Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives

Article abstract of DOI:10.1016/j.bmc.2009.05.020

Twelve aminoarylazocompounds (A-C) and 46 aryltriazene 7 derivatives (D-G) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. Eight aminoazocompounds and 27 aryltriaz

Full text of DOI:10.1016/j.bmc.2009.05.020

Bioorganic & Medicinal Chemistry 17 (2009) 4425–4440
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiviral and cytotoxic activities of aminoarylazo compounds
and aryltriazene derivatives
b
Michele Tonelli a, Iana Vazzana a, Bruno Tasso a, Vito Boido a, Fabio Sparatore a, , Maurizio Fermeglia ,
Maria Silvia Paneni b, Paola Posocco b, Sabrina Pricl b, Paolo La Colla c, Cristina Ibba c, Barbara Secci c,
Gabriella Collu c, Roberta Loddo c
*
a Dipartimento di Scienze Farmaceutiche, Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
b Dipartimento di Ingegneria Chimica, dell’Ambiente e delle Materie prime, Università di Trieste, Via Valerio 10, 34127 Trieste, Italy
c Dipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Cittadella Universitaria, S.S. 554, Km 4.500, 09042 Monserrato (Cagliari), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve aminoarylazocompounds (A–C) and 46 aryltriazene 7 derivatives (D–G) have been synthesized
and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and
DNA viruses.
Eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high
level, against one or more viruses. A marked activity against BVDV and YFV was prevailing among the
former compounds, while the latter type of compounds affected mainly CVB-2 and RSV. None of the
active compounds inhibited the multiplication of HIV-1, VSV and VV.
Received 19 March 2009
Revised 30 March 2009
Accepted 7 May 2009
Available online 15 May 2009
Keywords:
Aminoarylazo compounds
Aryltriazene derivatives
Antiviral activity
Arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are
the following; BVDV: 1 > 7 > 8 > 4; YFV: 7 > 5; CVB-2: 25 > 56 > 18; RSV: 14 > 20 > 55 > 38 > 18 > 19; HSV-
1: 2. For these compounds the EC50 ranged from 1.6
(2).
lM (1) to 12 lM (18), and the S. I. from 19.4 (1) to 4.2
BVDV RdRp targeting
Thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for
developing antiviral agents against ss RNA viruses, particularly against RSV and BVDV, which are impor-
tant human and veterinary pathogens.
Finally, molecular modeling investigations indicated that compounds of structure A–C, active against
BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a
good agreement between the trends of the estimated IC50 and the experimental EC50 values.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
proliferative activities have been found in a peculiar class of cyclic
aminoazocompounds (3,3-disubstituted-3,4-dihydro-1,2,4-benzo
We have recently shown that some arylazoenamines (Fig. 1) are
endowed with antiviral activity in vitro against RNA viruses, par-
ticularly CVB-2, RSV, BVDV, YFV and Sb-1, in many cases with
triazines), which, moreover, can display several other pharmacolog-
ical activities depending on the substituents that are present on the
bicyclic system.5,6 Very potent relaxant activity on intestinal and
uterine smooth muscles was displayed at nanomolar concentration
by N-dialkylaminoalkyl derivatives of another cyclic aminoazo sys-
tem, namely 11H-dibenzo-[1,2,5]-triazepine, which exhibited also
local anesthetic and antithrombotic activities.7
EC50 in the range from 0.8 to 10 l
M.1
Arylazoenamines can be considered as substructures of ortho-
(and para-) aminoazocompounds, many of which are endowed with
antimicrobial and antiparasitic activities, but additional pharmaco-
logical activities have been also shown in particular cases (Fig. 2).
2,6-Diamino-3-phenylazopyridine (Pyridium) inhibits various
cocci and coli bacilli, 5-(4-amino-1-naphthylazo)uracil is highly
effective against Schistosoma mansonii,2 while 1-[3-(4-phenylazo-
5,6,7,8-tetrahydronaphth-1-yl)amino]propylpiperidine3 and 1-[3-
(4-phenylazo-5,6,7,8-tetrahydro naphth-1-yl)amino]lupinane4 are
very active against Mycobacterium tuberculosis. Antifungal and anti-
Arylazoenamines could be also seen as vinylogues of aryltriaz-
enes (Fig. 2), a class of compounds which hold an important
R'
R'''
N
N
RIV
N
R
R''
Figure 1. General structure of the previously studied arylazoenamines.
* Corresponding author. Tel.: +39 010 512721; fax +39 010 3538358.
E-mail address: sparator@unige.it (F. Sparatore).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.020

4426
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
H
O
N
N
O
NH2
N
;
N
NH
N
N
N
N
NH2
R
N
H
R
N
H
Et
Et
CH2
R= (CH2)3
N
R= H; CH2CH2N
H
N
N
N
N
N
N
N
C2H5
N
N
H
N
H
N
CH2
N
CH3
CH3
(CH2)n
CH3
(CH2)3N
H N
2
O
H2N
O
CH3
C
C
N
N
N
CH3
N
N
N
N CH3
N
N
N
N
N
NH
C
O
CH
3
N
R
CH3
N
H C
NH
N
N
N
C
O
3
H
N
CH3
N
N
CH
3
N
CH3
N
N
N
C
CH3
O
R= H; CONH(CH 2)2
N
O
Cl
R
N
H
N
N
N
N
R
NH2
N
N
H N
NH
Cl
2
2
C
C
NH
NH
H2N
N
Et
N
N
Br
N
N
Br
Br
N
N
N
H
N
N
CH2
N
O
N
H
N
C
O
CH2CH2
Br
H
N
Figure 2. Some biologically active aminoarylazo compounds and aryltriazene derivatives.
position as carcinogens and/or anticancer agents. 3,3-Dimethyl-1-
phenyltriazene and, even better, dacarbazine are able to methylate
DNA; thus the former resulted a powerful carcinogen, while the
latter is in clinical use for the treatment of malignant melanoma
and Hodgkin’s lymphoma.8 Temozolamide,9 closely related to
dacarbazine, is currently used to treat malignant glyomas. A recent
development of this cyclic aryltriazene is represented by benzo-
triazepinones,10 that are very promising agents against breast can-
cer. It is worth noting that these compounds are weak alkylating
agents and may damage DNA by a novel mechanism.
known to inhibit the epidermal growth factor receptor tyrosine ki-
nase (EGFR-TK), and also to a benzophenone residue,12 that is pres-
ent in an inhibitor of pharnesyl-transferase and of tubulin-
polymerization.
The introduction of a triazene group on the molecule of pyri-
methamine, a dihydrofolate reductase inhibitor, generated a com-
pound that combines antitumoral potential with inhibition of
Pneumocystis jirovecii13 (P. carinii). The insertion of triazene group
between two benzamidine units produced diminazene (Berenil),
a powerful agent against the African trypanosomiasis.
The triazene pharmacophore has been linked to other bioactive
moieties in order to obtain chymeric compounds which should be
able to exert a double cytotoxic mode of action. Thus, the triazene
group was linked to 4-anilino-quinazoline backbone,11 which is
To the best of our knowledge, no antiviral activity has been seen
with either amino arylazocompounds or aryltriazenes, with the
only exception for a patent claiming the activity of 1-phenyl-3,3-
dimethyltriazene against the tobacco mosaic virus.14 However,

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4427
antiviral activity has been shown by several benzotriazole deriva-
tives, which could be formally considered as cyclic triazenes,
though the resonance stabilization of the heterocycle could imply
even substantial differences in the chemical and hence the biolog-
ical behavior of the two kinds of compounds. Interestingly, some
halogenated benzotriazoles have been shown to inhibit the
NTPase/helicase activities of hepatitis C and related viruses.15 Aro-
matic and heteroaromatic esters of 1-hydroxybenzotriazole were
found to strongly inhibit (Ki = 7.5 nM) the 3CL-protease, which is
essential for the replication of the coronavirus responsible of SARS
(severe acute respiratory syndrome).16 More recently, a set of ben-
zotriazole derivatives have been found endowed with potent activ-
ity against RSV and moderate activity against YFV, BVDV and CVB-
2.17
On this base we deemed interesting to investigate the possible
antiviral activity of some assorted aminoarylazo compounds and
aryltriazene derivatives, allotted in seven groups A–G (Fig. 3). On
the whole 12 amino arylazo compounds (A–C) and 46 triazene
derivatives (D–G) were evaluated in cell based assays for cytotox-
icity and antiviral activity against a large panel of RNA and DNA
viruses.
derivative 57 see.q 1-(3-Nitrophenylazo)piperidine (48) is reported
as known,r but we failed to find out any data concerning its
characterization.
References,a-r concerning the previously described compounds,
are available as Supplementary data.
The 28 novel compounds were prepared according to the fol-
lowing Schemes 1 and 2.
All new compounds were characterized by elemental analyses
and 1H NMR spectra.
The N-(3’-dimethylaminopropyl)-3-trifluoromethylaniline and
the N-(lupinyl)-3-trifluoromethylaniline, required by Scheme 1,
were already described by some of us.18
It is observed that the presence of a 3-trifluoromethyl group
prevented completely the coupling of diazonium salt on the para
position of the N-substituted anilines giving place only to the tria-
zene derivatives E.
3. Results and discussion
3.1. Biological activity—general considerations
The prepared compounds (12 amino arylazocompounds A–C
and 46 triazene derivatives D–G) were evaluated in vitro in parallel
cell-based assays for cytotoxicity and antiviral activity (Tables 3–5)
against viruses representative of two of the three genera of the Fla-
viviridae family, that is, Flaviviruses (YFV) and Pestiviruses (BVDV),
as Hepaciviruses can hardly be used in routine cell-based assays.
Title compounds were also tested against representatives of other
virus families. Among ssRNA+ were a Retrovirus (Human Immuno-
deficiency Virus type 1, HIV-1), two Picornaviruses (Coxsackie
2. Chemistry
Thirty out of the 58 compounds, that were evaluated for antivi-
ral activity, were already known and were prepared according to
the literature. Compounds 7–12 were already described by some
of us.4 For 1,3-diaryltriazenes 13, 15–17, 20–24, see;a–h for arylazo-
pyrrolidines 34, 36, 38–42, see;i–l for arylazopiperidines 45, 49–51,
see;m,n for arylazopiperazine 52, 54, 55 see;o,p finally, for cytisine
CH3
NH CH
2
NH(CH 2)3N
CH3
H
N
N
N
N
N
CH3
R
R
CH3
A : 1 - 6
B : 7, 8
NH CH
2
H
N
N
N
R
C : 9 - 12
X
X
N
N
N
N
N
N
R
R
R'
F (X = nil; CH2; CO; N R') :
34 - 56
D (X = H;
) :
CH3 13 - 24
CH3
E (X =(CH2)3 N
;
lupinyl): 25 - 33
CH3
O
N
N
N
N
R
G : 57, 58
Figure 3. Structures of the investigated compounds.

