Synthesis of substituted benzamides as anti-inflammatory agents that inhibit preferentially cyclooxygenase 1 but do not cause gastric damage

Article abstract of DOI:10.1016/S0223-5234(01)01251-X

Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) benzamide) (5a), is a non-steroidal anti-inflammatory drug (NSAID), commercialised in Italy until 1985 with the brand name of Synovial, that has been widely used to treat arthritic patient. In addition, it was shown to spare gastric mucosa. Here we have synthesised a series of novel substituted benzamides, related to Parsalmide, and have evaluated their activity in vitro on COX-1 and COX-2 as well as in vivo in the carrageenin-induced rat paw edema, a classical in vivo anti-inflammatory assay. Compounds 5b, 11a and 11b, which showed a favourable profile in vitro and in vivo, were screened in comparison with Parsalmide for gastrointestinal (GI) tolerability in vivo in the rat. Results obtained showed that Parsalmide and compound 11b inhibited both COX-1 and COX-2 in vitro as well as they were active in vivo. Both compounds were devoid of gastric effect at the efficacious dose. In addition, both prevented indomethacin-induced gastric damage. Thus, these compounds may guide the definition of a new leading structure with anti-inflammatory activity that may allow designing new safer NSAIDs.

Full text of DOI:10.1016/S0223-5234(01)01251-X

Eur. J. Med. Chem. 36 (2001) 517–530
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01251-X/FLA
Original article
Synthesis of substituted benzamides as anti-inflammatory agents that inhibit
preferentially cyclooxygenase 1 but do not cause gastric damage
Giuseppe Caliendoa,*, Vincenzo Santagadaa, Elisa Perissuttia, Beatrice Severinoa,
Ferdinando Fiorinoa, Timothy D. Warnerb, John L. Wallacec, Demian Rocha Ifad,
Edson Antunesd, Giuseppe Cirinoe, Gilberto de Nuccid
aDipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli ‘Federico II’, Via Domenico Montesano 49,
I-80131 Naples, Italy
bThe William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry,
Charterhouse Square, London EC1M 6BQ, UK
cIntestinal Disease Research Unit, Faculty of Medicine, University of Calgary, Alberta, Canada
dDepartment of Pharmacology, Faculty of Medical Sciences, P.O. Box 6111, UNICAMP, Campinas SP, Brazil
eDipartimento di Farmacologia Sperimentale, Universita` di Napoli ‘Federico II’, Via Domenico Montesano 49,
I-80131 Naples, Italy
Received 11 February 2001; revised 11 June 2001; accepted 12 June 2001
Abstract – Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) benzamide) (5a), is a non-steroidal anti-inflammatory drug (NSAID),
commercialised in Italy until 1985 with the brand name of Synovial®, that has been widely used to treat arthritic patient. In addition,
it was shown to spare gastric mucosa. Here we have synthesised a series of novel substituted benzamides, related to Parsalmide, and
have evaluated their activity in vitro on COX-1 and COX-2 as well as in vivo in the carrageenin-induced rat paw edema, a classical
in vivo anti-inflammatory assay. Compounds 5b, 11a and 11b, which showed a favourable profile in vitro and in vivo, were screened
in comparison with Parsalmide for gastrointestinal (GI) tolerability in vivo in the rat. Results obtained showed that Parsalmide and
compound 11b inhibited both COX-1 and COX-2 in vitro as well as they were active in vivo. Both compounds were devoid of gastric
effect at the efficacious dose. In addition, both prevented indomethacin-induced gastric damage. Thus, these compounds may guide
the definition of a new leading structure with anti-inflammatory activity that may allow designing new safer NSAIDs. © 2001
´
Editions scientifiques et me´dicales Elsevier SAS
anti-inflammatory agents / COX-2/COX-1 inhibition / benzamide derivatives / gastric damage
1. Introduction
prostaglandins are produced by the inducible isoform
COX-2 while physiological prostaglandins are pro-
duced by the constitutive isoform COX-1. On this
premise several new inhibitors have been developed
and some are now commercially available [1]. How-
ever, there are clearly identified areas of the body
where COX-2 expression is beneficial and in some
cases COX-1 is relevant for pathological effects [2].
Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) ben-
zamide) is a non-steroidal anti-inflammatory drug
(NSAID), originally synthesised in Italy in the 1970s
[3–5]. Parsalmide has been commercially available
since 1985 with the brand name Synovial® and it has
Non-steroidal anti-inflammatory drugs are a main-
stay in the treatment of inflammation and they owe
their therapeutic and side effects in large part to the
inhibition of cyclooxygenase (COX). The separation
of the therapeutic effects from the side effects has
been a major goal in the design and synthesis of these
drugs. The discovery of a second isoform of cyclooxy-
genase, namely COX-2, has opened a new line of
research based on the assumption that pathological
* Correspondence and reprints:
E-mail address: caliendo@unina.it (G. Caliendo).

518
G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
2. Chemistry
been widely used to treat arthritic patients [6–10].
This drug has a peculiar basic structure and it has
been also shown to spare gastric mucosa and also to
prevent gastric lesions induced by other NSAIDs [11–
16]. Due to these peculiar actions we have synthesised
Parsalmide and designed a new series of analogues in
the attempt to obtain COX inhibitors sparing the
gastrointestinal (GI) tract. In particular, the struc-
tures synthesised bear in the place of the propynyloxy
residue in 2-position present in Parsalmide a few
aromatic or non-aromatic pharmacophoric portions
present in some drugs that have been shown to be
COX-2 selective (i.e. Nimesulide, NS-398, RWJ-
63356). The aim of this approach was to verify if
these structural changes would have shifted selectivity
towards COX-2. In addition we investigated if the
pharmacological activity was modulated when the
amino group in 5-position was transformed into an
acidic group by protecting it with the methanesul-
fonyl- or, in neutral group, through protection with
the acetyl-moiety. Indeed, the methanesulfonylamide
moiety was fundamental for the activity of the above-
mentioned drugs, as well as for celecoxib.
The general procedure used for the synthesis of the
N-(n-butyl)-benzamide derivatives 4a,b, 5a,b, 6a,b is
shown in figure 1 while the physical and H-NMR
1
data are reported, respectively, in tables I and II.
Commercially, 5-amino-salicylic acid (1) was acylated
with acetic hydride to give the intermediate 2. Con-
densation of 5-acetamide-2-hydroxy-benzoic acid (2)
with n-butylamine in the presence of 1-hydroxybenzo-
triazole (HOBt) and N,N%-dicyclohexylcarbodiimide
(DCC) in anhydrous dimethylformamide (DMF) gave
5-acetamide-N-butylsalicylamide (3), which was alky-
lated with the appropriate alkyl halide in the presence
of sodium metal in anhydrous isopropyl alcohol to
provide the desired compounds 4a,b. Compound 4a
was converted into the corresponding amine 5a
(Parsalmide) by treatment with sulphuric acid. The
last step did not give the corresponding amine when a
3-methyl-2-butenyloxy residue (4b) was present in the
2-position. In fact, in this case the decomposition of
the compound was observed. Therefore, the hydroly-
sis of 4b to the corresponding amine 5b was con-
ducted employing 3 N NaOH solution. Finally, the
amines 5a and 5b were converted into the methanesul-
fonamides 6a and 6b by treatment with methanesul-
fonyl chloride and pyridine in toluene.
All synthesised compounds were screened in vitro
for COX-1 and COX-2 inhibitory effect and then
tested in vivo in carrageenin-induced rat paw edema.
The more active compounds were screened for GI
tolerability in vivo in the rat.
The general strategy for the synthesis of amines
11a–e, acetamides 12a–e and methanesulfonamides
13a–e is summarised in figure 2 while the physical
Figure 1. Synthetic procedure of compounds 4a,b; 5a,b; 6a,b.

