Benzodiazepine receptor ligands. 8: Synthesis and pharmacological evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide 3- and 8-disubstituted: High affinity ligands endowed with inverse-agonist pharmacological efficacy

Bioorganic & Medicinal Chemistry 14 (2006) 758–775

Benzodiazepine receptor ligands. 8: Synthesis and pharmacological

evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide

3- and 8-disubstituted: High aﬃnity ligands endowed with

inverse-agonist pharmacological eﬃcacy

Gabriella Guerrini,^a,*Annarella Costanzo,^aGiovanna Ciciani,^aFabrizio Bruni,^a

Silvia Selleri,^aCamilla Costagli,^aFranc¸ois Besnard,^bBarbara Costa,^cClaudia Martini,^c

Gaetano De Siena^dand Petra Malmberg-Aiello^d

a

`

Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via U. Schiﬀ,

6, 50019 Polo Scientiﬁco, Sesto Fiorentino, Firenze, Italy

b

´

Department of Molecular and Functional Genomics, Sanoﬁ-Synthelabo, 10 rue des Carrieres, 92500 Rueil-Malmaison, France

c

`

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita degli Studi di Pisa,

via Bonanno 6, 56126 Pisa, Italy

Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Universita degli Studi di Firenze,

Viale Pieraccini 6, 50139 Firenze, Italy

d

`

Received 4 May 2005; revised 25 August 2005; accepted 30 August 2005

Available online 7 October 2005

Abstract—The synthesis and the binding study of new 3-arylesters and 3-heteroarylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide

8-substituted are reported. The nature of these substituents (in terms of lipophilic and electronic features) seems to inﬂuence the

binding aﬃnity. High-aﬃnity ligands were studied in mice in vivo for their pharmacological eﬀects, considering six potential

benzodiazepine actions: anxiolytic-like eﬀects, muscle relaxant eﬀects, motor coordination, anticonvulsant action, spontaneous

motor activity, and ethanol-potentiating action. Compounds 4d and 6d showed an inverse-agonist proﬁle. These compounds were

evaluated also for their binding at benzodiazepine site on GABA_Areceptor complex (GABA_A/BzR complex) subtype to evaluate

their subtype selectivity.

1. Introduction

GABA_Areceptor complex composed of a-, b-, and c-

subunits, arranged in a pentameric structure.^1,2Sensitiv-

ity to Bz is conferred by the c-subunit (c₂genetic vari-

ant) and adjacent a-subunit (a₁, a₂, a₃, and a₅genetic

variants). The heterogeneity of GABA_A/BzR complex

subtypes associated with regionally distinct distribution

in the central nervous system has been indicated as the

major factor responsible for the therapeutic actions dis-

played by BZs.^3–6

The ability of benzodiazepines to strengthen the inhibi-

tory action of GABA in the brain oﬀers a clinical ther-

apy of primary importance in treating neurological

disorders such as anxiety, sleep disturbance, muscle

spasms and epilepsy. However, some side eﬀects limit

the clinical usefulness of BZs, such as memory impair-

ment, cognition and motor disturbances, potentiation

of the eﬀects of alcohol, tolerance, and physical depen-

dence. Classical benzodiazepines (Bz) act as positive

allosteric modulators, binding a speciﬁc site on the

Based on a vivo point mutation strategy recent studies⁷

have demonstrated that sedation, anterograde amnesia,

and part of seizure protection are mediated through

a₁-subtypes, anxiolysis through a₂-subtypes and muscle

relaxant eﬀects through a₂-, a₃-, and a₅-subtypes.

Keywords: GABA_A/BzR complex ligands; Pyrazolo[5,1-c][1,2,4]benzo-

triazines; Inverse-agonists; Tricyclic heteroaromatic system.

*

The search for subtype speciﬁc ligands is a promising

approach in order to clarify the pharmacological role

Corresponding author. Tel.: +39 055 4573766; fax: +39 055

4573671; e-mail: gabriella.guerrini@uniﬁ.it

doi:10.1016/j.bmc.2005.08.058

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

759

of subtype receptors and to obtain new drugs with

higher therapeutic selectivity and a reduced side-eﬀect

proﬁle. In light of this, our studies on pyrazolo [5,1-

c][1,2,4]benzotriazines have been directed toward the

search for new selective nonbenzodiazepine ligands at

the GABA_A/BzR complex with preferential aﬃnity or

preferential eﬃcacy.