4428
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
CH
3
NH
N
CH
3
N
N
R
H
N
CH
3
A : 1-6
HN
only for
R'= H
+
+
-
CH
3
N
Cl
-
2
AcO
a), b)
+
R
CH
3
N
R'
CH
3
N
N
R'
N
R
E : 25-32
N
H
HN
+
-
H
N
N
Cl
2
N
N
N
a)
+
CF
3
CF
3
E : 33
Scheme 1. Reagents and conditions: (a) pH 7 (CH3COONa); Et2O; (b) CC (Al2O3/Et2O): E, yields 13–68%; then Et2O + 5%MeOH: A, yields 3–10%.
NH
2
H
N
N
R'
N
a)
b)
R'
R
R
D : 14, 18, 19
X
+
-
N
Cl
+
N
N
2
N
HN
X
R
F : 35, 37, 43, 44, 46-48, 53, 56
O
N
O
N
N
N
N
b)
HN
G : 58
NO
2
Scheme 2. Reagents and conditions: (a) oily anilines added to the diazonium salt solution, followed by CH3COONa; yields: 30–92%; (b) pH 7–8 (1 N NaOH); yields: 25–90%.
Virus type B2, CVB2, and Poliovirus type-1, Sabin strain, Sb-1);
among ssRNA- were a Paramyxoviridae (Respiratory Syncytial
Virus, RSV) and a Rhabdoviridae (Vesicular Stomatitis Virus, VSV)
representative. Among double-stranded RNA (dsRNA) viruses was
a Reoviridae representative (Respiratory Enteric Orphan Virus
type-1, Reo-1). Two representatives of DNA virus families were
also included: Herpes Symplex type 1, HSV-1 (Herpesviridae) and
Vaccinia Virus, VV (Poxviridae).
Interestingly, 35 (8 aminoazocompounds and 27 triazene deriv-
atives) over 58 tested compounds exhibited antiviral activity
against one or more viruses; in particular 16 compounds exhibited
a selective activity against a single virus, while 13, 4 and 2 were,
respectively, active against two, three and four viruses. On the
other hand, 23 compounds (4 aminoarylazo and 19 triazene deriv-
atives) were not able to inhibit the multiplication of any virus at
concentrations up to 100 lM.
AZT (30-azido-thymidine), NM 108 (20-C-methyl-guanosine), NM
176 (20-C-ethynyl-cytidine), Ribavirin, NM 299 (6-azauridine), M
5255(mycophenolic acid)and ACG (acyclovir)wereused asreference
inhibitors of ssRNA+, ssRNAꢀ and DNA viruses, respectively.
None of the active compounds inhibited the multiplication of
HIV-1, VSVand VV, butan increasingnumber of themexhibited anti-
viral activity against, in the order, Reo-1 (4), Sb-1 (7), HSV-1 (8),
BVDV(9), YFV(9), RSV (12) and CVB-2(13) (Tables 1 and 2). Nineteen

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4429
Table 1
3.3. Structure–activity relationships
Number of active compounds of structures A–C on susceptible viruses and range of
their EC50
As already seen, eight [(dialkylaminoalkyl)amino]azobenzenes
(A–C) and 27 triazene derivatives (D–G) have shown antiviral
activity, sometimes of high level against one or more viruses.
From Tables 1 and 3 appears that compounds of structure A–C
have a marked activity against BVDV and YFV, with only occa-
sional, though still of high level, activity against HSV-1, Reo-1,
CVB-2 and RSV.
On the whole the triazene derivatives D–G are characterized by
the most frequent activity against CVB-2 and RSV, to which are
associated, with decreasing frequency, the activity on Sb-1, HSV-
1, YFV, Reo-1 and BVDV (Tables 2, 4 and 5).
However, considering separately each subgroup of triazene
derivatives, it is observed that the activity against Sb-1 is mainly
associated to the pyrrolidine-related triazenes F, while activity
against HSV-1 is associated with the diaryltriazenes that bear a
dimethylaminopropyl chain. The importance of a basic chain for
the activity against HSV-1 is underlined by the high activity shown
by compounds 2 and 4, bearing the same dimethylaminopropyl
chain, even linked to the aminoazobenzene pharmacophore (A),
but also by the moderate activity of triazene 53, which contains
a basic N-methylpiperazine residue.
Virusa
No. of active aminoazocompounds (A–C) No. of active
over 12 tested compoundsb
compounds(range
of EC50, lM)
Reo-1c
HSV-1d
BVDVe
YFVe
1
2
7
4
1
1
/
1 (12)
1 (12)
1 (16)
1 (12)
/
/
/
1 (6)
5 (1, 6–9)
3 (7–10)
/
/
1 (30)
/
1 (25)
/
RSVf
CVB-2e
1 (11)
a
HIV-1, VSV, VV and Sb-1 were unaffected by all tested compounds of structure
A–C.
b
Compounds with EC50 > 100 lM, or higher than CC50 for the host cells are
considered inactive.
c
Double stranded RNA virus.
DNA virus.
Single-stranded, positive RNA virus.
Single-stranded, negative RNA virus.
d
e
f
compounds have shown an EC50 6 10
lM, 28 had EC50 between 11
and 30 M and only 15 had EC50 in the range 31–90
l
l
M.
Therefore, both the [(dialkylaminoalkyl)amino]azobenzene and
the aryltriazene molecular patterns represent interesting pharma-
cophore for developing novel antiviral agents, particularly against
ssRNA viruses.
Cytotoxicity and antiviral activities of tested and reference com-
pounds are reported in Tables 3–5. In these Tables, viruses for
which no active compounds have been found are not indicated.
Conversely it is worth noting that the activity against BVDV, lar-
gely diffused among the amino azo compounds A–C, is very rarely
present (2 over 46 compounds) among the triazene derivatives D–
G, even among those (E) sharing the basic chain of the former groups.
Active and inactive compounds are found side by side in all
groups A–G, thus their molecular patterns seem to address the
activity against the different type of viruses, while the substituents
on the aromatic nucleus are the determinants for activity or inac-
tivity. However, attempts to define general structure-activity rela-
tionships resulted rather frustrating, since observations that hold
for the activity against most viruses may not hold for some others.
Indeed the meta substitution with halogens, trifluoromethyl
and nitro groups favors the display of activity in the groups A–E
and G, but in compounds of group F is the para substitution that
promotes the activity; however occasional exceptions are observed
in both cases.
An unsubstituted aromatic ring can be found in active (1, 7, 9,
21, 25, 52, 54–56) and inactive (13, 34, 45) compounds, while
the presence of electron-releasing groups as CH3 and OCH3 (32,
42) or of a benzyl residue (15, 22) is always detrimental for the
expression of activity.
Despite the high antiviral activity of many compounds, the corre-
sponding selectivity index (S. I.) are generally low, due to the rather
high cytotoxicity exerted on the host cells. Only few compounds
exhibited a S. I. higher than 10 for one or more viruses, while 13
3.2. Cytotoxicity on host cells
Test compounds showed different degrees of cytotoxicity
against the confluent cell monolayers (in stationary growth) used
to support the multiplication of the different viruses.
The most susceptible to toxicity were the exponentially grow-
ing lymphoblastoid human cells (MT-4) used to grow HIV-1, while
the non-human host cell lines exhibited a progressively reduced
sensitivity in the order Vero-76 > BHK > MDBK. On the other hand,
it is observed that the toxicity is not evenly distributed among the
different types of test compounds, being mainly shown by the tria-
zene derivatives of structure E and by the amino azo compound A–
C, which are characterized by the presence of the basic dimethyl-
aminopropyl and lupinyl moieties. Indeed the most toxic com-
pounds were 29 and 12, with a mean of the CC50 values for the
host cells of 17.5 and 21.5
lM, respectively.
The other groups of triazene derivatives of structure D, F and G
are relatively non toxic, exhibiting in most cases CC50 P 100 lM.
Table 2
Number of active compounds of structures D–G on susceptible viruses and range of their EC50
Virusa
No. of active triazene derivatives (D–G)
No. of active compounds
(range of EC50, lM)
over 46 tested compoundsb
Reo-1c
Sb-1d
3
7
6
2
5
11
12
/
/
/
/
/
1 (30)
3 (25–30)
1 (30)
/
2 (23, 24)
/
1 (33)
2 (42, 44)
/
/
1 (50)
/
2 (35, 50)
1 (80)
1 (51)
2 (55, 64)
1 (90)
1 (80)
1 (15)
3 (12–14)
1 (19)
1 (20)
3 (15–20)
3 (12–14)
HSV-1e
BVDVd
YFVd
/
RSVf
6 (3–10)
4 (6–10)
2 (60, 73)
1 (71)
CVB-2d
2 (25, 30)
a
HIV-1, VSV and VV were unaffected by all tested compounds of structure D–G.
b
c
d
e
f
Compounds with EC50 > 100
Double stranded RNA virus.
Single-stranded, positive RNA virus.
DNA virus.
lM, or higher than CC50 for the host cells are considered inactive.
Single-stranded, negative RNA virus.

4430
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
Table 3
Cytotoxicity against MT-4, MDBK, BHK and Vero-76 cell lines and BVDV, YFV, Reo-1, CVB-2, RSV and HSV-1 inhibitory activity of amino azocompounds of structure A–C
Compda
R
MT-4
CC50
MDBK
CC50
BVDV
EC50
BHK-21
CC50
YFV
EC50
Reo-1
EC50
VERO-76
CC50
CVB-2
EC50
RSV
EC50
HSV-1
EC50
b
c
d
e
f
g
h
i
j
k
1
H
17
14
19
16
35
17
31
77
100
15
40
51
>100
2
>100
31
P100
0.2
58
33
>100
59
20
>100
48
52
>100
30
64
1.6
16
>100
9
P20
>100
2.5
7
31
20
>100
33
43
>100
47
P100
>100
14
38
9
90
>100
>100
>100
>100
>100
P31
7
>100
12
10
>100
9
>100
P100
>14
>38
>9
1.8
26
>100
>100
>100
>100
>31
>20
>100
>33
12
>100
>47
>100
>100
>14
>38
>9
2.4
>100
>100
>100
>100
>100
30
25
60
30
65
50
60
80
80
50
80
15
>100
20
>100
>100
>100
P13
>30
11
25
>30
6
>60
12
2l
3
3-NO2
4-NO2
2,5-diF
2,6-diF
4-CH3
H
3-CF3
H
3-CF3
2,5-diF
2,4-diF
>25
>60
>30
>65
>50
>60
>80
>80
>50
>80
>15
>100
1.2
>100
7
>100
0.6
>60
>30
>65
>50
>60
>80
>80
>50
>80
>15
20
4l
5
6
7
8
>65
>50
>60
>80
>80
>50
>80
>15
>100
>20
3
9
30
7
10
11
12
>64
>11
1.7
>100
>100
7
11
NM 108 (20-C-methylguanosine)
NM 299 (6-azauridine)
ACG (acycloguanosine)
Ribavirin
>100
>100
>100
>100
>100
42
>20
>100
>100
24
>100
>100
>13
NM 176 (20-C-ethynylcytidine)
M 5255 (mycophenolic acid)
38
>42
>13
a
None of these compounds inhibited the multiplication of HIV-1, VSV, VV and Sb-1 viruses.
b
Compound concentration (
l
M) required to reduce the viability of mock-infected MT-4 (CD4+ human T cells containing an integrated HTLV-1 genome) cells by 50%, as
determined by the MTT method.
c
Compound concentration (
Compound concentration (
lM) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the MTT method.
d
l
M) required to achieve 50% protection of MDBK cells from BVDV (Bovine Viral Diarrhea Virus) induced cytopathogenicity, as determined by
M) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT
lM) required to achieve 50% protection of BHK cells (kidney fibroblast) from YFV (Yellow Fever Virus) induced cytopathogenicity, as deter-
the MTT method.
e
Compound concentration (
l
method.
f
Compound concentration (
mined by the MTT method.
g
Compound concentration (
lM) required to achieve 50% protection of BHK cells (kidney fibroblast) from Reo-1 induced cytopathogenicity, as determined by the MTT
method.
h
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Tested as hydrochloride.
l
M) required to reduce the viability of mock-infected VERO-76 (monkey normal kidney) monolayers by 50%.
M) required to reduce the plaque number of CVB-2 (Coxsackie Virus B 2) by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of RSV (Respiratory Syncytial Virus) by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of HSV-1 (Herpes Simplex virus, Type-1) by 50% in VERO-76 monolayers.
i
j
l
l
l
k
l
compounds had a S. I. in the range from 9 to 5 and the remaining
had S. I. below this value.
EC50 = 0.9
ble with that of compound 14.
lM, while racemate had IC50 = 3.5 lM, a value compara-
Taking into account the EC50 and S. I. values, the best com-
pounds were the following, which are arranged in the order of
decreasing potency and selectivity; BVDV: 1 > 7 > 8 > 4; YFV:
7 > 5; CVB-2: 25 > 56 > 18; RSV: 14 > 20 > 55 > 38 >18 > 19 > 23;
Sb-1: 41; HSV-1: 2. For these compounds the EC50 ranged from
On the other hand, BVDV is the prototype of pestiviruses, which
commonly affect cloven-hoofed animals (cattle, swine, sheep).
BVDV infection is responsible of reduced dairy production and in-
creased (even up to 30%) cattle mortality throughout the world.21
Thus, the availability of effective and inexpensive anti-pestivi-
rus drugs is of importance to relieve such an heavy economic
burden.
1.6 lM (1) to 15 lM (23, 41), while the corresponding S. I. values
were in the range from 19.4 (1) to 4.2 (2).
Though the activity shown by the above compounds may appear
as not particularly impressive, it must be noted that, at the present,
very few experimental agents are known to act against those viruses
and that, moreover, they often display high cytotoxicity.
A particular interest is deserved by compounds displaying good
activity against RSV and BVDV. RSV, is responsible of serious respi-
ratory tract infections in infants, elderly and immunocompromised
patients with high mortality rate.
Very potent anti-RSV agents, interfering with the virus-host fu-
sion process, have been developed in the last years, as the benzimid-
azole derivative BMS-433771 which exhibited an EC50 as low as
21 nM; however, attempts to demonstrate therapeutic efficacy were
not successful,19 probably due to the rapid emergence of resistance.
Quite recently a chiral 1,4-benzodiazepine derivative, acting
through a different mechanism, though much less potent than
BMS-433771, showed very promising characteristics and is being
examined in phase II clinical studies.20
The development of anti-BVDV drugs and the understanding of
their mechanism of action is of further interest because it may pro-
vide valuable informations for the design of agents active against
hepatitis C virus (HCV).22 HCV, which like BVDV belongs to the Fla-
viviridae family, infects about 3% of world population. Since chronic
HCV infection can progress to fatal cirrhosis and hepatocellular
carcinoma, the development of effective and safe anti-HCV agents
is urgently needed. The current combination therapy with pegylat-
ed interferon and ribavirin is effective in less than 60% of the trea-
ted patients and is associated with heavy side effects.
For the understanding of some aspects of virus replication,
BVDV has been considered even more advantageous than the cur-
rently used HCV subgenomic replicon system.23
In the last years, several potent anti-BVDV agents have been
developed and shown to target the RNA-dependent RNA-polymer-
ase (RdRp), even if resulting almost inactive on the purified
enzyme. Compounds VP32947 (3-[(dipropylaminoethyl)thio]-5H-
1,2,4-triazino[5,6-b]indole)24 and BPIP (5-[4-bromobenzyl]-2-phe-
nyl-5H-imidazo[4,5-c]pyridine)23 exhibited EC50 as low as 30–
40 nM. Other compounds (as the thiazolylurea DPC-A69280-29,25
Nevertheless the chirality-associated developability issue can-
not be underestimate, since the antiviral activity was found to re-
side mainly in the S-enantiomer (RSV-604), that exhibited an

