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U. Kessler et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5575–5577
Figure 1. Commercially available antiviral compounds and the hit compound 5.
interaction domain between the PB1 and PA subunits using a
small peptide derived from the PA-binding domain of PB1.
In order to identify chemical compounds that efficiently block
the PA–PB1 interaction, the novel biochemical ELISA-based screen-
ing approach described for influenza peptides10,11 was adapted to
screen a set of chemical libraries and natural and antiviral com-
pounds collections. A total of 15,000 molecules was subjected to
binding assays to PA subunit.10,12 The inhibitory concentration
(IC50) was determined only for compounds that reported a positive
outcome in the ELISA prescreen. A Plaque Reduction assay on
NO2
0-1
N
N
NO2
Cl
HX
R
O
N
N
O
i.
X
0-1
7a-v
X = S, O
6
R
ii., iii.
MDCK cells infected with the virus A/WSN/33 (H1N1) was used
NHR1
Cl
S
13
for the determination of IC50
.
This approach lead us to the iden-
N
N
N
N
O
tification of the hit benzofurazan compound 5 that showed inhibi-
tion of viral replication at micromolar level (IC50 = 5 M) (Fig. 1).
O
iv.
l
R1 = H 8
R
R
S
Benzofurazans are a class of compounds known for their fluores-
cent properties thus possessing broad application in medicinal
chemistry and biology. However, no examples of benzofurazans
endowed with anti-influenza activity have been reported so far.
Thus, in order to explore the chemical space around the benzofura-
zan core, evaluate the structure–activity relationships (SARs) and
increase the activity of the hit compound 5, the synthesis of a ser-
ies of analogues was planned. In particular, modifications at posi-
tion C4, such as the displacement of nitro group with amines or
chlorine, and at C7, such as the introduction of different benzylic
and (thio)phenolic substituents, were carried out (Fig. 1). Herein,
we report our preliminary studies on the evaluation of the anti-
influenza A activity and cytotoxicity of a series of novel benzofura-
zan compounds.
The hit compound 5 and the corresponding analogues 7a–v were
rapidly obtained in high yields through an aromatic nucleophilic
substitution reaction on the commercially available 4-chloro-7-ni-
tro benzofurazan 614 under fairly mild conditions. Refluxing of 6
with an equimolar amount of the appropriate nucleophile
(benzylthiol, thiophenol, phenol) for 20 min in presence of cata-
lytic amount of pyridine or KOAc afforded the desired compounds
7a–v (Scheme 1). Unsurprisingly, the reaction of nucleophiles
2-aminophenol, 4-aminophenol and 2-aminothiophenol with 6
led to the formation of the corresponding amino-derivatives 7t,
7u, and 7v in place of the desired O or S benzofurazan derivatives,
presumably due to the higher nucleophilicity of the amine group
under these reaction conditions. The synthesis of the O-benzyl ben-
zofurazan 11 proved to be challenging (Table 1). Attempts to syn-
1 = CH3 9a
10
1 = Bn 9b
OMe
OMe
Scheme 1. Reagents and conditions: (i) catalytic amount of pyridine or KOAc, EtOH
reflux, 20 min; (ii) Fe, HCl, DCM/MeOH, rt, 25 min; (iii) (a) R1 = CH3: paraformal-
dehyde, Na, NaBH4, MeOH, reflux, 1 h; (b) R1 = Bn/benzaldehyde, NaBH4/PTSA, DCE,
rt, 16 h; (iv)8, CuCl2, tert-butyl nitrite, CH3CN, 65 °C, 3 h.
Cs2CO3 under microwave irradiation, affording desired derivative
11 in acceptable 24% yield (entry 7). Comparable yields were ob-
tained reacting 6 with BnOH in the presence of 2% Pd(AcO)2 (entry
8). Finally, modifications at C4 were carried out treating thiobenzyl
derivative 5 with Fe in HCl which led to the formation of the cor-
responding amine 8. Reductive amination of 8 with benzaldehyde
or paraformaldehyde afforded alkyl derivatives 9a–b, whilst treat-
ment of 8 with CuCl2 in the presence of tBuONO led to the chloro-
derivative 10.
The synthesized compounds were then assayed to evaluate
their inhibitory activity toward influenza virus strain A/WSN/33
(H1N1). Results are reported in Table 2.
Compounds 7a–c (entries 2–4), bearing a fluoro/chloro-benzyl
or benzyl chain, resulted inactive, while the bromo derivative com-
pound 7d prove to be as active as the hit compound 5. However, 7d
showed higher cytotoxicity than 5. On the other hand, the presence
of a nitro group on the benzylic chain of compound 7e (entry 6)
produced an increase of the anti-viral activity. Compound 7e
thesize 11 by nuclophilic substitution on
6 under standard
proved to be active at 1–2.5 lM and showed a cytotoxic profile
conditions were unsuccessful (entry 1). Neither the use of benzyl
alcohol sodium salt as nucleophile led to desired 11 (entries 2
and 3). Finally, a Ullman type reaction was planned: benzofurazan
6 was reacted with benzyl alcohol in the presence of 20% CuI and
similar to 5. In general the replacement of the thiobenzyl chain
with substituted thiophenol moieties resulted in compounds (en-
tries 9–14) displaying medium/good activity. Interestingly, all
thiophenol derivatives 7h–m showed a better cytotoxic profile
Kessler, Ulrich