In the present study, with the aim to investigate if chem-

ical modiﬁcation of the 8-substituent of the pyrazolo-

[5,1-c][1,2,4]benzotriazine 5-oxide system could yield

new ligands, with the same high aﬃnity but improved

eﬃcacy and receptor subtype selectivity as compared

to lead compounds 6 and 11, we replaced the chlorine

atom of molecules of two series, respectively, with sub-

stituents of diﬀerent lipophilic (p) and electronic (r) fea-

tures and bulkiness (Es),¹⁵leaving the favorable

substituent at the position 3 unchanged. Therefore, we

synthesized new derivatives of both series, bearing at po-

sition 8 bromine or iodine atoms (ﬂuorine atom has

resulted in being detrimental for binding¹⁶), a methyl-,

an ethoxy- a methoxy-, and a methylthio group. Some

3-arylester and 3-heteroaryl deoxy derivatives were also

synthesized to conﬁrm the diﬀerent role of a 5-oxide

group in the binding of ligands of two series, as previ-

ously evidenced.^9,10

Recently, we reported two new series of 8-chloropyraz-

olo[5,1-c][1,2,4]benzotriazine 5-oxide 3-arylester (A ser-

ies) and 3-heteroaryl derivatives (B series), respectively,

which showed high aﬃnity for BZR (K_i= 36.3–

1.00 nM range) (Chart 1).^8–11

For the sake of clarity, reference compounds listed in

Chart 1 follow the same numerical succession of newly

synthesized derivatives.

On the basis of structure–activity relationship (SAR)

data from numerous derivatives, it was proposed that

the 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine system,

the same in the two series (A and B), binds the GA-

BA_A/BzR complex through N1 and N4 atoms by means

of a hydrogen bond involving H₂and H₁donor receptor

sites for this class of ligands.¹²The presence at the posi-

tion 3 of a p-electron rich ring, providing a p–p interac-

tion with receptor protein, improves the aﬃnity of these

ligands in both series.^9,10The pharmacophoric descrip-

tors are in accordance with those of the model formulat-

ed by other researchers.^13,14

2. Chemistry

The crucial synthetic step to obtain the pyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide system is the intramolecu-

lar cyclization between the nitro and amino group under

basic conditions of the suitable 1-(2-nitrophenyl)-5-

aminopyrazole, following a procedure reported in our

previous papers.¹⁷The synthetic pathways which yielded

desired ﬁnal compounds are illustrated in Schemes 1 and

2; chemical and physical data of new compounds are

shown in Tables 7 and 8 in Section 6.

The lead compounds of two series, 3-(2-thienylmethoxy-

carbonyl)- and 3-(3-thienyl)-derivative, 6 and 11, respec-

tively, were found to have a selective in vivo activity. In

particular, the ester 6 shows anxiolytic-like activity sim-

ilar to diazepam without any sedative or amnesic prop-

erties, or interference from alcohol. The 3-heteroaryl

derivative 11 displays selective anticonvulsant proper-

ties. Nevertheless, compounds 6 and 11, tested for

binding at a_x(1-3)b₂c₂, a₅b₃c₂GABA_A/BzR complex sub-

types, do not display pronounced preferential subtype

selectivity.⁹Based on pharmacological results, it was

proposed that 6 and 11 are functionally selective ligands

with a mixed agonist/antagonist proﬁle for various

receptor subtypes.

In this work, the appropriate 3-carboxy-8-substituted

pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides (1a,b,c,¹⁷

d,¹⁷e, and f¹⁸see Table 7 in Section 6) were the starting

material for the synthesis of 3-ester derivatives. Acids 1a

and b, (Scheme 1) 8-bromo- and 8-iodio-substituted,

were achieved from the corresponding ethyl 1-(2-nitro-

5-bromophenyl)-, and the ethyl 1-(2-nitro-5-iodiophe-

nyl)-5-aminopyrazol-4-carboxylate Ia and b (see Table

8 in Section 6), which were, in turn, obtained from the

suitable

5-bromo-2-nitro-,¹⁹

or

5-iodio-2-nitro-

phenylhydrazine²⁰and ethyl 2-cyano-3-ethoxypropen-

eate in ethanol.¹⁷Acid 1e, 8-methoxy- was obtained

from 3-carboxy-8-chloropyrazolo[5,1-c][1,2,4]benzotri-

azine 5-oxide exploiting the aromatic nucleophilic sub-

stitution of chlorine atom, as reported to obtain 1d¹⁷

and 1f.¹⁸

R₃=COOCH₂-2-MeO-Phenyl

R₃=COOCH₂-2-thienyl

B Series

The desired ester derivatives (see Table 7 in Section 6

and Scheme 2) 2a–b, 3c, and 10c were prepared by treat-

ing the suitable 3-carboxylic acid (1a–c) with thionyl

chloride, and the intermediate 3-carbonyl chloride was

dissolved in 2-methyl-3-butene stabilized chloroform

and added to the suitable alcohol. All other esters 4a–

f, 5c, 6a–f, 7c, 8c, and 9c were obtained from acids

1a–f by treatment with triethyl amine/ethyl chlorocar-

bonate in tetrahydrofurane (THF) to achieve the not

isolated, mixed anhydride, which was reacted with suit-

able alcohol.

R₃= 3-thienyl 11 ¹⁰

R₃= 2-thienyl 12 ¹⁰

11

R₃= 3-furyl 13

To evaluate the inﬂuence on biological activity of the

8-substituent (bromine, iodine, methyl-, ethoxy-,

Chart 1. .

