New anti-viral drugs for the treatment of the common cold

Article abstract of DOI:10.1016/j.bmc.2007.12.030

Human Rhinovirus (HRV) is the most important aetiologic agent of common cold in adults and children. HRV is a single-stranded, positive sense RNA virus and, despite the high level of conservation among different serotypes, sequence alignment of viral protease 3C with mammalian protease reveals no homology. Thus, protease 3C is an optimal target for the development of anti-HRV agents. In the present work we investigated the design, the synthesis and the development of new potential reversible inhibitors against HRV protease 3C. Docking studies on the crystallized structure of HRV2 protease 3C led us to the design and the synthesis of a series of 3,5 disubstituted benzamides able to act as analogues of the substrate. We also developed 1,3,5 trisubstituted benzamides where aromatic substitutions on the aryl ring led us to investigate the importance of π-π interaction on the stabilization of protease 3C-inhibitor complex. All structures were tested for enzymatic inhibition on HRV14 protease 3C. Results highlighted the inhibitory activity of compounds 13, 14, and 20 (91%, 81%, and 85% at 10 μM, respectively), with the latter exhibiting an ID₅₀ (dose that inhibits 50% of the viral cytopathic effect) on HRV-14 = 25 μg/ml.

Full text of DOI:10.1016/j.bmc.2007.12.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3091–3107
New anti-viral drugs for the treatment of the common cold
Caterina Maugeri,a,* Maria A. Alisi,a Claudia Apicella,a Luciano Cellai,b
Patrizia Dragone,a Elena Fioravanzo,c Saverio Florio,d Guido Furlotti,a
Giorgina Mangano,a Rosella Ombrato,a Renzo Luisi,d Raffaello Pompei,e Vito Rincicotti,a
Vincenzo Russo,a Marco Vitielloa and Nicola Cazzollaa
aAngelini Farmaceutici, A.C.R.A.F. SpA, P.le della Stazione snc, 00040 S. Palomba, Roma, Italy
bCNR Istituto di Cristallografia, Via Salaria km. 29,3—C.P. 10—16 Monterotondo Scalo, Roma, Italy
cS.IN Soluzioni Informatiche, Via G. Salvemini 9, 36100 Vicenza, Italy
d
`
Dipartimento Farmaco Chimico, Universita di Bari, Via E. Orabona, 4I-70125 Bari, Italy
`
Dipartimento di Scienze Mediche, Universita di Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy
e
Received 25 September 2007; revised 7 December 2007; accepted 14 December 2007
Available online 23 December 2007
Abstract—Human Rhinovirus (HRV) is the most important aetiologic agent of common cold in adults and children. HRV is a sin-
gle-stranded, positive sense RNA virus and, despite the high level of conservation among different serotypes, sequence alignment of
viral protease 3C with mammalian protease reveals no homology. Thus, protease 3C is an optimal target for the development of
anti-HRV agents. In the present work we investigated the design, the synthesis and the development of new potential reversible
inhibitors against HRV protease 3C. Docking studies on the crystallized structure of HRV2 protease 3C led us to the design
and the synthesis of a series of 3,5 disubstituted benzamides able to act as analogues of the substrate. We also developed 1,3,5 tri-
substituted benzamides where aromatic substitutions on the aryl ring led us to investigate the importance of p–p interaction on the
stabilization of protease 3C-inhibitor complex. All structures were tested for enzymatic inhibition on HRV14 protease 3C. Results
highlighted the inhibitory activity of compounds 13, 14, and 20 (91%, 81%, and 85% at 10 lM, respectively), with the latter exhib-
iting an ID50 (dose that inhibits 50% of the viral cytopathic effect) on HRV-14 = 25 lg/ml.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
protease 2A and 3C.3 Protease 2A accomplishes the
cleavage of Tyr-Gly pairs but is not essential for the via-
bility of the virus. Despite its overall architecture corre-
lating with trypsine-like serine protease, protease 3C
(3CP) is a cysteinyl protease with His-Glu-Cys being
the catalytic triad. It hydrolyses Gln-Gly, Gln-Ser and
Gln-Ala pairs so it is essential for viral replication.4 Re-
cent DNA sequencing of 3CP of different serotypes re-
vealed a high degree of homology within the 3C
coding region including active site residues.5 However,
sequence alignment of viral 3CP with mammalian prote-
ase reveals no homology.6 Thus, 3CP is an optimal tar-
get for the development of anti-viral agents.
Human Rhinovirus (HRV) is the most important aetio-
logic agent of the common cold in adults and children.
At present, no anti-HRV agent exists and the large num-
ber of different serotypes (ꢀ105)1 makes unlikely the
development of vaccines. HRV clinical manifestations
include nasopharyngeal syndrome with signs as sneez-
ing, rhinorrhea and malaise; usually fever is not present
and cough is only mild.2 HRV is a single-stranded, po-
sitive sense RNA virus. First step in replication involves
its attachment to the membrane of the host cell via
ICAM-1 receptors and its penetration by endocytosis.
Following the uncoating of the viral RNA from the cap-
sid, the positive strand is translated into a long polypro-
tein which undergoes proteolysis by virus-encoded
Elucidation of 3CP crystal structure together with sub-
strate cleavage specificity studies have facilitated the ra-
tional design of inhibitors.
Keywords: Human rhinovirus; Protease 3C; Cysteinyl protease; Tri-
substituted benzamides; Aloketones.
Several peptidic and non-peptidic inhibitors acting as
analogues of the substrate have been reported. They
*
Corresponding author. E-mail: c.maugeri@angelini.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.030

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C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
Figure 1. Examples of known HRV 3CP protease inhibitors.
all contain groups which can be attacked by SH of the
cysteine of the catalytic triad of the protease, thus gen-
erating a reversible analogue of the transition state. It
has been found that 3CP needs at least four aminoacids
upstream the scissile bond, so tetrapeptides containing
electrophiles as aldehydic groups have been synthes-
ised.7–9 Tetrapeptides, but also tri and dipeptides,
reversibly inhibited 3CP with good IC50 values. Short
polypeptides were also combined with thiol-reactive
groups such as methyl and halomethyl ketones10 (e.g.,
1, IC50 = 48 nM, Fig. 1), Michael acceptors11,12 (e.g.,
2, EC50 = 0.54 lM, Fig. 1),13 iodoacetamides, b-lactones
and pseudoxazolones.14 Peptidic lactam AG7088 was
tested in Phase II trial with intranasal delivery. Unfortu-
nately, peptide-based inhibitors show rapid metabolism
and poor bioavailability so they are not likely to be good
drug candidates.
synthesis and the pharmacological evaluation of new
potential reversible inhibitors.
2. Drug design
Starting point of molecular modelling studies was the al-
ready available X-ray crystal structure of the covalent
adduct between HRV-2 3CP and AG708823 (pdb code:
1CQQ).
AG7088 is an effective anti-viral agent and inhibits all 48
serotypes of rhinoviruses with Kobs/I = 1,470,000 Mꢁ1 sꢁ1
on HRV-14. It is also equally effective on HRV-serotype
14 (EC50 = 0.013lM) and HRV- serotype 2 (EC50
=
0.012 lM) in HeLa cells.23 The catalytic triad of 3CP
is represented by Cys 147, Glu 71 and His 40 with the
nucleophilic SH of the Cys in the active site covalently
linked to the inhibitor’s electrophilic b-carbon of the
Michael acceptor.23 Analysis of the HRV-2 3CP-
AG7088 X-ray crystal structure suggested that in the
catalytic site there are some pockets. A brief exploration
highlights that the Michael acceptor is in S3 pocket, the
lactamic group leans forward S1 and the benzyl group
positions itself in S2 pocket. The lactamic group of the
inhibitor is hydrogen bonded both with His 161 and
Thr 142 (Fig. 2). Comparing the structure of AG7088
with the structure of 2HN-Thr-Gly-Leu-Phe-Gln-Gly-
Non-peptidic 3CP inhibitors include b-lactams,15 isa-
tines,16 homophthalimides17 (e.g., 3, IC50 = 55.4 lM,
Fig. 1), alkyl halides, ketones and quinones.18–20 Vari-
ous substituted benzamides are also present, in particu-
lar, 5-substituted a,b-unsaturated keto benzamides
showed good inhibitory properties.21,22
The aim of the present study was to identify new non-
peptidic low molecular weight broad-spectrum inhibi-
tors of HRV 3CP. Here, it is described the design, the
Figure 2. (A) Schematic diagram of AG7088 (green) bound in the HRV-2 3CP active site. Hydrogen bonds are shown as dashed lines (black),
covalent bond is shown as red dashed lines and the residues which make up the enzyme binding subsites are depicted. (B) AG7088 bound to HRV-2
3CP. The catalytic triad residues (His40, Glu71, and Cys147 in yellow), the inhibitor AG7088 (green) and hydrogen bonded residues (His161 and
Thr142 in turquoise) are shown in tube.

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3093
Figure 5. Interaction between 3CP and inhibitor.
The covalent inactivation of 3CP by halomethyl ketones
was modelled as shown in Figure 5.
Figure 3. Glutamine-Glycine cleavage site in comparison with 3-
formylbenzamide and a Michael acceptor benzamide.
The inhibitor (I) forms first a reversible complex with
the enzyme (E) and only then it can undergo a nucleo-
philic attack by Cys 147, leading to a covalent complex.
The observed biological activity depends upon both the
equilibrium binding constants (k2/k1) and the chemical
rate of covalent bond formation. In this study we ex-
plored the reversible complex formation as dominated
by non-covalent molecular recognition by means of vir-
tual docking and the subsequent chemical reaction as
dominated by the reactivity of the halomethyl ketones
by means of quantum chemical calculations.
Pro-COOH, a known 3CP substrate that shows high
specificity cleavage between Gln (P1) and Gly (P01), the
lactamic group obviously mimics the side chain of Gln
(P1).
On the basis of interactions between AG7088 and 3CP
other authors designed substituted benzamides.22 Meta
disubstitution allows to mimic the natural substrate
Gln (P1) of 3CP as the substituents are at the appropri-
ate distance and orientation (Fig. 3). Amide corresponds
to lactamic group in AG7088 and the electrophilic reac-
tive group is always an aldehyde, a ketone or a 1,4-
unsaturated ester (Michael acceptor). X-ray crystal
structure of a complex between 3-(3-carbamoyl-phe-
nyl)-acrylic acid 2-hydroxyethyl ester and 3CP21 con-
firms interactions of carboxamide group with Thr 142
and His 161. Further studies show that inhibitory activ-
ity is increased when compounds can bind recognition
sites S1–S3 on the 3CP.
2.1. Virtual docking
All the docking and scoring calculations were performed
using the Schro¨dinger suite of softwares (Schro¨dinger,
LLC, New York, NY). 1CQQ, the co-crystal structure
of the covalent adduct between 3CP and AG7088, was
used. In order to create the conditions prior to the bind-
ing, the covalent bond between the two structures was
broken. The side chain of Cys 147, originally bound to
AG7088, was shifted to avoid steric clashes and the
complex 3CP-AG7088 was energetically minimized with
MACROMODEL24 with the OPLS-AA force field.25
The minimized complex retains all the original non-
bonding interactions.
We developed a virtual library employing three different
core structures: 3-(difluoroacetyl) benzamide (a), 4-
(difluoroacetyl)-2,3-dihydro-1H-isoindol-1-one (b) and
4-(difluoroacetyl)-1,3-dihydro-2H-indol-2-one
(c)
(Fig. 4). The electrophilic reactive group is a difluoro-
methyl ketone that mimics the Michael acceptor group
of AG7088. Positions ortho and meta to difluoromethyl
ketone were explored to determine which one is able to
place a substituent R, formed by a linker and an aro-
matic group (Table 1), into the S2 pocket which seems
to be essential for good inhibitory activity.
Docking was performed using GLIDE26 program
(Schro¨dinger, LLC, New York, NY). Grids for molecu-
lar docking were calculated employing different Van der
Waals scaling parameters in order to allow the site to
‘breathe’. The virtual library of 120 structures previ-
ously designed was docked into 3CP structure. For each
structure three different poses were sampled and visually
inspected to determine which one is able to retain origi-
nal interactions with Thr 142 and His 161 and to place
the aromatic substituent into the S2 lipophilic pocket.
Best poses were retrieved with 3-(difluoroacetyl) benz-
amide core structure, meta substitution, R group formed
by a two-atom linker and phenyl or naphthyl as aro-
matic group. These results guided chemical synthesis in
our laboratory.
Figure 4. Core structures of the virtual library.
2.2. Quantum mechanical calculations
Table 1. Proposed substituents R1/R2 and R3 on the core structures a,
b, and c
Inactivation power of halomethyl ketones was studied
by means of quantum mechanical calculations. We as-
sumed that their biological activity depends upon the
chemical rate of covalent bond formation. In fact non-
bonded interactions that stabilize EI form and influence
the equilibrium binding constants (k2/k1) are more or
less the same if just the halomethyl ketone structure
changes.
Compound
R1/R2
R3
Core a, b, and c
NH–CH2–R3
CO–R3
a-Naphthyl
Ph
CH2–R3
Diphenyl
b-Naphthyl
CH2–CO–R3
CH2–CH2–R3

