Original article

1,8-Naphthyridines V. Novel N-substituted5-amino-N,N-diethyl-9-isopropyl [1,2,4]triazolo[4,3-a]

[1,8]naphthyridine-6-carboxamides, as potent anti-inflammatoryand/or analgesic agents completely devoid of acute gastrolesivity

Giancarlo Grossi a, Mario Di Braccio a, Giorgio Roma a,*, Vigilio Ballabeni b,Massimiliano Tognolini b, Elisabetta Barocelli b

a Dipartimento di Scienze Farmaceutiche, Università di Genova, Viale Benedetto XV, 3, 16132 Genova, Italyb Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy

Received 16 July 2004; accepted 27 September 2004

Available online 22 December 2004

Abstract

Most N,N-disubstituted 5-amino-N,N-diethyl-9-isopropyl [1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamides 9 (compounds 9a, c–i)and the N-monosubstituted one 8c were obtained by treating with excess amine the corresponding 5-chloroderivative 7a, which was in turnprepared by cyclocondensation of the 2,4-dichloro-N,N-diethyl-1,8-naphthyridine-3-carboxamide (4a) with isobutyrohydrazide. Compounds8a,b and 9b,j–m were obtained according with the methods shown in Scheme 1. The above now synthesized compounds, along with thepreviously described 8d and 8e, were tested for their anti-inflammatory, analgesic and antipyretic properties, and most compounds also fortheir effect on spontaneous mice locomotor activity and their acute gastrolesivity in rats. Several compounds showed potent anti-inflammatoryand/or analgesic activities, and all the compounds tested proved to be completely lacking in acute gastrolesivity. In many cases compounds 8and 9 produced hypothermic effect, usually at high doses. On the whole, the N-monosubstituted 5-aminoderivatives 8 appeared to be morepotent anti-inflammatory agents than the corresponding N,N-disubstituted 9, whereas these latter compounds exhibited higher analgesicactivity.© 2004 Elsevier SAS. All rights reserved.

Keywords: [1,2,4]Triazolo[4,3-a] [1,8]naphthyridines; Anti-inflammatory; Analgesic; Gastrolesivity; Structure–activity relationships

1. Introduction

The search for new anti-inflammatory agents devoid of thelimitations and side effects of the classical non steroidal anti-inflammatory drugs (NSAIDs) has significantly increased inrecent years.

In the course of our previous studies on the design andsynthesis of novel anti-inflammatory agents in the 1,5-benzodiazepine and 1,8-naphthyridine structural fields, thebest pharmacological results were shown by tricyclic com-pounds resulting from the fusion of 1,2,4-triazole ring withthe proper bicyclic moieties. Actually, on one hand, starting

from the inactive 1,5-benzodiazepines 1, the substituted 4H-[1,2,4]triazolo[4,3-a] [1,5]benzodiazepin-5-amines 2 wereobtained (Fig. 1), some of which endowed with appreciableand statistically significant anti-inflammatory and/or analge-sic properties: these compounds showed in mice low acutetoxicity and, contrary to classical NSAIDs, no acute gastrole-sivity (at the 400 mg kg–1 os dose) [1–3].

* Corresponding author. Tel.: +39 10 353 8374; fax: +39 10 353 8358.E-mail address: roma@unige.it (G. Roma).

Fig. 1. Structures of 1,5-benzodiazepine derivatives 1 and 4H- [1,2,4]tria-zolo[4,3-a] [1,5]benzodiazepine derivatives 2.

European Journal of Medicinal Chemistry 40 (2005) 155–165

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0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.ejmech.2004.09.022

On the other hand, among the 1,8-naphthyridine deriva-tives 3–6, previously synthesized by us, some compounds 4and 5 exhibited significant anti-inflammatory activity and/oranti-aggressive properties, depending on the structure [4,5](Fig. 2). Considering these and the above described results,we more recently prepared the substituted [1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamides 7 [6] and 8 [7] withthe aim of separating the anti-inflammatory from anti-aggressive activity, and/or obtaining more potent anti-inflammatory compounds (Fig. 2). Indeed, chloroderivatives7 proved to be devoid of anti-aggressive activity, but only afew of them showed statistically significant anti-inflamma-tory properties [6], whereas many of the aminoderivatives 8exhibited potent anti-inflammatory activity sometimes accom-

panied, only at high doses, by notable anti-aggressive prop-erties [7].

On the basis of the very interesting pharmacological prop-erties of compounds 8, we have now regarded it useful towiden our knowledge of structure–activity relationships(SAR) in this structural field by synthesizing and testing anumber of compounds 9, in whose position 5 a N,N-disubstituted amino group replaces the N-monosubstituted oneof their analogs 8, thus eliminating the possible formation ofhydrogen bond between the carbonyl and amino groups. The6- and 9-substituents of compounds 9a–m now prepared aresteadily the diethylcarbamoyl and isopropyl groups, respec-tively, which proved to be the most effective ones for the anti-inflammatory activity of compounds 8 [7]. Also few furthercompounds 8 (8a–c) have been now prepared as intermedi-ates of 9j,l,m, respectively, and tested for comparison (seeScheme 1).

The new substituted 5-amino [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides 8 and 9 described in thispaper have been tested for their analgesic, anti-inflammatoryand antipyretic properties, as well as for acute gastrolesivityand their effect on spontaneous mice locomotor activity, inorder to complete their in vivo pharmacological profile.Besides, aiming at effectively comparing the pharmacologi-cal properties of compounds 8 and 9, also 8d and 8e, recentlydescribed by us as potent anti-inflammatory agents [7], havenow been submitted to this wide pharmacological evaluation

Fig. 2. Structures of 1,8-naphthyridine-3-carboxamide derivatives 3–6 and[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide derivatives 7–9.

Scheme 1. Synthetic routes to the substituted 5-amino [1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamides 8a–c and 9a–m.

156 G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

under the experimental conditions reported in the presentpaper.

2. Chemistry

In order to obtain satisfactory yields of appreciably purecompounds 9a–m, different synthetic routes were used,depending on the structure of their N,N-disubstituted 5-aminogroup (Scheme 1).

Thus, compounds 9a,c–i (whose 5-amino groups are “sym-metrically” disubstituted) were obtained by treating the cor-responding 5-chloroderivative (7a) with an excess of theproper amine (120–140 °C; in a closed vessel for compounds9a,c). The starting compound 7a was prepared by cyclocon-densation of 2,4-dichloro-N,N-diethyl-1,8-naphthyridine-3-carboxamide (4a) [4] with isobutyrohydrazide (155 °C).