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4431
Sb-1
Table 4
Cytotoxicity against MT-4, MDBK, BHK and Vero-76 cell lines and BVDV, YFV, Reo-1, CVB-2, RSV, HSV-1 and Sb-1 inhibitory activity of 1,3-diaryltriazenes of structure D and E
Compda
X
R
R0
MT-4
CC50
MDBK
CC50
BVDV
EC50
BHK-21
CC50
YFV
EC50
Reo-1
EC50
VERO-76
CC50
CVB-2
EC50
RSV
EC50
HSV-1
EC50
b
c
d
e
f
g
h
i
j
k
l
EC50
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
H
H
H
H
H
H
H
H
H
H
47
>100
42
>100
>100
>100
94
>100
>100
>100
>100
>100
>100
82
>100
95
38
37
39
25
>100
45
>100
22
>100
>100
>100
>100
>100
42
>100
>100
>100
19
>100
>100
>100
>100
>100
>100
>82
>100
P95
>38
>37
P39
>25
90
>45
>100
>22
1.7
>100
>100
7
38
>42
>100
>100
>100
38
>100
>100
>100
>100
>100
>100
>100
>100
50
49
54
36
25
54
52
>100
40
90
>100
P100
>100
>38
>100
>100
80
>100
>100
>100
>100
>100
>50
P49
24
P36
>25
>54
20
>100
>100
>100
>38
>100
>100
>100
>100
>100
>100
33
>100
>50
>49
>54
>36
90
15
100
33
>100
80
80
P100
50
P100
>100
58
35
20
40
40
7
30
25
100
75
>90
8
>100
>33
>100
12
P90
3
>100
>33
>100
10
10
8
20
>100
15
>58
>35
>20
>40
>40
>7
>30
P25
>100
>75
>100
1.2
>90
>15
>100
>33
>100
55
>80
>100
>50
>100
>100
>58
12
14
30
>40
>7
>30
12
>100
>75
>100
>20
3
>100
>100
>13
>90
>15
>100
>33
>100
>80
>80
>100
>50
>100
29
>58
>35
>20
>40
>40
>7
>30
>25
>100
>75
>100
>20
>100
>100
20
3-CF3
CH2-Phm
3-NO2
3-NO2
2,5-diF
2,5-diF
2,5-diF
H
H
3-NO2
H
3-CF3
40
>100
100
>100
P100
55
P100
>100
45
33
14
14
20
13
16
23
18
17
>100
2
>100
31
P100
0.2
>80
50
2,5-diF
CH3
CH3
CH3
CH3
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
(CH2)3-NMe2
Lupinyln
H
>50
>100
>100
>58
6
P20
>40
>40
>7
>30
9
>100
>75
20
H
CH2-Phm
3-NO2
4-CH3
H
H
H
H
3-CF3
H
H
H
3-CF3
H
H
H
3-NO2
4-NO2
2,5-diF
2,5-diF
2,6-diF
4-CH3
H
29
30
31
32
>25
>54
>52
>100
>40
>100
23
1.8
33
3-CF3
NM 108
NM 299
ACG
Ribavirin
NM 176
M 5255
2.4
>100
20
>100
>100
>100
P13
>100
>100
>100
>100
>100
26
>100
>100
>100
>100
>100
>20
>100
>100
24
>100
>100
>100
>100
>100
7
>100
0.6
>13
>13
a
None of compounds 13–33 inhibited the multiplication of HIV-1, VSV and VV viruses.
b-k
For the meaning see Table 3.
Compound concentration ( M) required to reduce the plaque number of Sb-1 (Poliovirus Type-1, Sabin strain) by 50% in Vero-76 monolayers.
l
l
m
n
In compounds 15 and 22 the R0-substituted phenyl ring is replaced by a benzyl residue.
(1S,9aR-Octahydro-2H-quinolizin-1-yl)methyl.
the benzimidazolone 145326 and 7-amino-1,3-dihydroxy-10-
methyl-6-[4-(2-pyridinyl)-1-piperazinyl]-9(10H)acridone27
have
EC50 in the range 0.6–1.5 M, that are comparable with those of
the presently described aminoaryl azocompounds 1 and 7 (1.6
and 2.5 M, respectively). The structural simplicity of these com-
pounds, which allows a wide range of easy and inexpensive molec-
ular modifications, make them an attractive model to develop
more active and less toxic anti-pestivirus agents.
ing, nucleotides recruitment, and polymerization reaction (see
Fig. 4a). In addition, there is an N-terminal region (residues 71–
138) of which residues 71–91 are disordered in the relevant crystal
structure. A thorough search of a putative binding site for our mol-
ecules onto BVDV RdRp was conducted following our successful
recipe developed for studying allosteric inhibitors of Polio-virus
helicase.33 The portion of the enzyme making up the binding site
interacting with the inhibitors is located in the fingers domain
)
l
l
In view of these considerations, we deemed interesting to per-
form some molecular modeling investigations to study wether
the BVDV RdRp could be the target of also our compounds 1, 2, 4
and 7–10.
(residues 139–313 and 351–410), consisting of 12 a-helices and
11 b-strands (see Fig. 4a).
In BVDV RdRp, as in other viral RdRps, the N terminus of the fin-
gers domain, together with a long insert in the fingers domain (res-
idues 260–288), form the fingertip region that associates with the
thumb domain. This region is characterized by a three-strand con-
formation, and since the fingers and the thumb domains are asso-
ciated through this fingertip region, the conformational change
induced by the RNA template binding into the central channel is
somewhat limited. The remainder of the fingers domain is com-
3.4. Modeling of the A–C series of compounds versus BVDV
RdRp
BVDV, the best-studied pestivirus, has a genome that consists of
an approximately 12.6-kb positive sense ssRNA. The BVDV genome
is translated into a single polyprotein which is processed into at
least four structural and six nonstructural (NS) proteins required
for viral assembly and replication. Among the nonstructural pro-
teins, the NS5B is an RNA-dependent RNA-polymerase (RdRp) en-
zyme responsible for genome replication as a part of a larger,
membrane associated replicase complex. The crystal structure of
RdRp from several families of single- or double-strand RNA viruses,
including HCV28–30 and BVDV,31,32 have been recently made avail-
able in the Protein Data Bank (PDB) repository.
Despite the low sequence identity between different polymer-
ases, the crystal structures of these proteins (from BVDV, HCV
and other families of ss and dsRNA viruses) all present the shape
of a right hand with fingers, palm, and thumb domains. In particu-
lar, the BVDV RNA-dependent RNA-polymerase core domain (resi-
dues 139–679) has a dimension of approximately 74 ꢁ 60 ꢁ 58 Å
around a central cavity,31 which serves as for RNA template bind-
prised of a b-strand rich region (b-fingers) and an
gion ( -fingers) close to the palm domain.
a-helix rich re-
a
According to the procedure adopted, all compounds were char-
acterized by a similar docking mode in the putative binding site of
the BVDV RdRp, as exemplified by compound 1 in Figure 5.
In particular, the residues lining the pocket (see Fig. 5d) in-
clude the side chains of residues from N217 to F224 belonging
to a loop between the two strands b3 and b4, and those of resi-
dues from K263 to E265 of a loop between strands b5 and b6.
Amino acids from I287 to E291 contribute to binding from the
b-sheet portion b8, whilst another loop, connecting two
motifs ( 19 and 20, respectively), concurs with residues from
R529 and L530.
a-helical
a
a
Going into details, the aminoalkyl part of the molecule is en-
gaged in stabilizing nonbonded interactions with the apolar side
chains of residues I261, I287, A221, and A222, while the diarylazo