760

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

N

R₈

R₃

N

i

N

N⁺

O^-

R₄

N

R₅'

1a-c

R₃= COOH

NH₂

R₈

a = Br

NO₂

b = I

c = Me

R₃= 3-tienyl 11a-c

R₃= 2-tienyl 12c

Ia-b

R_5'a = Br

b = I

R₄= COOEt

R₄= 3-tienyl

R₄= 2-tienyl

R_5'= Cl

IIa-c

IIIc

c = Me

i

N

Cl

R₃

N

R₈

R₃

ii

N

N⁺

N

N⁺

O^-

1

R₃= COOH

R₃= 3-tienyl

R₃= 2-tienyl

R₃= COOH 1d-f

R₃= 3-tienyl 11d-g

R₃= 2-tienyl 12e-f

R₈

d = OEt

11

12

e = OMe

f = SMe

g = OH

Scheme 1. Reagents: (i) NaOH 10% solution; (ii) NaOH/EtOH (d compounds) or NaOH/MeOH (e compounds) or NaSMe/EtOH (f compounds) or

NaOH 40% (g compound). 1c,¹⁷1d¹⁷and 1f.¹⁸

N

R₈

R₃

N

R₈

R₃

i, ii

N

N⁺

O^-

N

N⁺

O^-

R₈a = Br

b = I

1a-f

R₃= COOH

c = Me

2a-d, 3c, 4a-f, 5c, 6a-f, 7c, 8c, 9c, 10c

R₃= COOR

d = OEt

e = OMe

f = SMe

N

R₈

I

R₃

iii, iv

N

N⁺

O^-

N⁺

O^-

13c-f

R₃= 2-furyl 14c-d

R₃= 3-furyl

16c-f

Scheme 2. Reagents: (i) SOCl₂; (ii) ROH for compounds 2a and b, 3c, and 10c. (i) NEt₃; (ii) ClCOOEt/THF, ROH for compounds 3c, 4a–f, 5c, 6a–f,

7c, 8c, and 9c. For compound 2c, see Ref. 18 and for 2d, see. Ref. 17 In compounds 2, R₃= COOEt; 3, R₃= COOCH₂Ph; 4, R₃= COOCH₂-2-

OMePh; 5, R₃= COOCH₂-2-NO₂Ph; 6, R₃= COOCH₂-2-tienyl; 7, R₃= COOCH₂-3-tienyl; 8, R₃= COOCH₂-2-furyl; 9, R₃= COOCH₂-3-furyl; 10,

R₃= COO-2-OMePh. (iii) Toluene, Tetrakis (Pd(PPh₃)₄); (iv) 2-furyl- or 3-furylboronic acid, Na₂CO₃2 M.

methoxy-, and methylthio-group) in the 3-heteroaryl

series, compounds 11a–g, 12c, 12e–f, 13c–f, and 14c–d

(Table 7 in Section 6 and Schemes 1 and 2 ) were synthe-

sized. Compounds 11a–c, 3-(thien-3-yl)- and 12c, 3-

(thien-2-yl)- were obtained reacting, as the ﬁrst reaction

step, the 5-bromo-2-nitro-,¹⁹5-iodio-2-nitro-,²⁰and

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

761

5-methyl-2-nitrophenylhydrazine¹⁸with 2-(thien-3-yl)-

and 2-(thien-2-yl)-3-oxapropanenitrile^21,22following a

reported method.¹⁰The obtained 4-(thien-3-yl)-5-amin-

opyrazoles IIa–c, and 4-(thien-2-yl)-5-aminopyrazole

IIIc (see Table 8 in Section 6) were then cyclized to pyr-

azolobenzotriazine 5-oxide system in alkaline medium.¹⁰

Compounds 11d–f, 8-ethoxy-, 8-methoxy-, and 8-meth-

ylthio derivative, were obtained in ethanol/sodium

hydroxide, methanol/sodium hydroxide, and ethanol/so-

dium thiomethoxide, exploiting the aromatic nucleophil-

ic substitution of the 3-(3-thienyl)-8-chloropyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxides.¹⁰The 8-hydroxy deriva-

tive 11g, isolated as a by-product in the 11d synthesis,

was also characterized and used for the SAR. Com-

pounds 12e–f were obtained from 3-(2-thienyl)-8-chloro-

Only the 8-bromo derivative 6a displayed an aﬃnity val-

ue twofold higher than that of reference compound 6⁹

(6a: K_i= 3.6 nM vs 6: K_i= 6.8 nM). The order of sub-

stituents on binding aﬃnity is Cl > Br > I. Therefore,

the substitution at the position 8 of the chlorine atom

of reference compounds (Chart 1, compounds 4⁸and

6,⁹) with bromine (2a, 4a, and 6a) or iodine (2b, 4b,

and 6b), more lipophilic atoms than chlorine (Br

p = 1.19, I p = 1.43 vs Cl p = 0.71),¹⁵slightly reduced

the BZR recognition of ligands (see Table 1). Taking

into account the similar electronic parameter of three

halogen atoms (Br r = 0.23, I r = 0.28, Cl r = 0.23)¹⁵

it seems reasonable to hypothesize that the lipophilic

and/or steric hindrance features (Br E_s= ꢀ1.16, I

E_s= ꢀ1.40, and Cl E_s= ꢀ0.97)¹⁵may play an important

role in anchoring ligands to receptor. A more lipophilic

atom could alter the right lipophilic/hydrophilic balance

of the whole ligand–receptor protein system; a bulkier

halogen would not permit the perfect interaction of

the molecule with receptor.

pyrazolo[5,1-c][1,2,4]benzotriazine

5-oxides¹⁰

and

methanol/sodium hydroxide or ethanol/sodium thio-

methoxide, respectively, in the same manner as com-

pounds 11e–f (Scheme 1).