Due to the unsatisfactory results afforded by the treatmentof 7a with diethylamine under various experimental condi-tions, compound 9b was prepared through a three-step pro-cedure, starting from the treatment with excess diethylamineof the more active bicyclic 4-chloroderivative 3a [4] (140 °C,in a closed vessel) to afford the corresponding 4-(diethyl-amino)derivative 10 in high yield. By heating compound 10in refluxing phosphorus oxychloride, the 2-chloro-4-(diethylamino)-N,N-diethyl-1,8-naphthyridine-3-carboxamide (11) was then obtained, which in turn under-went cyclocondensation with isobutyrohydrazide (155 °C) togive the desired 5-(diethylamino)-N,N-diethyl-9-isopropyl[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide (9b).

On the other hand, compounds 9j,l,m,k were preparedthrough the N-methylation with CH3I (80 °C; in the presenceof anhydrous KOH/K2CO3) of the correspondingN-monosubstituted 5-aminoderivatives 8a–d, respectively.The starting compounds 8a,b were obtained by the cyclocon-densation of the corresponding bicyclic chloroderivatives 6a[5], 6b [4], respectively, with isobutyrohydrazide (155 °C),and 8c from the reaction of 7a with excess aniline (160 °C),whereas compound 8d was previously described by us [7].

The results of elemental analyses, and IR and 1H-NMRspectral data are consistent with the structures attributed tothe compounds described in this paper (see Section 5.1 andTable 2), and the spectral data agree with the ones previouslyreported by us for analogous compounds [4–7].

For instance, as we previously remarked for compounds 7[6] and 8 [7], also the patterns of N-CH2

1H-NMR signals(CDCl3) of the now described compounds 9a–m clearly indi-cate that frequently the methylene protons are chemical shiftnonequivalent and coupled with each other, thus suggestingthe chirality of these molecules (atropisomerism about theirhindered heteroaryl-carbonyl bond). In such compounds alsothe CH3 groups of CH–(CH3)2 moieties are not chemical shiftequivalent and give two doublets at different d values as1H-NMR signals (e.g. see compounds 7a, 8a, and 9j). Con-cerning then the IR spectra (KBr) of the N,N-disubstituted5-aminoderivatives 9, the low frequencies of the tertiary

amides m CO bands (1620–1638 cm–1) can be attributed tothe conjugation of the carbonyl group with the 5-amino sub-stituent. The further lowering of m CO band frequency shownby the N-monosubstituted 5-aminoderivatives 8a–c (m CO1606–1614 cm–1) is reasonably attributable to the additionalpresence of intermolecular H-bonding between the CO andNH groups.

3. Pharmacological results and discussion

Compounds 8a–e and 9a–m were tested in vivo for theiranalgesic, anti-inflammatory and antipyretic activities at theinitial dose of 200 mg kg–1 os. Compounds exhibiting a sta-tistically significant activity at this dose were further tested atdoses decreasing by a factor of two, until statistically signifi-cant activity was no longer observed. For most compoundsalso the acute gastrolesivity in rats (at the 100 mg kg–1 dose)and the effect on spontaneous mice locomotor activity (atproperly chosen doses) were evaluated after oral administra-tion (see Section 5.2). The pharmacological results obtainedare listed in Table 1.

Except for the 5-[(N-methyl,N-phenyl)amino]derivative9m, all the above compounds at the 200 mg kg–1 dose (inhi-bition range 57–100%), and 12 of them at the 100 mg kg–1

dose (inhibition range 58–100%), showed statistically signifi-cant antinociceptive activity in the acetic acid induced writh-ing test in mice. The most potent compound, i.e. the 5-(4-methyl-1-piperazinyl)derivative 9h, produced 92% and 85%inhibitions (P < 0.01) at the 12.5 and 3.12 mg kg–1 doses,respectively (indomethacin: 84% inhibition, P < 0.01, at the10 mg kg–1 dose [3]).

The anti-inflammatory activity (carrageenan induced ratpaw edema assay) proved to be statistically significant, at the200 mg kg–1 dose, for 13 of the 18 compounds tested (edemainhibition range 22–60%), and still at the 25 mg kg–1 dose forcompounds 8a, 8d, 8e and 9l (edema inhibition range41–55%). The N-monosubstituted 5-aminoderivatives 8a, 8dand 8e (i.e. the most interesting anti-inflammatory agents inthis structural class) produced a statistically significant pro-tection down to the 6.25 mg kg–1 dose (35%, 35% and 32%,respectively). The most potent of them, i.e. the 5-(ethyl-amino)derivative 8e, showed a 48% edema inhibition(P < 0.01) at the 12.5 mg kg–1 dose (indomethacin: 51%,P < 0.01, at the 10 mg kg–1 dose [3]).

Only compounds 8b, 9l and 9g showed statistically sig-nificant antipyretic activity in rats (LPS induced fever test),but the 5-(cyclopropylamino)derivative 8b (67% protection,P < 0.05, at the 100 mg kg–1 dose) and the 5-[(N-cyclopropyl,N-methyl)amino]derivative 9l (98% protection,P < 0.01, at the 50 mg kg–1 dose) produced hypothermic effectat higher doses. Only the 5-(1-piperazinyl)derivative 9g regu-larly showed antipyretic activity from the 200 mg kg–1 dose(100% protection, P < 0.01) down to the 50 mg kg–1 dose(23% protection), without any hypothermic effect. Actually,10 further compounds at the 200 mg kg–1 dose, and some ofthem also at lower doses, exhibited hypothermic effect.