4432
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
Table 5
Cytotoxicity against MT-4, MDBK, BHK and Vero-76 cell lines and YFV, Reo-1, CVB-2, RSV, HSV-1 and Sb-1 inhibitory activity of triazene derivatives of structure F and G
Compda
X
R
MT-4
CC50
MDBK
CC50
BHK-21
CC50
YFV
EC50
Reo-1
EC50
VERO-76
CC50
CVB-2
EC50
RSV
EC50
HSV-1
EC50
Sb-1
EC50
b
c
e
f
g
h
i
j
k
l
34
35
36
37
38
39
40
41
42
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
H
>100
>100
>100
>100
66
>100
>100
P100
68
>100
>100
>100
>100
>100
>100
>100
>100
60
P100
>100
>100
>100
42
>100
>100
P100
P100
>100
>100
>100
>100
>42
>100
>100
>100
>100
>100
>100
>100
>100
>42
>100
>100
>100
>100
>100
P100
P100
>100
80
>100
P100
90
>100
>100
>100
>100
>80
>100
>100
>90
P100
>100
>100
>100
8
>100
>100
60
>100
>100
>100
>100
>80
>100
>100
>90
>100
P100
>100
>100
30
42
51
15
>90
3-Cl
3-Br
3-CF3
3-NO2
4-Cl
4-Br
4-NO2
4-
90
>90
>90
>90
OCH3
43
44
Nil
Nil
2,5-diF >100
>100
70
>100
50
>100
>50
>100
>50
>100
58
35
>58
>100
20
>100
>58
44
25
3,4-
diCl
H
3-Cl
3-Br
3-NO2
4-Cl
4-Br
H
75
45
46
47
48
49
50
51
52
53
54
55
56
CH2
CH2
CH2
CH2
CH2
CH2
CO
N-CH3
N-CH3
N-COOC2H5
N-Ph
N-2-
>100
>100
>100
>100
>100
>100
>100
65
>100
>100
>100
>100
>100
>100
P100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
90
>100
>100
>100
>100
>100
>100
>90
>70
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>90
>70
30
>100
>100
>100
>100
>100
85
>100
>100
>85
>83
>100
25
13
14
71
30
>100
>100
>85
>83
>100
73
>60
>45
>100
>90
8
>100
>100
>85
>100
>100
>85
83
>83
>83
>100
>100
60
45
P100
90
>100
>100
>60
>45
64
>90
>100
>65
>100
>100
>60
>45
>100
>90
H
70
2,5-diF >100
>100
>100
>100
>100
H
H
H
100
33
90
P100
65
>100
10
>100
>65
>65
Pyrimidinyl
57
58
4-Cl
3-NO2
46
97
85
>100
90
>100
>100
>100
>100
>100
>85
>100
1.8
>85
80
2.4
>100
>100
>100
>100
>100
55
90
>55
>90
20
>20
>100
>100
24
>55
P90
>100
1.2
>100
7
>55
>90
>100
>20
3
>100
>100
>13
>55
>90
>100
>20
>100
>100
20
>100
>100
2
>100
31
>100
>100
>100
>100
>100
>100
42
NM-108
NM 299
ACG
Ribavirin
NM 176
M 5255
>100
20
>100
>100
>100
P13
26
>100
>100
>100
>100
P100
0.2
>100
0.6
>13
>13
a
None of compounds 34–58 inhibited the multiplication of HIV-1, BVDV, VSV and VV viruses.
For the meaning see Tables 3 and 4.
b-l
moiety is nicely encased in a subsite lined by the side chains of
N217, N264, E265, K266, R529, and L530. More importantly, how-
ever, this molecular scaffold is anchored in place by three persis-
Accordingly, it follows that the association between the ligands
and the RdRp is mainly driven by more favorable nonpolar interac-
tions in the complex than in the solution, in harmony with a pro-
posed general scheme for noncovalent association.33,34 However,
as indicated for instance by the energy components of compound
2, this driving force can be weakened when the polar groups on
the molecule do not find an adequate bonding pattern in the pro-
tein compared to water. The free energy penalty for this
tent hydrogen bonds (HBs).
A first HB bridge involves the
nitrogen atom of the N(CH3)2 substituent group on 1 and the –
OH group of Y289 side chain, with an average dynamic length
(ADL) of 3.08 0.3 Å. The second HB interaction engages the termi-
nal amino group of K263 and the N atom of the inhibitor secondary
amino group (ADL = 3.18 0.2 Å). Finally, one of the nitrogen
atoms belonging to the azo group characterizing compound 1 is in-
volved in the third HB interaction with the backbone –NH group of
E265 (ADL = 2.92 0.3 Å).
(DEel + DGPB) term is least for compound 1 and this, together with
its substantial van der Waals contribution and moderate entropic
unfavorable term, consequently leads to one of the highest binding
affinity in this set of inhibitors.
Importantly, quantitative information about the affinities of our
ligands towards BVDV RdRp can be inferred by applying the MM/
One of the most important benchmark in this study, however, is
the correspondence between the estimated free energies of bind-
ing and the experimental measured EC50 values. Indeed, we can ob-
serve a good agreement between the trend exhibited by the IC50
values reported in Table 6 and the corresponding biological activity
determined for these compounds in BVDV infected cell line (see
Table 3). Although we obviously cannot directly compare the com-
puted binding free energy (and hence the corresponding IC50) with
the EC50 values deriving from experiment, we can observe that, in
most cases, the rank of the inhibitors with respect to their activity
towards their putative targets, the RdRps of BVDV, is maintained,
although with some discrepancies. In fact, all estimated IC50 values
are systematically higher than those determined by in vitro exper-
iments with infected cell lines.
PBSA methodology to estimate the free energy of binding,
DGbind,
and its components. The calculated Gbind values for the dimethyl-
D
aminopropyl derivatives 1, 2, 4, and the quinolizidinylmethyl
derivatives 7–10 are listed in Table 6. Generally speaking, and in
harmony with our previous findings,33,34 both the nonbonded
mechanical energy components of
DGbind, DEvdW and DEel, afford
a substantial, favorable contribution to binding for both series of
compounds. On the other hand, due the polar character of the
azo group, the desolvation penalty paid by these molecules upon
binding (DGPB) is also quite substantial, so that the net, resulting
electrostatic contribution to the affinity of these inhibitors to their
enzyme receptor are notably unfavorable. Specifically, for this ser-
ies of compounds, the mean value of the electrostatic energy
The overall results obtained from molecular modeling deserve
some more comments. First of all, the binding site identified by
our procedure is very close to the putative binding site proposed
for two allosteric inhibitors of BVDV RdRp, VP32947 and BPIP,23
(
D
Eel
of the van der Waals and hydrophobic overall interaction energies
EvdW
Gnp) are ꢀ46 kcal/mol.
+ DGPB) is 26 kcal/mol, whilst the corresponding mean values
(D
+ D

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4433
Figure 4. (a) Overview of the entire structure of the RdRp of BVDV. The protein domains are colored as follows: pink, N-terminal domain (residues 71–138); light green,
fingers domain (residues 139–313 and 351–410); palm domain, kaki (residues 314–350 and 411–500); sienna, thumb domain (residues 501–679). (b) Overlay of the 3D
models of RdRp of BVDV (purple) and HCV (red). (c) BVDV and HCV polymerase amino acid alignment: top line, BVDV (residues 92–679); bottom line, HCV (residues 1–531).
The alignment of the fingers domain is highlighted in purple (BVDV) and red (HCV).
and involves residue F224 (see Fig. 5d), a residue found mutated into
S224 in BVDV BPIP-resistant clones. The binding pocket is located in
a turn of the fingers domain between two b-sheets which is believed
to be involved in finger flexibility for RNA template/product translo-
cation,35 dimerization of the RdRp in the replication complex, or pro-
tein/protein interactions,36,37 enabling the assembly of an active
replication complex. According to our docking results, then, the
hypothetical binding of our compounds to the polymerase could af-
fect the finger flexibility or impair the capacity of the protein to
translocate its template/product during RNA polymerization. Alter-
natively, the diarylazo moiety could possibly act by hampering the
entrance of the template RNA in the template binding cavity.
Although our in silico data clearly show the same trend of the
corresponding experimental EC50 values, at the same time they
yield no evidence that binding of these inhibitors onto the BVDV
RdRp result in a real allosteric inhibition of the enzyme or in a re-
duced template-binding ability. One of the explanations why we
find IC50 values higher than the corresponding EC50 could be ex-
plained by the following rationale. Recently an attempt of co-crys-
tallize the BVDV RdRp with the inhibitor VP32947 failed do to a
dimer interface near the putative binding site of VP32947 in the
BVDV RdRp crystal. The authors of the work proposed that the for-
mation of this dimer could constitute an important point in the
replication complex. Moreover, it was also hypothesized that the
top of the fingers domain could be a protein-docking site pivotal
to the interactions with other enzymes of the replicase complex.
As the VP32947 binding site is part of the putative binding pocket
for our molecular series, it could be that, upon binding, our com-
pounds hinder the interactions among different proteins making
up the replicase complex.
Last but not least, it is important to recall that RdRp functions in
virus-infected cells in the context of membrane-bound replication
complexes, that usually consist of several virus-encoded proteins,
host proteins, and various forms of viral RNA. The RNA-dependent
RNA polymerase may then have a role in interacting with these
other components and/or in the stability of the replication com-
plexes. If one or more of these putative actions of the RdRp in in-
fected cells is sensitive to our inhibitors, viral RNA synthesis may
be disrupted in a manner which is distinct from the one estimated
by our in silico assays.
Now, given the similarity among the polymerases of pestivirus-
es, hepaciviruses, and flaviviruses, any knowledge gained from any
one of these viral systems is likely to foster progress in the others.
Given the high importance of the hepatitis C pandemic and the

4434
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
Figure 5. Overall (a, top left) and detailed (b, top right) space filling representation of the BVDV RdRp molecular surface and compound 1 docked into the protein putative
binding site. The inhibitor is in CPK representation, with carbons in cyan, nitrogens in blue, and hydrogens in white. (c, bottom left) Ribbon diagram of BVDV RdRp/1 complex
structure as resulting from the applied docking/MD procedure. The protein is colored light blue. The inhibitor 1 is represented as a stick model with carbons in gray and
nitrogens in blue. (d, bottom right) Details of compound 1 (in a stick representation) in the binding pocket in the enzyme fingers domain. Color scheme as above. The side
chains of all residues that form the primary binding pocket interacting with compound 1 are shown as stick models, and the atom color-coding is as follows: N217, firebrick;
A221, orange; A222, dark kaki; F224, red; E258, dim gray; T259, rosy brown; I261, green; K263, hot pink; N264, tan; E265, sienna; K266, dark magenta; I287, gold; Q288, navy
blue; Y289, purple; P290, dark slate blue; E291, pink; R295, olive drab; R529, coral; L530, kaki. Hydrogen bonds are highlighted as light gray broken lines. Hydrogen atoms,
counterions, and water molecules are omitted for clarity.
stringent need for efficient therapeutics for this pathology, it is ex-
pected that new molecular entities such as those described in this
work can provide meaningful insights towards the identification of
possible anti-HCV agents. Interestingly, the overall sequence iden-
tity between BVDV and HCV RdRp proteins is quite low, approxi-
mately 30%. If we consider the alignment of BVDV and HCV RdRp
reported in Figure 4c, and in particular focus on the highlighted fin-
gers domain part, we can immediately speculate that: (i) the F224
residue in the BVDV protein has no counterpart in the correspond-
ing HCV enzyme; accordingly, if the drug would bind in the same
binding pocket with the same binding mode, there should in prin-
ciple be no problem of resistant mutant at this position. (ii) The
important region of K263–K266 in the BVDV RdRp is highly con-
served in the corresponding HCV polymerase (KNEK in BVDV and
KNEV in HCV, respectively, see Fig. 4c); importantly, both K263
and E265 are engaged in fundamental stabilizing hydrogen bonds
with the inhibitors in the BVDV case but, being conserved, they
are able to establish the same interactions with the compounds
also in the case of HCV. (iii) The residue Y289 of BVDV RdRp is
not conserved in the corresponding HCV polymerase, being re-
placed by a phenylalanine. Therefore, the anchoring hydrogen
bond between the Y289 –OH group and, for instance, the tertiary
nitrogen of 1 can no longer take place. Taken all together, these evi-
dences can justify a minor potency of this molecular series towards
the HCV RdRp as a target with respect to the BVDV counterpart. At
the same time, in the case of both viruses, the binding pockets on
the respective RdRps do share a sufficient degree of structural and
sequence homology; thus, since these regions involve the fingers
domains, which overhang the palm domains of the enzymes, they
might be involved in RNA substrate recognition and, hence, the
Table 6
Free energy components and total binding free energies for compounds of the series A–C on BVDV RdRp
1
2
4
7
8
9
10
D
D
D
D
D
D
D
EvdW
Eel
EMM
GPB
Gnp
Gsol
GMM=PBSA
Ssolute
ꢀ40.2 0.3
ꢀ32.5 0.2
ꢀ72.7 0.2
57.5 0.5
ꢀ6.3 0.0
51.2 0.6
ꢀ21.5 0.5
14.3
ꢀ38.6 0.2
ꢀ34.2 0.3
ꢀ72.8 0.2
60.0 0.4
ꢀ7.1 0.0
52.9 0.4
ꢀ19.9 0.4
14.0
ꢀ39.5 0.4
ꢀ33.9 0.3
ꢀ73.4 0.3
52.6 0.4
ꢀ6.7 0.1
52.9 0.5
ꢀ20.5 0.3
14.3
ꢀ39.5 0.3
ꢀ29.3 0.3
ꢀ68.8 0.3
59.6 0.3
ꢀ9.1 0.0
46.6 0.2
ꢀ22.2 0.2
15.2
ꢀ39.8 0.2
ꢀ29.8 0.1
ꢀ69.6 0.2
55.7 0.4
ꢀ8.7 0.1
48.4 0.4
ꢀ21.2 0.3
14.8
ꢀ39.3 0.3
ꢀ28.7 0.3
ꢀ68.0 0.2
55.3 0.4
ꢀ7.9 0.1
47.4 0.3
ꢀ20.7 0.4
15.0
39.4 0.2
ꢀ29.4 0.3
ꢀ68.8 0.3
55.6 0.3
ꢀ7.8 0.0
47.8 0.3
ꢀ21.0 0.3
14.7
TD
D
IC50
Gbind
ꢀ7.2
ꢀ5.9
ꢀ6.2
ꢀ7.0
ꢀ6.4
ꢀ5.7
ꢀ6.3
a
5
38
28
7
22
66
25
All values are in kcal/mol. IC50 values are in
l
M.
a
33,34
IC50 values were obtained using the following relationship:
D
Gbind = RT ln IC50.