The synthetic path to obtain the 8-methyl- and 8-ethoxy

derivatives bearing at the position 3 a furyl ring, 13c–f

and 14c–d (Scheme 2) exploited the palladium(0)cata-

lyzed cross-coupling reaction between 3-iodio-8-meth-

yl—16c, 3-iodio-8-ethoxy—16d, 3-iodio-8-methoxy—

16e, and 3-iodio-8-methylthio pyrazolo[5,1-c][1,2,4]ben-

zotriazine 5-oxide 16f (see below), and 3-furyl- and 2-

furylboronic acid.¹¹For this purpose, 3-iodo derivatives

16c–f were achieved from corresponding 8-substituted

pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide, 8-methyl-¹⁸

8-ethoxy-¹⁷8-methoxy-, 15e, and 8-methylthiopyrazol-

o[5,1-c][1,2,4]benzotriazine 5-oxide¹⁸by treatment with

iodine monochlorine (ICl) in chloroform following the

classical halogenation reaction. Compound 15e, 8-meth-

oxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide, was ob-

tained by decarboxylation from the corresponding 3-

carboxylic derivative 1e.

When the position 8 was substituted with a methyl- (3c,

4c, 6c, 7c, 8c, and 9c), an ethoxy group (4d, 6d), a meth-

oxy group (4e, 6e) or a methylthio group (4f, 6f), high

aﬃnity ligands were in general obtained (K_irange

0.31–9.5 nM). The exception of compounds 5c 3-(2-

nitrobenzyloxycarbonyl)- and 10c 3-(2-methoxyphen-

oxycarbonyl)-derivatives conﬁrms that when at the posi-

tion 3 there is an electron-poor ring such as 2-

nitrobenzyloxy-, or too brief a spacer such as 2-meth-

oxyphenoxy, complete loss of receptor recognition is ob-

tained.⁸In particular, 8-ethoxy derivatives (4d, 6d) and

8-methyl derivatives (6c, 7c, 8c, and 9c) show the best

aﬃnity values, 3-8-fold higher with respect to corre-

sponding 8-chlorosubstituted reference compounds (see

Table 1). In general, the order with which diﬀerent

groups inﬂuence the binding aﬃnity is EtO > Me >

MeS > MeO. A lipophilic value (p) slightly lower than

or comparable to that of chlorine (EtO p = 0.38, Me

p = 0.60 vs Cl p = 0.71)¹⁵supported by electronic fea-

tures (r) opposite of chlorine (EtO r = ꢀ0.24, Me

r = ꢀ0.17 vs Cl r = 0.23)¹⁵increases binding aﬃnity,

as in the case of compounds 4d, 6c, and d, or maintains

a good BzR aﬃnity (ꢁ2-fold lower than that of 8-chloro

derivative) as in the case of 4c. When the lipophilic (p)

and electronic (r) values are opposite of chlorine

(MeO p = ꢀ0.02 vs Cl p = 0.71; MeO r = ꢀ0.27 vs Cl

r = 0.23)¹⁵, the aﬃnity value decreases as in the case

of compounds 4e and 6e. The substitution of chlorine

with a thiomethyl group (MeS p = 0.60 vs Cl p = 0.71;

MeS r = 0.00 vs Cl r = 0.23)¹⁵as in compounds 4f

and 6f seems negligible.

The synthesis of the 5-deoxide derivatives 2bR, 3cR,

4aR, and 11cR (see Table 7) useful for structure–activity

relationships (SAR) was performed by treatment of cor-

responding 5-oxide compounds 2b, 3c, 4a, and 11c with

triethylphosphite (TEP).¹⁸

3. Biological results

The BZR binding aﬃnity of new 3-ester- and 3-heteroa-

ryl derivatives was evaluated by their ability to displace

[³H]ﬂumazenil ([³H]Ro15-1788) from its speciﬁc binding

in bovine brain membrane. Binding data for 3-ester-

(2a–b, 3c, 4a–f, 5c, 6a–f, 7c, 8c, 9c, and 10c), 3-heteroa-

ryl derivatives (11a–g, 12c, e–f, 13c–f, and 14c–d) and

corresponding 5-deoxide compounds (2bR, 3cR, 4aR,

and 11cR), and for reference compounds (see Chart 1),

useful for the SAR discussion, are reported in Tables 1

and 2, respectively.

As far as the 3-heteroaryl series (3-/2-tienyl, 3-/2-furyl) is

concerned (Table 2), in general good aﬃnity values are

shown (K_irange 10–37 nM). Also in this series the sub-

stitution of chlorine atom of position 8 with methyl-,

ethoxy-, and methoxy-electron-donating groups be-

comes favorable for binding (see compounds 11c, 11e,

13c–d, and 14c–d).