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
Table I. Physical data for N-(-n-butyl)-benzamide derivatives.
519

Table II. 1H-NMR and MS data of N-(-n-butyl)-benzamide derivatives.
1H-NMR (CDCl3); l (ppm); J (Hz)
MS m/z
4a
8.77 (s, 1H, ArNH); 8.22 (dd, 1H, J=2.9 Hz, ArH); 7.97 (d, 1H,
J=2.9 Hz, ArH); 7.89 (br, 1H, CONH); 7.00 (d, 1H, J=7.9 Hz,
289.0 (base, M++1); 249.9 (M+−CH2CꢀCH); 215.8
(M+−NH(CH2)3CH3); 189.9 (M+−CONH(CH2)3CH3); 173.8
ArꢁH); 4.79 (s, 2H, OCH2); 3.47 (q, 2H, J=6.9 Hz, CH2NH); 2.60 (M+−NH(CH2)3CH3; −COCH3); 147.8 (M+−CONH(CH2)3CH3;
(s, 1H, CꢀCH); 2.18 (s, 3H, COCH3); 1.57 (q, 2H, J=6.9 Hz,
CH2CH2NH); 1.44 (m, 2H, J=7.9 Hz, CH2CH3); 0.95 (t, 3H,
J=6.9 Hz, CH2CH3)
−COCH3)
4b
8.57 (br, 1H, CONH); 8.22 (dd, 1H, J=2.9 Hz, ArH); 8.20 (br, 1H, 319.0 (base, M++1); 251.0 (M+−CH2ꢁCHꢂC(CH3)2); 177.5
CONH); 7.98 (d, 1H, J=2.9 Hz, ArH); 6.96 (d, 1H, J=7.9 Hz,
ArH); 5.51 (t, 1H, J=5.5 Hz,CHꢂC); 4.62 (d, 2H, J=3.5 Hz,
OCH2); 3.44 (q, 2H, J=6.9 Hz, CH2NH); 2.18 (s, 3H, COCH3);
1.82 (s, 3H, C(CH3)2; 1.79 (s, 3H, C(CH3)2); 1.55 (q, 2H, J=6.9
Hz, CH2CH2NH); 1.42 (m, 2H, J=7.9 Hz, CH2CH3); 0.95 (t, 3H,
J=6.9 Hz, CH2CH3)
(M+−CH2CHꢂC(CH3)2; −NH(CH2)3CH3); 151.8
(M+−CH2ꢁCHꢂC(CH3)2; −CONH(CH2)3CH3)
5a
5b
7.83 (br, 1H, CONH); 7.50 (d, 1H, J=2.9 Hz, ArH); 6.83 (d, 1H,
J=7.9 Hz, ArH); 6.72 (dd, 1H, J=2.9 Hz, ArH); 4.67 (s, 2H,
OCH2); 3.57 (br, 2H, ArNH2); 3.41 (q, 2H, J=6.9 Hz, CH2NH);
2.55 (s, 1H, CꢀCH); 1.54 (q, 2H, J=6.9 Hz, CH2CH2NH); 0.91 (t,
3H, J=6.9 Hz, CH2CH3)
8.20 (br, 1H, CONH); 7.58 (d, 1H, J=2.9 Hz, ArH); 6.92 (d, 1H,
J=7.9 Hz, ArH); 6.78 (dd, 1H, J=2.9 Hz, ArH); 5.50 (t, 1H,
J=5.6 Hz, CHꢂC); 4.55 (d, 2H, J=3.5 Hz, OCH2); 3.44 (q, 2H,
J=6.9 Hz, CH2NH); 3.15 (br, 2H, ArNH2); 1.82 (s, 3H, C(CH3)2);
1.75 (s, 3H, C(CH3)2); 1.55 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.40
(m, 2H, J=7.9 Hz, CH2CH3); 0.95 (t, 3H, J=6.9 Hz, CH2CH3)
8.54 (s, 1H, ArNH); 8.36 (d, 1H, J=2.9 Hz, ArH); 7.98 (br, 1H,
CONH); 7.67 (dd, 1H, J=2.9 Hz, ArH); 7.06 (d, 1H, J=7.9 Hz,
ArH); 4.82 (s, 2H, OCH2); 3.56 (q, 2H, J=6.9 Hz, CH2NH); 2.93
(s, 3H, SO2CH3); 2.62 (s, 1H, CꢀCH); 1.62 (q, 2H, J=6.9 Hz,
CH2CH2NH); 1.46 (m, 2H, J=7.9 Hz, CH2CH3); 0.97 (t, 3H,
J=6.9 Hz, CH2CH3)
8.90 (s, 1H, ArNH); 8.52 (d, 1H, J=2.9 Hz, ArH); 8.33 (br, 1H,
CONH); 7.68 (dd, 1H, J=2.9 Hz, ArH); 7.00 (d, 1H, J=7.9 Hz,
ArH); 5.55 (t, 1H, J=5.5 Hz, CHꢂC); 4.65 (d, 2H, J=3.5 Hz,
OCH2); 3.58 (q, 2H, J=6.9 Hz, CH2NH); 2.90 (s, 3H, COCH3);
1.90 (s, 3H, C(CH3)2); 1.78 (s, 3H, C(CH3)2); 1.59 (q, 2H, J=6.9
Hz, CH2CH2NH); 1.48 (m, 2H, J=7.9 Hz, CH2CH3); 0.98 (t, 3H,
J=6.9 Hz, CH2CH3)
247.0 (base, M++1); 207.8 (M+−CH2CꢀCH); 173.7
(M+−NH(CH2)3CH3); 147.8 (M+−CONH(CH2)3CH3); 134.8
(M+−CH2CꢀCH; −NH(CH2)CH3)
277.0 (base, M++1); 208.9 (M+−CH2ꢁCHꢂC(CH3)2); 177.8
(M+−CONH(CH2)3CH3); 135.7 (M+−CH2ꢁCHꢂC(CH3)2;
−NH(CH2)3CH3)
6a
6b
325.0 (base, M++1); 251.8 (M+−NH(CH2)3CH3); 245.9
(M+−SO2CH3); 206.9 (M+−CH2CꢀCH; −SO2CH3); 172.9
(M+−NH(CH2)3CH3; −SO2CH3); 145.6 (M+−CONH(CH2)3CH3;
−SO2CH3); 117.8 (M+−OCH2CꢀCH; −NH(CH2)3CH3; −SO2CH3)
355.0 (base, M++1); 286.9 (M+−CH2ꢁCHꢂC(CH3)2); 214.8
(M+−CH2CHꢂC(CH3)2; NH(CH2)3CH3); 207.6
(M+−CH2ꢁCHꢂC(CH3)2; −SO2CH3); 135.8 (M+−CH2ꢁCHꢂC(CH3)2;
−NH(CH2)3CH3; −SO2CH3)