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4435
inhibitor molecules may affect the RdRps interactions with the
incoming RNA molecule.
Erba EA-1110 CHNS-O instrument in the Microanalysis Laboratory
of the Department of Pharmaceutical Sciences of Genoa University.
5.2. Aminoazocompounds of structure A and triazenes of
structure E. General method
4. Conclusions
Twelve aminoarylazocompounds of structures A–C, and 46 aryl-
triazene derivatives D–G have been synthesized and assayed for
antiviral activity against a panel of 10 RNA and DNA viruses.
Thirty-five (8 aminoazocompounds and 27 triazene derivatives)
over 58 tested compounds exhibited antiviral activity against one
or more viruses and many of them had shown an EC50 6 10 lM
against at least one virus. None of the active compounds inhibited
the multiplication of HIV-1, VSV and VV.
Aminoazocompounds A–C have a marked activity against BVDV
and YFV, while the triazene derivatives D–G are characterized by
the most frequent activity against CVB-2 and RSV.
Despite the high antiviral activity of many compounds, the cor-
responding selectivity index (S. I.) are generally low, due the rather
high cytotoxicity exerted on the host cell lines.
Taking into account the EC50 and S. I. values, the best com-
pounds are the following, arranged in order of decreasing potency
and selectivity; BVDV: 1 > 7 > 8 > 4; YFV: 7 > 5; CVB-2:
25 > 56 > 18; RSV: 14 > 20 > 55 > 38 > 18; HSV-1: 2. For these
A solution of arylamine (8 mmol) in 1.56 mL of conc. hydrochlo-
ridric acid and 1 mL of water was cooled to 0 °C and slowly diazo-
tized with a solution of sodium nitrite (8 mmol) in 2 mL of water.
The diazonium salt solution was added dropwise, during about
10–20 min, to a cold water solution of the N-(3-dimethylaminopro-
pyl/lupinyl)aniline monoacetate (8 mmol), and maintaining care-
fully pH 7 with the simultaneous addition of a saturated solution
of sodium acetate. After stirring the reaction mixture for 45 min at
0–5 °C, the oil product was extracted with Et2O, dried, then vac-
uum-distilled (0.1–0.2 torr) at 80–90 °C to remove the unreacted
N-substituted aniline. The residue was fractionated by CC, eluting
firstly with Et2O the triazene derivative (E) and then with Et2O + 5%-
MeOH the isomeric azocompound (A) in quite lower yield.
The isolated compounds were further purified either by crystal-
lization or, when oily, by a second CC or distillation.
5.2.1. [N,N-Dimethyl-N0-(4-phenylazophenyl)]propane-1,3-
diamine (1)
compounds the EC50 ranged from 1.6 lM (1) to 12 lM (18), while
the corresponding S. I. values were in the range from 19.4 (1) to
4.2 (2).
Yield: 6%. Oil (CC: Al2O3, Et2O). Bp (p = 0.1–0.2 torr) 100–105°.
TLC: Rf = 0.4 (Al2O3, Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.74 (q, 2H,
C(2)); 2.20 (s, N(CH3)2); 2.37 (t, J = 6.2, 2H, C(3)); 3.24 (t, J = 7.0, 2H,
C(1)); 5.19 (s, NH); 6.58 (d, J = 10.0, 2 arom. H); 7.28–7.48 (m, 5 arom.
H); 7.75 (d, J = 10.0, 2 arom. H). Anal. Calcd for C17H22N4: C, 72.31; H,
7.85; N, 19.84. Found: C, 72.15; H, 8.05; N, 19.74.
A particular interest is deserved by compounds displaying good
activities against RSV (as 14 and 20) and BVDV (as 1 and 7). The
former virus is responsible of serious respiratory tract infections
in humans, with high mortality rate, while the latter is responsible
of severe epidemic outbreaks in livestock, but is also a surrogate
model for the evaluation of novel, urgently needed agents against
HCV, an emerging threat to human health worldwide.
The above compounds represent valuable starting models for
developing better (more potent and less toxic) and inexpensive
antiviral agents through the variation of the dialkylaminoalkyl
chain and/or the substituents on the aromatic rings.
In view of these considerations, molecular modeling investiga-
tions were performed to study wether the active compounds of
structures A-C could target the BVDV RNA-dependent RNA-poly-
merase (RdRp), which shares some structural similarity with HCV
RdRp.
Indeed a good agreement was observed between the trend
exhibited by the IC50 (calculated from the estimated free energies
of binding) and the corresponding biological activities determined
for these compounds in BVDV infected MDBK cell line.
The overall sequence identity between BVDV and HCV RdRp
proteins is approximately 30%, but the important region K263-
K266 in the BVDV RdRp is highly conserved in the corresponding
HCV polymerase, thus targeting of HCV RdRp by the A-C molecular
series could be expected, even if with a minor potency in respect to
the BVDV counterpart.
5.2.2. N,N-Dimethyl-N0-[4-(3-nitrophenylazo)phenyl]propane-
1,3-diamine (2)
Yield: 4%. Mp 205–208 °C (EtOH abs.). TLC: Rf = 0.3 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.91 (q, 2H, C(2)); 2.07 (s,
NH); 2.25 (s, N(CH3)2); 2.37 (t, J = 6.9, 2H, C(3)); 4.34 (t, J = 7.6,
2H, C(3)); 7.11–8.40 (m, 8 arom. H). Anal. Calcd for C17H21N5O2.
HCl: C, 56.12; H, 6.09; N, 19.25. Found: C, 56.31; H, 6.20; N, 18.88.
5.2.3. N,N-Dimethyl-N0-[4-(4-nitrophenylazo)phenyl]propane-
1,3-diamine (3)
Yield: 3%. Mp 116–119 °C (hexane). TLC: Rf = 0.3 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.76 (q, 2H, C(2)); 2.21 (s,
N(CH3)2); 2.38 (t, J = 6.3, 2H, C(3)); 3.24 (t, J = 6.9, 2H, C(1)); 5.72
(s, NH); 6.54 (d, J = 10.0, 2 arom. H); 7.75–7.90 (m, 4 arom. H);
8.25 (d, J = 10.1, 2 arom. H). Anal. Calcd for C17H21N5O2: C, 62.37;
H, 6.47; N, 21.39. Found: C, 62.06; H, 6.68; N, 21.00.
5.2.4. N0-[4-(2,5-Difluorophenylazo)phenyl]-N,N-
dimethylpropane-1,3-diamine (4)
Yield: 11%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.4 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.76 (q, 2H, C(2)); 2.20 (s,
N(CH3)2); 2.38 (t, J = 6.7, 2H, C(3)); 3.23 (t, J = 7.0, 2H, C(1)); 5.49
(s, NH); 6.55 (d, J = 9.0, 2 arom. H); 6.88–7.44 (m, 3 arom. H);
7.78 (d, J = 9.2, 2 arom. H). Anal. Calcd for C17H20F2N4ꢂHCl: C,
57.54; H, 5.97; N, 15.79. Found: C, 57.43; H, 6.10; N, 15.73.
5. Experimental
5.1. General
5.2.5. N0-[4-(2,6-Difluorophenylazo)phenyl]-N,N-
dimethylpropane-1,3-diamine (5)
Chemicals, solvents and reagents used for syntheses were pur-
chased from Sigma–Aldrich, Fluka or Lancaster, and were used
without any further purification.
Column chromatography (CC): neutral alumina (Al2O3), activity 1
(Merck). Mps: Büchi apparatus, uncorrected. 1H NMR spectra: Varian
Gemini-200 spectrometer; CDCl3; d in ppm rel. to Me4Si as internal
standard. J in hertz. Elemental analyses were performed on a Carlo
Yield: 15%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.3 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.70 (q, 2H, C(2)); 2.19 (s,
N(CH3)2); 2.36 (t, J = 6.5, 2H, C(3)); 3.23 (t, J = 6.7, 2H, C(1)); 5.43
(s, NH); 6.56 (d, J = 10.0, 2 arom. H); 6.85–7.20 (m, 3 arom. H);
7.75 (d, J = 9.0, 2 arom. H). Anal. Calcd for C17H20F2N4: C, 64.14;
H, 6.33; N, 17.60. Found: C, 63.90; H, 6.61; N, 17.52.