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C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
Figure 6. Reactivity of halomethyl ketones.
Halomethyl ketones in water exist in equilibrium with
their hydrated form [d]. It is clear that only the unhy-
drated form [e] is the active form of the inhibitor that
can undergo the nucleophilic attack of the enzyme. As
more reactive is the halomethyl ketone and more sta-
ble is the hydrated form, the ideal R group should
show a compromise: reactive enough to be able to un-
dergo the nucleophilic attack of the Cys, but not too
much not to stabilize the inactive hydrated form
(Fig. 6).
3. Chemistry
The compounds evaluated were isoindolinone deriva-
tives 4 and 5 and benzamide derivatives 6–26 (Chart 1
and Table 3).
Compound 4 was prepared in five steps starting from
phthalimide 27 which was reduced to isoindolinone 28
with Sn/HCl.28 Bromination of 28 with Br2 in the pres-
ence of AlCl3 followed by Pd catalyzed coupling with
ethyl-1-trimethylstannylvinyl ether and hydrolysis in
HCl gave the 6-acetylisoindolinone 30. Mono bromina-
tion of the acetyl group gave the desired compound 4
(Scheme 1).
Ab initio geometry optimization and energy calculations
at HF 3-21G* level were performed with Spartan27 on
systems shown in Figure 7 to investigate the chloro
and bromo influence on reactivity and subsequently on
biological activity of these systems.
Compound 5 was prepared from the commercially avail-
able 3-bromo-2-methylbenzoic acid 31 which was trans-
formed to the corresponding methyl ester that was
brominated at the benzylic position with NBS under
Energy and density of LUMO (Lowest Occupied Molec-
ular Orbital) was calculated to evaluate the reactivity of
the system towards nucleophilic attack and energy dif-
ference between forms [d] and [e] was employed to eval-
uate the stabilization of the hydrated form.
CHX2 and CH2X pairs with X = Cl or Br were con-
sidered. Results are reported in Table 2. Compounds
containing chloro show lower LUMO energy and den-
sity, that is to say higher reactivity and so hypotheti-
cal better biological activity. Moreover, CH2Br
substituent shows a high stability of the hydrated
form (about 8 kcal/mol), that means worse biological
activity while CHBr2 has a similar stability as CHCl2.
According to obtained results, chloro and dichloro
methyl ketone should exhibit the best activity, being
the right compromise between reactivity and tendency
to hydration.
Chart 1.
Table 3.
Compound
X
R1
R
7
8
H
—
—
CF3
H
CHF2
CHF2
CHF2
CHCl2
CH2Cl
CH2Cl
CH2Cl
CHBr2
CHBr2
CHBr2
CHBr2
CHBr2
CH2Br
CH2Br
CH2Br
CH2Br
CH2Br
OCH3
OCH3
9
CH2
CONH
H
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
—
—
H
CH2
CH2
H
Ph
1-Naphthyl
—
Ph
Figure 7. System of ab initio calculations.
CH2
CH2
CONH
CONH
H
1-Naphthyl
Ph
1-Naphthyl
—
Table 2. Hypothesized substituents X and relative energy and density
values
CH2
CH2
CONH
CONH
NH
Ph
1-Naphthyl
SUBST X
LUMO (eV)
q LUMO
DHF (kcal/mol)
CHCl2
CHBr2
CH2Cl
CH2Br
1.51265
1.58944
1.76881
1.81562
0.02815
0.02774
0.02732
0.02722
ꢁ22.2
ꢁ21.6
ꢁ16.5
ꢁ24.7
Ph
1-Naphthyl
Bn
Bn
NBn

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3095
Scheme 1. Reagents and conditions: (i) Sn, HCl, EtOH, 70 ꢁC; (ii) Br2, AlCl3, ClCH2CH2Cl, 70 ꢁC (45% yield); (iii) CH2@C(OEt)SnMe3, Pd(PPh3)4,
NMP; (iv) HCl (50% yield); (v) Br2, CHCl3(76% yield).
Scheme 2. Reagents and conditions: (i) MeOH, HCl, Et2O; (ii) NBS, AIBN, CH2Cl2; (iii) NH3, MeOH (95% yield); (iv) CH2@C(OEt)SnMe3,
Pd(PPh3)4, NMP; (v) HCl (50% yield); (vi) Br2, CHCl3 (50% yield).
reaction with aqueous ammonia and further benzylation
gave 6 (Scheme 3).
Trifluoromethyl derivative 7 was prepared by nucleo-
philic trifluoromethylation of 3-cyanobenzoic acid 37 fol-
lowed by oxidation of 3831 to benzamide 7 (Scheme 4).
Scheme 3. Reagents and conditions: (i) HCOOH, Et3N, Pd/C 10%,
Difluoromethyl derivatives 8–10 were prepared starting
20 min.; (ii) NaNO2, 0 ꢁC, 20 min, CH3CHNOH, CuSO4 Æ 5H2O, 10ꢁ
from 3-bromo-5-iodobenzoic acid 39, 3-iodobenzoic acid
41 and methyl ester of 3,5-dihydroxybenzoic acid 42
C, 1.5 h; (iii) SnCl2 Æ 2H2O, EtOH, 70 ꢁC (82% yield); (iv) NH3, 24 h;
(v) BnCl, K2CO3, reflux (34% yield).
using a palladium catalyzed coupling reaction as a key
step for the introduction of the difluoroacetyl group via
the corresponding methoxyethoxymethyl (MEM) 2,2-di-
radical condition (AIBN, CH2Cl2)29 to furnish 32, which
fluoro-1-tributylstannilvinyl ether32 (Schemes 5 and 6).
in the presence of NH3 in MeOH gave the 4-bromoiso-
indolinone 33.30 As for compound 4 the Pd catalyzed
coupling of 33 with ethyl-1-trimethylstannylvinyl ether
followed by hydrolysis with HCl gave the acetyl deriva-
tive 34, which was further brominated to give the desired
compound 5 (Scheme 2).
The Stille-type reaction with difluorovinylstannane
[F2C@C(OMEM)SnBu3] on the methyl ester of 41 fol-
lowed by hydrolysis and amidation gave derivative 8.
The methyl ester of 39 was coupled with difluorovinylst-
annane [F2C@C(OMEM)SnBu3] to give 40 that with a
further palladium catalyzed Negishi-type coupling with
a suitable organozinc derivative furnished target com-
pound 9. The triflate of methyl ester 42 was regioselec-
tively coupled first with difluorovinylstannane
[F2C@C(OMEM)SnBu3] to give 43 which reacted with
benzamide under the Buchwald Xantphos catalyzed
conditions33 to furnish 44. Hydrolysis and amidation
gave derivative 10.
Benzamide derivative 6 was obtained starting from
ethyl-3,5-dinitrobenzoate 35 which was selectively re-
duced with formic acid to the monoamino derivative
that was diazotized and reacted with acetaldehyde
oxime to furnish the corresponding oxime. Further
reduction of the nitro group gave the oxime 36 whose
Derivatives 11, 12, 15, and 20 were obtained starting
from 3-acetylbenzonitrile 45 (Scheme 7) that was con-
verted into the amide 4634 and brominated to give 15
and 20.34 Treatment of 45 with TMSCl and MnO2 in
AcOH gave a mixture of mono- and dichloro derivatives
that were converted into amides 11, 12 by hydrolysis
with H2SO4.
Scheme 4. Reagents and conditions: (i) DMAP, DCC, MeOH (73%
yield); (ii) (CH3)3SiCF3, CsF, dimethoxyethane (50% yield); (iii) H2O2
30%, NaOH 6 N, EtOH, 95 ꢁC (47% yield).
Scheme 5. Reagents and conditions: (i) HCl, Et2O, MeOH (89% yield); (ii) F2C@C(OMEM)SnBu3, CuI, PPh3, Pd(AcO)2, DMF, 60 ꢁC (67% yield);
(iii) Pd(PPh3)4, R1ZnBr, THF, 70 ꢁC; (iv) Me3SiCl, MeOH; (v) KOH, EtOH; (vi) SOCl2, 80 ꢁC; (vii) NH3 (8 = 70% yield, 9 = 80% yield).

3096
C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
Scheme 6. Reagents and conditions: (i) Tf2O, Et3N, CH2Cl2 (80% yield); (ii) F2C@C(OMEM)SnBu3 Pd2(dba)3, LiCl, AsPh3, NMP, 60 ꢁC (50%
yield); (iii) Pd(OAc)2, Xantphos, PhCONH2, K2CO3, dioxane, 100 ꢁC (33% yield); (iv) Me3SiCl, MeOH; (v) KOH, EtOH; (vi) SOCl2, 80 ꢁC; (vii)
NH3 (49% yield).
Scheme 7. Reagents and conditions: (i) H2O2 30%, NaOEt (67% yield); (ii) Br2, AcOH (15 = 15%, 20 = 54%); (iii) MnO2, (CH3)3SiCl, AcOH; (iv)
H2SO4 (11 = 11%, 12 = 10%).
Scheme 8. Reagents and conditions: (i) HCl 37%, MeOH, 70 ꢁC, 4 h; (ii) R0CH2ZnBr, Pd(PPh3)4, THF, 60 ꢁC, 12 h (47a = 60%; 47b = 60%); (iii)
BVE, Pd(OAc)2, PPh3, Et3N, CH3CN, 100 ꢁC; HCl 3 N, THF; (iv) NH3 32%, THF, 80 ꢁC (48a = 50%; 48 b = 50%); (v) Br2 or Cl2, CHCl3, 50 ꢁC
(13 = 15%; 14 = 45%; 16 = 65%; 17 = 15%; 21 = 68%; 22 = 52%).
Compounds 13, 14, 16, 17, 21 and 22 were obtained
(Scheme 8) starting from 3-bromo-5-iodobenzoic acid
39. The Negishi-type palladium catalyzed coupling with
organozinc furnished methyl esters 47a, b which were
coupled with butylvinyl ether (BVE) in the presence of
PPh3 and Pd(OAc)2 to get after amidation derivatives
48a, b. The last step was the reaction with Br2 or Cl2
in CHCl3 to get the desired compounds.
transformed into the corresponding benzamide deriva-
tives 52 which reacted with Br2 to furnish the target
compounds 18, 19, 23, 24 (Scheme 9).
Finally methyl esters 25, 26 were obtained from nitro
benzoic acid 53 by amidation, reduction of the nitro
moiety and further benzylation of 5436 (Scheme 10).
Starting from 5-aminoisophthalate methyl ester 49,
derivative 5035 was obtained by a diazotation/acetyla-
tion sequence. Basic hydrolysis of 50 followed by mono
amidation gave benzoic acid derivatives 51 that were
4. Results and discussion
The aim of the present study was the identification of
new potential reversible non-peptidic inhibitors of
Scheme 9. Reagents and conditions: (i) NaNO2, HCl concd, 0 ꢁC, 200/CH3CHNOH, CuSO4.5H2O, 10 ꢁC, 1.5 h/HCl 37%, reflux (42%); (ii) NaOH
2N, reflux; (iii) R0NH2, DCC, HOBt, DIEA, DMF, 1.300 (51a = 52%; 51b = 50%); (iv) SOCl2, toluene/THF, NH3(g) (52a = 40%; 52b = 32%); (v) Br2,
CHCl3 (18 65%; 19 45%; 23 60%; 24 68%).