Table II. (Continued)
1H-NMR (CDCl3); l (ppm); J (Hz)
MS m/z
11a a 8.68 (br, 1H, CONH); 7.95 (d, 1H, J=2.9 Hz, ArH); 7.50 (d, 1H,
J=7.9 Hz, ArH); 7.37 (dd, 1H, J=2.9 Hz, ArH); 4.72 (m, 1H,
cyclohexyl); 3.54 (br, 2H, ArNH2); 3.50 (q, 2H, J=6.9 Hz, CH2NH);
2.10–1.83 (m, 8H, cyclohexyl); 1.69 (q, 2H, J=6.9 Hz, CH2CH2NH);
1.58 (m, 2H, cyclohexyl); 1.50 (m, 2H, J=7.9 Hz, CH2CH3); 1.02 (t,
3H, J=6.9 Hz, CH2CH3)
291.0 (base, M++1); 208.9 (M+−C6H11); 190.8
(M+−CONH(CH2)3CH3); 135.8 (M+−C6H11; −NH(CH2)3CH3)
11b 8.35 (br, 1H, CONH); 7.75 (d, 1H, J=2.9 Hz, ArH); 4.44 (d, 1H,
J=7.9Hz, ArH); 7.30–6.95 (m, 5H, ArH); 3.35 (q, 2H, J=6.9 Hz,
CH2NH); 1.44 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.28 (m, 2H, J=7.9
Hz, CH2CH3); 0.89 (t, 3H, J=6.9 Hz, CH2CH3)
284.9 (base, M++1); 211.9 (M+−NH(CH2)3CH3)
303.0 (base, M++1); 229.9 (M+−NH(CH2)3CH3)
11c a 8.50 (br, 1H, CONH); 7.84 (d, 1H, J=2.9 Hz, ArH); 7.50 (d, 1H,
J=7.9 Hz, ArH); 7.10–7.28 (m, 4H, ArH); 7.06 (dd, 1H, J=2.9 Hz,
ArH); 3.40 (q, 2H, J=6.9 Hz, CH2NH); 3.35 (br, 2H, ArNH2); 1.56
(q, 2H, J=6.9 Hz, CH2CH2NH); 1.40 (m, 2H, J=7.9 Hz, CH2CH3);
0.95 (t, 3H, J=6.9 Hz, CH2CH3)
11d 8.25 (br, 1H, CONH); 7.22 (d, 1H, J=2.9 Hz, ArH); 7.60 (d, 1H,
319.0 (base, M++1); 245.9 (M+−NH(CH2)3CH3); 210.8
J=7.9 Hz, ArH); 7.29–6.96 (m, 4H, ArH); 6.83 (d, 1H, J=2.9 Hz, (M+−NH(CH2)3CH3; −Cl)
ArH); 3.32 (q, 2H, J=6.9 Hz, CH2NH); 3.01 (br, 2H, ArNH2); 1.46
(q, 2H, J=6.9 Hz, CH2CH2NH); 1.25 (m, 2H, J=7.9 Hz, CH2CH3);
0.82 (t, 3H, J=6.9 Hz, CH2CH3)
11e 8.37 (br, 1H, CONH); 7.84 (d, 1H, J=2.9 Hz, ArH); 7.72 (d, 1H,
J=7.9 Hz, ArH); 7.25–6.96 (m, 4H, ArH); 6.82 (dd, 1H, J=2.9 Hz,
ArH); 3.38 (q, 2H, J=6.9 Hz, CH2NH); 2.92 (m, 1H, CH(CH3)2);
1.47 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.30 (m, 2H, J=7.9 Hz,
327.0 (base, M++1); 211.9 (M+−NH(CH2)3CH3; −CH(CH3)2)
CH2CH3); 1.27 (d, 6H, CH(CH3)2); 0.85 (t, 2H, J=6.9 Hz, CH2CH3)
12a 9.07 (s, 1H, ArNH); 8.25 (br, 1H, CONH); 8.14 (dd, 1H, J=2.9 Hz, 333.0 (base, M++1); 251.0 (M+−C6H11); 177.7 (M+−C6H11;
ArH); 7.98 (d, 1H, J=2.9 Hz, ArH); 6.92 (d, 1H, J=7.9 Hz, ArH); −NH(CH2)3CH3)
4.38 (m, 1H, cyclohexyl); 3.43 (q, 2H, J=6.9 Hz, CH2NH); 2.15 (s,
3H, COCH3); 2.01–1.73 (m, 8H, cyclohexyl); 1.57 (q, 2H,
CH2CH2NH); 1.50 (m, 2H, cyclohexyl); 1.39 (m, 2H, J=7.9 Hz,
CH2CH3; 0.92 (t, 3H, J=6.9 Hz, CH2CH3)
12b 8.40 (s, 1H, ArNH); 8.20 (dd, 1H, J=2.9 Hz, ArH); 8.00 (d, 1H,
J=2.9 Hz, ArH); 7.70 (br, 1H, CONH); 7.38–7.00 (m, 5H, ArH);
6.15 (d, 1H, J=7.9 Hz, ArH); 3.48 (q, 2H, J=6.9 Hz, CH2NH);
2.40 (s, 3H, COCH3); 1.40 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.33
(m, 2H, J=7.9 Hz, CH2CH3); 0.95 (t, 3H, J=6.9 Hz, CH2CH3)
12c 8.70 (s, 1H, ArNH); 8.17 (dd, 1H, J=2.9 Hz, ArH); 8.00 (d, 1H,
J=2.9 Hz, ArH); 7.69 (br, 1H, CONH); 7.08–6.97 (m, 4H, ArH);
6.82 (d, 1H, J=7.9 Hz, ArH); 3.43 (q, 2H, J=6.9 Hz, CH2NH);
2.18 (s, 3H, COCH3); 1.50 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.31
(m, 2H, J=7.9 Hz, CH2CH3); 0.87 (t, 3H, J=6.9 Hz, CH2CH3)
327.0 (base, M++1); 253.9 (M+−NH(CH2)3CH3); 211.6
(M+−NH(CH2)3CH3; −COCH3)
345.0 (base, M++1); 271.8 (M+−NH(CH2)3CH3); 229.9
(M+−NH(CH2)3CH3; −COCH3)