4436
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
5.2.6. N,N-Dimethyl-N0-4-(40-tolylazo)phenylpropane-1,3-
diamine (6)
5.2.14. N,N-Dimethyl-1-[3-phenyl-1-(p-tolyl)triazen-3-yl]-3-
propanamine (32)
Yield: 10%. Mp 78–79 °C (Et2O). TLC: Rf = 0.4 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.84 (q, 2H, C(2)); 1.96 (s, CH3);
2.34 (s, N(CH3)2); 2.61 (t, J = 6.6, 2H, C(3)); 3.21 (t, J = 7.1, 2H,
C(1)); 4.65 (s, NH); 6.56 (d, J = 10.1, 2 arom. H); 7.20 (d, J = 10.0, 2
arom. H); 7.62–7.78 (m, 4 arom. H). Anal. Calcd for C18H24N4: C,
72.94; H, 8.16; N, 18.90. Found: C, 72.68; H, 8.50; N, 19.20.
Yield: 64%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.8 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.82 (q, 2H, C(2)); 2.19 (s,
N(CH3)2); 2.24–2.37 (m, 2H, C(3) and 3H, CH3-Ph); 4.24 (t, J = 7.6,
2H, C(1)); 6.97–7.47 (m, 9 arom. H). Anal. Calcd for C18H24N4: C,
72.94; H, 8.16; N, 18.90. Found: C, 72.61; H, 8.24; N, 19.40.
5.2.15. 3-[(1S,9aR)-Octahydro-2H-quinolizin-1-ylmethyl]-1-
phenyl-3-(trifluoromethylphenyl)triazene (33)
5.2.7. N,N-Dimethyl-1-(1,3-diphenyltriazen-3-yl)-3-
propanamine (25)
Yield: 47%. Mp 102–103 °C (pentane). 1H NMR (CDCl3): 0.66–
Yield: 31%. Oil (CC: Al2O3, Et2O). Bp (p = 0.1–0.2 torr) 100–
105 °C. TLC: Rf = 0.7 (Al2O3, Et2O + 2%Et2NH). 1H NMR (CDCl3):
1.80 (q, 2H, C(2)); 2.20 (s, N(CH3)2); 2.31 (t, J = 7.1, 2H, C(3));
4.27 (t, J = 7.3, 2H, C(1)); 6.52–7.83 (m, 10 arom. H). Anal. Calcd
for C17H22N4: C, 72.31; H, 7.85; N, 19.84. Found: C, 72.31; H,
7.87; N, 19.88.
2.20 (m, 14H of quinolizidine); 2.80–2.96 (m, 2Ha near N of quin-
olizidine); 4.23 (dd, J = 14.8, 4.0, 1H of CH2-Quinolizidine); 4.96 (dd,
J = 15.0, 11.0, 1H of CH2-Quinolizidine); 7.23–7.76 (m, 9 arom. H).
Anal. Calcd for C23H27F3N4: C, 66.32; H, 6.54; N, 13.45. Found: C,
66.58; H, 6.59; N, 13.03.
5.3. Aryltriazenes of structure D and F. General method
5.2.8. N,N-Dimethyl-1-[1-(3-trifluoromethylphenyl)-3-
phenyltriazen-3-yl]-3-propanamine (26)
The aromatic amine (25 mmol) was dissolved in 7.3 mL of conc.
hydrochloridric acid and 10 mL of water, cooling in a ice bath. The solu-
tion was kept at 0–5 °C, while adding drop by drop a solution of sodium
nitrite (25 mmol) in 10 mL of water. The diazonium salt was then cou-
pled with the relevant amine (25 mmol) dissolved in 45 mL of 1 N
NaOH (pH 7–8). After 20 min of stirring, the precipitate was collected
by filtration. The solid was crystallized from the suitable solvent.
In the case of compounds 14, 18 and 19 the proper oily aniline
was added to the diazonium salt solution, followed by a solution of
sodium acetate to reduce acidity.
Yield: 13%. Oil (CC: Al2O3, Et2O). Bp (p = 0.1–0.2 torr) 80–85 °C.
TLC: Rf = 0.8 (Al2O3, Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.83 (q, 2H,
C(2)); 2.20 (s, N(CH3)2); 2.31 (t, J = 6.8, CH2(3)); 4.28 (t, J = 7.0,
CH2(1)); 7.17–7.78 (m, 9 arom. H). Anal. Calcd for C18H21F3N4: C,
61.70; H, 6.04; N, 15.99. Found: C, 61.54; H, 6.08; N, 15.93.
5.2.9. 1-[1-(3-Nitrophenyl)-3-phenyltriazen-3-yl]-N,N-
dimethyl-3-propanamine (27)
Yield: 58%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.7 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.86 (q, 2H, C(2)); 2.20 (s,
N(CH3)2); 2.32 (t, J = 6.9, 2H, C(3)); 4.29 (t, J = 7.5, 2H, C(1));
7.06–8.35 (m, 9 arom. H). Anal. Calcd for C17H21N5O2: C, 62.37;
H, 6.47; N, 21.39. Found: C, 62.55; H, 6.61; N, 21.46.
When the coupling-product separated as an oil (compounds 37,
43, 53) it was extracted with Et2O and after removing the solvent
the oil was purified by CC. The cytisine derivative 58 was extracted
with CHCl3, and the solid obtained was crystallized from acetone.
5.3.1. 1-Phenyl-3-(30-trifluoromethylphenyl)triazene (14)
Yield: 92%. Mp 95–96 °C (hexane). 1H NMR (CDCl3): 7.08–7.69
(m, 9 arom. H); 9.68 (s, NH). Anal. Calcd for C13H10F3N3: C, 58.87;
H, 3.80; N, 15.84. Found: C, 58.66; H, 4.20; N, 15.97.
5.2.10. N,N-Dimethyl-1-[1-(4-nitrophenyl)-3-phenyltriazen-3-
yl]-3-propanamine (28)
Yield: 51%. Mp 74–75 °C (Et2O). TLC: Rf = 0.8 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.86 (q, 2H, C(2)); 2.18 (s,
N(CH3)2); 2.32 (t, J = 7.1, 2H, C(3)); 4.30 (t, J = 7.8, 2H, C(1));
7.10–7.50 (m, 5 arom. H); 7.59 (d, J = 10.2, 2 arom. H); 8.19 (d,
J = 10.2, 2 arom. H). Anal. Calcd for C17H21N5O2: C, 62.37; H, 6.47;
N, 21.39. Found: C, 62.27; H, 6.59; N, 21.56.
5.3.2. 1-(2,5-Difluorophenyl)-3-phenyltriazene (18)
Yield: 30%. Mp 125–126 °C (pentane). 1H NMR (CDCl3): 6.60–
7.50 (m,
8
arom. H); 9.70 (s, NH). Anal. Calcd for
C12H9F2N3 + 0.5H2O: C, 57.37; H, 3.58; N, 16.76. Found: C, 57.69;
H, 3.52; N, 16.61.
5.2.11. 1-[1-(2,5-Difluorophenyl)-3-phenyltriazen-3-yl]-N,N-
dimethyl-3-propanamine (29)
5.3.3. 1-(2,5-Difluorophenyl)-3-(30-trifluoromethylphenyl)
triazene (19)
Yield: 48%. Mp 61–64 °C (pentane). TLC: Rf = 0.7 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.89 (q, 2H, C(2)); 2.18 (s,
N(CH3)2); 2.30 (t, J = 6.9, 2H, C(3)); 4.28 (t, J = 7.2, 2H, C(1));
6.70–7.60 (m, 8 arom. H). Anal. Calcd for C17H20F2N4: C, 64.14; H,
6.33; N, 17.60. Found: C, 64.30; H, 6.55; N, 17.35.
Yield: 61%. Mp 103–104 °C (hexane). 1H NMR (CDCl3): 6.64–
7.72 (m, 7 arom. H); 9.71 (s, NH). Anal. Calcd for C13H8F5N3: C,
51.84; H, 2.68; N, 13.95. Found: C, 52.14; H, 2.66; N, 13.99.
5.2.12. 1-[1-(2,5-Difluorophenyl)-3-(3-trifluoromethylphenyl)-
triazen-3-yl]-N,N-dimethyl-3-propanamine (30)
5.3.4. 1-(3-Chlorophenyl)azopyrrolidine (35)
Yield: 90%. Mp 49–50.5 °C (hexane). 1H NMR (CDCl3): 1.96 (br s,
4H, C(3, 4)); 3.73 (br s, 4H, C(2, 5)); 6.96–7.37 (m, 4 arom. H). Anal.
Calcd for C10H12ClN3: C, 57.28; H, 5.77; N, 20.04. Found: C, 57.30;
H, 5.65; N, 20.25.
Yield: 57%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.7 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.86 (q, 2H, C(2)); 2.18 (s,
N(CH3)2); 2.29 (t, J = 6.7, 2H, C(3)); 4.30 (t, J = 7.2, 2H, C(1));
6.77–7.80 (m, 7 arom. H). Anal. Calcd for C18H19F5N4: C, 55.96; H,
4.96; N, 14.50. Found: C, 55.95; H, 4.76; N, 14.66.
5.3.5. 1-(3-Trifluoromethylphenyl)azopyrrolidine (37)
Yield: 81%. Oil (CC: Al2O3, Et2O) 1H NMR (CDCl3): 1.97 (br s, 4H,
C(3, 4)); 3.66 (br s, 2H); 3.80 (br s, 2H); 7.20–7.70 (m, 4 arom. H).
Anal. Calcd for C11H12F3N3: C, 54.32; H, 4.97; N, 17.27. Found: C,
54.33; H, 5.14; N, 17.48.
5.2.13. 1-[1-(2,6-Difluorophenyl)-3-phenyltriazen-3-yl]-N,N-
dimethyl-3-propanamine (31)
Yield: 41%. Oil (CC: Al2O3, Et2O). TLC: Rf = 0.7 (Al2O3,
Et2O + 2%Et2NH). 1H NMR (CDCl3): 1.90 (q, 2H, C(2)); 2.19 (s,
N(CH3)2); 2.33 (t, J = 7.0, 2H, C(3)); 4.26 (t, J = 7.6, 2H, C(1));
6.81–7.47 (m, 8 arom. H). Anal. Calcd for C17H20F2N4: C, 64.14; H,
6.33; N, 17.60. Found: C, 63.98; H, 6.49; N, 17.55.
5.3.6. 1-(2,5-Difluorophenyl)azopyrrolidine (43)
Yield: 89%. Oil (CC: Al2O3, Et2O). 1H NMR (CDCl3): 1.97 (br s, 4H,
C(3, 4)); 3.64 (br s, 2H); 3.88 (br s, 2H); 6.60–7.20 (m, 3 arom. H).

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4437
Anal. Calcd for C10H11F2N3: C, 56.87; H, 5.25; N, 19.89. Found: C,
56.76; H, 5.54; N, 20.19.
at an initial density of 6 ꢁ 105 and 1 ꢁ 106 cells/mL, respectively,
in maintenance medium, without or with serial dilutions of test
compounds. Cell viability was determined after 48–96 h at 37 °C
in a humidified CO2 (5%) atmosphere by the 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method.38
Vero 76 cells were resuspended in 24 multiwell plates at an
initial density of 4 ꢁ 105 cells/mL. The cell number of Vero 76
monolayers was determined by staining with the crystal violet
dye.
5.3.7. 1-(3,4-Dichlorophenyl)azopyrrolidine (44)
Yield: 65%. Mp 63–65 °C (hexane). 1H NMR (CDCl3): 1.97 (br s,
4H, C(3, 4)); 3.59 (br s, 2H); 3.83 (br s, 2H); 7.06–7.49 (m, 3 arom.
H). Anal. Calcd for C10H11Cl2N4: C, 49.20; H, 4.54; N, 17.21. Found:
C, 49.18; H, 4.57; N, 17.12.
5.3.8. 1-(3-Chlorophenyl)azopiperidine (46)
For cytotoxicity evaluations, exponentially growing cells de-
rived from human hematological tumors [CD4+ human T-cells con-
taining an integrated HTLV-1 genome (MT-4)] were seeded at an
initial density of 1 ꢁ 105 cells/mL in 96 well plates in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 100
Yield: 30%. Mp 32–33°C (hexane). 1H NMR (CDCl3): 1.65 (br s,
6H, C(3, 4, 5)); 3.73 (br s, 4H, C(2, 6)); 7.00–7.44 (m, 4 arom. H).
Anal. Calcd for C11H14ClN3: C, 59.00; H, 6.58; N, 19.12. Found: C,
59.06; H, 6.31; N, 18.78.
units/mL penicillin G and 100 lg/mL streptomycin. Cell cultures
5.3.9. 1-(3-Bromophenyl)azopiperidine (47)
were then incubated at 37 °C in a humidified, 5% CO2 atmosphere
in the absence or presence of serial dilutions of test compounds.
Cell viability was determined after 96 h at 37 °C by the MTT
method.
Yield: 28%. Mp 30 °C (hexane). 1H NMR (CDCl3): 1.65 (br s, 6H,
C(3, 4, 5)); 3.73 (br s, 4H, C(2, 6)); 7.05–7.56 (m, 4 arom. H). Anal.
Calcd for C11H14BrN3: C, 49.27; H, 5.26; N, 15.67. Found: C, 49.28;
H, 4.99; N, 15.82.
5.4.4. Antiviral assay
5.3.10. 1-(3-Nitrophenyl)azopiperidine (48)
Activity of compounds against Human Immunodeficiency virus
type-1 (HIV-1) was based on inhibition of virus-induced cytopath-
ogenicity in MT-4 cells acutely infected with a multiplicity of infec-
Yield: 40%. Mp 33–35 °C (hexane). 1H NMR (CDCl3): 1.66 (br s,
6H, C(3, 4, 5)); 3.74 (br s, 4H, C(2, 6)); 7.32–8.20 (m, 4 arom. H).
Anal. Calcd for C11H14N4O2: C, 56.40; H, 6.02; N, 23.92. Found: C,
56.26; H, 6.16; N, 24.06.
tion (m.o.i.) of 0.01. Briefly, 50
l
L of RPMI containing 1 ꢁ 104 MT-4
were added to each well of flat-bottom microtitre trays containing
50
Then, 20
l
L of RPMI, without or with serial dilutions of test compounds.
L of a HIV-1 suspension containing 100 CCID50 were
5.3.11. 1-Methyl-4-(2,5-difluorophenylazo)piperazine (53)
Yield: 71%. Oil (CC: Al2O3, Et2O). 1H NMR (CDCl3): 2.31 (s, CH3);
2.50 (t, J = 5.2, 4H, C(2, 6)); 3.83 (t, J = 5.2, 4H, C(3, 5)); 6.65–7.22
(m, 3 arom. H). Anal. Calcd for C11H14F2N4: C, 54.99; H, 5.87; N,
23.32. Found: C, 54.88; H, 5.98; N, 23.61.
l
added. After a 4-day incubation, cell viability was determined by
the MTT method.
Activity of compounds against Yellow fever virus (YFV) and
Reo virus type-1 (Reo-1) was based on inhibition of virus-induced
cytopathogenicity in acutely infected BHK-21 cells. Activities
against Bovine viral diarrhoea virus (BVDV), in infected
MDBK cells, were also based on inhibition of virus-induced
cytopathogenicity.
BHK and MDBK cells were seeded in 96-well plates at a den-
sity of 5 ꢁ 104 and 3 ꢁ 104 cells/well, respectively, and were al-
lowed to form confluent monolayers by incubating overnight in
growth medium at 37 °C in a humidified CO2 (5%) atmosphere.
5.3.12. 4-Phenylazo-1-(pyrimidin-20-yl)piperazine (56)
Yield: 48%. Mp 74–75 °C (hexane). 1H NMR (CDCl3): 3.84 (t,
J = 5.5, 4H, C(2, 6)); 3.95 (t, J = 5.5, 4H, C(3, 5)); 6.48 (t, J = 4.8, 1H,
C(5’)); 7.07–7.46 (m, 5 arom. H); 8.28 (d, J = 4.8, 2H, C(40, 60)). Anal.
Calcd for C14H16N6: C, 62.67; H, 6.01; N, 31.32. Found: C, 62.39; H,
6.16; N, 31.63.
5.3.13. 3-(3-Nitrophenylazo)cytisine (58)
Cell monolayers were then infected with 50
dilution (in serum-free medium) to give a m.o.i = 0.01. One hour
later, 50 L of MEM Earle’s medium, supplemented with inacti-
vated fetal calf serum (FCS), 1% final concentration, without or
with serial dilutions of test compounds, were added. After 3–
4 days of incubation at 37 °C, cell viability was determined by
the MTT method.
lL of a proper virus
Yield: 25%. Mp 201–203 °C (acetone). 1H NMR (CDCl3): 1.95–
2.19 (m, 2H, C(13)); 2.58–2.75 (m, 1H, C(5)); 3.12–3.27 (m, 5H,
C(1,2,4)); 3.74–3.92 (m, 1H, C(6)); 4.20–4.30 (m, 1H, C(6)); 6.05
(d, J = 6.7, 1H, C(9)); 6.32 (d, J = 8.4, 1H, C(11)); 7.12–8.12 (m, 1H,
C(10) and 4 arom. H). Anal. Calcd for C17H17N5O3: C, 60.17; H,
5.05; N, 20.64. Found: C, 60.27; H, 5.33; N, 20.31.
l
Activity of compounds against Coxsackie Virus type B2 (CVB-
2), Polio Virus type-1 Sabin strain (Sb-1), Vesicular Stomatitis
Virus (VSV), Vaccinia Virus (VV), Herpes Virus 1 (HSV-1) and
Respiratory Syncytial Virus (RSV), A-2 strain, in infected Vero 76
cells, was determined by plaque reduction assays in Vero 76 cell
monolayers. To this end, Vero 76 cells were seeded in 24-well
plates at a density of 2 ꢁ 105 cells/well and were allowed to form
confluent monolayers by incubating overnight in growth medium
at 37 °C in a humidified CO2 (5%) atmosphere. Then, monolayers
5.4. Cell-based assays
5.4.1. Compounds
Compounds were dissolved in DMSO at 100 mM and then di-
luted in culture medium.
5.4.2. Cells and viruses
Cell lines were purchased from American Type Culture Collec-
tion (ATCC). The absence of mycoplasma contamination was
checked periodically by the Hoechst staining method. Cell lines
supporting the multiplication of RNA and DNA viruses were the
following: CD4+ human T-cells containing an integrated HTLV-1
genome (MT-4); Madin Darby Bovine Kidney (MDBK); Baby Ham-
ster Kidney (BHK-21) and Monkey kidney (Vero 76) cells.
were infected with 250
lL of proper virus dilutions to give 50–
100 PFU/well. Following removal of unadsorbed virus, 500
lL of
Dulbecco’s modified Eagle’s medium supplemented with 1% inac-
tivated FCS and 0.75% methyl-cellulose, without or with serial
dilutions of test compounds, were added. Cultures were incubated
at 37 °C for 2 (Sb-1 and VSV), 3 (CVB-2, VV and HSV-1) or 5 days
(RSV) and then fixed with PBS containing 50% ethanol and 0.8%
crystal violet, washed and air-dried. Plaques were then counted.
EC50 (50% effective concentration) was calculated by linear regres-
sion technique.
5.4.3. Cytotoxicity assays
For cytotoxicity tests, run in parallel with antiviral assays,
MDBK and BHK cells were resuspended in 96 multiwell plates