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3097
Quantum mechanical calculations indicated that com-
pounds containing chloro and dichloro methyl ketone
would exhibit the best activity, being the right compro-
mise between reactivity and tendency to hydration.
All synthesised compounds were tested on their inhibi-
tory activity against HRV-14 3CP and a colorimetric
method37 for in vitro screening was set up in our labo-
ratories. Prior to testing against 3CP, all compounds
were tested for their activity against dithiothreitol
(DTT), as many protease inhibitors could be susceptible
to free thiol and thus have an unspecific action. None of
the tested compounds were inactivated at two different
concentrations of DTT (6 and 0.12 mM, data not
shown). First set of compounds (11, 12, 15, 20) was then
screened at 10 lM and each experiment was performed
in duplicate. Mean inhibition became P50% using
1 mM leupeptin as control (unspecific protease inhibi-
tor, mean inhibition 57 6%) and this is shown in Fig-
ure 8. On the basis of the results, compound 7 was
newly synthesised and tested on 3CP in vitro system
at various doses, ranging from 0.16 to 100 lM. This
compound did not exhibit inhibition of 3CP at any used
concentration.
Scheme 10. Reagents and conditions: (i) SOCl2, NH3(g), 3 h; (ii)
SnCl2 Æ 2H2O, EtOH, 70 ꢁC (50%); (iii) BnCl, Et3N, reflux, 12 h
(25 = 21%; 26 = 7%).
HRV 3CP. The work started from the consideration
that 1,3 disubstituted benzamides behave as good and
potent inhibitors of HRV 3CP.21,22 The amidic group
seems to be essential for making hydrogen bonds within
the active site of the enzyme, also it resembles the side
chain of the natural substrate Gln P1 at the appropriate
distance and orientation. An electrophilic meta group is
necessary in order to react with the SH of the cysteine of
the catalytic triad of the protease, to generate a revers-
ible analogue of the transition state.
We designed a series of benzamides where the electro-
philic group is represented by a methyl ketone alpha
mono, di or tri halo-substituted (compounds 7, 8, 11,
12, 15, 20).
Second set of compounds (4–5, 6 and 8–10, 13, 14, 16–
19, 21–26) was screened in duplicate at 10 lM using
an Agouron aldehydic compound as reference38 (named
as AL21-01). Data obtained (Fig. 8) show that inhibi-
tion of 3CP enzymatic activity is comparable to those
of reference compounds 20 (85 6%) and AL21-01
(92 6%) and it was reached for most of the com-
pounds, being very interesting for molecules 4, 11, 12,
13, 20, 23, 24, 14, 17, 22 and 16 (inhibition P70%).
Molecular modelling supported the study generating a
virtual library where three main structures are repre-
sented: 1,3,5 trisubstituted benzamides (compounds 6,
9–10, 13, 14, 16–19, 21–26) and the cyclic derivatives
3,4 and 4,5 isoindolinones (compounds 4 and 5). Com-
pounds 6, 9–10, 13, 14, 16–19, 21–26 all contain an aro-
matic portion in position 5 that should be able to fill the
S2 pocket of the 3CP thus increasing the inhibitory
activity. They also contain an amide group in meta to
the electrophilic group (halomethyl ketone or oxime).
Isoindolinones 4 and 5 are more rigid structures where
the electrophile and the amide groups are kept in meta.
On the basis of these results, 10 molecules were chosen
to be further screened at three different concentrations
(0.1, 1, 10 lM). Two compounds, 13 and 14, show an
Figure 8. Screening of compounds 4, 5 and 6, 9–10, 11–13, 14, 15–19, 20–26 at 10 lM; 20 and AL21-01 are reference compounds.

3098
C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
Table 4. Screening at 0.1, 1, and 10 lM for selected compounds. 20
and AL21-01 are references
portions. We can hypothesize that presence of hydrogen
bond interactions in proximity of the S2 pocket is not
favorable for inhibition activity. An interesting result
is the high percent inhibition of compound 4 which will
be further studied.
% Inhibition
10ꢁ7 M
10ꢁ6 M
10ꢁ5 M
20
AL21-01
4
8
23
75
27
30
39
27
19
61
79
25
85
92
75
68
83
86
80
81
91
70
We decided to test few representative compounds, 12,
13, 14 and 20, for cytotoxicity on HeLa cells and for
anti-viral activity against HRV-14. As expected, com-
pounds 13 and 14 showed high cytotoxicity with
MNTD50 (maximum non-toxic dose which reduces
50% of cells) = 0.75 lg/ml and inhibitory activity on
HRV-14 at doses higher than the toxic ones
(ID50 > 0.75 lg/ml). Same result gave compound 12
with MNTD50 = 3.12 lg/ml and activity on HRV-14 at
higher doses. However, compound 20 proved to be quite
interesting having an MNTD50 > 100 lg/ml and
ID50 = 25 lg/ml (IP > 4). Compound 20 was also tested
on more cellular lines (VERO, MDCK) and exhibited
low toxicity and good anti-viral activity against HSV2
(Herpes Simplex-2) and WSN influenza A virus at doses
lower than the toxic ones (data not shown).
16
21
22
23
24
14
13
4
14
0
12
8
5
25
14
0
inhibition >50% at 1 lM, comparable with that ob-
tained for AL21-01 (Table 4).
From those preliminary results we could confirm some
of our hypothesis. First of all the presence of a reactive
electrophilic group seems to be necessary to react with
the cysteine of the active site of 3CP. Halo substituted
ketones are reactive, especially Br and Cl mono substi-
tuted. Also, results showed that the best compounds,
13 and 14, have a CH2Cl reactive group which was
hypothesised to be the best compromise between reactiv-
ity and tendency to hydration. Also, the presence of the
meta-amide and of a meta-aromatic group looks quite
interesting. The aromatic- not too bulky-group should
be able to fill the S2 pocket of the protease thus increas-
ing activity and the amide should contribute to form
hydrogen bonds within the active site of protease. The
type of aromatic group apparently did not make any dis-
tinction towards activity: compounds 13 and 14 contain,
respectively, a phenyl and a naphthyl group and show
equal activity. The nature of the linker seems to be
important for inhibition: almost all of the best molecules
contain a methylene group linking the two aromatic
Results so far obtained show the higher cytotoxicity of
CH2Cl group over CH2Br group, which prevented us
from performing all the scheduled tests on HeLa cells.
However, a good nucleophile keeps the inhibitory activ-
ity already reported from Reich et al.21 1,3,5 trisubsti-
tuted benzamides have again comparable activity on
HRV-14 but we need to investigate in more detail how
different groups that fill the S2 pocket can enhance
activity.
5. Virtual docking of compounds 13 and 20
We decided to explore the orientations of compounds 13
and 20 by virtual docking in the binding site of HRV-2.
This is shown in Figure 9A and B.
Figure 9. Representation of co-crystal structures of compounds 20 (A) and 13 (B) with HRV-2 3CP. Hydrogen bonds are represented in yellow.

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3099
Minimum energy pose of both compounds corresponds
to the orientation found in AG7088 co-crystallized with
HRV-2 3CP (Fig. 2) and matches the orientation of Mi-
chael-acceptor benzamides21 where the carboxamide
and the electrophile group are away from each other.
The amidic group of 13 and 20 lies in the S1 pocket
and it mimics the lactamic group of AG7088 as it is sta-
bilized by hydrogen bonds with Thr142 and His161. Ori-
entation of compound 20 is further stabilized by
formation of a hydrogen bond between the carboxamide
oxygen and Gly145. Also, the reactive group (aloketone)
of both compounds is placed in the S3 pocket towards
the catalytic site of 3CP.
inhibitors of 3CP21 were employed to establish the best
combination of scaling parameters to be used. Virtual li-
brary 3D structures were generated using the LigPrep
software, which generates a minimized conformation
of each ligand and multiply protonation/tautomeriza-
tion states when appropriate.
7.2. Biology
7.2.1. Enzymatic assay. A colorimetric assay was per-
formed, based on chromogen substrate (a p-nitroaniline
conjugated peptide) cleavage by protease. After hydroly-
sis the enzyme releases yellow-colored free p-nitroani-
line, whose absorbance can be measured at 405 nm
against a blank where either substrate or enzyme is
not included in the reaction mixture. Test was per-
formed on 96-well plates containing 200 ll/well reaction
mixture. Reaction mixture includes recombinant
HRV14-3C protease at 0.7 lM, protease substrate
(EALFQ-pNA) and compounds at 10 lM final concen-
tration (1% DMSO final concentration) in the reaction
buffer (25 mM Hepes, pH 7.5, 150 mM NaCl,
0.12 mM DTT and 1 mM EDTA). Reaction was initi-
ated by enzyme addiction and absorbance was read at
405 nm at time 0 and after 60 minutes of incubation at
30 ꢁC, using a spectrophotometer (Victor II, Wallac).
Leupeptin 1 mM was used as inhibition control. Blank
samples were prepared for each compound in order to
check for absorbance interferences.
Compound 13 is even more stabilized by additional
hydrogen bond between the carboxamide oxygen and
Cys147. It also contains a meta benzyl substituent,
which lacks in compound 20, that leans forward S2
pocket. This fact probably explains the better inhibitory
activity of compound 13 over compound 20 and under-
lies the need for additional interaction than 1,3 disubsti-
tuted benzamides do. However, we do not see any
chemical interaction between the benzyl group of com-
pound 13 and 3CP and this fact suggests us to consider
in more detail other possible groups that could interact
somehow with 3CP.
6. Conclusions
In this study we demonstrated the ability of some 1,3
disubstituted and 1,3,5 trisubstituted benzamides to in-
hibit HRV-14 3CP. We supported further evidence for
the need of an electrophilic reactive group, a primary
amide and an aromatic portion to increase the activity.
We also have encouraging results from 3,4-isoindoli-
none-like structures.
Percent inhibition was calculated for each sample with
respect to positive control in the same plate (mean of
duplicate), which represented 100% of reaction. Mean
value of inhibition was then calculated between com-
pound replicates. Data are expressed as means SDV.
7.2.2. Cytotoxicity and anti-viral activity. Test com-
pounds were dissolved in DMSO at a concentration of
25 mg/ml and kept in refrigerator until use.
Results so far obtained look quite promising and need a
better understanding of the mechanisms involved in the
reactivity of 3CP in order to have more detailed SAR
studies. Also, compounds here reported need further
investigations concerning cellular activity and, more
important, cytotoxicity.
Cytotoxic and anti-viral activities were examined in
HeLa cells (Ohio strain, human adenocarcinoma) incu-
bated at 33 ꢁC in a 3% CO2 atmosphere. They were cul-
tured in DMEM with 1% non-essential aminoacids and
10% foetal calf serum (Gibco Labs. Inc.). Anti-viral
activity was assayed on common cold viruses HRV-14
(type A) and HRV-39 (type B) obtained from ATCC
(USA).
7. Experimental
7.1. Molecular modelling
Cytotoxic activity was determined on HeLa cells cul-
tured on 24 multiwell plates at a density of about 104/
cells per ml and incubated in the presence of decreasing
concentrations of the tested compounds. After 5 days
the cytotoxic effect of the compounds was checked by
the incorporation of a vital dye (neutral red). The max-
imum non-toxic dose (MNTD50), which is the dose that
inhibits less than 50% cell replication compared to the
control, was calculated by regression analysis. All the
experiments were done in triplicate.
The initial minimization was performed using the Mac-
roModel program (Schro¨dinger, LLC, New York, NY).
Inhibitor and Cys 147 residue were fully flexible while
other residues were harmonically constrained to their
crystallographic positions. The employed force field
was OPLS-AA choosing an electrostatic treatment dis-
tance-dependent with the dielectric constant set to 4R.
Docking was performed using GLIDE26 program
(Schro¨dinger, LLC, New York, NY). Structure was pre-
pared using the default Protein Preparation. Grids for
molecular docking were calculated employing 0.8, 0.9,
and 1.0.Van der Waals scaling parameters. Known
The Rhinovirus inhibition was evaluated by the analysis
of the viral replication cycle on cell monolayers and sub-
sequent titration under nutrient agar. HeLa cell mono-