It appears from the results reported in Table 1 that the

introduction at the position 8 of the 3-ester derivatives

of a bromine (2a, 4a, and 6a) or iodine (2b, 4b, and

6b) yielded, in general, compounds with binding aﬃnity

lower than those of corresponding 8-chloroderivatives.

The lower aﬃnity binding of compound 11b, 8-iodine

derivative, than 8-chloro derivative could be due to the

762

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

Table 1. BZR ligand aﬃnity of pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 3-ester derivatives

N

R₈: a= Br

N

b= I

R₈

R₃

c=Me

d=OEt

e=OMe

f=SMe

N

X

Compound

R₃

R₈

X

I%^a

K_i^b(nM)

8-Cl^c

35

2a

2b

COOEt

Br

I

O

—

O

—

O

—

O

95

90

65.2 5.3

93.0

121 10

8

2bR

3c

COOEt

COOCH₂Ph

I

95

Me

Br

I

100

93

15

26.5

5

2

11.6

3cR

4a

3

0.3

COOCH₂-2-MeOPh

COOCH₂-2-NO₂Ph

COOCH₂-2-tienyl

COOCH₂-3-tienyl

COOCH₂-2-furyl

COOCH₂-3-furyl

COO-2-MeOPh

100

99

4 1.0

4aR

4b

7.3 0.5

10

100

98

1

4c

4d

Me

2.3 0.2

0.31 0.03

4.6 0.4

3.8 0.4

—

OEt

OMe

SMe

Me

Br

99

4e

4f

5c

100

61

6a

6b

99

3.6 0.3

11

6 6.8

I

100

99

2

6c

6d

Me

OEt

OMe

SMe

Me

1.41 0.2

0.85 0.05

9.5 1.0

4.0 0.7

3.97 0.4

4.57 0.5

4.39 0.6

—

100

99

6e

6f

7c

13.3

24.4

11.4

8c

9c

100

59

10c

Daz^d

Flu^d

10 1.1

0.9 0.5

^aPercent of inhibition of speciﬁc [³H]Ro15-1788 binding at 10 lM concentration are means SEM of ﬁve determinations.

^bK_ivalues are means SEM of ﬁve determinations.

^cThe ﬁrst column represents the number of reference compound 8-chloro derivatives; the second column represents K_ivalues (nM) of corresponding

3-aryl- heteroarylester-8-chloro derivatives: see Refs. 8–11,16,18.

^dSee Ref. 8.

diﬀerent dimension of iodine with respect to chlorine

(I E_s= ꢀ1.40, Cl E_s= ꢀ0.97).¹⁵

3.1. Binding of selected compounds at a_x(1,2,5)b₂c₂GA-

BA_A/BzR complex subtype

The signiﬁcant loss of aﬃnity of 11g (K_i= 690 nM),

bearing a hydroxyl group at the 8-position, conﬁrms

the unsuitable substitution with a hydrophilic group at

that position.¹⁸

Compound 4d and 6d, which showed a selective anxio-

genic activity (see Section 4), were tested, in comparison

with diazepam (full agonist) and zolpidem (a₁-selective

agonist), for their ability to displace [³H]Ro15-1788

from recombinant rat a_x(1,2,5)b₂c₂GABA_A/BzR com-

plex subtypes, which are stably expressed in human

embryonic kidney cells (HEK293). As can be observed

in Table 3, compound 4d displayed preferential subtype

selectivity a₁/a₂(K_i= 0.81 nM vs 70 nM) and no recog-

nition to a₅subtype receptor. Compound 6d recognized

all studied GABA_A/BzR complex subtypes and dis-

played preferential subtype selectivity a₁/a₂, a₅

(K_i= 2.3 nM vs 10 nM and 57 nM, respectively).

With regard to the importance of the 5-oxide, that

reinforces the hydrogen bond between N₄/H₂receptor

donor site,¹⁸it appears that in the 3-arylester series

the auxiliary role of the 5-oxide group to reinforce

binding is negligible because the p–p interaction

works together in a more signiﬁcant manner at

anchorage.⁹In fact, the aﬃnity values of 5-oxide

and 5-deoxide derivatives are comparable: 3cR

K_i= 26.5 nM vs 3c K_i= 15.2 nM, 4aR K_i= 7.3 nM

vs 4a K_i= 5 nM.

4. Pharmacological results

In the 3-heteroaryl series, instead, the decrease of

aﬃnity of 11cR, N-deoxy derivative, conﬁrms the need

Some of the newly synthesized molecules, 4d, 6a, c, d,

e, 7c (A series) 11a, c, and d (B series), were studied

in mice in vivo for their pharmacological eﬀects. Six

for

a 5-oxide group to recognize the receptor

protein.^9,10

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

763

Table 2. BZR ligand aﬃnity of pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 3-heteroaryl derivatives

N

R₈:

a= Br

b= I

c=Me

N

R₈

Het

d= OEt

e= OMe

f= SMe

N

X

Compound

Het

R₈

X

I%^a

K_i^b(nM)