157G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

Table 1Structures and pharmacological data of compounds 8a–e and 9a–m

Compound NHR′ Dose(mg kg–1 os)

Analgesicactivitya (%inhibition)

Anti-inflammatoryactivityb

(% inhibition)

Antipyreticactivityc

(% inhibition)

Effect on micelocomotoractivityd

(% change)

Acutegastrolesivityin ratse

8a – 200 85**f 42** #g

100 82** 45** # –97** 0/8

50 38 42** 6

25 – 41** –

12.5 – 35** –

6.25 – 35** –

3.12 – 0 –

8b – 200 84** 45** #

100 82** 39** 67* +38 0/8

50 26 10 12

8c – 200 100** 24 28

100 12 – – – –

8dh – 200 100** 59** #

100 100** 57** # –18 0/8

50 33 58** #

25 – 46** #

12.5 – 34** #

6.25 – 35* 9

3.12 – 21 –

8eh – 200 100** 60** #

100 100** 58** # –9 0/8

50 20 59** #

25 – 55** #

12.5 – 48** 21

6.25 – 32* –

3.12 – 18 –

9a – N(CH3)2 200 92** 54** #

100 41 48** # – 0/8

50 – 43** 27

25 – 12 –

9b – N(C2H5)2 200 100** 42** #

100 83** 46** 15 –32 0/8

50 61* 14 –

25 29 – –

9c – N(C2H4OH)2 200 81** 21 #

100 35 – 11 – –

9d – 200 57** 34** #

100 58* 19 0 –35 0/8

50 0 – –

(continued on next page)

158 G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

Table 1(continued)

Compound NHR′ Dose(mg kg–1 os)

Analgesicactivitya (%inhibition)

Anti-inflammatoryactivityb

(% inhibition)

Antipyreticactivityc

(% inhibition)

Effect on micelocomotoractivityd

(% change)

Acutegastrolesivityin ratse

9e – 200 84** 27 0

100 16 – – – –

9f – 200 79** 22* 0

100 69** 15 – –38 0/8

50 0 – –

9g – 200 95** 39* 100**

100 100** 0 53** –92** 0/8

50 63** – 23 –48

25 28 – –

9h – 200 86** 34** 41

100 88** 23 – –94** 0/8

50 85** – –

25 90** – –

12.5 92** – –

6.25 83** – –

3.12 85** – – –3

1.6 39* – –

0.8 3 – –

9i – 200 88* 24 #

100 20 – 26 – –

9j – 200 81** 59** #

100 78** 10 # –3 0/8

50 83** – 0

25 16 – –

9k – 200 86** 40** #

100 89** 44** # –57 0/8

50 50** 25** #

25 0 14 3

9l – 200 93** 37** #

100 96** 39** # –78* 0/8

50 32 48** 98**

25 – 42** 20

12.5 – 29* –

6.25 – 14 –

9m – 200 0 0 18 – –

Indomethacin 10 84**i 51**i 93**i – 7/8

Diazepam 10 – – – –98** –a Acetic acid induced writhing in mice.b Carrageenan induced rat paw edema.c LPS induced fever in rats.d Activity counted in 30 min.e Number of rats showing gastric lesions.f * P < 0.05, ** P < 0.01 significance as compared to controls (Student’s t-test).g Detection of hypothermic effect.h Previously synthesized by us, Ref. [7].i Data from Ref. [3].

159G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

The notable anti-aggressive activity showed in mice bysome previously described compounds 8 [7] has now inducedus to verify the possible connection between the analgesicand CNS depressant properties in this structural class, by aproper test. Thus, compounds 8 and 9 which exhibited statis-tically significant antinociceptive activity in the writhing testat the 100 mg kg–1 dose were evaluated, at the same dose, fortheir effect on mice spontaneous locomotor activity. Fourcompounds (8a, 9g, 9h, 9l) of the 12 tested produced a sta-tistically significant sedative effect (range of the spontaneousmotility change: –97%, –78%), whereas all the remainingcompounds did not practically modify the mice spontaneousmotility. Furthermore, only the results afforded by two of theabove four compounds (8a and 9l), suggested the occurrenceof a seeming antinociception in the writhing test (see Table 1).Actually, when 9g and 9h were tested, for their effect on loco-motor activity, at the lowest dose (50 and 3.12 mg kg–1,respectively) at which they showed highly significant(P < 0.01) antinociceptive activity (63% and 85% inhibition,respectively), these compounds did not significantly reducethe mice spontaneous motility (–48% and –3%, respec-tively).

Considering the above results, it seems rather unlikely thatthe antinociceptive activity of compounds 8 and 9 can mainlyderive from their CNS depressant properties.

Finally, it must be pointed out that none of the 13 com-pounds endowed with statistically significant anti-inflammatory activity displayed any acute gastrolesivity inrats at the 100 mg kg–1 dose. As regards the safety profile ofthe compounds 8 and 9 now studied, we can further remarkthat no lethal toxic effect was produced by them, up to thehighest dose administered (200 mg kg–1 os), in the animalstreated.

4. Conclusions

From the pharmacological data reported in Table 1 for com-pounds 8a–e and 9a–m the following conclusions can bedrawn.

Compounds 8 and 9 represent an interesting class of potentanalgesic and/or anti-inflammatory agents endowed with asatisfactory safety profile.

On the whole, the N-monosubstituted 5-aminoderivatives8 appear to be more potent as anti-inflammatory agents, whilethe N,N-disubstituted ones 9 show higher analgesic activity.The 5-(ethylamino)derivative 8e and the 5-(4-methyl-1-piperazinyl)derivative 9h proved to be very potent anti-inflammatory and analgesic agents, respectively, completelydevoid of acute gastrolesivity (see the data of 8e, 9h andindomethacin in Table 1).

The hypothermic effect frequently produced by these com-pounds is generally absent at low doses. Thus, it does notinterfere with the notable activities shown at these doses bythe most potent analgesic and/or anti-inflammatory agents.

The total absence of acute gastrolesivity shown in rats bythe compounds studied may suggest that they do not affect

the activity of the cyclooxygenase enzyme. The investigationon the action mechanism of these compounds is currently inprogress.

5. Experimental

5.1. Chemistry

Melting points (m.p.) were determined using a Fisher–Johns apparatus and are uncorrected. IR spectra were recordedon a Perkin–Elmer “Spectrum One” spectrophotometer(abbreviations relative to IR bands: br = broad, s = strong, w= weak, sh = shoulder). 1H-NMR spectra were recorded on aVarian Gemini 200 (200 MHz) spectrometer, using (CH3)4Sias an internal reference (d = 0), and chemical shifts arereported in ppm. Spin multiplicities are given as follows: s(singlet), d (doublet), dd (double doublet), t (triplet), q (quar-tet), m (multiplet). Analyses of all new compounds, indicatedby the symbols of the elements, were within ±0.4% of thetheoretical values and were performed by the Laboratorio diMicroanalisi, Dipartimento di Scienze Farmaceutiche, Uni-versity of Genoa. Thin layer chromatograms were run onMerck silica gel 60 F254 precoated plastic sheets (layer thick-ness 0.2 mm). Column chromatography was performed usingCarlo Erba silica gel (0.05–0.20 mm) or Carlo Erba neutralaluminum oxide (Brockmann activity I).