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C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
layers in 24-well plates were infected with the viruses at a
multiplicity of infection (MOI) of 0.1. Then decreasing
amounts of the test compounds were added and after
24 h of incubation the cells underwent a cycle of
freeze-thawing. The supernatant was centrifuged and
tested on cell monolayers under 2% nutrient agar to de-
tect the number of viral infectious units produced stain-
ing with neutral red and counting the visible viral
plaques.
7.3.2. 6-Acetylisoindolin1-one (30). To a solution of 29
(0.5 g, 2.34 mmol) and ethyl 1-(trimethylstannyl)vinyl
ether (0.827 g, 3.52 mmol) in NMP (10 mL), Pd(PPh3)4
(0.136 g, 0.12 mmol) was added and the mixture was re-
fluxed for 24 h. The reaction mixture was quenched by
addition of saturated aqueous ammonium chloride and
extracted with ethyl acetate, dried and concentrated.
The crude product was dissolved in THF (5 mL) and
HCl 2 N (0.6 mL) was added. The mixture was stirred
at r.t. for 30 min and the solution was concentrated
and filtered to obtain a white solid 30 (0.205 g,
The addition of test compounds at various times before
(ꢁ1 h and t0) and after (from +1 to +6 h) viral infection
was used for determining the inhibited virus cycle step.
Sub-confluent HeLa cells in 24-well plates were infected
by the Rhinoviruses at a MOI of 0.1 for 1 h. After 48 h
of incubation, the cytopathic effect was checked, the
plates were freeze-thawed and the viral particles pro-
duced were counted under nutrient agar.
1
1.17 mmol, 50%). H NMR (500 MHz, CDCl3) d: 8.41
(1H, s), 8.22 (1H, dd, J = 7.9, 1.6 Hz), 7.57 (1H, d,
J = 7.9 Hz), 6.60 (1H, s broad) 4.52 (2H, s), 2.66 (3H,
s). Anal. Calcd for C10H9NO2: C, 68.56; H, 5.18; N,
8.00. Found: C, 68.62; H, 5.12; N, 8.08.
7.3.3.
6-(Bromoacetyl)isoindolyn-1-one
(4).
Br2
(0.058 mL, 1.14 mmol) was added dropwise to a solu-
tion of 30 (0.2 g, 1.14 mmol) in chloroform (10 mL).
The reaction mixture was stirred at 70 ꢁC for 20 min,
cooled to rt and filtered to give 4 (0.22 g, 0.87 mmol,
The neutralizing activity of the test compounds on viral
infectivity was studied on HRV-14. A suspension of the
test virus with about 106 Infectious Units (IU) per ml
was challenged with decreasing doses of the compounds
to be assayed. After 1 h of contact at 37 ꢁC, the viral sus-
pensions were titrated on HeLa cell monolayers under
nutrient agar to determine the remained live viruses.
1
76%). H NMR (300 MHz, DMSO-d6) d ppm 4.49 (2
H, s) 5.02 (2 H, s) 7.76 (1 H, dd, J = 8.04, 0.73 Hz)
8.20 (1 H, dd, J = 7.97, 1.68 Hz) 8.27 (1 H, d,
J = 1.02 Hz) 8.75 (1 H, s broad); MS (ESI) m/e 254.0
[(M+H+)], 256 [(M+2+H+)]. HPLC tR 13.7 min; purity
93% Synergy hidro-RP 250X4.6 mm 4 lm, gradient elu-
tion of 10–40% CH3CN (0.01% TFA)/water(0.01%
TFA) for 30 min, k = 218 nm. Anal. Calcd for
C10H8BrNO2: C, 47.27; H, 3.17; N, 5.51. Found: C,
46.82; H, 2.92; N, 5.22.
Cytotoxicity on VERO and MDCK cultured cells was
determined on 24 multiwell plates in a similar manner.
Anti-viral activity was evaluated after 48 h of incubation
at 37 ꢁC and 5% CO2.
7.3. Chemistry
7.3.4. 4-Bromoisoindolin-1-one (33). NH3/MeOH was
added dropwise to a solution of methyl 3-bromo-2-(bro-
momethyl)benzoate (0.735 g, 2.38 mmol) in MeOH
(7 mL) and stirred at rt until the formation of a white
precipitate. The mixture was filtered and the solid was
washed with Et2O and dried to give the desired product
33 (0.479 g, 2.26 mmol, 95%). 1H NMR (500 MHz,
CD3OD) d: 7.78 (2H, d, J = 7.7 Hz), 7.46 (1H, t,
J = 7.7 Hz), 4.38 (2H, s); MS (ESI) m/e 212.0
[(M+H+)], 214 [(M+2+H+)].
All reactions were performed under nitrogen atmo-
sphere unless otherwise noted. Reagents and starting
materials were obtained from commercial sources and
used as received. Organic solutions were dried over
1
Na2SO4. H NMR were recorded on a Brucker Avance
300 instrument and chemical shifts (d) are reported rel-
ative to TMS and/or referenced to the solvent in which
they were measured. Combined HPLC-UV analysis
were recorded using a Waters Alliance 2795 Separa-
tions Module and Waters 2487 Dual k Absorbance
Detector. LC/MS were recorded on API 365 Applied
Biosystem equipped with ionSPRAY interface. Ele-
mental analysis was performed using a Carlo Erba
EA1108 instrument.
7.3.5. 4-Acetylisoindolil-1-one (34). Prepared following
the procedure described for 30. (0.205 g, 1.17 mmol,
50%). 1H NMR (500 MHz, CDCl3) d: 8.09 (1H, d,
J = 7.5 Hz), 8.07 (1H, d, J = 7.6 Hz), 7.62 (1H, t,
J = 7.6 Hz), 6.88 (1H, s broad) 4.79 (2H, s), 2.66 (3H,
s). Anal. Calcd for C10H9NO2: C, 68.56; H, 5.18; N,
8.00. Found: C, 68.64; H, 5.14; N, 8.11.
7.3.1. 6-Bromoisoindolin-1-one39 (29). Dry AlCl3 (2.5 g,
18.7 mmol) was placed in a three bottom flask under
N2 and a solution of isoindolin-1-one (1 g, 7.5 mmol)
in dry 1,2-dichloroethane (15 mL) was added. Br2
(0.50 mL, 9.7 mmol) was then added dropwise and the
mixture was refluxed for 15 h. After cooling to rt, the ex-
cess of bromine was destroyed with an aqueous solution
of sodium thiosulfate. The mixture was washed with
ethyl acetate and the organic layer dried and concen-
trated. Chromatography on silica gel (10% ethyl ace-
tate/hexane) gave 29 (0.640 g, 3.02 mmol, 45%). 1H
NMR (500 MHz, CDCl3) d: 7.91 (1H, m), 7.73 (1H,
dd, J = 8.1, 1.8 Hz), 7.47 (1H, d, J = 8.1 Hz), 4.41 (2H,
s); MS (ESI) m/e 212.0 [(M+H+)], 214 [(M+2+H+)].
7.3.6. 4-(Bromoacetyl)isoindolin-1-one (5). Prepared fol-
lowing the procedure described for 4. (0.045 g,
0.18 mmol, 50%). 1H NMR (300 MHz, DMSO-d6) d
ppm 8.75 (s, 1 H), 8.31 (dd, J = 7.75, 1.02 Hz, 1 H),
7.97 (dd, J = 7.60, 0.88 Hz, 1 H), 7.70 (t, J = 7.60 Hz,
1 H), 5.01 (s, 2 H), 4.62 (s, 2 H). MS (ESI) m/e 254.0
[(M+H+)], 256 [(M+2+H+)]. HPLC tR 10.6 min; purity
90% PKB-100 250 · 4.6 mm 5 lm, gradient elution of
5–55% CH3CN/CH3COONH4 10 mM for 20 min,
k = 250 nm. Anal. Calcd for C10H8BrNO2: C, 47.27;
H, 3.17; N, 5.51. Found: C, 47.02; H, 3.03; N, 5.42.