8-Cl^c

11a

11b

11c

11cR

11d

11e

11f

3-Tienyl

2-Tienyl

3-Furyl

2-Furyl

Br

O

—

O

99

93

95

90

93

98

96

75

95

98

94

100

97

96

100

99

40

88

3

7

11 36.3

I

Me

16 1.7

318 20

35.9 0.27

Me

OEt

OMe

SMe

OH

Me

19.0

37

2

7

11g

12c

12e

12f

690 53

22

17

2

12 10.3

13 12

OMe

SMe

Me

56

13c

13d

13e

13f

13.2

12.6

27

2

OEt

OMe

SMe

Me

39

14c

14d

Daz^d

Flu^d

10.7

10.2

2

14 20

OEt

10 1.1

0.9 0.5

^aPercent of inhibition of speciﬁc [³H]Ro15-1788 binding at 10 lM concentration are means SEM of ﬁve determinations.

^bK_ivalues are means SEM of ﬁve determinations.

^cThe ﬁrst column represents the number of reference compound 8-chloro derivatives; the second column represents K_ivalues (nM) of corresponding

3-heteroaryl-8-chloro derivatives: see Refs. 11,12.

^dSee Ref. 9.

potential benzodiazepine actions were considered: even-

tual anxiolytic-like eﬀects were screened using light/

dark choice test, muscle relaxant eﬀects with grip-

strength meter test, and motor coordination with the

rota-rod test. The anticonvulsant action was evaluated

using the new drugs against pentylenetetrazole-induced

convulsions and the hole-board test was adopted to

discover eventual alterations in mouse spontaneous

motor activity caused by drugs which previously had

a bend on anxiogenic-like properties. Finally, the drugs

were tested also for their ethanol-potentiating action.

Diazepam was used as the positive reference molecule

(see Tables 4–6 and Fig. 1).

expected, diazepam (0.3, 1, and 3 mg/kg po) 30 min

after the treatment dose-dependently increased the

number of falls from rotating rod, reaching statistical

signiﬁcance at the dose of 3 mg/kg in comparison

with the vehicle-treated group of mice (Table 4).

None of the newly synthesized substances induced

any eﬀect on mice endurance on the rota rod, as

reported in Table 4. Only compounds 6e and 11a

at the highest dose used (30 mg/kg po) induced some

excitation in the mice, demonstrating to have some

motor disturbance.

4.2. Muscle relaxant eﬀects

In all the tests, only molecules 4d, 6d, e, and 11d (8-eth-

oxy- and 8-methoxy derivatives of A and B series) dem-

onstrated to have selective anxiogenic-like eﬀects.

Compounds 6a and 11a (8-bromo derivatives of A and

B series), and compounds 6c, 7c, and 11c (8-methyl

derivative ligands whose in vivo data are not reported)

endowed with high aﬃnity binding value (K_irange

1.41–40 nM) showed no or negligible pharmacological

action. These in vivo results, which seem to be in con-

trast with binding data, may be attributed to poor oral

bioavailability and/or to rapid metabolization.

We then wanted to study in more detail eventual

myorelaxant eﬀects. In the grip-strength meter test,

diazepam at the dosage of 3 mg/kg po induced a sta-

tistically signiﬁcant muscle relaxant eﬀect (Table 5).

Signiﬁcant eﬀect was observed also with compound

11a at both doses (10 and 30 mg/kg po). Compounds

4d, 6a, 6d, 6e, and 11d showed no pharmacological

eﬀect.

4.3. Eﬀects on spontaneous motor activity

A very common eﬀect of all the old benzodiazepine li-

gands is some degree of sedation. The hole-board test

was more sensitive, with respect to the two previous tests

(rota-rod and grip-strength meter test), in checking this

kind of behavior: diazepam already at the lower dosage

4.1. Eﬀects on motor coordination

First of all, we studied the eﬀects of the substances

on mouse performance in the rota-rod test. As

764

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

Table 3. Aﬃnity value at recombinant a_x(1,2,5)b₂c₂GABA_A/BzR complex subtypes

N

R₈

N

R₃

N

O

Compound

R₃

R₈

K_i(nM) or inhibition (I) %^a(lM)

a₁

a₂

a₅

4d

6d

COOCH₂-2-MeOPh

COOCH₂-2-tienyl

OEt

Cl

0.81

2.3

70

10

I% 25%

57

8.6

6^b

3.00

14.1 2.1

26.7

26

7.80 1.1

156

Daz^c

Zolpidem^d

9.80 1.1

>10000

^aK_ivalues represent means SEM derived from three independent experiments, conducted in triplicate; I% Percent of inhibition of speciﬁc

[³H]Ro15-1788 binding at 10 lM concentration are means SEM of ﬁve determinations.

^bSee Ref. 9.

^cSee Ref. 10.

^dSee Ref. 13.

