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (25r).
N
N
N O
N
NH
O
N

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2H, CH2), 7.37 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.42 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2
Hz, 2H, H-3'',5''), 7.91 (m, 1H, H-4), 10.90 (s, 1H, NH); EIMS m/z 358 [M]+
(18), 344 (6), 343 (28),
341 (1), 316 (11), 286 (27), 259 (41), 244 (5), 117 (8), 116 (100), 89 (12), 43 (15); Anal. Calcd for
C19H14N6O2: C 63.68; H 3.94; N 23.45. Found: C 63.79; H 3.97; N 23.32.
3-Amino-4-(1-methyl-1H-benzimidazol-2-yl)-furazan (26).
N
N
Me
N
O
N
H2N
H NMR (DMSO-d6): δ 4.12 (s, 3H, Me), 6.88 (s, 2H,
NH2), 7.32 (t, J = 7.8 Hz, 1H, H-5), 7.40 (t, J = 7.8 Hz, 1H, H-6), 7.68 (d, J = 8.2 Hz, 1H, H-7), 7.79 (d,
J = 8.2 Hz, 1H, H-4); 13
C NMR (DMSO-d6): δ 31.9, 110.6, 119.5, 122.7, 124.1, 135.6, 138.2, 140.7,
141.4, 155.9; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.83; H 4.18; N 32.46;
IR (KBr): ν max 3410, 3311, 1633, 1598, 1577, 1549, 1472, 1457, 1422, 1327, 1290, 1262, 1231, 1157,
1132, 1071, 998, 905, 866, 813, 754, 740, 717, 604, 568, 546.
4-Fluoro-N-[4-(1-methyl-1H-benzimidazol-2-yl)- furazan-3-yl]benzamide (26t).
N
N
N O
N
NH
Me
O
F
H NMR (DMSO-d6): 4.23 (s, 3H, Me), 7.40 (t, J =
7.8 Hz, 1H, H-6), 7.48 (t, J = 7.8 Hz, 1H, H-5), 7.53 (t, J = 8.8 Hz, 2H, H-3'',5''), 7.78 (d, J = 7.8 Hz,
1H, H-7), 7.92 (d, J = 7.8 Hz, 1H, H-4), 8.15 (dd, J = 5.8 Hz, J = 8.8 Hz, 2H, H-2'',6''), 10.14 (s, 1H,
NH); EIMS m/z 337 [M]+
(11), 170 (8), 158 (100), 143 (5), 123 (32), 95 (21), 75 (6); Anal. Calcd for
C17H12FN5O2: C 60.53; H 3.59; N 20.76. Found: C 60.43; H 3.56; N 20.90.
3-Amino-4-(1-ethyl-1H-benzimidazol-2-yl)-furazan (27).
N
N
N O
N
NH2
Et
H NMR (DMSO-d6): δ 1.35 (t, J = 7.0 Hz, 3H,
CH3CH2), 4.63 (q, J = 7.0 Hz, 2H, CH2N), 7.01 (s, 2H, NH2), 7.30 (t, J = 7.7 Hz, 1H, H-5), 7.38 (t, J =

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7.7 Hz, 1H, H-6), 7.70 (d, J = 8.0, 1H, H-7), 7.78 (d, J = 8.0, 1H, H-4); 13
C NMR (DMSO-d6): δ 14.8,
2939, 1635, 1626, 1593, 1575, 1545, 1485, 1472, 1461, 1446, 1410, 1376, 1351, 1330, 1293, 1260,
1202, 1155, 1134, 1088, 1074, 998, 955, 907, 864, 784, 757, 742, 705, 565, 466.
N-(4-[1-(4-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (28r).
N
N
N O
N
NH
O
MeO
2H, OMe-4''), 5.89 (s, 2H, CH2), 6.80 (d, J = 8.5 Hz, 2H, H-3'',5''), 7.17 (d, J = 8.5 Hz, 2H, H-2'',6''),
7.36 (d, J = 7.6 Hz, 1H, H-6), 7.39 (t, J = 8.0 Hz, 1H, H-5), 7.64 (d, J = 7.6 Hz, 1H, H-7), 7.85 (d, J =
(5), 333 (1), 321 (3), 291 (15), 121 (100), 77
3-Amino-4-(1-(4-methoxybenzyl)-5-(trifluoromethyl)-1H-benzimidazol-2-yl)-furazan (29).
N
N
N O
N
NH2
MeO
F3C
H NMR (DMSO-d6): δ 3.69 (s, 3H, OMe-4''), 5.95 (s,
2H, CH2), 6.86 (d, J = 8.6 Hz, 2H, H-3'',5''), 7.02 (s, 2H, NH2), 7.16 (d, J = 8.6 Hz, 2H, H-2'',6''), 7.74
(dd, J = 1.6 Hz, J = 8.6 Hz, 1H, H-6), 7.98 (d, J = 8.6 Hz, 1H, H-7), 8.26 (d, J = 1.6 Hz, H-4); 13
C NMR
(DMSO-d6): δ 159.3, 156.7,143.3, 141.6, 138.4, 137.8, 128.7, 128.3, 126.9, 124.9, 124.4, 123.3, 121.6,
118.1, 114.6, 113.2, 55.3, 48.4; 19
F NMR (DMSO-d6): -60.24. 15
N NMR (DMSO-d6): 26.70, -18.27, -
131.45, -207.32, -332.63; EIMS m/z 389 [M]+
; Anal. Calcd for C18H14F3N5O2: C 55.53; H 3.62; N
17.99. Found: C 55.48; H 3.60; N 17.92; IR (KBr): ν max 3442, 3335, 1640, 1614, 1581, 1517, 1464,
1441, 1343, 1331, 1304, 1268, 1255, 1231, 1183, 1158, 1105, 1052, 1033, 1009, 972, 890, 867, 836,
816, 804, 780, 756, 699, 652, 631, 569.

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