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3101
7.3.7. Ethyl 3-amino-5-[1-(hydroxyamino)ethyl]benzoate
(36). Ethyl 3,5-dinitrobenzoate (15 g, 62.5 mmol), Pd/C
10% (0.685 g, 0.6 mmol) and Et3N (39.2 mL) were
placed in a three-bottom flask equipped with a con-
denser. When the mixture started to reflux formic acid
(10.1 mL, 269 mmol) was added dropwise. The solution
was stirred for 200. After being cooled at room temper-
ature the reaction mixture was diluted with dichloro-
methane and filtered off Celite. The organic layer was
washed with water, dried and concentrated. The crude
product was used for the next reaction without further
purification.
3H), 8.28 (br s, 1 H), 8.10 (t, J = 1.37 Hz, 1 H), 7.84
(ddd, J = 5.49, 1.92, 1.74 Hz, 2 H), 7.57 (br s, 1 H),
7.25–7.47 (m, 5 H), 5.25 (s, 2 H), 2.29 (s, 3 H). MS
(ESI) m/e 284.2 [(M+H+)]. HPLC tR 15.6 min; purity
92% pkb-100 254 · 4.6 mm 5 lm, gradient elution of
15–45% CH3CN (0.01% TFA)/H2O (0.01% TFA) for
30 minutes, k = 206 nm. Anal. Calcd for C16H17N3O2:
C, 67.83; H, 6.05; N, 14.83. Found: C, 67.23; H, 5.88;
N, 14.66.
7.3.9. 3-(Trifluoroacetyl)benzonitrile (38). 3-Cyanoben-
zoic acid (4.87 g, 33 mmol) dissolved in dichlorometh-
ane (35 mL), MeOH (4 mL) and DMAP (3.22 g,
26 mmol) were placed in a two bottom flask The solu-
tion was stirred at 0 ꢁC and then DCC (7.5 g,
36.4 mmol) was added in 300. The reaction mixture
was stirred at 0 ꢁC for 150 and was then warmed to rt
and stirred for 4 h. The precipitate was filtered off and
the filtrate was washed with HCl 0.5 N, NaHCO3 and
NaCl. The organic layer was dried and concentrated
to obtain methyl 3-cyanobenzoate (3.9 g, 24.2 mmol,
73%), which was used for the next reaction without fur-
ther purification.
Ethyl 3-amino-5-nitrobenzoate (5.54 g, 26.4 mmol) was
dissolved in water (33 mL) and HCl 37% (2.1 mL).
The mixture was stirred at 0 ꢁC and sodium nitrite
(2 g, 29 mmol) was added. The reaction mixture was
stirred for 20 min more at 0 ꢁC. In the meanwhile acet-
aldehyde oxime (3.12 g, 52.8 mmol) was dissolved in
5 mL of water and CuSO4 pentahydrate (1.98 g,
7.92 mmol) and sodium acetate trihydrate (19.1 g,
140 mmol) were added. The solution was cooled to 0–
5 ꢁC and the diazonium salt was added. The reaction
mixture was stirred at 10–15 ꢁC for 1.5 h. HCl 37%
(12.1 mL) was added and the solution was refluxed for
2 h. After being cooled at room temperature the reaction
mixture was extracted with ethyl acetate and concen-
trated. The crude product was used for the next reaction
without further purification.
Methyl 3-cyanobenzoate (1.6 g, 10 mmol) was placed in
a three bottom flask under N2 and trimethylsilyil triflu-
oromethane (1.85 mL, 12.5 mmol) in dry CH2Cl2
(25 mL) and dry CsF (0.076 g, 0.5 mmol) were added.
The solution was stirred at rt for 2;days. HCl 6 N
(10 mL) was added and the reaction mixture was stirred
for 5 h. The mixture was separated and the water was
extracted with DCM. The organic layer was washed
with NaCl, dried and concentrated. The residue was
purified by flash chromatography on silica gel (ethyl ace-
To a solution of ethyl 3-[(1-N-hydroxyethanimidoyl)]-5-
nitrobenzoate (1.8 g, 7.1 mmol) in abs EtOH (20 mL)
SnCl2 H O (8.5 g, 37.7 mmol) was added. The solution
2
*
was stirred at 70 ꢁC for 1 h and poured in to a NaHCO3
ice bath. The precipitate was filtered off and the alkaline
solution was extracted with ethyl acetate. The organic
layer was dried and concentrated. The residue was puri-
fied by flash chromatography on silica gel (chloroform/
methanol 95/5) to obtain 36 (1.3 g, 5.8 mmol, 82%).
GC–MS m/z 222.1 (8) [M+], 206.2 (38), 177.1 (61),
135.1 (100). Anal. Calcd for C11H14N2O3: C, 59.45; H,
6.35; N, 12.60. Found: C, 59.22; H, 6.29; N, 12.58.
1
tate/hexane 1/9) to obtain 38 (1 g, 5 mmol, 50%). H
NMR (300 MHz, DMSO-d6) d ppm 8.39–8.43 (m, 1
H), 8.28–8.35 (m, 1 H), 7.85–7.97 (m, 2 H). GC–MS
m/z 199 (5.3) [M+], 130.1 (100), 102.1 (92), 75 (58).
7.3.10. 3-(Trifluoroacetyl)benzamide (7). 38 (1 g,
5.02 mmol), H2O2 30% (1.8 mL, 17.5 mmol) and EtOH
95ꢁ (5 mL) were placed in a three bottom flask equipped
with a condenser and a magnetic stirrer. The resulting
mixture was stirred at 50 ꢁC and NaOH 6 N (0.21 mL,
1.26 mmol) was added. The solution was stirred for
21 h and, after that, the reaction mixture was neutralized
with HCl 6 N. The solution was concentrated, diluted
with water and extracted with DCM. The white precip-
itated obtained was filtered off to give 7 (0.52 g,
2.39 mmol, 47%). 1H NMR (300 MHz, DMSO-d6) d
ppm 8.52 (s, 1 H), 8.33 (dt, J = 7.76, 1.40 Hz, 1 H),
8.29 (br s, 1 H), 8.18 (dt, J = 7.84, 0.87 Hz, 1 H), 7.78
(t, J = 7.84 Hz, 1 H), 7.65 (br s, 1 H). GC–MS m/z
217.1 (23) [M+], 201.0 (38), 173.0 (40), 148.0 (100).
HPLC tR 8.5 min; purity 99.9% pkb-100 254 · 4.6 mm
5 lm, gradient elution of 10–30% CH3CN/H2O for
20 min, k = 224 nm. Anal. Calcd for C9H6F3NO2: C,
49.78; H, 2.79; N, 6.45. Found: C, 49.79; H, 2.61; N,
6.45.
7.3.8. 3-(Benzylamino)-5-[1-N-hydroxyethanimidoyl]benz-
amide (6). Compound 36 (1.3 g, 5.8 mmol) was placed in
a two bottom flask and 40 mL of NH3 32% were added.
The reaction mixture was stirred for 48 h. The solution
was diluted with water and extracted with dichlorometh-
ane. The organic layer was dried and concentrated. The
crude residue was used for the next reaction without fur-
ther purification.
3-amino-5-[1-N-hydroxyethanimidoyl]benzamide (0.35 g,
1.81 mmol), K2CO3 (0.27 g, 1.95 mmol), i-BuOH
(10 mL) and EtOH (10 mL) were placed in a three bot-
tom flask equipped with a condenser and a magnetic
stirrer. Benzyl chloride (0.23 mL, 2 mmol) was added
and the mixture was refluxed o.n. After being cooled
at room temperature the solution was filtered and the
solvent was evaporated in vacuo. The residue was puri-
fied by flash chromatography on silica gel (chloroform/
methanol 95/5) to obtain 6 (0.175 g, 0.6 mmol, 34%).
1H NMR (300 MHz, DMSO-d6) d ppm 9.77 (br s,
7.3.11. Methyl 3-bromo-5-{2,2-difluoro-1-[(2-methoxy-
ethoxy)methoxy]ethenyl}benzoate (40). To a solution of
3-bromo-5-iodobenzoic acid (1 g, 3.06 mmol) in dry

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C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
MeOH (5 mL) dry HCl (0.306 mL of a 1 M solution in
diethyl ether) was added. The reaction mixture was stir-
red at room temperature for 24 h. The solvent was evap-
orated in vacuo. Chromatography on silica gel (20%
ethyl acetate/petroleum ether) gave the ester intermedi-
ate (0.930 g, 2.72 mmol, 89%).
reaction was evaluated by IR spectroscopy of a sample of
the crude reaction mixture. After being cooled at room
temperature SOCl2 was evaporated in vacuo and the crude
residue was dissolved in dry THF. NH3(g) was bubbled
for 1 h into the solution. Formation of amide bond was
evaluated by IR spectroscopy. The solvent was evapo-
rated in vacuo and the residue was purified by chromatog-
raphy on silica gel (ethyl acetate). Crystallization by ethyl
acetate/hexane yield 9 (0.280 g, 0.96 mmol, 80%). 1H
NMR (300 MHz, DMSO-d6) d ppm 8.34 (s, 1 H), 8.19
(br. s., 1 H), 8.13 (s, 1 H), 8.04 (s, 1 H), 7.56 (br s, 1 H),
6.93–7.40 (m, 6 H), 4.11 (s, 2 H). GC–MS (70 eV; rel
Int): 289 [M+], 238, 165, 91. HPLC tR 6.5 min; purity
95% Platinum 100 150 · 4.6 mm 3 lm, gradient elution
5–25% CH3CN/CH3COONH4 for 20 min, k = 207 nm.
Anal. Calcd for C16H13F2NO2: C, 66.43; H, 4.53; N,
4.84. Found: C, 66.12; H, 4.30; N, 4.98.
In a reaction vessel equipped with a condenser and a mag-
netic stirrer Pd(OAc)2 (25 mg, 0.11 mmol), CuI (172 mg,
0.932 mmol) and PPh3 (108 mg, 0.41 mmol) were placed
under a stream of argon. Dry DMF (5 mL) and the aryl
bromide intermediate (1.4 g, 4.106 mmol) were then
added. The resulting mixture was stirred at 60 ꢁC and
the stannane F2C@C(OMEM)SnBu3 was added. The
mixture was stirred at 60 ꢁC for 16 h. After being cooled
at room temperature the reaction mixture was quenched
with a saturated aqueous NH4Cl solution and the result-
ing mixture was extracted with AcOEt. The organic ex-
tract was washed with water, dried, filtered through
Celite and the filtrate was concentrated under reduced
pressure. The residue was purified by chromatography
on silica gel (20% ethyl acetate/petroleum ether) obtaining
40 (1.016 g, 2.66 mmol, 67%). 1H NMR (200 MHz,
CDCl3) d ppm 8.18 (2 H, s) 8.12 (2 H, s) 7.85 (1 H, s)
4.91 (1 H, s) 3.97 (3 H, s) 3.93–3.85 (2 H, m) 3.62–3.51
(2 H, m) 3.41 (3 H, s). Alal. Calcd for C15H19BrF2O5: C,
45.36; H, 4.82. Found: C, 45,22; H, 4.82.
7.3.13. 3-(Difluoroacetyl)benzamide (8). Prepared follow-
ing the procedure described for 9 from 3-iodobenzoic
acid as starting material (0.280 g, 1.4 mmol, 70%). H
1
NMR (300 MHz, DMSO-d6) d ppm 8.50 (s, 1H), 8.11–
8.28 (m, 3 H), 7.72 (t, J = 7.75 Hz, 1 H), 7.59 (br s, 1
H), 7.20 (t, J = 52.50 Hz, 1 H). MS (ESI) m/e 198
[(Mꢁ)]. HPLC tR 8.6 min; purity 98% Pentium 100
150 mm 3 lm, gradient elution 5–45% CH3CN/
CH3COONH4 10 mM for 20 min, k = 226 nm. Anal.
Calcd for C9H7F2NO2: C, 54.28; H, 3.54; N, 7.03.
Found: C, 53.95; H, 3.54; N, 6.76.
7.3.12. 3-Benzyl-5-(difluoroacetyl)benzamide (9). Dry
Pd(PPh3)4 (0.151 g, 0.131 mmol) was placed in a three
bottom flask under N2. Dry THF (15 mL), 40 (1 g,
2.62 mmol) and BnZnBr (13 mL, 6.5 mmol) were added.
The resulting mixture was stirred at 70 ꢁC. The degree of
completion of the reaction mixture was evaluated by GC
analyses of a sample of the crude reaction mixture. After
being cooled at room temperature the reaction was
quenched with a saturated aqueous NH4Cl solution
and the resulting mixture was extracted with AcOEt.
The organic extract was washed with water, dried, fil-
tered through Celite and the filtrate was concentrated
under reduced pressure. The residue was purified by
chromatography on silica gel (20% ethyl acetate/petro-
leum ether) obtaining the benzylated compound.
7.3.14. Methyl-3-{2,2-difluoro-1-[(2-methoxyethoxy)metho-
xy]ethenyl}-5-{[(trifluoromethyl)sulfonyl]oxy}benzoate (43).
Methyl 3,5-dihydroxybenzoate (5 g, 29.7 mmol) was
placed in a three bottom flask under N2 and was dissolved
in dry dichloromethane (30 mL). Et3N (1.79 g,
178.2 mmol) was added. The mixture was stirred for 50
at rt and then was cooled at 0 ꢁC. Fifteen millilitres of a
solution of trifluoromethansulfonic anhydride in CH2Cl2
was added dropwise. The mixture was stirred for 4 h at rt,
then was quenched with water and extracted with dichlo-
romethane. The organic layer was dried and the solvent
was evaporated in vacuo. The residue was purified by
chromatography on silica gel (ethyl acetate/petroleum
ether 1/9) to obtain methyl 3,5-bis(trifluoromethyl)sulfo-
nyloxy benzoate (10.3 g, 23.8 mmol, 80%).
To a solution of the benzylated compound (0.667 g,
1.7 mol) in MeOH at 0 ꢁC Me3SiCl (0.4 mL, 3 mmol)
was added and the mixture was stirred overnight. The
solvent was evaporated and the crude product was used
for the next step without further purification. The resi-
due was dissolved in KOH/EtOH 10% (10 mL) and
the mixture was stirred until the TLC showed the end
of the reaction. The solvent was evaporated, the crude
residue was dissolved in water and the solution was ex-
tracted with ethyl acetate. The organic layer was neu-
tralized with HCl 3 N and the aqueous phase was
extracted with ethyl acetate; the organic layer was dried
and evaporated in vacuo to obtain the acid (0350 g,
1.2 mmol), that was used for the next reaction without
further purification.
CuI (0.044 g, 0.231 mmol), Pd(dba)3 (0.043 g,
0.046 mmol), AsPh3 (0.057 g, 0.185 mmol), LiCl
(0.294 g, 6.9 mmol) and the intermediate ester (1 g,
2.3 mmol) were placed in a three bottom flask under N2.
NMP (8 mL) and the stannane (1.47 g, 3.18 mmol) were
added and the mixture was stirred at 60 ꢁC for 2 h. After
being cooled to room temperature the reaction mixture
was quenched with water and extracted with ethyl acetate;
the organic layer was dried and concentrated in vacuo.
The residue was purified by chromatography on silica
gel (ethyl acetate/petroleum ether 1/1) to obtain 43
1
(0.5 g, 1.1 mmol, 50%). H NMR (500 MHz, CDCl3) d:
8.18 (1H, s), 7.84 (1H, s), 7.57 (1H, s), 4.88 (2H, s) 3.97
(3H, s), 3.90-3.80 (2H, m), 3.50–3.40 (2H, m), 3.55 (3H,
s). GC-MS (70 eV; rel Int): 419(2) [M+ꢁ31], 348 (8), 311
(13), 89 (96), 59 (100). Anal. Calcd for C16H19F5O8S: C,
41.21; H, 4.11. Found: C, 41.18; H, 4.11.
3-Benzyl-5-(difluoroacetyl)benzoic
1.21 mmol) was dissolved in SOCl2 (35 mL) and the mix-
acid
(0.350 g,
ture was stirred at 80 ꢁC. The degree of completion of the