Table 4. Motor coordination, anticonvulsant, and anxiolytic-like eﬀects of new compounds in comparison with those of diazepam

Treatment^a

mg/kg po

Motor coordination

rota-rod test

Anticonvulsant activity

Anxiety activity light–dark box

n

No. of falls in 30 s

Against PTZ-induced attacks (%)

n

No. transfer in 5⁰Time (s) in light in 5⁰

CMC 1%^b

Diazepam

0.1 ml

20

10

15

6

0.30 0.12

0.5 0.27

0.6 0.21

1.2 0.4^*

5

20

15.6 1.9

66.1 6.5

0.3

1

70^***

100^***

9

17

23.4 4.5^**

22.7 2.3^*

79.7 9.7

147.5 14.2^**

3

Flumazenil

4d

100

10

30

10

30

10

30

10

100

10

0.3

10

30

10

30

10

0.3

10

1

16

17.0 2.7

66.5 6.7

10

9

0.1 0.1

0.4 0.24

0.6 0.22

0.3 0.15

0.13 0.09

0.35 0.11

0

5

11

10

9

10.2 2.2

10.8 2.2

8.1 2.6

5.7 2.2^**

6.9 1.6^**

14.4 1.9

69.2 10.9

69.2 14.1

81.4 14.9

39.6 9.7^*

42.7 8.4

66.8 10.7

6a

10

11

10

20

6.6

17.6

6d

17

19

6d+Flu

6d+daz

6e

12

0.58 0.19

41.6

10

12

10

0.2 0.13

0.5 0.22^c

0.25 0.13

0.6 0.16^c

0.5 0.17

20

0

8

6.9 2.5^*

8.9 1.9^*

7.15 2.26

9.7 2.2

37.7 9.7^*

63.0 11.6

45.7 8.8

11a

16

0

13

10

69.7 11.78

11a+daz

11d

40

10

7.2 2.3

128.7 36.2

46.2 8.2

3

10

0.7 0.2

0.6 0.26

0.3 0.15

0

10

0

10

30

5

6.6 1.3^*

^aTreatment with new compounds and diazepam (po) was performed 30 min and ﬂumazenil (ip) 40 min before the test.

^bCarboxymethylcellulose 1%.

^cExcitation.

^*P < 0.05 versus cont rol mice.

^**P < 0.01 versus control mice.

^***P < 0.001 versus control mice.

of 1 mg/kg po diminished mouse movements on the

board signiﬁcantly and at the dose of 3 mg/kg also curi-

osity toward the holes.

Additional experiments were carried out for compounds

4d and 6d to evaluate their eﬀects on mice spontaneous

motor activity. Neither compound 4d nor 6d was found

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

765

Table 5. Myorelaxant activity of compounds 4d, 6a, 6d, 6e, 11a,

and11d in the grip-strength meter test

Treatment^a

mg/kg po

Myorelaxant activity

grip-strength meter

n

Test 35⁰(g)

CMC 1%

Diazepam

0.1 ml

0.3

1

16

5

64.5

1

66.0 3.1

61.9 1.74

44.2 4.4^***

64.6 1.9

63.2 2.5

60.5 2.3

62.9 2.3

64.2 1.5

63.9 1.4

63.1 2.3

60.4 1.7

57.6 3.0^*

56.0 3.4^*

63.3 1.5

59.2 1.4

11

13

10

11

10

13

10

8

3

4d

10

30

10

30

10

30

10

30

10

30

10

30

6a

6d

Figure 1. Eﬀect of compounds 4d and 6d (30 mg/kg) on ethanol-

induced sleeping time, in comparison with that of diazepam (po). Each

column represent the mean SEM of 10 mice. Substances were

administered 30 min before ethanol (4 g/kg ip). P < 0.001 (StudentÕs t

test).

6e

11a

11d

8

^aThe test was performed 30 and 20 min after administration of com-

pounds and diazepam (po), respectively.

^*P < 0.05 versus control mice.

4.4. Eﬀects on mouse anxiety

With the reference molecule, diazepam, at the dose of

3 mg/kg po, we observed a strong increase not only in time

spent in the lit compartment of the light/dark apparatus,

but the number of mouse transfers were augmented al-

ready at the dose of 1 mg/kg po, too. This molecule, at

the same doses, as demonstrated before (Tables 5 and

6), caused reduction of motor coordination, myorelax-

ation, and sedation, eﬀects also reported by many other

authors.^4,24Subsequently, the increased parameters in

the light/dark choice test should be considered reliable

indicators of anxiolytic-like behavior as suggested by

other authors.^25,26In the opposite manner, all the newly

synthesized compounds did not aﬀect the mouse sponta-

neousmotor activity (Table6), butin the light/dark choice

test the compounds 6d and e decreased the time spent in

the lit compartment and the number of transfers from

one compartment into the other, in a statistically signiﬁ-

cant way (Table 4), while the compounds 4d and 11d

(30 mg/kg po) behaved in the same manner but not statis-

tically signiﬁcantly, and the compounds 6a and 11a be-

came inactive. So, not only the decreased time, but also

the diminished number of transfers seems to indicate an

anxiogenic-like action of our molecules.^26,27The anxio-

genic-like eﬀect of 6d was completely antagonized by the

nonselective benzodiazepine antagonist, ﬂumazenil (Ta-

ble 4), conﬁrming the inverse agonist action of 6d on the

GABA_A/BzR complex. The activation of a₂subtype

receptors has been proposed as being responsible for the

anxiolytic-like eﬀect of benzodiazepine ligands.^28,29Since

compound 6d in the hole-board test and in PTZ-induced

convulsion test (see below) resulted to antagonize diaze-

pam eﬀects and then block a₁subtype receptor (for which

it has a very high aﬃnity), anxiogenic-like behavior seems

to depend on inverse antagonism for a₂subtype receptors,

for which the aﬃnity is also high (K_i= 10 nM).