5.1.1. General procedure for the synthesis of compounds7a, 8a,b

A mixture of 6.0 mmol of 4a [4] (1.79 g), 6a [5] (1.91 g),or 6b [4] (2.01 g), 12.0 mmol (1.22 g) of isobutyrohydrazideand 10 ml of Dowtherm A was stirred at 155 °C for 20 min.After cooling, 10% aqueous Na2CO3 (50 ml) and dichlo-romethane (50 ml) were added and the mixture was furtherstirred at room temperature for 30 min. After discarding someinsoluble impurities by filtration, the mixture was transferredin a separatory funnel, then the organic layer was collectedand the aqueous one was exhaustively extracted with dichlo-romethane. The combined organic phases were dried (anhy-drous Na2SO4) then evaporated to dryness in vacuo, and theresidue was subjected to column chromatography (silica gelfor 7a; neutral aluminum oxide for 8a,b), in all cases elutingfirst with dichloromethane in order to remove Dowtherm A.The reaction products were then recovered eluting with ethylacetate (compound 7a) or with the mixture ethylacetate/tetrahydrofuran (1:1) (compounds 8a,b). The eluatecollected, after removal of solvents gave an oily or solid resi-due from which compounds 7a, 8a, or 8b were obtained asbelow described.

5.1.1.1. 5-Chloro-N,N-diethyl-9-isopropyl [1,2,4]triazolo-[4,3-a] [1,8]naphthyridine-6-carboxamide (7a). The thickoil obtained from the reaction performed with 4a, treated witha little isopropyl ether afforded the nearly pure 7a as a pink–orange crystalline solid (1.14 g, 55%); white crystals, m.p.

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153–154 °C, after crystallization from ethyl acetate/petroleum ether with charcoal. 1H-NMR (CDCl3): d 1.18 and1.38 [2t, 3H + 3H, N(CH2CH3)2], 1.53 and 1.60 [2d, 3H +3H, 9-CH(CH3)2], 3.31 [q, 2H, 2H of N(CH2CH3)2], 3.56–3.88 [m, 2H, 2H of N(CH2CH3)2], 4.49 [m, 1H, 9-CH(CH3)2],7.62 (dd, J3,4 = 8.1 Hz, J3,2 = 4.7 Hz, 1H, H-3), 8.55 (dd, J4,3

= 8.1 Hz, J4,2 = 1.7 Hz, 1H, H-4), 8.78 (dd,J2,3 = 4.7 Hz, J2,4 = 1.7 Hz, 1H, H-2); IR (KBr): 1638 s (CO),1602, 1590, 1556 w, br, 1520 w cm–1. Anal. (C17H20ClN5O)C, H, N, Cl.

5.1.1.2. 5-(Butylamino)-N,N-diethyl-9-isopropyl [1,2,4]tria-zolo[4,3-a] [1,8]naphthyridine-6-carboxamide (8a). The oilyresidue deriving from the reaction carried out with 6a, treatedwith a little isopropyl ether, afforded pure 8a as pale yellowsolid (1.43 g, 62%); m.p. 180–181 °C after crystallizationfrom isopropyl ether. 1H-NMR (CDCl3): d 0.97 (t, 3H,NCH2CH2CH2CH3), 1.18 [t, 3H, 3H of N(CH2CH3)2], 1.26–1.52 [m, 8H, 3H of N(CH2CH3)2 + 3H of 9-CH(CH3)2 +NCH2CH2CH2CH3], 1.62 [d, 3H, 3H of 9-CH(CH3)2], 1.70–1.89 (m, 2H, NCH2CH2CH2CH3), 3.00 (m, 1H, 1H ofNCH2CH2CH2CH3), 3.30–3.65 [m, 4H, 3H of N(CH2CH3)2

+ 1H of NCH2CH2CH2CH3], 3.84 [m, 1H, 1H ofN(CH2CH3)2], 4.13 [m, 1H, 9-CH(CH3)2], 6.11 (m, 1H, NH;disappeared with D2O), 6.94 (dd, J3,4 = 8.3 Hz, J3,2 = 4.6 Hz,1H, H-3), 8.13 (dd, J4,3 = 8.3 Hz, J4,2 = 1.5 Hz, 1H, H-4),8.31 (dd, J2,3 = 4.6 Hz, J2,4 = 1.5 Hz, 1H, H-2); IR (KBr):3265 (NH), 1606 s, br (CO), 1548, 1520 cm–1. Anal.(C21H30N6O) C, H, N.

5.1.1.3. 5-(Cyclopropylamino)-N,N-diethyl-9-isopropyl[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide(8b). The whitish solid residue obtained from 6b was takenup in a little ethyl ether and filtered to give pure 8b (1.45 g,64%); after crystallization from tetrahydrofuran, white crys-tals, m.p. 198–199 °C, were obtained. 1H-NMR (CDCl3): d0.35–0.66 and 0.86 (2m, 2H + 2H, cyclopropyl CH2’s),1.26 and 1.35 [2t, 3H + 3H, N(CH2CH3)2], 1.37 and 1.65[2d, 3H + 3H, 9-CH(CH3)2], 3.07 (m, 1H, cyclopropyl CH),3.38 and 3.51–3.80 [2m, 1H + 3H, (NCH2CH3)2], 4.11 [m,1H, 9-CH(CH3)2], 6.85 (dd, J3,4 = 8.3 Hz, J3,2 = 4.6 Hz, 1H,H-3), 7.07 (s, 1H, NH; disappeared with D2O), 8.10 (dd,J4,3 = 8.3 Hz, J4,2 = 1.5 Hz, 1H, H-4), 8.28 (dd, J2,3 = 4.6 Hz,J2,4 = 1.5 Hz, 1H, H-2); IR (KBr): 3218 (NH), 1630 sh, 1609 s(CO), 1542, 1519 cm–1. Anal. (C20H26N6O) C, H, N.