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3103
7.3.15.
Methyl
3-benzamido-5-{2,2-difluoro-1-[(2-
7.3.19. 3-(Chloroacetyl)benzamide (12), 3-(dichloroace-
tyl)benzamide (11). (CH3)3SiCl (7.95 mL, 18 mmol) was
added to a solution of 3-acetylbenzonitrile (1.45 g,
10 mmol) and MnO2 (1.56 g, 15 mmol) in AcOH
(15 mL) and the mixture was stirred at 0 ꢁC o.n. The
solution was poured into water and extracted with a
Et2O/eptane 1:1. The organic layer was washed with
NaOH 0.025 M, dried and concentrated. The mixture
of mono and dichloro compounds (0.741 g) was used
for the next reaction without further purification.
methoxyethoxy)methoxy]ethenyl}benzoate (44). Pd(A-
cO)2 (0.021 g, 0.022 mmol), Xantphos (0.039 g,
0.066 mmol), benzamide ( 0.175 g, 1.44 mmol) and
K2CO3 (0.215 g, 1.55 mmol) were placed in a three bot-
tom flask under N2. 1.5 mL of a 1 M solution of 43 in
dioxane was added and the reaction mixture was stirred
for 20 h. The mixture was diluted with dichloromethane,
filtered through Celite and concentrated. The residue
was purified by chromatography on silica gel (ethyl ace-
tate/petroleum ether 1:1) to obtain 44 (0.150 g,
1
0.35 mmol, 33%). H NMR (500 MHz, CDCl3) d: 8.21
The mixture was placed in a three bottom flask equipped
with a condenser and H2SO4 (2 mL) was added. The
mixture was stirred at 50 ꢁC for 2 h. The solution was di-
luted with water and extracted with chloroform, dried
and concentrated. The residue was purified by flash
chromatography on silica gel (chloroform/methanol
9:1) to obtain two isolated compounds 12 (0.14 g,
0.71 mmol) and 11 (0.262 g, 1.13 mmol).12 1H NMR
(300 MHz, DMSO-d6) d ppm 8.46 (t, J = 1.53 Hz, 1
H), 8.05–8.22 (m, 3 H), 7.58–7.71 (m, 1 H), 7.54 (br s,
1 H), 5.24 (s, 2 H). GC–MS m/it z 148 (100)
[M+ꢁCH2Cl], 130.1 (30). HPLC tR 9.2 min; purity
75.7% pkb 100 254 · 4.6 mm 5 lm, gradient elution of
10–40% CH3CN/H2O for 30 min, k = 284 nm. Anal.
Calcd for C9H8ClNO2: C, 54.70; H, 4.08; N, 7.09.
Found: C, 54.79; H, 4.10; N, 7.02. 11 1H NMR
(300 MHz, DMSO-d6) d ppm 8.54 (t, J = 1.65 Hz, 1
H), 8.18–8.24 (m, J = 8.03, 2.46, 1.20, 1.20 Hz, 2 H),
8.17 (br s, 1 H), 7.91 (s, 1 H), 7.70 (t, J = 7.76 Hz, 1
H), 7.59 (br s, 1 H). GC–MS m/z 148.1 (100)
[M+ꢁCHCl2]. HPLC tR 17.1 min; purity 99.8% pkb
100 254 · 4.6 mm 5 lm, gradient elution of 10–40%
CH3CN/H2O for 30 min, k = 251 nm. Anal. Calcd for
C9H7Cl2NO2: C, 46.58; H, 3.04; N, 6.04. Found: C,
46.43; H, 2.97; N, 5.92.
(2H, s), 8.05 (1H, s), 7.88–7.70 (3H, m), 7.53 (1H, t,
J = 8.3 Hz) 7.45 (2H, t, J = 7.7 Hz). Anal. Calcd for
C22H25F2NO6: C, 60.41; H, 5.76; N, 3.20. Found:
60.55; H, 5.81; N, 3.25.
7.3.16. 3-(Difluoroacetyl)-5-[(phenylcarbonyl)amino]benz-
amide (10). Prepared following the procedure described
for 9 from44 as starting material (0.055 g, 0.17 mmol,
1
49%). H NMR (300 MHz, DMSO-d6) d ppm 10.65 (s,
1H), 8.64 (d, J = 1.65 Hz, 2 H), 8.24 (s, 1 H), 8.20 (br
s, 1 H), 7.98–8.07 (m, 2 H), 7.48–7.68 (m, 4 H), 7.15
(t, J = 52.50 Hz, 1 H). MS (ESI) m/e 317.1 [(Mꢁ)],
349.1 [(Mꢁ+MeOH)]. HPLC tR 4.6 min; purity 90%
Platinum 100 150 mm 3 lm, gradient elution of 2–18%
CH3CN/CH3COONH4 10 mM for 16 min, k = 205 nm.
Anal. Calcd for C16H12F2N2O3: C, 60.38; H, 3.80; N,
8.80. Found: C, 60.21, H, 3.70, N, 8.73.
7.3.17. 3-Acetylbenzamide (46). Prepared following the
procedure described for 7 (1.5 g, 9.2 mmol, 67%) from
3-acetylbenzonitrile as starting material. 1H NMR
(300 MHz, CDCl3) d: 2.67 (3H, s), 5.93 (1 H, br s), 6.30
(1 H, br s), 7.58 (1 H, t, J = 7.8 Hz), 8.06 (1 H, dt,
J = 7.8, 1.5 Hz), 8.12 (1 H, dt, J = 7.8, 1.5 Hz), 8.40 (1
H, t, J = 1.5 Hz); MS (EI) m/z 164.0671 [M+], 148 (100).
7.3.20. Methyl 3-benzyl-5-bromobenzoate (47a). HCl
37% (0.255 mL, 3.059 mmol) was added to a solution
of 3-bromo-5-iodobenzoic acid (10 g, 30.5 mmol) in
MeOH (200 mL). The reaction mixture was stirred at
70 ꢁC o.n. The solution was poured into water and ex-
tracted with ethyl acetate; the organic layer was dried
and concentrated to obtain the methyl 3-bromo-5-iodo-
benzoate. The crude residue was dissolved in 20 mL of
THF and placed in a three bottom flask under N2.
Pd(PPh3)4 (0.560 g, 48.5 mmol) was added and the solu-
tion was stirred at room temperature for 50. A solution
0.5 M of benzylzinc bromide (21.3 mL, 10.7 mmol) was
added and the reaction mixture was stirred at 60 ꢁC
o.n. The solution was cooled at 0 ꢁC and water
(30 mL) and HCl 2 N (15 mL) were added. The aqueous
phase was extracted with ethyl acetate; the organic layer
was filtered through Celite, dried and concentrated. The
residue was purified by flash chromatography on silica
gel (hexane/ethyl acetate 9/1) to obtain 47a (1.8 g,
5.8 mmol, 60%). GC–MS m/z: 304(77) [M+], 165 (100).
Anal. Calcd for C15H13BrO2: C, 59.04; H, 4.29; Found:
C, 59.17; H, 4.35.
7.3.18. 3-(Dibromoacetyl)benzamide (15), 3-(bromoace-
tyl)benzamide (20). Br2 (0.31 mL, 6.1 mmol) was added
to a solution of 46 (1 g, 6.1 mmol) in AcOH (90 mL).
The reaction mixture was stirred at 60 ꢁC for 4 h. The
solvent was evaporated in vacuo and the crude residue
was purified by chromatography on silica gel (hexane/
ethyl acetate 2:8). Two products were isolated 15
(0.3 g, 0.93 mmol, 15%) and 20 (0.8 g, 3.3 mmol, 54%).
15 1H NMR (300 MHz, DMSO-d6) d ppm 8.53 (t,
J = 1.75 Hz, 1H), 8.15–8.26 (m, 2 H), 8.14 (br s, 1 H),
7.86 (s, 1 H), 7.57 (br s, 1 H) 7.68 (t, J = 7.75 Hz, 1
H). GC–MS m/z 163.1 (1) [M+ꢁ2Br], 148.0 (100). HPLC
tR 21.3 min; purity 92% 18-DB 254 · 4.6 mm 5 lm, gra-
dient elution of 10–45% CH3CN/H2O (HCOOH 0.1%)
for 35 min, k = 219 nm. Anal. Calcd for C9H7Br2NO2:
C, 33.68; H, 2.20; N, 4.36. Found: C, 33.45; H, 2.11;
N, 4.26. 20 1H NMR (300 MHz, DMSO-d6) d ppm
8.48 (t, J = 1.53 Hz, 1 H), 8.10–8.19 (m, 3 H), 7.65 (t,
J = 7.82 Hz, 1 H), 7.54 (br s, 1 H), 4.97 (s, 1 H). GC–
MS m/z 241.0 (2) [M+] 163.1 (13) [M+ꢁBr], 147.9
(100). HPLC tR 12.6 min; purity 91% 18-DB
254 · 4.6 mm 5 lm, gradient elution of 10–45%
CH3CN/H2O (HCOOH 0.1%) for 35 min, k = 219 nm.
Anal. Calcd for C9H8BrNO2: C, 44.66; H, 3.33; N,
5.79. Found: 44.52; H, 3.32; N, 5.71.
7.3.21. Methyl 3-bromo-5-(5,8-dihydronaphthalen-2-
ylmethyl)benzoate (47b). Prepared following the proce-
1
dure described for 47a (3 g, 8.4 mmol, 60%). H NMR

