Synthesis and structure-activity relationships of novel furazan-3,4-diamide analogs as potent anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2009.12.017

This study describes the synthesis and structure-activity relationships of a series of furazan-3,4-diamide analogs. 1,2,5-Oxadiazole ring and electron-withdrawing substituent on the phenyl ring are proposed to be the important elements which contribute to a significant extent maximal potency of anti-proliferation effect.

Full text of DOI:10.1016/j.bmcl.2009.12.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1148–1152
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of novel furazan-3,4-diamide
analogs as potent anti-cancer agents
Wen-Shan Li a, , Shivaji V. More a,b, Chie-Hong Wang a, Ya Ching Jen a, Ching-Fa Yao b,
,
*
*
Tein-Fu Wang a, Chin-Chun Hung c, Shu-Chuan Jao c
a Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
b Department of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan
c Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
This study describes the synthesis and structure–activity relationships of a series of furazan-3,4-diamide
analogs. 1,2,5-Oxadiazole ring and electron-withdrawing substituent on the phenyl ring are proposed to
be the important elements which contribute to a significant extent maximal potency of anti-proliferation
effect.
Received 8 October 2009
Revised 1 December 2009
Accepted 3 December 2009
Available online 6 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anti-cancer agents
Drug discovery
Synthesis
Structure–activity relationship
The search of biologically promising new chemical entity
(NCE)1–6 against deadly cancer disease remains great attention in
drug discovery. Major sources of bioactive NCE are identified from
or inspired by natural products,7,8 marine metabolites9,10 and ran-
dom screening of chemical library.11–13 Furoxan 1 and benzofuro-
xan analogs 2 (Fig. 1) were recently found to be potent as the
anti-cancer, anti-microbial, anti-aggregating, anti-ulcer, and
immunosuppressive agents.14–18 Similarly, biological studies re-
vealed that oxadiazolopyrazines 3, furazans 4, and diaminofuraz-
ans 5 (Fig. 1) exhibited significant anti-bacterial properties and
also showed the active use in the treatment of cancer, atheroscle-
rosis, angiogenesis, neurodegenerative diseases, and inflammatory
diseases.19–24 Given that the potential of compounds 1–5 is con-
firmed as the therapeutic candidates, the 1,2,5-oxadiazole (fura-
zan) moiety might represent a satisfactory pharmacophore to
design anti-cancer agents. To test our initial hypothesis, we syn-
thesized a set of furazan-3,4-diamide analogs 6 and evaluated
the tumor cell growth inhibitory activity in two human cancer cell
lines.
the desired aliphatic furazan-3,4-diamides 7–11/33 and aromatic
furazan-3,4-diamides 12–22 in high yields,25 respectively. This
method displays a significant improvement over the original syn-
thesis of compound 7, which required 2–6 equiv of 2,2,2-trichloro-
acetyl chloride in the presence of Et3N and gave the product in low
yield at room temperature.
The HPLC-purified furazan-3,4-diamides 7–22 were dissolved in
DMSO and tested for growth-inhibitory potency in U-87 MG hu-
man glioblastoma and SW480 human colon adenocarcinoma cell
Initial synthesis of aliphatic and aromatic furazan-3,4-diamides,
7–11, 33, and 12–22 (Fig. 2), started with 3,4-diaminofurazan 34
and the corresponding acyl chloride (Scheme 1). Lewis acid
(BF3ÁEt2O)-mediated coupling reaction in dioxane at reflux gave
* Corresponding authors.
E-mail address: wenshan@gate.sinica.edu.tw (W.-S. Li).
Figure 1. Six 1,2,5-oxadiazole derivatives.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.017

W.-S. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1148–1152
1149
Figure 2. Rationale for the design of aliphatic and aromatic furazan-3,4-diamides (7–11 and 12–22).
Scheme 1. Reagents and conditions: (a) BF3ÁEt2O, dioxane, reflux, 10 h, 55–90%; (b) MW, 5–10 min, 70–85%.
lines. The assay results, summarized in Table 1, show that unlike
compounds 7–11 which exhibited weak activities, compound 33
having two chloroacetyl moieties possessed a significantly lower
whereas, compound 13 bearing two furanyl groups displayed weak
activity, suggesting that the phenyl moiety is an acceptable phar-
macophore. Compound 21 having two 4-(trifluoromethyl)phenyl
substituents was the most active compound with IC50 values of
IC50 in both cell lines (IC50 = 17.1 and 7.4
lM) as compared to other
aliphatic furazan-3,4-diamides (IC50 >200
l
M). Efforts to analyze
14.6 and 11.2 lM against two cancer cell lines (Table 1). Similar
compound 33-induced cellular pathway required for effective inhi-
bition of cell growth are in progress.
In comparison with aliphatic furazan-3,4-diamides 7–11, eleven
aromatic furazan-3,4-diamides 12–22 showed similar or greater
growth-inhibitory potency against U-87 MG and SW480 cells.
Compound 12 with two phenyl substituents moderately inhibited
to compound 21, compounds 17, 18, and 22, which bear the elec-
tron-withdrawing groups in the phenyl ring, gave promising re-
sults in inhibition of cell growth with IC50 values between 10 and
39 lM. Compounds 19 and 20 having the trifluoromethyl group
at the C-2 and C-3 position on the phenyl ring possessed higher
IC50 values in both cell lines and lower solubility as compared to
the parent compound 21. In comparison with 21, three electron-
cell growth with IC50 values of 121.6 and 137.0 lM, respectively,