5.1.2. N,N-Diethyl-9-isopropyl-5-(phenylamino)[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide(8c)

The mixture of 5.0 mmol (1.73 g) of 7a, 20 ml of aniline,and 20 ml of ethylene glycol was stirred at 160 °C for 24 h.After cooling, the nearly black solution obtained was pouredinto water (200 ml) and the resulting emulsion was exhaus-tively extracted with chloroform. The combined extracts (driedover anhydrous Na2SO4), after removal of solvent, gave adark, tarry oil which was dissolved in a little dichloromethane

and chromatographed on a silica gel column, eluting first withthe mixture dichloromethane/ethyl acetate (1:1) in order toremove the excess aniline. The elution was then pursued withthe mixture ethyl acetate/acetone (4:1). The eluate collectedwas decolorized with charcoal, then evaporated to dryness invacuo to give the nearly pure 8c as a yellowish amorphoussolid which was taken up in a little isopropyl ether and fil-tered (1.19 g, 59%); yellow crystals, m.p. 198–199 °C, aftercrystallization from isopropyl ether with charcoal. 1H-NMR(CDCl3): d 1.04 and 1.17 [2t, 3H + 3H, N(CH2CH3)2],1.57 and 1.59 [2d, 3H + 3H, 9-CH(CH3)2], 3.19–3.73 [m,4H, N(CH2CH3)2], 4.44 [m, 1H, 9-CH(CH3)2], 6.79 (near d,2H, phenyl H-2′,6′), 6.92 (near t, 1H, phenyl H-4′), 7.15–7.29 (m, 3H, phenyl H-3′,5′ + H-3), 7.32 (s, 1H, NH; disap-peared with D2O), 8.11 (dd, J4,3 = 8.3 Hz, J4,2 = 1.5 Hz, 1H,H-4), 8.60 (dd, J2,3 = 4.6 Hz, J2,4 = 1.5 Hz, 1H, H-2); IR(KBr): 3200 w, br (NH), 1614 s (CO), 1599, 1534, 1497 cm–1.Anal. (C23H26N6O) C, H, N.

5.1.3. Synthesis of 5-(dialkylamino)-N,N-diethyl-9-isopropyl [1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamides 9a,c–i

Compounds 9a,c–i were prepared by the reaction of the5-chloroderivative 7a with an excess of the proper aminethrough the below described experimental procedures,depending on the amine used.

5.1.3.1. Compound 9a. A mixture of 5.0 mmol (1.73 g) of 7aand 25 ml of 30% dimethylamine in anhydrous ethanol washeated at 120 °C in a closed vessel for 8 h. After cooling, theresulting solution was evaporated to dryness in vacuo and theresidue was partitioned between 10% aqueous Na2CO3

(100 ml) and dichloromethane (100 ml). The organic layerwas collected and the aqueous one was further extracted twicewith dichloromethane. The combined organic phases weredried (anhydrous Na2SO4), then evaporated to dryness invacuo to give a thick oil from which, after treatment with alittle ethyl ether, pure 9a separated out as a whitish solid whichwas then crystallized from ethyl acetate/petroleum ether.

5.1.3.2. Compound 9c. A mixture of 5.0 mmol (1.73 g) of 7aand 10 ml of diethanolamine was heated at 140 °C in a closedvessel for 16 h. After cooling, the viscous mixture obtainedwas dissolved in a little ethanol and poured into water(100 ml): the mixture was then subjected to an exhaustiveextraction with chloroform/isopropanol (9:1). The combinedextracts were dried (anhydrous Na2SO4) and evaporated todryness in vacuo: the resulting thick oil was chromato-graphed on a silica gel column, eluting first with ethyl acetateto recover a moderate amount of starting compound 7a(0.42 g). The fraction subsequently collected by elution withacetone, after removal of solvent, afforded pure 9c as a yel-lowish solid which was then crystallized from ethylacetate/isopropyl ether to give pale yellow crystals.

5.1.3.3. Compounds 9d–i. A mixture of 5.0 mmol (1.73 g) of7a, 5 ml of the proper liquid dialkylamine (pyrrolidine, pip-

161G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

eridine, 1-methylpiperazine or morpholine) or 5 g of solidamine (4-hydroxypiperidine or piperazine), and 7 ml of dim-ethyl sulfoxide was stirred at 130 °C for 2 h. The orange solu-tion obtained was then poured into water (200 ml) and theresulting emulsion was exhaustively extracted with dichlo-romethane. The combined extracts, dried (anhydrous Na2SO4)and evaporated to dryness in vacuo, afforded an oily or nearlysolid residue from which, after treatment with a little ethylether or isopropyl ether and standing, the expected com-pound 9 separated out as a pink–orange solid. After crystal-lization from the proper solvent with charcoal, whitish crys-tals were obtained.

Data for compounds 9a,c–i are reported in Table 2.

5.1.4. General procedure for the N-methylationof compounds 8a–d to compounds 9j,l,m,k

To the suspension of 3.0 mmol of 8a (1.15 g), 8b (1.13 g),8c (1.21 g), or 8d (1.15 g) [7] in 80 ml of 2-butanone, 24 mmol(1.34 g) of finely powdered KOH mixed with 2.0 g of anhy-drous K2CO3 were added, then the mixture was refluxed(80 °C) for 5 min. A solution of 4.0 mmol (0.25 ml) ofiodomethane in 10 ml of 2-butanone was then slowly addedand the resulting mixture was further refluxed for 1 h, withstirring. After cooling, the solvent was removed in vacuo andthe residue was partitioned between water (200 ml) anddichloromethane (200 ml). The organic layer was collectedand the aqueous one was further extracted twice with dichlo-romethane. The combined organic phases were dried (anhy-drous Na2SO4), then evaporated to dryness in vacuo to givean oil which was chromatographed on a silica gel columneluting first with ethyl acetate. The first fraction of this elu-ate, containing some impurities, was discarded. Compound9m (prepared starting from 8c) was recovered pursuing theelution with ethyl acetate, whereas compounds 9j,l,k (pre-pared starting from 8a,b,d, respectively) were recovered elut-ing then with the mixture ethyl acetate/acetone (4:1). The elu-ates collected, evaporated to dryness in vacuo, gave oilyresidues from which, after treatment with a little isopropylether, pure compounds 9j–m separated out as whitish or paleyellow solids which were then crystallized from the suitablesolvents.

Data for compounds 9j–m are reported in Table 2.