3104
C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
(300 MHz, DMSO-d6) d ppm 7.72–7.93 (m, 7 H), 7.44–
7.53 (m, 2 H), 7.41 (dd, J = 8.33, 1.75 Hz, 1 H), 4.21 (s, 2
H), 3.83 (s, 3 H). GC–MS m/z: 356 (44) [M+], 215 (100),
141 (46), 107 (48). Anal. Calcd for C19H15BrO2: C,
64.24; H, 4.26. Found: C, 64.13; H, 4.18.
7.3.25. 3-Acetyl-5-(phenylcarbamoyl)benzoic acid (51a).
50 (11 g, 46.6 mmol) was placed in an open flask
equipped with a condenser and it was dissolved in
NaOH 2 N (250 mL). The mixture was subjected to
microwave irradiation at 400 W for 6 min and then at
300 W for 10 min. At the end of irradiation, the reaction
mixture was cooled at room temperature and HCl 2 N
was added until the complete precipitation of a solid.
The precipitate was filtered and used for the next step
without further purification. The compound (5 g,
24 mmol) was dissolved in DMF (130 mL) and the solu-
tion was stirred at 0 ꢁC; HOBt (3.57 g, 26.4 mmol) and
DCC (5.45 g, 26.4 mmol) were added. The reaction mix-
ture was stirred at 0 ꢁC for 30 min, then aniline
(2.41 mL, 26.4 mmol) was added and the solution was
stirred for 1.5 h. The solution was stirred for 3 h more
at room temperature and filtered. The filtrated was di-
luted with HCl 2 N and extracted with ethyl acetate.
The organic layer was neutralized with NaOH 2 N and
extracted with water. HCl 2 N was added until the com-
plete precipitation of the product. The precipitated was
filtered and recrystallized from abs. EtOH obtaining 51a
7.3.22. 3-Acetyl-5-benzylbenzamide (48a). Pd(OAc)2
(0.06 g, 0.27 mmol), PPh3 (0.14 g, 0.5 mmol), butylviny-
lether (2.3 mL, 17.8 mmol), 47a (1.08 g, 3.5 mmol) and
Et3N (0.592 mL, 4.3 mmol) in dry CH3CN were placed
in a two bottom flask under N2. The reaction mixture
was stirred at 100 ꢁC o.n. After being cooled at room
temperature the solution was filtered, washed with water
and concentrated in vacuo. The residue was dissolved in
a mixture 1:1 HCl 10%/THF (80 mL) and stirred at
room temperature for 3 h. The mixture was diluted with
water and extracted with ethyl acetate. The organic layer
was dried and concentrated. The crude residue was used
for the next step without further purification.
To a solution of the crude residue (0.45 g, 1.7 mmol) in
THF (3 mL) 6 mL of NH3 32% was added and the mix-
ture was stirred at 80 ꢁC for 5 h. The solution was di-
luted with water and extracted with ethyl acetate. The
organic layer was dried and concentrated obtaining
48a (215 g, 0.85 mmol, 50%). 1H NMR (300 MHz,
CDCl3) d ppm 8.21 (t, J = 1.61 Hz, 1 H), 7.88 (s, 1 H)
7.94 (s, 1 H), 7.11–7.38 (m, 5 H), 5.97 (br s, 2 H), 2.61
(s, 3 H) 4.09 (s, 2 H). MS (ESI) m/e 254.0 [(M+H+)].
Anal. Calcd for C14H12BrNO: C, 57.95; H, 4.17; N,
4.83. Found: C, 57.98; H, 4.18; N, 4.85.
1
(3.56 g, 12.6 mmol, 52%). H NMR (300 MHz, DMSO-
d6) d ppm 13.55 (br s, 1H), 10.60 (s, 1H), 8.74 (t,
J = 1.65 Hz, 1 H), 8.71 (t, J = 1.74 Hz, 1 H), 8.61 (t,
J = 1.56 Hz, 1 H), 7.74–7.84 (m, 2 H), 7.39 (t,
J = 7.87 Hz, 2 H), 7.09–7.20 (m, 1 H), 2.72 (s, 3 H).
MS (ESI) m/e 284.3 [(M+H+)]. Anal. Calcd for
C16H13NO4: C, 67.84; H, 4.63; N, 4.94. Found: 67.80;
H, 4.66; N, 4.99.
7.3.26. 3-Acetyl-5-(naphth-1-ylcarbamoyl)benzoic acid
(51b). Prepared following the procedure described for
51a (0.7 g, 2.1 mmol, 50%). 1H NMR (300 MHz,
DMSO-d6) d ppm 13.55 (br s, 1 H), 10.81 (s, 1 H),
8.86 (d, J = 1.61 Hz, 2 H), 8.65 (t, J = 1.61 Hz, 1 H),
7.96–8.04 (m, 2 H), 7.86–7.93 (m, 1 H), 7.51–7.67 (m,
4 H), 2.74 (s, 3 H). MS (ESI) m/e 334.4 [(M+H+)]. Anal.
Calcd for C20H15NO4: C, 72.06; H, 4.54; N, 4.20.
Found: 71.89; H, 4.41; N, 4.09.
7.3.23. 3-Acetyl-5-(5,8-dihydronaphthalen-2-ylmethyl)benz-
amide (48b). Prepared following the procedure described
for 48a (0.5 g, 1.6 mmol, 50%). 1H NMR (300 MHz,
DMSO-d6) d ppm 8.30 (t, J = 1.61 Hz, 1H), 8.15 (br s,
1 H), 8.06 (t, J = 1.61 Hz, 1 H), 8.01 (t, J = 1.61 Hz, 1
H), 7.75–7.91 (m, 4 H), 7.34–7.54 (m, 4 H), 4.25 (s, 2
H), 2.60 (s, 3 H). MS (ESI) m/e 304.0 [(M+H+)]. Anal.
Calcd for C18H14BrNO: C, 63.55; H, 4.15; N, 4.12.
Found: C, 63.59; H, 4.15; N, 4.13.
7.3.27. 5-Acetyl-N-phenylbenzene-1,3-dicarboxamide (52a).
51a (1.5 g, 5.3 mmol) was dissolved in thionyl chloride
(15 mL) and the mixture was stirred at 50 ꢁC for 3 h. After
being cooled at room temperature the thionyl chloride
was evaporated in vacuo and the crude residue was dis-
solved in dry THF. NH3(g) was bubbled into the solution
for 2 h. The solvent was evaporated in vacuo and the res-
idue was purified by chromatography on silica gel
(CHCl3/MeOH 98:2) obtaining 52a (0.6 g, 2.1 mmol,
7.3.24. Dimethyl 5-acetylbenzene-1,3-dicarboxylate (50).
Dimethyl
5-aminobenzene-1,3-dicarboxylate
(1 g,
4.8 mmol) was dissolved in water (6 mL) and HCl 37%
(0.38 mL). The mixture was stirred at 0 ꢁC and sodium
nitrite (0.36 g, 5.2 mmol) was added. The reaction mix-
ture was stirred for an other 20 min at 0 ꢁC. In the
meanwhile acetaldehyde oxime (0.6 g, 10.2 mmol) was
dissolved in 5 mL of water and CuSO4 pentahydrate
(0.4 g, 1.6 mmol) and sodium acetate trihydrate (3.5 g,
125.7 mmol) were added. The solution was cooled to
0–5 ꢁC and the diazonium salt was added. The reaction
mixture was stirred at 10–15 ꢁC for 1.5 h. HCl 37%
(2.2 mL) was added and the solution was refluxed for
2 h. After being cooled at room temperature the reaction
mixture was extracted with ethyl acetate and concen-
trated. The residue was purified by flash chromatogra-
phy on silica gel (hexane/ethyl acetate 8/2) to obtain
50 (0.36 g, 1.52 mmol, 42%). 1H NMR (300 MHz,
CDCl3) d ppm 8.87 (t, J = 1.65 Hz, 0 H), 8.78 (d,
J = 1.65 Hz, 2 H), 4.00 (s, 6 H), 2.70 (s, 3 H). MS
(ESI) m/e 237.1 [(M+H+)], 194.9, 193.1.
1
40%). H NMR (300 MHz, DMSO-d6) d ppm 13.55 (br
s, 1 H), 10.60 (s, 1 H), 8.74 (t, J = 1.65 Hz, 1 H), 8.71 (t,
J = 1.74 Hz, 1 H), 8.61 (t, J = 1.56 Hz, 1 H), 7.74–7.84
(m, 2 H), 7.39 (t, J = 7.87 Hz, 2 H), 7.09–7.20 (m, 1 H),
2.72 (s, 3 H). MS (ESI) m/e 284.3 [(M+H+)]. Anal. Calcd
for C16H14N2O3: C, 68.07; 5.00; N, 9.92. Found: C, 67.86;
H, 4.94; N, 9.81.
7.3.28. 5-Acetyl-N-naphthalen-1-ylbenzene-1,3-dicarbox-
amide (52b). Prepared following the procedure described
for 52a (0.23 g, 0.69 mmol, 32%).1H NMR (300 MHz,
DMSO-d6) d ppm 13.55 (br s, 1 H), 10.60 (s, 1 H),
8.74 (t, J = 1.65 Hz, 1 H), 8.71 (t, J = 1.74 Hz, 1 H),