Table II. (Continued)
1H-NMR (CDCl3); l (ppm); J (Hz)
MS m/z
12d 9.10 (s, 1H, ArNH); 8.24 (dd, 1H, J=2.9 Hz, ArH); 8.04 (d, 1H,
J=2.9 Hz, ArH); 7.77 (br, 1H, CONH); 7.31–6.94 (m, 4H, ArH);
6.86 (d, 1H, J=7.9 Hz, ArH); 3.42 (q, 2H, J=6.9 Hz, CH2NH);
2.18 (s, 3H, COCH3); 1.48 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.29
(m, 2H, J=7.9 Hz, CH2CH3); 0.87 (t, 3H, J=6.9 Hz, CH2CH3)
12e 8.86 (s, 1H, ArNH); 8.18 (d, 1H, J=2.9 Hz, ArH); 8.04 (d, 1H,
J=2.9 Hz, ArH); 7.84 (br, 1H, CONH); 7.27–6.89 (m, 4H, ArH);
6.85 (d, 1H, J=7.9 Hz, ArH); 3.43 (q, 2H, J=6.9 Hz, CH2NH);
2.92 (m, 1H, CH(CH3)2); 2.19 (s, 3H, COCH3); 1.52 (q, 2H, J=6.9
Hz, CH2CH2NH); 1.32 (m, 2H, J=7.9 Hz, CH2CH3); 1.27 (d, 6H,
CH(CH3)2); 0.89 (t, 3H, J=6.9 Hz, CH2CH3)
361.0 (base, M++1); 287.8 (M+−NH(CH2)3CH3); 245.8
(M+−NH(CH2)3CH3; −COCH3)
369.0 (base, M++1); 253.9 (M+−NH(CH2)3CH3; −CH(CH3)2); 211.9
(M+−NH(CH2)3CH3; −CH(CH3)2; −COCH3)
13a 8.68 (s, 1H, ArNH); 8.57 (br, 1H, CONH); 8.40 (d, 1H, J=2.9 Hz, 369.0 (base, M++1); 286.9 (M+−C6H11); 213.9 (M+−C6H11;
ArH); 7.62 (dd, 1H, J=2.9 Hz, ArH); 6.98 (d, 1H, J=7.9 Hz, ArH);−NH(CH2)3CH3); 206.8 (M+−C6H11; −SO2CH3); 132.7 (M+−C6H11;
4.61 (m, 1H, cyclohexyl); 3.56 (q, 2H, J=6.9 Hz, CH2NH); 2.91 (s, −NH(CH2)3CH3; −SO2CH3)
3H, COCH3); 1.79–1.78 (m, 8H, cyclohexyl); 1.58 (q, 2H, J=6.9 Hz,
CH2CH2NH); 1.57 (m, 2H, cyclohexyl); 1.46 (m, 2H, J=7.9 Hz,
CH2CH3); 0.96 (t, 3H, J=6.9 Hz, CH2CH3)
13b 8.40 (s, 1H, ArNH); 7.92 (d, 1H, J=2.9 Hz, ArH); 7.85 (br, 1H,
CONH); 7.58 (dd, 1H, J=2.9 Hz, ArH); 7.42–7.00 (m, 5H, ArH);
6.92 (d, 1H, J=7.9 Hz, ArH); 3.58 (q, 2H, J=6.9 Hz, CH2NH);
2.97 (s, 3H, SO2CH3); 1.55 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.30
(m, 2H, J=7.9 Hz, CH2CH3); 0.95 (t, 3H, J=6.9 Hz, CH2CH3)
13c 8.77 (s, 1H, ArNH); 8.40 (d, 1H, J=2.9 Hz, ArH); 7.95 (br, 1H,
CONH); 7.65 (dd, 1H, J=2.9 Hz, ArH); 7.18–6.98 (m, 4H, ArH);
6.85 (d, 1H, J=7.9 Hz, ArH); 3.62 (q, 2H, J=6.9 Hz, CH2NH);
3.00 (s, 3H, COCH3); 1.62 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.35
(m, 2H, J=7.9 Hz, CH2CH3); 0.96 (t, 3H, J=6.9 Hz, CH2CH3)
13d 8.62 (s, 1H, ArNH); 8.31 (d, 1H, J=2.9 Hz, ArH); 7.73 (br, 1H,
CONH); 7.54 (dd, 1H, J=2.9 Hz, ArH); 7.30–6.91 (m, 4H, ArH);
6.79 (d, 1H, J=7.9 Hz, ArH); 3.64 (q, 2H, J=6.9 Hz, CH2NH);
2.91 (s, 3H, COCH3); 1.46 (q, 2H, J=6.9 Hz, CH2CH2NH); 1.25
(m, 2H, J=7.9 Hz, CH2CH3); 0.81 (t, 3H, J=6.9 Hz, CH2CH3)
13e 8.77 (s, 1H, ArNH); 8.46 (d, 1H, J=2.9 Hz, ArH); 8.06 (br, 1H,
CONH); 7.61 (dd, 1H, J=2.9 Hz, ArH); 7.27–6.98 (m, 4H, ArH);
6.86 (d, 1H, J=7.9 Hz, ArH); 3.55 (q, 2H, J=6.9 Hz, CH2NH);
2.98 (s, 3H, COCH3); 2.93 (m, 1H, CH(CH3)2); 1.57 (q, 2H, J=6.9
Hz, CH2CH2NH); 1.34 (m, 2H, J=7.9 Hz, CH2CH3); 1.28 (d, 6H,
CH(CH3)2); 0.89 (t, 3H, J=6.9 Hz, CH2CH3)
363.0 (base, M++1); 289.9 (M+−NH(CH2)3CH3); 209.9
(M+−NH(CH2)3CH3; −SO2CH3)
381.0 (base, M++1); 307.8 (M+−NH(CH2)3CH3); 227.8
(M+−NH(CH2)3CH3; −SO2CH3)
397.0 (base, M++1); 323.9 (M+−NH(CH2)3CH3); 318.0
(M+−SO2CH3); 243.9 (M+−NH(CH2)3CH3; −SO2CH3); 209.9
(M+−NH(CH2)3CH3; −SO2CH3; −Cl)
405.0 (base, M++1); 289.9 (M+−NH(CH2)3CH3; −CH(CH3)2); 211.9
(M+−NH(CH2)3CH3; −CH(CH3)2; −SO2CH3)
a These compounds were determined using CD3OD as solvent.