5.1.5. 4-(Diethylamino)-N,N-diethyl-1,2-dihydro-2-oxo-1,8-naphthyridine-3-carboxamide (10)

A mixture of 15.0 mmol (4.20 g) of 4-chloro-N,N-diethyl-1,2-dihydro-2-oxo-1,8-naphthyridine-3-carboxamide (3a) [4],10 ml of diethylamine and 20 ml of ethanol was heated at140 °C in a closed vessel for 16 h. After cooling, the resultingsolution was evaporated to dryness in vacuo and the residuewas partitioned between 5% aqueous NaHCO3 (100 ml) anddichloromethane (100 ml). The organic layer was collectedand the aqueous one was further extracted twice with dichlo-romethane. The combined organic phases, dried (anhydrousNa2SO4) and evaporated to dryness in vacuo, gave a solidresidue which was taken up in a little ethyl acetate and fil-

tered to give pure compound 10 (4.04 g, 85%); white crys-tals, m.p. 226–227.5 °C, after crystallization from ethylacetate. 1H-NMR (CDCl3): d 1.06–1.40 [m, 12H,N(CH2CH3)2 + CON(CH2CH3)2], 3.04–3.56 and 3.79 [2m,7H + 1H, N(CH2CH3)2 + CON(CH2CH3)2], 7.16 (dd,J6,5 = 8 Hz, J6,7 = 4.8 Hz, 1H, H-6), 8.13 (dd, J5,6 = 8 Hz,J5,7 = 1.6 Hz, 1H, H-5), 8.72 (dd, J7,6 = 4.8 Hz, J7,5 = 1.6 Hz,1H, H-7), 12.11 (s, 1H, NH; disappeared with D2O); IR (KBr):3450 w and 3220–2530 (NH), 1650 s and 1633 s (CO), 1595 s,1547 cm–1. Anal. (C17H24N4O2) C, H, N.

5.1.6. 2-Chloro-4-(diethylamino)-N,N-diethyl-1,8-naphthyridine-3-carboxamide (11)

A mixture of 6.5 mmol (2.06 g) of compound 10 and 20 mlof phosphorus oxychloride was refluxed (110 °C) for 6 h,with stirring. After cooling, the excess phosphorus oxychlo-ride was removed in vacuo and the sticky residue was dis-solved in ice-cold water; after careful addition of excessNaHCO3, the mixture was exhaustively extracted with dichlo-romethane. The combined extracts were dried (anhydrousNa2SO4), then evaporated to dryness in vacuo: the resultingthick oil was chromatographed on a neutral aluminum oxidecolumn eluting with dichloromethane/ethyl acetate (1:1). Theeluate collected, after removal of solvents, gave an yellowthick oil from which, after treatment with a little isopropylether and standing, pure compound 11 separated out as anyellowish solid (1.14 g, 52%); pale yellow needles, m.p. 110–111 °C, after crystallization from isopropyl ether. 1H-NMR(CDCl3): d 1.10–1.38 [m, 12H, N(CH2CH3)2 +CON(CH2CH3)2], 3.12–3.72 [m, 8H, N(CH2CH3)2 +CON(CH2CH3)2], 7.44 (dd, J6,5 = 8.5 Hz, J6,7 = 4.2 Hz, 1H,H-6), 8.44 (dd, J5,6 = 8.5 Hz, J5,7 = 2 Hz, 1H, H-5), 9.02 (dd,J7,6 = 4.2 Hz, J7,5 = 2 Hz, 1H, H-7); IR (KBr): 1635 s (CO),1594, 1555 s cm–1. Anal. (C17H23ClN4O) C, H, N, Cl.

5.1.7. 5-(Diethylamino)-N,N-diethyl-9-isopropyl[1,2,4]triazolo[4,3-a] [1,8]naphthyridine-6-carboxamide(9b)

A mixture of 6.0 mmol (2.01 g) of compound 11,12.0 mmol (1.22 g) of isobutyrohydrazide and 10 ml ofDowtherm A was stirred at 155 °C for 30 min. After cooling,the reaction was worked up exactly as those for the prepara-tion of compounds 7a, 8a,b: the oily residue finally obtainedwas subjected to chromatography on a neutral aluminumoxide column, eluting first with dichloromethane in order toremove Dowtherm A, then with ethyl acetate. The eluate col-lected, after removal of solvent, afforded an oil which wastreated with a little isopropyl ether to give the nearly purecompound 9b as a pink–orange crystalline solid (0.99 g, 43%);pale-yellow crystals, m.p. 122–123 °C, after crystallizationfrom isopropyl ether with charcoal. 1H-NMR (CDCl3): d1.09–1.39 [m, 12H, N(CH2CH3)2 + CON(CH2CH3)2],1.50 and 1.58 [2d, 3H + 3H, 9-CH(CH3)2], 3.08–3.88 [m,8H, N(CH2CH3)2 + CON(CH2CH3)2], 4.45 [m, 1H,9-CH(CH3)2], 7.47 (dd, J3,4 = 8.1 Hz, J3,2 = 4.7 Hz, 1H, H-3),8.48 (dd, J4,3 = 8.1 Hz, J4,2 = 1.7 Hz, 1H, H-4), 8.66 (dd,

162 G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

Table 2Physical and chemical data of compounds 9a,c–m

Compound Yield(%)

M.p. (°C)(solvent)a

Molecularformulab

IRc (cm–1) 1H-NMRd (d, ppm)

9a 85 181–182 (A) C19H26N6O 1632 s (CO),1601, 1585,1552, 1515 w

1.25 and 1.34 [2t, 3H + 3H, N(CH2CH3)2], 1.48 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 2.96 [s, 6H, N(CH3)2], 3.30–3.78 [m, 4H, N(CH2CH3)2], 4.43 [m, 1H, 9-CH(CH3)2],7.47 (dd, 1H, H-3), 8.42 (dd, 1H, H-4), 8.67 (dd, 1H, H-2)

9c 25 180–181 (B) C21H30N6O3 3346 s, br (OH),1620 s (CO),1599, 1586,1549, 1518 w

1.26 and 1.36 [2t, 3H + 3H, N(CH2CH3)2], 1.52 and 1.58[2d, 3H + 3H, 9-CH(CH3)2], 2.19e (broad s, 1H, OH), 3.20–4.09 [m, 12H, N(CH2CH2OH)2 + N(CH2CH3)2], 4.42 [m,1H, 9-CH(CH3)2], 5.86e (broad s, 1H, OH), 7.48 (dd, 1H,H-3), 8.37 (dd, 1H, H-4), 8.69 (dd, 1H, H-2)

9d 71 133–134 (A) C21H28N6O 1638 s (CO),1602, 1584,1550, 1510 w

1.22 and 1.34 [2t, 3H + 3H, N(CH2CH3)2], 1.49 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 2.00 (m, 4H, pyrrolidineb-CH2’s), 3.12–3.26 (m, 2H, 2H of pyrrolidine a-CH2’s),3.37–3.77 [m, 6H, 2H of pyrrolidine a-CH2’s +N(CH2CH3)2], 4.42 [m, 1H, 9-CH(CH3)2], 7.47 (dd, 1H,H-3), 8.38 (dd, 1H, H-4), 8.67 (dd, 1H, H-2)