1150
W.-S. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1148–1152
Table 1
diminished solubility in cellular solution was also found with com-
In vitro antitumor activity for compounds 7–33
pounds 23 and 24. Compound 25 with a hydrophilic 3,4-diamino-
1H-pyrazol-5-ol resulted in at least 14–18-fold less potent than 21,
indicating that the 1,2,5-oxadiazole core structure is essential for
potency. Our results suggest that the core structure of aromatic
3,4-diamides would be preferred for the 1,2,5-oxadiazole ring.
To enhance the potency, we synthesized numerous analogs 27–
30 (Fig. 4) with alterations of the trifluoromethyl substituent.26 In
addition, we next evaluate the effect of carbonyl group of amide
bond in compound 21 for growth-inhibitory activity. The prepara-
tion starting with 3,4-diaminofurazan 34, and a 4-(trifluoro-
methyl)benzoyl group was introduced by pyridine-mediated
acylation to obtain a 4-aminofurazan-3-amides 26 in high yield
(with trace 21, Scheme 2). Amide 26 was subjected to reduction
with lithium aluminum hydride followed by selective acylation
on primary amine and purification through column chromatogra-
phy, affording the asymmetrical 4-aminofurazan-3-amides 31.
For the reduction of the amide moiety of 31, an excess of lithium
aluminum hydride was used to give symmetrical furazan-3,4-dia-
mine 32.
a
Compds
IC50 (lM)
U-87 MG
SW480
7
8
9
>200
>200
>200
>200
>200
>200
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
>20b
>20b
>200
>200
121.6 4.2
>200
137.0 2.3
>200
>40b
>40b
70.7 15.3
71.0 7.5
>40b
>40b
24.0 3.8
25.5 1.2
>100
39.7 3.2
17.2 1.1
89.3 6.4
>10b
>10b
14.6 2.3
17.7 0.8
>8b
11.2 1.3
10.8 1.0
>8b
>8b
>200
>200
>8b
>200
>200
As expected, compound 26 displayed a significant loss of
growth-inhibitory activity compared to 21 (Table 1). The results
suggest that the introduction of two 4-(trifluoromethyl)benzoyl
groups into the 3,4-diaminofurazan 34 scaffold is clearly beneficial
for activity. The replacement of the 4-trifluoromethyl group of 21
with other electron-withdrawing substituents (F, Cl, Br, and I) led
to the improvement of activity. For example, the 4-fluorobenzoyl
6.0 1.0
14 (70%)c
14 (77%)c
1 (70%)c
18.2 1.0
20.6 1.0
17.1 2.7
10 (50%)c
8 (50%)c
4 (50%)c
2 (50%)c
18.5 1.1
22.9 2.0
7.4 1.1
a
derivative 27 exhibited an IC50 of 6.0 lM against U-87 MG cells
Amount of compound necessary to inhibit the growth of cancer cells by 50% in
48 h. Values are means of three experiments (IC50, mean SEM, n = 3).
(Table 1, Fig. 5), representing a 2.4-fold improvement over 21
and 20-fold improvement over 12. The concentrations of com-
pounds 28–30 required to inhibit U-87 MG cells growth by 50%
were lower (Table 1) as compared to compound 21. Similar results
were observed for 27–30 against SW480 cell line. Unfortunately,
these compounds reached a plateau of effective potency and could
not completely inhibit cancer cell growth under high dose condi-
tion or dose-dependent manner. For example, compound 30 hav-
ing two 4-iodobenzoyl moieties possessed a significant anti-
proliferation effect to inhibit 50% U-87 MG cells growth at the level
of nanomolar range; whereas it became inactive at concentration
b
Due to poor solubility, the value is expressed as the maximum concentration
used in study.
c
Compound became inactive at indicated concentration or greater. The percent
(%) inhibition, in parentheses, is expressed as the maximum percent (%) inhibition
of cells growth.
donating compounds 14–16 displayed weaker growth-inhibitory
activities against two human cancer cell lines. Thus, these results
indicate that the electron-withdrawing functionalities (Cl, F or
CF3) identified by screening were clearly required for the improved
antitumor activity.
of 1 lM or greater (inhibiting U-87 MG cells growth at the maxi-
mum of 70%, Table 1). Similar results were found for 27–30 against
SW480 cell line. These observations gave us a clue that the hydro-
phobic trifluoromethyl group holds promise for effective potency.
To examine whether the carbonyl groups of 21 could affect the
growth-inhibitory activity, we tested the anti-proliferation effect
of compounds 31 and 32. Slight diminished activity was observed
with 31 and 32, in which a carbonyl or two carbonyl groups were
reduced into methylene moiety. For instance, compounds 31 and
32 are 1.2–1.4- and 1.7–2.0-fold less potent than 21 against U-87
MG and SW480 cells, respectively.
As shown in Table 1, two 4-(trifluoromethyl)phenyl substitu-
ents in the aromatic furazan-3,4-diamide series is critical for
strong activity. To elucidate an initial structure–activity profile,
our first aim was the design and synthesis of the suitable core
structures of aromatic 3,4-diamides 23–25 (Fig. 3), for which we
chose thiophene, benzene and 1H-pyrazol-5-ol to replace the
1,2,5-oxadiazole group. Employing the method of Lewis acid-med-
iated coupling reaction in Scheme 1, compounds 23–25 were syn-
thesized by reacting 4-(trifluoromethyl)benzoyl chloride with
thiophene-3,4-diamine, benzene-1,2-diamine, and 3,4-diamino-
1H-pyrazol-5-ol, respectively, under reflux condition. Unluckily,
this exercise did not improve the growth-inhibitory activity and
Altogether, the structure–activity relationship (SAR) study
(Fig. 6) revealed that two aromatic amide substituents of analogs
Figure 3. The core structures of aromatic 3,4-diamides 23–25 used in this study.