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
523
and 1H-NMR data are reported, respectively, in tables
I and II. The condensation reaction of the commer-
cially available 2-chloro-5-nitrobenzoic acid (7) with
n-butylamine performed in anhydrous DMF in the
presence of DCC/HOBt produced the corresponding
intermediate 8. Subsequent dissolution of 8 in anhy-
drous dioxane and treatment with a solution of the
appropriate alcohol and NaH in anhydrous dioxane
gave the corresponding intermediates 9a–e. Reduc-
tion of the nitro group of intermediates 9a–e, carried
out with sodium borohydride reagent, gave the corre-
sponding intermediates 10a–e. Free bases 10a–e were
converted into the corresponding hydrochloride salts
11a–e by treatment with an excess of diethyl ether
saturated with dry, gaseous HCl. The acetamides
12a–e were prepared by reaction of the corresponding
amines 10a–e in anhydrous DMF with acetic anhy-
dride. Finally, the methanesulfonamides 13a–e were
also prepared by reaction of the corresponding inter-
mediates 10a–e with methanesulfonyl chloride and
pyridine in toluene. All compounds had satisfactory
1
elemental analyses, and their H-NMR and/or mass
spectra were consistent with the proposed structures.
The pKa values of compounds 5b, 11a and 11b were
performed potentiometrically using a pH meter: mi-
cro pH 2002 — Crison Instruments, S.A.
3. Biological assays
All synthesised compounds (series 4–6, 11–13)
were screened in vitro for COX-1 and COX-2 in-
Figure 2. Synthetic procedure of compounds 11a–e, 12a–e, 13a–e.

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
hibitory effect by evaluating the ability of the com-
pounds to inhibiting COXs in a purified enzyme as-
say. Then all compounds were tested in vivo in
carrageenin-induced rat paw edema, a classical in vivo
assay used to characterise NSAIDs. The reference
compounds used were indomethacin (3, 10, 30 mg/kg)
and celecoxib (3, 10, 30 mg/kg) given orally. Follow-
ing the preliminary screening, the compounds that
showed a favourable profile in vitro and in the paw
edema assay (5a, 5b, 11a and 11b) were screened for
GI tolerability in vivo in the rats as acute gastric
damage and indomethacin-induced gastric damage. A
dose response in carrageenin oedema was performed
also for compounds 11a, 11b and Parsalmide (10, 30,
100 mg/kg).
pounds 4b and 6b caused a loss of the effect both in
vivo and in vitro (table III). On the basis of these
results we synthesised a few analogues where the
propynyloxy residue in 2-position was replaced by a
cyclohexyloxy- 11a, 12a, 13a, phenyloxy- 11b, 12b,
13b and phenyloxy- para-substituted with fluorine
11c, 12c, 13c, chlorine 11d, 12d, 13d or isopropyl
group 11e, 12e, 13e. Modulation in the amino group
was performed by inserting an acetyl group 12a–e or
the methanesulfonyl moiety 13a–e. The presence of a
cyclohexyloxy group (11a) caused a reduction of the
in vitro ratio COX-2/COX-1 but the in vivo activity
was comparable to Parsalmide (table III). Modifica-
tion in compound 11a of the amino group either by
inserting an acetyl (12a) or a methanesulfonyl group
(13a) caused a loss of activity both in vivo and in
vitro (table III). Insertion of an unsubstituted aro-
matic ring in the place of the cyclohexyl moiety (11b)
slightly reduced both the COX-2/COX-1 ratio and the
anti-inflammatory activity in vivo (table III). Again,
modification of the amino group (12b, 13b) caused a
loss of effect both in vitro and in vivo. The presence
of substituent in the aromatic ring such as fluorine
(11c, 12c, 13c), chlorine (11d, 12d, 13d) or isopropyl
group (11e, 12e, 13e) caused a loss of activity in vivo
(table III). It is of interest to note that compounds
11c, 11e, and 12e displayed in vitro a COX-2/COX-1
ratio of 19, 30 and 67, respectively, but they were
ineffective in the in vivo assay (table III).
On the basis of the in vivo and in vitro results we
selected basic compounds 5b, 11a and 11b (pKa values
4.20, 3.80 and 3.15, respectively) and they were tested
in comparison with Parsalmide in an in vivo model of
gastric damage. The results obtained (figure 3A)
showed that Parsalmide and compound 11b were de-
void of gastric effect while compounds 5b and 11a
showed some gastric damage. The ED50 for
Parsalmide, indomethacin, celecoxib and compounds
11a and 11b were 82 7, 6.5 1.4, 15 3.1, 75 6, 68 6
mg/kg, respectively.
Since Parsalmide has been shown to protect from
gastric damage induced by NSAIDs [11–13] we com-
pared compound 11b with Parsalmide in an assay of
gastric damage induced by indomethacin. Both
Parsalmide and compound 11b clearly and signifi-
cantly prevented the gastric damage induced by in-
domethacin (figure 3B). These data indicated that
modification in the 2-position with a phenyl ring (11b)
preserved activity in vitro and in vivo and left intact
the gastric sparing properties. The other modification
with a 3-methyl-2-butenyloxy (5b) or with a cyclo-
4. Results and discussion
Parsalmide is a 5-amino-N-butyl-2-(2-propynyloxy)
benzamide derivative with anti-inflammatory proper-
ties that has a peculiar basic structure (pKa=4.6 [5]).
Parsalmide, which displayed the same chemical profile
originally described [3–5], was synthesised and tested
in vitro to assess the COX-2/COX-1 selectivity as well
as in vivo to evaluate the anti-inflammatory activity.
Parsalmide (5a) inhibited about 83% edema forma-
tion in vivo and showed in vitro a COX-2/COX-1
ratio of 16 (table III). This ratio shows that
Parsalmide is more selective for COX-1. In order to
acquire new insights into the structure–activity rela-
tionships in Parsalmide, we synthesised and screened
a new series of derivatives where the propynyloxy
moiety in the 2-position of the phenyl ring was re-
placed by a few alkyl- and (un) substituted aryl-oxy
residues bearing electron-donating or -withdrawing
substituents. In addition, the amine moiety was free
or protected by acetyl- or methanesulfonyl-group. In
vitro data showed that the presence of a methanesul-
fonamide group in 5-position (6a) caused a loss of the
in vitro COX-2/COX-1 activity that paralleled a re-
duction of the anti-inflammatory activity in vivo
(table III). Similarly, protection of the amino group
by acetyl 4a resulted in a loss of activity both in vitro
and in vivo (table III). Replacing the propynyloxy
group with a 3-methyl-2-butenyloxy moiety (5b)
slightly modified the COX-2/COX-1 ratio, which was
13, but the in vivo anti-inflammatory activity was
reduced by about 50% (table III). Also, in compound
5b modifications of the amino group leading to com-

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
Table III. Enzyme and in vivo activities of N-(-n-butyl)-benzamide derivatives.
525