9e 79 161–163 (A) C22H30N6O 1638 s (CO),1602, 1586,1550, 1510 w

1.24 and 1.34 [2t, 3H + 3H, N(CH2CH3)2], 1.48 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 1.60–1.98 (m, 6H, piperidine b+ c-CH2’s), 2.88-3.82 [m, 8H, piperidine a-CH2’s +N(CH2CH3)2], 4.44 [m, 1H, 9-CH(CH3)2], 7.50 (dd, 1H,H-3), 8.43 (dd, 1H, H-4), 8.68 (dd, 1H, H-2)

9f 84 221–221.5(C)

C22H30N6O2 3338 s, br (OH),1623 s (CO),1600, 1584,1546, 1515 w

1.25 and 1.34 [2t, 3H + 3H, N(CH2CH3)2], 1.49 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 1.66–1.93 (m, 2H, 2H of pipe-ridine b-CH2’s), 1.96–2.24 (m, 3H, 2H of piperidineb-CH2’s + OH; 2H after treatment with D2O), 2.90–3.95[m, 9H, N(CH2CH3)2 + piperidine a-CH2’s + piperidineCHOH], 4.44 [m, 1H, 9-CH(CH3)2], 7.51 (dd, 1H, H-3),8.40 (dd, 1H, H-4), 8.68 (dd, 1H, H-2)

9g 75 186–187 (A) C21H29N7O 3307 (NH),1637 s (CO),1601, 1585,1552, 1514 w

1.25 and 1.35 [2t, 3H + 3H, N(CH2CH3)2], 1.49 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 1.94e (s, 1H, NH), 2.88–3.60[m, 10H, piperazine CH2’s + 2H of N(CH2CH3)2], 3.69 [q,2H, 2H of N(CH2CH3)2], 4.44 [m, 1H, 9-CH(CH3)2], 7.51(dd, 1H, H-3), 8.47 (dd, 1H, H-4), 8.69 (dd, 1H, H-2)

9h 78 172–173 (A) C22H31N7O 1634 s (CO),1602, 1586,1552, 1513 w

1.22 and 1.35 [2t, 3H + 3H, N(CH2CH3)2], 1.49 and 1.57[2d, 3H + 3H, 9-CH(CH3)2], 2.25–3.60 [m, 10H, piperazineCH2’s + 2H of N(CH2CH3)2], 2.38 (s, 3H, NCH3), 3.69 [q,2H, 2H of N(CH2CH3)2], 4.43 [m, 1H, 9-CH(CH3)2], 7.50(dd, 1H, H-3), 8.44 (dd, 1H, H-4), 8.69 (dd, 1H, H-2)

9i 80 182.5–183(A)

C21H28N6O2 1632 s (CO),1603, 1584,1550, 1510 w

1.24 and 1.36 [2t, 3H + 3H, N(CH2CH3)2], 1.49 and 1.58[2d, 3H + 3H, 9-CH(CH3)2], 2.78–4.02 [m, 12H, morpho-line CH2’s + N(CH2CH3)2], 4.44 [m, 1H, 9-CH(CH3)2],7.50 (dd, 1H, H-3), 8.46 (dd, 1H, H-4), 8.70 (dd, 1H, H-2)

(continued on next page)

163G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

J2,3 = 4.7 Hz, J2,4 = 1.7 Hz, 1H, H-2); IR (KBr): 1638 s (CO),1598, 1583, 1546, 1511 w cm–1. Anal. (C21H30N6O) C, H, N.

5.2. Pharmacology

Wistar rats (250–300 g) and Swiss mice (25–35 g) of eithersex were used. The test compounds were suspended in 0.5%methylcellulose and administered at the initial dose of200 mg kg–1 by oral gavage (1 ml/100 g body weight) in ani-mals fasted overnight. Compounds that exhibited a statisti-cally significant effect at this dose were further tested at dosesdecreasing by a factor of two. In these experimental tests forthe evaluation of analgesic, anti-inflammatory and anti-pyretic activity, indomethacin (10 mg kg–1 os) was used asreference drug. The same dose of indomethacin was used inthe test for the study of the acute gastrolesivity of the com-pounds under investigation (100 mg kg–1 os), while diaz-epam (10 mg kg–1 os) was the reference drug in the study ofthe effects of the new compounds (100 mg kg–1 os) on spon-taneous locomotor activity. Groups of 8–10 animals were used

and control animals were orally treated with an equivalentvolume of vehicle alone.

The ethical guidelines for investigation of experimentalpain in conscious animals were followed and all of the testswere carried out according to the EEC ethical regulation (EECCouncil 86/609; D.L.27/01/1992, No.116).

5.2.1. Anti-inflammatory activityAnti-inflammatory activity was studied by inducing paw

edema according to Winter’s method [8]. 1% Carrageenan(0.1 ml) was injected into the plantar surface of the rat hindpaw simultaneously with the oral administration of the testcompounds. Paw volume was determined immediately afterthe injection of phlogogen agent and again 3, 4, 5 h later bymeans of a plethysmometer.

5.2.2. Analgesic activityAntinociceptive activity was studied by writhing test [9],

through the intraperitoneal injection of 0.2 ml per mouse ofacetic acid solution (0.6%) 1 h after oral treatment with the

Table 2(continued)

Compound Yield(%)

M.p. (°C)(solvent)a

Molecularformulab

IRc (cm–1) 1H-NMRd (d, ppm)

9j 55 141–142 (D) C22H32N6O 1632 s (CO),1598 s, 1583,1548, 1514 w

0.92 (t, 3H, NCH2CH2CH2CH3), 1.19–1.42 (m, 2H,NCH2CH2CH2CH3), 1.25 and 1.33 [2t, 3H + 3H,N(CH2CH3)2], 1.49 and 1.58 [2d, 3H + 3H, 9-CH(CH3)2],1.68 (m, 2H, NCH2CH2CH2CH3), 2.90 (s, 3H, NCH3),3.02–3.84 [m, 6H, NCH2CH2CH2CH3 + N(CH2CH3)2],4.44 [m, 1H, 9-CH(CH3)2], 7.49 (dd, 1H, H-3), 8.44 (dd,1H, H-4), 8.68 (dd, 1H, H-2)