W.-S. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1148–1152
1151
Figure 4. Symmetrical and asymmetrical 3,4-diaminofurazan derivatives 26–32 used in this study.
Scheme 2. Reagents and conditions: (a) pyridine, DCM, 0 °C to rt, 5 h, 70%; (b) LAH, THF, 0 °C to rt, 2 h, 70%; (c) MW, 4 min, 72%.
6 play an important role for inhibition of cancer cell growth. Elec-
tron-withdrawing substituents (CF3 and F) in the para-position of
phenyl ring are favorable for achieving anti-proliferation effect. A
1,2,5-Oxadiazole ring core structure is essential for the effective
activity, whereas the existence of carbonyl groups is not critical
for potency (Fig. 6).
In summary, we have identified bioactive NCEs from a set of
furazan-3,4-diamide analogs by random screening and SAR ap-
proaches. This work not only established an effective route to syn-
thesize diverse analogs of aliphatic and aromatic furazan-3,4-
diamides but also provided a general strategy for discovering po-
tent anti-cancer agents.

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W.-S. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1148–1152
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25.
A representative experiment for acid catalyzed reaction: Benzoyl chloride
Figure 6. Schematic representation of structure–activity relationships based on
diverse furazan-3,4-diamide analogs.
(281 mg, 2 mmol) was mixed with dioxane (3 mL) and followed by addition
of 3,4-diaminofurazan 34 (100 mg, 1 mmol) in dioxane (2 mL) and BF3ÁEt2O
6.78 mg (10 mol %). The reaction mixture was refluxed for 10 h (monitored by
TLC). The crude residue was diluted with DCM (15 mL) and washed with water
(2 Â 10 mL). Organic layer was dried over anhydrous MgSO4, filtered,
evaporated, and the crude product was purified by column chromatography
(SiO2, 20% EtOAc/hexane) to afford compound 12 (262 mg, 85%).
Acknowledgments
26.
A
representative experiment for MW condition: In 5 mL sample vial, 3,4-
We are grateful to Ping-Yu Lin and Dr. Mei-Chun Tseng for
assistance with mass spectrometry. We thank the National Science
Council(NSC 95-2113-M-001-018-MY3) and Academia Sinica (pro-
gram project AS-95-TP-AB1) for financial support.
diaminofurazan 34 (100 mg, 1 mmol) and 4-fluorobenzoyl chloride (317 mg,
2 mmol) were mixed. The sample vial was closed with Teflon cap and allowed
to react at 100 °C in microwave for 5 min (monitored by TLC). The crude
residue was directly purified by column chromatography (SiO2, 20% EtOAc/
hexane) to afford compound 27 (275 mg, 80%).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.017.