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
hexyloxy (11a) caused a loss of the gastric sparing
properties.
challenged by the present data. In fact, Parsalmide
and compound 11b are both basic structures that
preferentially inhibit COX-1 and COX-2 in vitro and
are active in vivo in the paw edema assay. The better
inhibitory activity on the COX-isoforms of the com-
pounds with a free aniline function is probably medi-
ated by non-covalent bonds to the enzyme [18]. In
addition and most importantly, both compounds ap-
pear to be not only devoid of gastric effects but are
also able to prevent GI damage induced by in-
domethacin. Thus, the data obtained imply that the
introduction in 2-position of structural features pos-
sessed by the reference drugs (Nimesulide, NS-398,
RWJ-63356) allowed to maintain COX inhibitory ef-
fects and in vivo activity. Thus, biological activity was
retained when the amine group in 5-position was free.
In conclusion we have demonstrated that
Parsalmide is 16 times more selective for COX-1 and
that structural modification performed in its structure
by inserting groups known to increase the anti-inflam-
matory activity led to compound 11b. Compound
11b, even while displaying a reduced COX-2/COX-1
ratio, showed a comparable anti-inflammatory effect.
Parsalmide (5a) and compound 11b may guide the
definition of a new leading structure to design GI safe
drugs that will not lack the COX-1 component. In-
deed, powerful NSAIDs such as indomethacin are
already available in the market as well as efficacious
COX-2 selective inhibitors but preferential NSAIDs
that will be GI safe but still inhibiting COX-1 may
turn to be important for all the beneficial biological
activities, such as the anti-aggregatory effect, where
COX-1 inhibition is required.
5. Conclusions
The present data further suggest that to inhibit
COX-1 and COX-2 an acidic structure is not neces-
sary. However, structures inhibiting COX-1 selec-
tively, such as ketorolac, which is highly selective for
COX-1 [17], have an acidic nature. In this respect, our
data shed a new light on the chemical requisite to
design new COX inhibitors. Indeed, the paradigm of
an acidic structure that has been already challenged
by the development of COX-2 inhibitors is further
6. Experimental protocols
Capillary melting points were determined on a
Buchi SPM-20 apparatus and are reported uncor-
rected. Elemental analyses were performed on a
Perkin–Elmer 2400 and were within 0.4% of the
1
theoretical values unless indicated otherwise. The H-
NMR measurements were performed on a Bruker
AMX 500 spectrometer using CDCl3 or CD3OD as
solvents and tetramethylsilane as internal standard.
Chemical shifts (l) are expressed in ppm downfield
from tetramethylsilane and coupling constants J are
expressed in Hertz. Significant 1H-NMR data are
reported in order: multiplicity (s, singlet; d, doublet;
dd, double doublet; t, triplet; q, quartet; m, multiplet;
br, broad). Mass spectrometric detection was per-
Figure 3. (A) Gastric damage induced by a single oral admin-
istration of 100 mg/kg per os of compounds 5a, 5b, 11a and
11b. (B) Protective effect of compounds 5a and 11b given
orally at the dose of 100 mg/kg on gastric damage induced by
indomethacin.

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
527
formed using a MicroMass (Manchester, UK) Qua-
troII LC triple-quadrupole mass spectrometer,
equipped with a heated nebuliser as the APCI source.
Thin layer chromatography was performed on pre-
coated silica gel plates Kieselgel 60F254 (Merck,
A.G., Darmstadt, Germany) and the spots were de-
tected by UV light.
Silica gel column chromatography was performed
on Kieselgel 60 (230–400 mesh). Extraction in usual
manner refers to washing the organic layer with wa-
ter, drying it over anhydrous magnesium sulphate,
and evaporating the solvent under reduced pressure.
Dimethylformamide (DMF) was distilled immedi-
ately before use over CaH2. All solvents were pur-
chased from Carlo Erba Reagents. Reagent grade
materials were purchased from Aldrich Chemical Co.
and from Fluka and were used without further
purification.
m.p. 150–151 °C. 1H-NMR (CDCl3, l, ppm): 12.35 (br,
1H, OH); 8.02 (s, 1H, ArNH); 7.47 (d, 1H, J=2.9 Hz,
ArH); 7.09 (dd, 1H, J=2.9 Hz, ArH); 6.90 (d, 1H,
J=7.9 Hz, Ar-H); 6.73 (br, 1H, CONH); 3.39 (q, 2H,
J=6.9 Hz, CH2NH); 2.15 (s, 3H, COCH3); 1.57 (q, 2H,
J=6.9 Hz, CH2CH2NH); 1.38 (m, 2H, J=7.9 Hz,
CH2CH3); 0.94 (t, 3H, J=6.9 Hz, CH2CH3). Anal.
(C13H18N2O3): C, H, N.
6.1.3. 5-Acetamide-N-butyl-2-(2-propynyloxy)
benzamide (4a) and 5-acetamide-N-butyl-2-
(3-methyl-2-butenyloxy) benzamide (4b)
Sodium metal (1.75 g, 76.5 mmol) was dissolved in
warm anhydrous isopropanol (40 mL). To this freshly
made solution was added 3 (9 g, 38.2 mmol) and
propargyl bromide or isopenthenyl bromide (49.7
mmol). The resulting reaction mixture was stirred under
reflux overnight. The sodium bromide was filtered off
and the solvent was removed in vacuum. Recrystallisa-
tion of the crude product from diethyl ether gave,
respectively, 4a or 4b as a white solid.
6.1. Chemistry
6.1.1. 5-Acetamide-2-hydroxy-benzoic acid (2)
6.1.4. 5-Amino-N-butyl-2-(2-propynyloxy) benzamide
(5a)
To a solution of 5-amino-salicylic acid (15 g, 98.0
mmol) (1) in water (40 mL) heated at 50 °C, was added
acetic anhydride (11.25 mL, 120.0 mmol). After the
mixture was stirred for 15 min, the insoluble residue
containing compound 2 was filtered and washed with ice
water. The residue was crystallised from water to yield 2
(15 g, 79%) as a white solid, m.p. 229–230 °C. 1H-
NMR (CDCl3, l, ppm): 9.28 (br, 1H, OH); 8.10 (br, 1H,
CONH); 8.08 (d, 1H, J=2.9 Hz, ArH); 7.61 (dd, 1H,
J=2.9 Hz, ArH); 6.91 (d, 1H, J=7.9 Hz, ArH). Anal.
(C9H9NO4): C, H, N.
A mixture of 4a (2.02 g, 7.0 mmol) and 4 N sulphuric
acid solution (30 mL) was heated at 95 °C for 2 h. The
reaction solution was cooled to ambient temperature
and the pH adjusted to 10 with 2 N NaOH solution.
The resulting white precipitate was filtered off, washed
with water, and dried. Recrystallisation from diethyl
ether afforded 1.5 g (90%) of the pure product 5a as a
white solid.
6.1.5. 5-Amino-N-butyl-2-(3-methyl-2-butenyloxy)
benzamide (5b)
6.1.2. 5-Acetamide-N-butylsalicylamide (3)
A mixture of 4b (10 g, 31 mmol) and 3 N NaOH
solution (60 mL) was heated at 95 °C for 2 h. The
reaction solution was cooled to ambient temperature.
The resulting white precipitate was filtered off, washed
with water, and dried. Recrystallisation from diethyl
ether afforded 5.5 g (65%) of the pure product 5b as a
white solid.
To a solution of 2 (15 g, 76.9 mmol) in DMF (50 mL)
was added 1-hydroxybenzotriazole (HOBt) (11.28 g,
83.5 mmol) and N,N%-dicyclohexylcarbodiimide (DCC)
(17.21 g, 83.5 mmol) in the above order at 0 °C. After
the mixture was stirred for 30 min, n-butylamine (7.6
mL, 76.9 mmol) was added. The resulting reaction
mixture was stirred at 0 °C for 2 h and overnight at
room temperature (r.t.). N,N%-Dicyclohexylurea (DCU)
was filtered off and the DMF was evaporated in vac-
uum. The residue was dissolved in CH2Cl2, washed
consecutively with brine, 1 N NaOH, brine, 1 N HCl
and brine. The organic phase was dried over magnesium
sulphate, filtered, and evaporated to dryness. The ob-
tained residue was purified by chromatography on a
silica gel column (elution with diethyl ether–ethanol
(8:2), v/v) to yield pure 3 (15.6 g, 86%) as a white solid,
6.1.6. 5-Methanesulfonamide-N-butyl-2-(2-propynyloxy)
benzamide (6a) and 5-methanesulfonamide-N-butyl-2-
(3-methyl-2-butenyloxy) benzamide (6b)
To a solution of 5a or 5b (5.4 mmol) and pyridine (0.5
mL) in toluene (30 mL) was added dropwise methane-
sulfonyl chloride (0.4 mL, 5.0 mmol). The resulting
reaction mixture was stirred at 70 °C for 4 h and then
cooled to ambient temperature and concentrated in