C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
3105
8.61 (t, J = 1.56 Hz, 1 H), 7.82–7.91 (m, 3H), 7.45–7.53
(m, 3H), 7.42 (dd, 1H), 2.72 (s, 3 H). Anal. Calcd for
C20H16N2O3: C, 72.28; H, 4.85; N, 8.43. Found: C,
72.54; H, 4.89; N, 8.47.
d6) d ppm 10.53 (s, 1 H), 8.71 (t, J = 1.57 Hz, 1 H),
8.65 (d, J = 1.74 Hz, 1H), 8.29 (br s, 1 H), 7.79 (d,
J = 7.49 Hz,
2 H), 7.68 (br s, 1 H), 7.39 (t,
J = 7.93 Hz, 2 H), 7.14 (t, J = 7.32 Hz, 1 H), 5.06 (s,
2H). MS (ESI) m/e 361.2 [(M+H+)]. HPLC tR
14.3 min; purity 82% pkb-100 254 · 4.6 mm 5 lm, gradi-
ent elution of 20–50% CH3CN/CH3COONH4 10 mM
for 30 min, k = 216 nm. Anal. Calcd for C16H13BrN2O3:
C, 53.21; H, 3.63; N, 7.76: Found: C, 53.49; H, 3.71; N,
7.62.
7.4. General procedure for monohalogenation reaction
A solution of the intermediate 48 or 52 (1.1 mmol) in
CHCl3 was treated with 1 mmol of the halogen X2 and
the reaction mixture was stirred at 50 ꢁC for 2 h. The
solution was poured into water and extracted with
dichloromethane. The organic layer was dried and con-
centrated. The residue was purified by flash chromatog-
raphy on silica gel (hexane/ethyl acetate 1:1) to obtain
the corresponding product.
7.4.6. 5-(Bromoacetyl)-N-2-naphthylisophthalamide (24).
(0.087 g, 0.21 mmol, 68%) 1H NMR (300 MHz, DMSO-
d6) d ppm 10.71 (s, 1 H), 8.83 (t, J = 1.65 Hz, 1 H), 8.79
(t, J = 1.65 Hz, 1 H), 8.64–8.72 (m, 2 H), 8.32 (br s, 1 H),
7.95–8.06 (m, 2 H), 7.90 (d, J = 8.05 Hz, 1 H), 7.52–7.65
(m, 4 H), 5.08 (s, 2 H). MS (ESI) m/e 411.1 [(M+H+)].
HPLC tR 12.4 min; purity 53% pkb-100 254 · 4.6 mm
5 lm, gradient elution of 25–55% CH3CN (0.01%
TFA)/H2O (0.01% TFA) for 30 min, k = 218 nm. Anal.
Calcd for C20H15BrN2O3: C, 58.41; H, 3.68; N, 6.81.
Found: C, 58.50; H, 3.72; N, 6.69.
7.4.1. 3-Benzyl-5-(bromoacetyl)benzamide (21). (0.07 g,
1
0.21 mmol, 68%). H NMR (300 MHz, CDCl3) d ppm
8.24 (t, J = 1.68 Hz, 0 H), 7.96 (t, J = 1.68 Hz, 1 H),
7.91 (t, J = 1.68 Hz, 1 H), 7.12–7.38 (m, 5 H), 6.04 (br
s, 2 H), 4.43 (s, 2 H), 4.10 (s, 2 H). MS (ESI) m/e
332.2 [(M+H+)]. HPLC tR 13.8 min; purity 98% pkb-
100 254 · 4.6 mm 5 lm, gradient elution of 30–60%
CH3CN (0.01% TFA)/H2O (0.01% TFA) for 30 min,
k = 216 nm. Anal. Calcd for C16H14BrNO2: C, 57.85;
H, 4.25; N, 4.22. Found: C, 57.71; H, 4.18; N, 4.17.
7.5. General procedure for dihalogenation reaction
A solution of the intermediate 48 or 52 (1 mmol) in
CHCl3 was treated with 2 mmol of the halogen X2 and
the reaction mixture was stirred at 50 ꢁC for 2.5 h. The
solution was poured into water and extracted with
dichloromethane. The organic layer was dried and con-
centrated. The residue was purified by flash chromatog-
raphy on silica gel (hexane/ethyl acetate 1:1) to obtain
the corresponding product.
7.4.2. 3-(Bromoacetyl)-5-(naphth-2-ylmethyl)benzamide
(22). (0.13 g, 0.34 mmol, 52%) 1H NMR (300 MHz,
DMSO-d6) d ppm 8.35 (t, J = 1.61 Hz, 1 H), 8.14 (br s, 1
H), 8.06–8.11 (m, 2 H), 7.78–7.90 (m, 4 H), 7.38–7.56
(m, 4 H), 4.96 (s, 2 H), 4.26 (s, 2 H). MS (ESI) m/e 382.1
[(M+H+)]. HPLC tR 12.4 min; purity 93% pkb-100
254 · 4.6 mm 5 lm, gradient elution of 35–65% CH3CN
(0.01% TFA)/H2O (0.01% TFA) for 30 min,
k = 225 nm. Anal. Calcd for C20H16BrNO2: C, 62.84; H,
4.22; N, 3.66. Found: C, 62.71; H, 4.24; N, 3.53.
7.5.1. 3-Benzyl-5-(dibromoacetyl)benzamide (16). (0.06 g,
0.145 mmol, 65%) 1H NMR (300 MHz, DMSO-d6) dppm
8.38 (s, 1 H), 8.11 (s, 2 H), 8.07 (s, 1 H), 7.82 (s, 1 H), 7.54
(br s, 1 H), 7.12–7.39 (m, 5 H), 4.08 (s, 2 H). MS (ESI) m/e
441.9 [(M+H+)]. HPLC tR 14.2 min; purity 98% pkb-100
254X4.6 mm 5 lm, gradient elution of 35–65% CH3CN
(0.01% TFA)/H2O (0.01% TFA) for 30 min, k =
258 nm. Anal. Calcd for C16H10Br2NO2: C, 46.75; H,
3.19; N, 3.41. Found: C, 46.93; H, 3.04; N, 3.41.
7.4.3. 3-Benzyl-5-(chloroacetyl)benzamide (13). (0.065 g,
0.22 mmol, 15%) 1H NMR (300 MHz, DMSO-d6) d
ppm 8.31 (s, 1 H), 8.12 (br s, 1 H), 8.05 (s, 1 H), 8.00
(s, 1 H), 7.51 (br s, 1 H), 7.14–7.38 (m, 5 H), 5.21 (s, 2
H), 4.08 (s, 2 H). MS (ESI) m/e 288.1 [(M+H+)]. HPLC
tR 12.6 min; purity 96% synergy hydro-RP 250 · 4.6 mm
4 lm, gradient elution of 70–40% CH3CN (0.01% TFA)/
H2O (0.01% TFA) for 30 min, k = 216 nm. Anal. Calcd
for C16H14ClNO2: C, 66.79; H, 4.90; N, 4.87. Found: C,
67.18; H, 5.11; N, 4.54.
7.5.2. 3-(Dibromoacetyl)-5-(naphth-2-ylmethyl)benzam-
ide (17). (0.04 g, 0.09 mmol, 15%) H NMR (300 MHz,
1
DMSO-d6) d ppm 8.38 (s, 1 H), 8.11 (s, 2 H), 8.07 (s,
1 H), 7.82 (s, 1 H), 7.78–7.90 (m, 3H), 7.54 (br s, 1
H), 7.38–7.56 (m, 4H), 4.08 (s, 2 H). MS (ESI) m/e
461.9 [(M+H+)]. HPLC tR 16.5 min; purity 94% pkb-
100 254 · 4.6 mm 5 lm, gradient elution of 35–65%
CH3CN (0.01% TFA)/H2O (0.01% TFA) for 30 min,
k = 225 nm. Anal. Calcd for C20H15Br2NO2: C, 52.09;
H, 3.28; N, 3.04. Found: C, 51.89; H, 3.28; N, 2.73.
7.4.4. 3-(Chloroacetyl)-5-(naphth-2-ylmethyl)benzamide
(14). (0.02 g, 0.06 mmol, 45%). 1H NMR (300 MHz,
DMSO-d6) d ppm 8.31 (t, J = 1.53 Hz, 1 H), 8.12 (br s, 1
H), 8.09 (t, 1 H), 8.06 (t, J = 1.61 Hz, 1 H), 7.82–7.91
(m, 3 H), 7.80 (s, 1 H), 7.45–7.53 (m, 3 H), 7.42 (dd,
J = 8.48, 1.75 Hz, 1 H), 5.21 (s, 2 H), 4.25 (s, 2 H). MS
(ESI) m/e 338.2 [(M+H+)]. HPLC tR 15.5 min; purity
97% pkb-100 254 · 4.6 mm 5 lm, gradient elution of
30–60% CH3CN (0.01% TFA)/H2O (0.01% TFA) for 30
minutes, k = 225 nm. Anal. Calcd for C20H16ClNO2: C,
71.11; H, 4.77; N, 4.15. Found: C, 69.95; H, 4.67; N, 3.97.
7.5.3. 5-(Dibromoacetyl)-N-phenylisophthalamide (18).
(0.003 g, 0.007 mmol, 65%) 1H NMR (300 MHz,
DMSO-d6) d ppm 10.53 (s, 1 H), 8.71 (t, J = 1.57 Hz,
1 H), 8.65 (d, J = 1.74 Hz, 1H), 8.29 (br s, 1 H), 7.79
(d, J = 7.49 Hz, 2 H), 7.68 (br s, 1 H), 7.39 (t,
J = 7.93 Hz, 2 H), 7.14 (t, J = 7.32 Hz, 1 H), 7.6 (s,
1H). MS (ESI) m/e 441.1 [(M+H+)]. HPLC tR
11.3 min; purity 93% pkb-100 254 · 4.6 mm 5 lm, gradi-
7.4.5. 5-(Bromoacetyl)-N-phenyslisophthalamide (23).
(0.09 g, 0.25 mmol, 60%) H NMR (300 MHz, DMSO-
1

3106
C. Maugeri et al. / Bioorg. Med. Chem. 16 (2008) 3091–3107
ent elution of 20–65% CH3CN (0.01% TFA)/H2O
(0.01% TFA) for 30 min, k = 208 nm. Anal. Calcd for
C16H12Br2N2O3: C, 43.67; H, 2.75; N, 6.37. Found:
43.75; H, 2.80; N, 6.41.
Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85.
Found: C, 67.42; H, 5.61; N, 9.78. 26 1H NMR
(300 MHz, DMSO-d6) d ppm 8.01 (br s, 1H), 7.70 (s,
1H), 7.47 (dd, J = 2.56, 1.39 Hz, 1H9, 7.18–7.41 (m,
12H), 4.78 (s, 4H), 3.78 (s, 3H). MS (ESI) m/e 375.3
[(M+H+)]. HPLC tR 16.7 min; purity 95% pkb-100
254 · 4.6 mm 5 lm, gradient elution of 30–60% CH3CN
(0.01% TFA)/H2O (0.01% TFA) for 30 min, k = 285 nm.
Anal. Calcd for C23H22N2O3: C, 73.78; H, 5.92; N, 7.48.
Found: C, 73.44; H, 6.03; N, 7.34.
7.5.4. 5-(Dibromoacetyl)-N-2-naphthyliphthalamide (19).
(0.04 g, 0.08 mmol, 45%) H NMR (300 MHz, DMSO-
1
d6) d ppm 10.70 (s, 1 H), 8.82–8.87 (m, 3 H), 8.72–8.76
(m, 1 H), 8.46 (br s, 0 H), 7.97–8.08 (m, 3 H), 7.86–
7.94 (m, 2 H), 7.51–7.63 (m, 4 H). MS (ESI) m/e 489.2
[(M+H+)]. Anal. Calcd for C20H14Br2N2O3: C, 49.01;
H, 2.88; N, 5.72. Found: C, 49.32; H, 3.01; N, 5.63.
Acknowledgments
7.5.5. Methyl 3-amino-5-carbamoylbenzoate (54). 3-
(methoxycarbonyl)-5-nitrobenzoic
acid
(11.2 g,
We thank Mr. G. Ciocci for his valid support during
chemical experiments and Dr. B. Garofalo for her con-
tribution in writing this paper. A special thanks also
goes to Dr. C. Milanese who supervised the entire
project.
49.7 mmol) and toluene (90 mL) were placed in a three
bottom flask equipped with a condenser and a magnetic
stirrer. The resulting mixture was vigorously stirred at
room temperature and thionyl chloride (12.7 mL,
175 mmol) was slowly added. The solution was refluxed
for 3 h. After being cooled at room temperature the sol-
vent was evaporated in vacuo. The crude residue was
used for the next reaction without further purification.
References and notes
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50%). H NMR (300 MHz, DMSO-d6) d ppm 7.91 (br
s, 1 H), 7.58 (t, J = 1.53 Hz, 1 H), 7.31 (dd, J = 2.34,
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1H), 7.18–7.40 (m, 8H), 6.73 (t, J = 6.07 Hz, 1H), 4.35
(d, J = 5.99 Hz, 2H), 3.82 (s, 3H). MS (ESI) m/e 285.2
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