4438
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
5.5. Molecular modeling
procedure;33,34 accordingly, it will be briefly reported below. The
software AUTODOCK 3.044 was employed to estimate the possible
binding orientations of all compounds in the receptor. In order to
encase a reasonable region of the protein surface and interior vol-
ume, centered on the binding site, the grids were 60 Å on each side.
Grid spacing (0.375 Å), and 120 grid points were applied in each
Cartesian direction so as to calculate mass-centered grid maps. AM-
BER 12-6 and 12-10 Lennard-Jones parameters were used in model-
ing van der Waals interactions and hydrogen bonding (N–H, O–H
and S–H), respectively. In the generation of the electrostatic grid
maps, the distance dependent relative permittivity of Mehler and
Solmajer was applied.45 For the docking of each compound to the
proteins, three hundred Monte Carlo/Simulated Annealing (MC/
SA) runs were performed, with 100 constant temperature cycles
for simulated annealing. For these calculations, the GB/SA implicit
water model41 was used to mimic the solvated environment. The
rotation of the angles u and /, and the angles of side chains were
set free during the calculations. All other parameters of the MC/
SA algorithm were kept as default. Following the docking proce-
dure, the structure of all compounds were subjected to cluster
analysis with a tolerance of 1 Å for an all-atom root-mean-square
deviation from a lower-energy structure representing each cluster
family. In the absence of any relevant crystallographic information
for our compounds, the structure of each resulting complex charac-
terized by the lowest interaction energy in the prevailing cluster
was selected for further evaluation.
All molecular dynamics simulations were carried out using the
sander and pmemd module within the AMBER 9 suite of programs,39
and the parm99 all-atom force field,40 working in parallel on 32
processors of the Tartaglia cluster at the University of Trieste (Trie-
ste, Italy). The crystallographic coordinates of the RNA-dependent
RNA-polymerase (RdRp) of BVDV (PDB entry 1S48.pdb)31 and
HCV (PDB entry 1CSJ.pdb)28 were employed as starting geometries
for protein simulations in complex with the most active com-
pounds (1, 2, 4, 7–10). Missing hydrogen atoms were added to
the protein backbone and side chains with LEaP module of AMBER
9. All ionizable residues were considered in the standard ionization
state at neutral pH. The geometry of added hydrogens and ions was
refined for 200 steps (steepest descent) in vacuum using the
parm94 force field.40 Further protein geometry refinement was car-
ried out using the sander module of AMBER 9 via a combined steepest
descent—conjugate gradient algorithm, using as a convergence cri-
terion for the energy gradient the root-mean-square of the Carte-
sian elements of the gradient equal to 0.01 kcal/(mol Å). The
generalized Born/surface area (GB/SA) continuum solvation mod-
el41 was used to mimic a water environment. As expected, no rel-
evant structural changes were observed between RdRp relaxed
models and the original 3-D structure.
The putative binding site for our compounds on the BVDV RdRp
was determined using the ActiveSite_Search option of the Binding
Site module of InsightII (v. 2001, Accelrys, San Diego, USA).33 Active-
Site_Search identifies protein active sites or binding sites by locat-
ing cavities in the protein structure. According to the Site_Search
algorithm employed, the protein is first mapped onto a grid which
covers the complete protein space. The grid points are then defined
as free points and protein points. The protein points are grid points,
within 2 Å from a hydrogen atom or 2.5 Å from a heavy atom. Then,
a cubic eraser moves from the outside of the protein toward the
center to remove the free points until the opening is too small
for it to move forward. Those free points not reached by the eraser
will be defined as site points. After a site is located, it can be mod-
ified by expanding or contracting the site. One layer of grid points
at the cavity opening site will be added or removed by each expand
or contract operation, respectively.
Each best substrate/RdRp complex resulting from the auto-
mated docking procedure was further refined in the AMBER 9 suite
using the quenched molecular dynamics (QMD) method.33,34 In
this case, 1 ns MD simulation at 310 K were employed to sample
the conformational space of the substrate–enzyme complex in
the GB/SA continuum solvation environment. The integration step
was equal to 1 fs and the parm99 force field parameters were ap-
plied in the simulations. After each ps, the system was cooled to
0 K, the structure was extensively minimized, and stored. To pre-
vent global conformational changes of the enzyme, the backbone
of the protein binding site was constrained by a harmonic force
constant of 100 kcal/Å, whereas the amino acid side chains and
the ligands were allowed moving without any constraint.
The best energy configuration of each complex resulting from
the previous step was allowed to relax in a 55-Å radius sphere of
TIP3P water molecules.46 The resulting system was minimized
with a gradual decrease in the position restraints of the protein
atoms. At the end of the relaxation process, all water molecules be-
yond the first hydration shell (i.e., at a distance > 3.5 Å from any
protein atom) were removed. Finally, to achieve electroneutrality,
a suitable number of counterions were added to neutralize the sys-
tem using the leap module of AMBER 9, removing eventually overlap-
ping water molecules. To reduce computational time to reasonable
limits, all proteins residues with any atom closer than 20 Å from
the center of mass of each bounded ligand were chosen to be flex-
ible in the dynamic simulations. Subsequently, a spherical TIP3P
water cap of radius equal to 20 Å was centered on each inhibitor
in the corresponding RdRp complex, including the hydrating water
molecules within the sphere resulting from the previous step. After
energy minimization of the new water cap for 10 ps, keeping the
protein, the ligand, and the pre-existing waters rigid, followed by
a MD equilibration of the entire water sphere with fixed solute
for 1 ns, further unfavorable interactions within the structures
were relieved by progressively smaller positional restraints on
the solute (from 25 to 0 kcal/(mol Å2) for a total of 2 ns. Each sys-
tem was gradually heated to 310 K in three intervals, allowing a
1 ns interval per each 100 K, and then equilibrated for 5 ns at
310 K, followed by 10 ns of data collection runs, necessary for the
estimation of the free energy of binding (vide infra). The MD sim-
ulations were performed at constant T = 310 K using the Berendsen
The model structures of all ligand molecules were built and
subjected to an initial energy minimization. The convergence crite-
rion was set to 10ꢀ4 kcal/(mol Å). A conformational search was car-
ried out using a well-validated, ad-hoc developed combined
molecular mechanics/molecular dynamics simulated annealing
(MDSA) protocol.33,34 Accordingly, the relaxed structures were
subjected to five repeated temperature cycles (from 310 K to
1000 K and back) using constant volume/constant temperature
(NVT) MD conditions. At the end of each annealing cycle, the struc-
tures were again energy minimized to converge below 10ꢀ4 kcal/
(mol Å), and only the structures corresponding to the minimum
energy were used for further modeling. The atomic partial charges
for the geometrically optimized compounds were obtained using
the RESP procedure,42 and the electrostatic potentials were pro-
duced by single-point quantum mechanical calculations at the
*
Hartree–Fock level with a 6-31G basis set, using the Merz–
Singh–Kollman van der Waals parameters.43 Eventual missing
force field parameters for the compounds considered were gener-
ated as follows: AM1 geometry optimization of the structure was
*
followed by RHF/6-31G single point calculation to obtain the elec-
trostatic potentials. Next, the RESP method was used for charge fit-
ting. The missing bond, angle torsion or van der Waals parameters
not included in the parm99 were generated using the antechamber
module of AMBER 9.
The optimized structures of the inhibitors were docked into the
putative enzyme allosteric binding site by applying a consolidated