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G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
vacuum to afford a solid. The crude residue was dis-
solved in CH2Cl2, washed with brine and the organic
phase was dried over magnesium sulphate and then
concentrated in vacuum. The crude products were fur-
ther purified by chromatography on a silica gel column
(elution with diethyl ether–ethanol (8:2), v/v) followed
by recrystallisation from diethyl ether–ethanol to give,
respectively, 6a (68%) or 6b (65%) as white solid.
m.p. 64–65 °C. 1H-NMR (CDCl3, l, ppm): 9.09 (d, 1H,
J=2.9 Hz, ArH); 8.44 (d, 1H, J=7.9 Hz, ArH); 7.88
(br, 1H, CONH); 7.05 (dd, 1H, J=2.9 Hz, ArH); 4.62
(m, 1H, cyclohexyl); 3.49 (q, 2H, J=6.9 Hz, CH2NH);
2.10–1.85 (m, 8H, cyclohexyl); 1.67 (q, 2H, J=6.9 Hz,
CH2CH2NH); 1.58 (m, 2H, cyclohexyl); 1.48 (m, 2H,
J=7.9 Hz, CH2CH3); 0.98 (t, 3H, J=6.9 Hz,
CH2CH3). Anal. (C11H23N2O4): C, H, N.
Using the procedure described for preparing 9a the
following compounds were prepared: 2-phenyloxy-5-ni-
tro-N-butylbenzamide (9b), 2-p-fluorophenyloxy-5-ni-
tro-N-butylbenzamide (9c), 2-p-chlorophenyloxy-5-
nitro-N-butylbenzamide (9d), and 2-p-isopropylpheny-
loxy-5-nitro-N-butylbenzamide (9e) (yields ranging be-
tween 70 and 80%). Similar 1H-NMR data occur in
intermediates 9b–e and were consistent with the pro-
posed structures.
6.1.7. 2-Chloro-5-nitro-N-butylbenzamide (8)
To a solution of 2-chloro-5-nitrobenzoic acid (7) (20
g, 99.0 mmol) in anhydrous DMF (150 mL) was added
HOBt (14.7 g, 109 mmol) and DCC (22.5 g, 109 mmol)
in the above order at 0 °C. The resulting reaction
mixture was stirred for 30 min and then n-butylamine
(10.8 mL, 109 mmol) was added. The reaction mixture
was stirred at 0 °C for 2 h and overnight at r.t. N,N%-
Dicyclohexylurea (DCU) was filtered off and the DMF
was evaporated in vacuum. The residue was dissolved in
CH2Cl2, washed consecutively with brine, 1 N NaOH,
brine, 1 N HCl and brine. The organic phase was dried
over magnesium sulphate, filtered, concentrated in vac-
uum and the residue was purified by chromatography
on a silica gel column (elution with diethyl ether–
petroleum ether (7:3), v/v). Recrystallisation by diethyl
ether–ethanol afforded 19.6 g (77%) of pure 8 as a
6.1.9. 2-Cyclohexyloxy-5-amino-N-butylbenzamide
(10a)
To a suspension of Pd/C (0.15 g) in 15 mL of water
was added a suspension (20 mL) of NaBH4 (1.0 g, 26.4
mmol) in water. The resulting reaction mixture was
stirred at r.t. under nitrogen atmosphere for 10 min.
Then, 9a (2.89 g, 8.1 mmol) in 100 mL of methanol was
added dropwise and the mixture was stirred for 30 min.
The reaction mixture was filtered through a Celite bed.
The solution was acidified with 1 N HCl to remove the
excess of NaBH4. The acidified solution was adjusted to
alkaline pH with 2 N NaOH solution and the resulting
suspension was extracted with diethyl ether. The organic
phase was dried over magnesium sulphate, filtered, and
evaporated to dryness to yield intermediate 10a as a
1
yellow solid, m.p. 134 °C. H-NMR (CDCl3, l, ppm):
8.46 (d, 1H, J=2.9 Hz, ArH); 8.19 (dd, 1H, J=2.9 Hz,
ArH); 7.59 (d, 1H, J=7.9 Hz, ArH); 6.23 (br, 1H,
CONH); 3.48 (q, 2H, J=6.9 Hz, CH2NH); 1.89 (q, 2H,
J=6.9 Hz, CH2CH2NH); 1.44 (m, 2H, J=7.9 Hz,
CH2CH3); 0.98 (t, 3H, J=6.9 Hz, CH2CH3). Anal.
(C11H13ClN2O3): C, H, Cl, N.
1
white solid (2.7 g, 87%). H-NMR data were reported as
6.1.8. 2-Cyclohexyloxy-5-nitro-N-butylbenzamide (9a)
A solution of sodium hydride (60% in mineral oil,
3.35 g, 84.0 mmol) in dry dioxane (150 mL) was added
to cyclohexanol (7.91 g, 79.0 mmol) in an ice-water bath
under nitrogen atmosphere. After the evolution of hy-
drogen had ceased the mixture was stirred at 70 °C for
2 h. Then intermediate 8 (19.6 g, 76.4 mmol) in anhy-
drous dioxane (100 mL) was added dropwise and the
resulting reaction mixture was stirred at 70 °C for 4 h
and overnight at ambient temperature. The solvent was
removed under reduced pressure and the residue was
dissolved in ethyl acetate and washed with brine. The
organic phase was dried over magnesium sulphate,
filtered, concentrated in vacuum and the residue purified
by chromatography on a silica gel column (elution with
diethyl ether–hexane (6:4), v/v) to give 9a (9.4 g, 75%),
hydrochloride salts.
Using the procedure described for preparing 10a the
following compounds were prepared: 2-phenyloxy-5-
amino-N-butylbenzamide (10b), 2-p-fluorophenyloxy-5-
amino-N-butylbenzamide (10c), 2-p-chlorophenyloxy-5-
amino-N-butylbenzamide (10d), and 2-p-isopro-
phylphenyloxy-5-amino-N-butylbenzamide (10e) (yields
ranging between 65 and 70%). Similar H-NMR data
occur in intermediates 10b–e and were consistent with
the proposed structures.
1
6.1.10. 5-Amino-N-butyl-2-cyclohexyloxy-benzamide
hydrochloride (11a)
Free base 10a (2.5 g, 8.6 mmol) was dissolved in
ethanol (50 mL) and treated with an excess of diethyl
ether saturated with dry, gaseous HCl. Recrystallisation