9k 65 145.5–147(E)

C22H32N6O 1637 s (CO),1602, 1585,1551, 1512 w

0.96 [d, 6H, NCH2CH(CH3)2], 1.27 and 1.34 [2t, 3H + 3H,N(CH2CH3)2], 1.49 and 1.57 [2d, 3H + 3H, 9-CH(CH3)2],2.07 [m, 1H, NCH2CH(CH3)2], 2.70 [near t, 1H, 1H ofNCH2CH(CH3)2], 2.88 (s, 3H, NCH3), 3.11 [m, 1H, 1H ofNCH2CH(CH3)2], 3.27–3.67 [m, 3H, 3H of N(CH2CH3)2],3.80 [m, 1H, 1H of N(CH2CH3)2], 4.44 [m, 1H,9-CH(CH3)2], 7.50 (dd, 1H, H-3), 8.50 (dd, 1H, H-4), 8.68(dd, 1H, H-2)

9l 66 198–199 (F) C21H28N6O 1632 s (CO),1599, 1585,1551, 1518 w

0.30–0.77 (m, 4H, cyclopropyl CH2’s), 1.29 and 1.31 [2t,3H + 3H, N(CH2CH3)2], 1.49 and 1.58 [2d, 3H + 3H,9-CH(CH3)2], 2.85 (m, 1H, cyclopropyl CH), 2.97 (s, 3H,NCH3), 3.21–3.42 [m, 1H, 1H of N(CH2CH3)2], 3.53–3.87[m, 3H, 3H of N(CH2CH3)2], 4.45 [m, 1H, 9-CH(CH3)2],7.46 (dd, 1H, H-3), 8.29 (dd, 1H, H-4), 8.67 (dd, 1H, H-2)

9m 58 177–178 (F) C24H28N6O 1623 s (CO),1600, 1588,1556

0.96 and 1.04 [2t, 3H + 3H, N(CH2CH3)2], 1.54 and 1.65[2d, 3H + 3H, 9-CH(CH3)2], 2.80–2.99 [m, 1H, 1H ofN(CH2CH3)2], 3.11–3.41 [m, 2H, 2H of N(CH2CH3)2], 3.37(s, 3H, NCH3), 3.63 [m, 1H, 1H of N(CH2CH3)2], 4.52 [m,1H, 9-CH(CH3)2], 6.68 (near d, 2H, phenyl H-2′,6′), 6.80(near t, 1H, phenyl H-4′), 7.19 (near t, 2H, phenyl H-3′,5′),7.43 (dd, 1H, H-3), 8.06 (dd, 1H, H-4), 8.72 (dd, 1H, H-2)

a Crystallization solvent: A = ethyl acetate/petroleum ether, B = ethyl acetate/isopropyl ether, C = ethyl acetate, D = isopropyl ether/ petroleum ether, E= petroleum ether, F = isopropyl ether.

b Anal. C, H, N.c In KBr pellets. Abbreviations: s = strong , w = weak, br = broad.d In CDCl3 solutions. Abbreviations: s = singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet. J values for H-2, H-3, H-4 signals

(dd) of all compounds : J2,3 = J3,2 = 4.7 Hz, J2,4 = J4,2 = 1.7 Hz, J3,4 = J4,3 = 8.1 Hz.e Disappeared with D2O.

164 G. Grossi et al. / European Journal of Medicinal Chemistry 40 (2005) 155–165

test drugs. Complete extension of either hind limb wasregarded as a writhing response. The number of writhings ofeach mouse was recorded every 5 min in the 45 min periodafter the injection of the noxious agent.

5.2.3. Antipyretic activityAntipyretic activity was determined in rats in which fever

was induced by intraperitoneal injection of 100 µg kg–1

Escherichia coli lipopolysaccharides (LPS) (Serotype0111:B4 Sigma, Milan, Italy), according to Romanovsky etal. [10]. The test compounds were administered orally 1 hbefore the injection, and rectal temperature was recorded inan air conditioned room (23 °C) immediately before and 3, 4,5, 6 h after pyretogen injection.

5.2.4. GastrolesivityThe acute gastric mucosal damage of the test compounds

(100 mg kg–1) and the reference drug indomethacin(10 mg kg–1) was evaluated by examining the stomachsexcised 5 h after oral administration of the drugs in rats. Thestomachs, fixed in 2% formalin, were opened and examinedwith a stereomicroscope by an observer unaware of the treat-ment the rats received. The presence of gastric lesions wasdetected by means of an image analyzer and consideredindicative of gastrolesivity.Acute gastrolesivity was expressedas the number of animals with gastric damage over the num-ber of treated animals.

5.2.5. Spontaneous locomotor activityThe test was performed after treatment of mice with

100 mg kg–1 of the compounds that proved to be significantlyeffective in writhing test at this dose, to verify the occurrenceof possible sedative effect confounding analgesia studies.Compounds 9g and 9h were also tested at the lowest dose atwhich they showed a statistically highly significant analgesicactivity (50 and 3.12 mg kg–1, respectively). Locomotor activ-ity was measured by means of an activity cage (height 35 cm,width 23 cm, depth 19 cm, Model 7401, Ugo Basile, Com-erio (VA), Italy). After oral administration of the compoundsunder study or vehicle, mice were placed singularly into theactivity cage and locomotor activity was automaticallyrecorded at time intervals of 5 min for 90 min. Mice sponta-neous motility was evaluated considering the final 30 minperiod. All experiments were conducted from 9:00 to 13:00.

5.2.6. Data analysisResults were calculated as mean ± S.E.M. Differences

between treated and control groups were determined by Stu-dent’s t-test (* P < 0.05 or ** P < 0.01 being considered asstatistically significant or highly significant, respectively).

The pharmacological activities of the compounds wereexpressed as the percentage of inhibition calculated from thedifference between the responses of the treated and the con-trol groups at the time of the peak response to noxious stimuli.Indeed, the antiphlogistic activity was considered at 3 h fromphlogogen agent, the analgesic activity at 10–15 min fromalgogen agent, and antipyretic activity at 5 h from pyretogenagent administration.

Acknowledgements

The authors wish to thank Dr. G. Domenichini for skillfultechnical assistance in pharmacological experiments, O.Gagliardo for elemental analyses, F. Tuberoni for I.R. spec-tra and Dr. R. Raggio for 1H-NMR spectra. The financial sup-port from University of Genoa and University of Parma isgratefully acknowledged.

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