M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
4439
et al. coupling algorithm47 with separate coupling of the solute and
Acknowledgments
solvent to the heat, an integration time step of 2 fs, and the appli-
cations of the Shake algorithm to constrain all bonds to their equi-
librium values, thus removing high frequency vibrations. Long-
range nonbonded van der Waals interactions were truncated by
using a dual cutoff of 9 and 13 Å, respectively, where energies
and forces due to interactions between 9 and 13 Å were updated
every 20 time steps. The particle mesh Ewald method was used
to treat the long-range electrostatics. For the calculation of the
binding free energy between the RdRp and each inhibitor in water,
a total of 100 snapshots were saved during the MD data collection
period described above, one snapshot per each 0.1 ns of MD
simulation.
Financial support from Italian MIUR (FIRB RBNE01J3SK01) and
BIOMEDICINE PROJECT is gratefully acknowledged. The authors
thanks O. Gagliardo for performing the elemental analyses.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.05.020.
References and notes
1. Tonelli, M.; Boido, V.; Canu, C.; Sparatore, A.; Sparatore, F.; Paneni, M. S.;
Fermeglia, M.; Pricl, S.; La Colla, P.; Casula, L.; Ibba, C.; Collu, D.; Loddo, R.
Bioorg. Med. Chem. 2008, 16, 8447.
2. Elslager, E. F.; Worth, D. F. J. Med. Chem. 1963, 6, 444.
3. Werbel, L. M.; Elslager, E. F.; Fisher, M. V.; Gavrilis, Z. B.; Phillips, A. A. J. Med.
Chem. 1968, 11, 411.
4. Vazzana, I.; Sparatore, F.; Fadda, G.; Manca, C. Farmaco 1993, 48, 737.
5. Novelli, F.; Sparatore, F. Farmaco 2002, 57, 871. and references cited therein.
6. Barbieri, F.; Sparatore, A.; Alama, A.; Novelli, F.; Bruzzo, C.; Sparatore, F. Oncol.
Res. 2003, 13.
7. Sparatore, F.; Sommovigo, P. G.; Boido, V. Boll. Chim. Farm. 1974, 113, 219.
8. Gesher, J. A.; Hickman, J. A.; Simmonds, R. G.; Stevens, M. F. G.; Vaughan, K.
Biochem. Pharmacol. 1981, 30, 89.
The binding free energy
DGbind of each RdRp/drug complex in
water was calculated according to the procedure termed Molecular
Mechanic/Poisson–Boltzmann Surface Area (MM/PBSA), and origi-
nally proposed by Srinivasan et al.48 In the MM/PBSA framework of
theory, the binding free energy of a given ligand to its receptor pro-
tein can be evaluated as
D
Gbind ¼ Gcomplex ꢀ ðGprotein þ Gligand
The individual terms of the MM/PBSA approach that contribute
Þ
ð1Þ
to the free energy of a molecule are
9. Prous, J.; Graul, A.; Castañer, J. Drugs Fut. 1994, 19, 746.
10. Dumont-Hornebeck, B.; Strube, Y. N.; Vasilescu, D.; Jean-Claude, B. J. Bioorg.
Med. Chem. Lett. 2000, 10, 2325.
11. Matheson, S. L.; Mc Namee, J.; Jean-Claude, B. J. Cancer Chemother. Pharmacol.
2003, 51, 11.
12. Manolov, J.; Machulla, H.-J.; Momekov, G. Pharmazie 2006, 61, 6.
13. Stevens, M. F. G.; Phillips, K. S.; Rathbone, D. L.; O’ Shea, D. M.; Queener, S. F.;
Schwalbe, C. H.; Lambert, P. A. J. Med. Chem. 1997, 40, 1886.
14. Yamada, Y.; Saito, J.; Kudamatsu, A.; Katsumata, O. Ger. Offen. 2147259, 1972;
Chem. Abstr. 1972, 77, 5145a.
15. Borowski, P.; Deinert, J.; Schalinski, S.; Bretner, M.; Ginalski, K.; Kulikowski, T.;
Shugar, D. Eur. J. Biochem. 2003, 270, 1645.
16. Wu, C.-Y.; King, K.-J.; Kuo, C.-J.; Fang, J.-M.; Wu, J.-T.; Ho, M.-Y.; Liao, C.-L.; Shie,
J.-J.; Liang, P.-H.; Wong, C.-H. Chem. Biol 2006, 13, 261.
17. Tonelli, M.; Paglietti, G.; Boido, V.; Sparatore, F.; Marongiu, F.; Marongiu, E.; La
Colla, P.; Loddo, R. Chem. Biodiversity 2008, 5, 2386.
18. Boido, V.; Sparatore, F. Farmaco Ed. Sci. 1974, 29, 526.
19. Meanwell, N. A.; Krystal, M. Drugs Fut. 2007, 32, 441. and references cited
therein.
Gmol ¼ EMM þ Gsolv ꢀ TSsolute
ð2Þ
where EMM denotes the sum of intra- and intermolecular mechani-
cal (MM) energies of a molecule in the gas phase, Gsolv is its solva-
tion free energy, and –TSsolute represents an estimate of the solute
entropy. EMM can be further divided into terms arising from electro-
static (Eel), van der Waals (EvdW), and internal (Eint) (i.e., bond, angle,
and torsional energies):
EMM ¼ Eel þ EvdW þ Eint
The solvation free energy:
Gsolv ¼ GPB þ Gnp
consists of a polar solvation energy component, GPB, which is calcu-
lated in a continuum solvent, usually a finite-difference Poisson-
Boltzmann (PB) model, and a nonpolar term, Gnp, which is propor-
tional to the solvent-accessible surface area (SA).
ð3Þ
ð4Þ
20. Henderson, E. A.; Alber, D. G.; Baxter, R. C.; Bithell, S. K.; Budworth, J.; Carter, M.
C.; Chub, A.; Cockerill, G. S.; Dowdell, V. C. L.; Fraser, I. J.; Harris, R. A.; Keegan, S.
J.; Kelsey, R. D.; Lumley, J. A.; Stables, J. N.; Weerasekera, N.; Willson, L. J.;
Powell, K. L. J. Med. Chem. 2007, 50, 1685.
Theensembleofstructuresfortheuncomplexedreactantsaregen-
erated either running separate MD simulations for them, or by using
the trajectory of the complex, simply removing the atoms of the
protein or ligand. In this work we applied the latter variant. Accord-
ingly, the term Eint in Eq. 2 cancels out in the calculation of the free en-
ergy of binding. The calculations of the polar solvation term GPB were
done with the DelPhi package,49 with interior and exterior dielectric
constants equal to 1 and 80, respectively. A grid spacing of 2/Å,
extending 20% beyond the dimensions of the solute, was employed.
The non-polar component GNP was obtained using the following
21. Houe, H. Biologicals 2003, 31, 137.
22. Buckwold, V. E.; Beer, B. E.; Donis, R. O. Antiviral Res. 2003, 60, 1.
23. Paeshuyse, J.; Leyssen, P.; Mabery, E.; Boddeker, N.; Vrancken, R.; Froeyen, M.;
Ansari, I. H.; Dutartre, H.; Rozenski, J.; Gil, L. H. V. G.; Letellier, C.; Lanford, R.;
Canard, B.; Koenen, F.; Kerkhofs, P.; Donis, R. O.; Herdewijn, P.; Watson, J.; De
Clercq, E.; Puerstinger, G.; Neyts, J. J. Virol. 2006, 80, 149.
24. Baginski, S. G.; Pevar, D. C.; Seipel, M.; Sun, S. C.; Benetatos, C. A.; Chunduru, S.
K.; Rice, C. M.; Collett, M. S. Proc. Natl. Acd. Sci. U.S.A. 2000, 97, 7981.
25. King, R. W.; Scarnati, H. T.; Priestley, E. S.; De Lucca, I.; Bansal, A.; Williams, J. K.
Antivir. Chem. Chemother. 2002, 13, 315.
26. Sun, J. H.; Lemm, J. A.; O’Boyle, D. R., II; Racela, J.; Colonno, R.; Gao, M. J. Virol.
2003, 77, 6753.
27. Tabarrini, O.; Manfroni, G.; Fravolini, A.; Cecchetti, V.; Sabatini, S.; De Clercq, E.;
Rozenski, J.; Canard, B.; Dutartre, H.; Paeshuyse, J.; Neyts, J. J. Med. Chem. 2006,
49, 2621.
28. Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.; Mathieu, M.; De
Francesco, R.; Rey, F. A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13034.
29. Ago, H.; Adachi, T.; Yoshida, A.; Yamamoto, M.; Habuka, N.; Yatsunami, K.;
Miyano, M. Structure 1999, 7, 1417.
30. Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino, A.; Weber, P. C. Nat.
Struct. Biol. 1999, 6, 937.
31. Choi, K. H.; Groarke, J. M.; Young, D. C.; Kuhn, R. J.; Smith, J. L.; Pevear, D. C.;
Rossmann, M. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4425.
32. Choi, K. H.; Gallei, A.; Becher, P.; Rossmann, M. G. Structure 2006, 14, 1107.
33. Carta, A.; Loriga, M.; Paglietti, G.; Ferrone, M.; Fermeglia, M.; Pricl, S.; Sanna, T.;
Ibba, C.; La Colla, P.; Loddo, R. Bioorg. Med. Chem. 2007, 15, 1914.
34. Mazzei, M.; Nieddu, E.; Miele, M.; Balbi, A.; Ferrone, M.; Fermeglia, M.; Mazzei,
M. T.; Pricl, S.; La Colla, P.; Marongiu, F.; Ibba, C.; Loddo, R. Bioorg. Med. Chem.
2008, 16, 2591.
relationship: GNP = cSA + b, in which c
= 0.00542 kcal/(mol Å2),
b = 0.92 kcal/mol, and the surface area was estimated by means of
the MSMS software.50 The last parameter in Eq. 1, that is, the change
in solute entropy upon association –TSsolute, was calculated through
normal-mode analysis. In the first step of this calculation, an 8-Å
sphere around the ligand was cut out from an MD snapshot for each
ligand-protein complex. This value was shown to be large enough to
yield converged mean changes in solute entropy. On the basis of the
size-reduced snapshots of the complex, we generated structures of
the uncomplexed reactants by removing the atoms of the protein
and ligand, respectively. Each of those structures was minimized,
using a distance-dependent dielectric constant e = 4r, to account for
solvent screening, and its entropy was calculated using classical sta-
tistical formulas and normal mode-analysis. To minimize the effects
due to different conformations adopted by individual snapshots we
averaged the estimation of entropy over 50 snapshots.
35. Lai, V. C.; Kao, C. C.; Ferrari, E.; Park, J.; Uss, A. S.; Wright-Minogue, J.; Hong, Z.;
Lau, J. Y. J. Virol. 1999, 73, 10129.
36. Dimitrova, M.; Imbert, I.; Kieny, M. P.; Schuster, C. J. Virol. 2003, 77,
5401.

4440
M. Tonelli et al. / Bioorg. Med. Chem. 17 (2009) 4425–4440
37. Piccininni, S.; Varaklioti, A.; Lardelli, M.; Dave, B.; Raney, D. A.; McCarthy, J. E. J.
Biol. Chem. 2002, 277, 45670.
42. Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Phys. Chem. 1993, 97,
10269.
38. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Meth. 1988, 20, 309.
43. Besler, B. H.; Merz, K. M.; Kollman, P. A. J. Comput. Chem. 1990, 11, 431.
44. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
45. Mehler, E. L.; Solmajer, T. Protein Eng. 1991, 4, 903.
46. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J.
Chem. Phys. 1983, 79, 926.
47. Berendsen, H. J. C.; Postma, J. P. M.; Van Gunsteren, W. F.; DiNola, A.; Haak, J. R.
J. Chem. Phys. 1984, 81, 3684.
48. Srinivasan, J.; Cheatham, T. E.; Cieplak, P.; Kollman, P. A.; Case, D. A. J. Am.
Chem. Soc. 1998, 120, 9401.
39. Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.;
Duke, R. E.; Luo, R.; Merz. K. M.; Pearlman, D. A.; Crowley, M.; Walker, R.
C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G; Wong, K. F.;
Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.;
Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.;
Ross, W. S.; Kollman, P. A., AMBER 9, University of California, San Francisco, CA
USA, 2006.
40. Wang, J.; Cieplak, P.; Kollman, P. A. J. Comput. Chem. 2000, 21, 1049.
41. Feig, M.; Onufriev, A.; Lee, M.; Im, W.; Case, D. A.; Brooks, C. L., III J. Comput.
Chem. 2004, 25, 265.
49. Gilson, M. K.; Sharp, K. A.; Honig, B. H. J. Comput. Chem. 1988, 9, 327.
50. Sanner, M. F.; Olson, A. J.; Spehner, J. C. Biopolymers 1996, 38, 305.