G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
529
from diethyl ether–ethanol (8:2, v/v) provides 11a (2.5
g, 89%) as a white solid.
sulfonamide-N-butyl-2-(4-fluorophenyloxy)benzamide
(13c), 5-methanesulfonamide-N-butyl-2-(4-chloropheny-
loxy)benzamide (13d), and 5-methanesulfonamide-N-bu-
tyl-2-(4-isopropylphenyloxy)benzamide (13e).
Using the procedure described for preparing 11a the
following compounds were prepared: 5-amino-N-butyl-
2-phenoxy-benzamide hydrochloride (11b), 5-amino-N-
butyl-2-(4-fluorophenyloxy) benzamide hydrochloride
(11c), 5-amino-N-butyl-2-(4-chlorophenyloxy) benza-
mide hydrochloride (11d), and 5-amino-N-butyl-2-(4-
isopropylphenyloxy) benzamide hydrochloride (11e).
6.1.13. Determination of pKa values: general procedure
The pKa values of compounds 5b, 11a and 11b have
been evaluated by reading the pH at the midpoint (50%
neutralisation) of the plot versus millilitres of titrant.
The titrations have been performed potentiometrically
on the hydrochlorides of compounds 5b, 11a and 11b
with NaOH standard solution in water–ethanol (2:1,
v/v); pKa values were 4.20, 3.80 and 3.15, respectively.
6.1.11. 5-Acetamide-N-butyl-2-cyclohexyloxy-benzamide
(12a)
To a solution of amine 10a (2.71 g, 7.0 mmol) in
anhydrous DMF (70 mL) was added acetic anhydride
(0.75 mL, 8.0 mmol). The resulting reaction mixture was
stirred at r.t. for 30 min and then the solvent removed in
vacuum. The crude residue was dissolved in CH2Cl2,
washed with 0.5 N NaOH and brine. The organic layer
was dried over magnesium sulphate, filtered, and con-
centrated in vacuum. The crude residue was purified by
chromatography on a silica gel column (elution with
diethyl ether–hexane (5.5:4.5), v/v). Recrystallisation
from diethyl ether–ethanol provides 1.6 g (71%) of 12a
as a white solid.
Using the procedure described for preparing 12a the
following compounds were prepared: 5-acetamide-N-bu-
tyl-2-phenoxy-benzamide (12b), 5-acetamide-N-butyl-2-
(4-fluorophenyloxy) benzamide (12c), 5-acetamide-N-
butyl-2-(4-chlorophenyloxy) benzamide (12d), and 5-
acetamide-N-butyl-2-(4-isopropylphenyloxy) benzamide
(12e).
6.2. Enzyme assay
6.2.1. Pure ovine COX-1 and COX-2 enzyme assay
Purified ovine COX-1 and COX-2 were obtained from
Cayman Chemicals (Ann Arbor, MI). Pure enzymes and
co-factors adrenaline (5 mM) and hematin (1 mm) were
dissolved in 50 mM Tris-buffer (pH 7.5). Hematin was
first dissolved as a concentrated stock of 100 mM in 1
M NaOH before being further diluted in Tris-buffer.
Enzymatic reactions were carried in individual wells of
96-well plates with a final reaction volume of 200 mL.
Drugs were diluted in Tris-buffer to obtain different
concentrations (10−11, 10−10, 10−9, 10−8, 10−7, 10−6
,
10−5, 10−4 M) and were added to the plate, followed by
co-factors and 10 U mL−1 of the enzyme (180 mL). The
plates were incubated at 37 °C for 30 min before addi-
tion of AA (10 mM). The reaction was stopped after a
further 15 min by the addition of 10 mL 1 M HCl, then
the pH 7.5 was balanced with 10 mL 1 M NaOH. By
measuring the formation of PGE2, a non-enzymatic
metabolite of PGH2, the activity of the enzymes and the
inhibitory effects of different compounds were assessed.
The content of PGE2 in the incubates was measured by
using radioimmunoassay. Data are presented as a per-
centage of control PGE2 production. The inhibition
graphs for each COX inhibitor were plotted as a mean
S.E.M. and were constructed from data collected on at
least four experimental days. Data were analysed by
using a computerised software package PRISM 3.0
(Graphpad software, USA).
6.1.12. 5-Methanesulfonamide-N-butyl-2-
cyclohexyloxy-benzamide (13a)
To a solution of 10a (2.71 g, 7.0 mmol) and pyridine
(0.6 mL) in toluene (60 mL) was added dropwise
methanesulfonyl chloride (0.9 mL, 7.6 mmol) in 10 mL
of toluene. The resulting reaction mixture was stirred at
70 °C for 5 h. After cooling at ambient temperature the
solvent was removed in vacuum. The crude residue was
dissolved in CH2Cl2, washed with brine and the organic
phase was dried over magnesium sulphate and concen-
trated in vacuum. The obtained residue was purified by
chromatography on a silica gel column (elution with
diethyl ether–hexane (5.5:4.5), v/v). Recrystallisation
from diethyl ether–ethanol affords 1.52 g (59%) of pure
13a as a white solid.
6.2.2. Carrageenin footpad edema
Male Wistar rats (120–140 g) were injected in the
right hind paw with 0.1 mL of a 1% solution of l-car-
rageenin in saline. Compounds were suspended in car-
boxymethylcellulose 0.5% and administered orally 1 h
Using the procedure described for preparing 13a the
following compounds were prepared: 5-methanesulfon-
amide-N-butyl-2-phenoxy-benzamide (13b), 5-methane-

530
G. Caliendo et al. / European Journal of Medicinal Chemistry 36 (2001) 517–530
prior to carrageenin injection. Paw volume was mea-
sured by using a hydroplethysmometer (Ugo Basile,
Italy) immediately before carrageenin injection and at
hourly intervals for 5 h. Control animals received an
equal volume of vehicle. Results were analysed by
ANOVA followed by Dunnett’s test.
Acknowledgements
The work was supported by a grant from Fundac¸a˜o
de Amparo a Pesquisa do Estado de Sa˜o Paulo
(FAPESP).
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