^***P < 0.001 versus control mice.

Table 6. Eﬀect of compounds 4d and 6d on animal spontaneous

motility, in the comparison of diazepam, in the mouse hole-board test

Treatment^a

mg/kg po

Sedative activity

Hole board

n

Hole

Plane

CMC 1%

Diazepam

0.1 ml

21

10

11

8

49.8 3.1

39.7 4.2

23.4 6.3^**

55.5 2.9

58.8 6.2

51.8 5.0

135.9 7.6

96.9 10.3^*

59.7 8.6^***

155.8 11.6

159.2 13.1

106.1 9.1

1

3

4d

10

30

10

1

8

4d+daz

6d

8

3

8

13

9

58.3 4.3

58.3 4.4

56.8 4.7

52.8 6.6

154.0 9.2

135.5 10.4

154.0 10.9

125.4 9.3^{^}

10

30

10

0.3

6d+daz

10

^aThe test was performed 30 and 20 min after administration of com-

pounds and diazepam (po), respectively.

^*P < 0.05 versus control mice.

^**P < 0.01 versus control mice.

^***P < 0.001 versus control mice.

^{^}P < 0.05 versus diazepam 1 mg/kg treated mice (T_stest).

to be active (Table 6). For this reason, we decided to

evaluate the antagonistic activity of these two com-

pounds as they have very high aﬃnity values for GA-

BA_Areceptor a₁subtype (0.81 and 2.3 nM,

respectively). In fact, 4d (10 mg/kg po) diminished,

although not signiﬁcantly, the sedative eﬀect of diaze-

pam (1 mg/kg po) in this test, while the eﬀect of com-

pound 6d reached statistic signiﬁcance (Table 6); the

opinion that sedation is caused by activation of the GA-

BA_Areceptor a₁subtype was thus conﬁrmed.^6,23

4.5. Study against chemically induced convulsions

A further approach to study the newly synthesized

molecules was to investigate their potency against

766

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

pentylenetetrazole (PTZ)-induced convulsions. At the

dose used (90 mg/kg sc), PTZ caused convulsions in

95% of mice. Diazepam already at the dosage of

0.3 mg/kg po signiﬁcantly protected 70% of mice

from clonic generalized convulsions (Table 4), while

none, among the newly synthesized molecules, dif-

fered statistically from carboxymethylcellulose vehicle

in causing protection from convulsions. Compound

11a (30 mg/kg po) caused some excitation and no

protection from convulsions; it has been used as a

potential antagonist toward diazepam-induced protec-

tion and it actually partially antagonized diazepam-

induced protection, but not in a statistically signiﬁ-

cant way (Table 4). Compound 6d also behaved in

the same manner.

EtO- which has a r value opposite (r = ꢀ0.24),¹⁵the

high aﬃnity ligand 6d with preferential a₁/a₂subtype

selectivity and no recognition at a₅subtype receptor

was obtained.

The same 8-substitution in the A and B series com-

pounds yielded the ethoxy derivatives 4d, 6d, and 11d,

all showing inverse agonist proﬁle in vivo as anxiogenic

eﬀect, opposite the lead compounds (4, 6, and 11 in

Chart 1).^9,10Recently, it has been described

32,33

that

new molecules with a semirigid heterocyclic structure

bearing various ethereal groups are endowed with in-

verse-agonist eﬃcacy.

Taken together, the data above reported suggest that an

8-substituent with suitable lipophilic and steric features

supports the hydrophobic interaction of the 3-ligand

substituent with receptor protein in the lipophilic pocket

(L₁/L₂) inﬂuencing the aﬃnity binding; on the other

hand, dependent on its electronic parameter, the same

8-substituent is also capable per se of interacting with

Ôcritical residuesÕ^34,35on the same pocket of various sub-

type receptors, eliciting diﬀerent pharmacological

eﬀects.

4.6. Eﬀect on ethanol-induced sleeping time

The potency with which diazepam is able to potentiate

ethanol-induced sleeping time is surprising (Fig. 1). Al-

ready at the lowest dose, diazepam (0.3 mg/kg po),

together with ethanol (4 g/kg ip), caused a statistically

signiﬁcant additive action: six mice out of eight lost

the righting reﬂex for the entire 2.5-h observation time,

while the control mice slept only for 66.0 9.5 min.

Conversely, the eﬀect of 6d at the highest anxiogenic-like

dose was comparable to that of the control group and

the other compound, 4d, demonstrated no signiﬁcant

loss of the righting reﬂex, as well (Fig. 1). Ethanol-rein-

forcing actions are often connected to the activation of

the a₅subunit of the GABA_Areceptor.^30,31As far as

the a₅subunit is concerned, 6d binds to it with an inter-

mediate (K_i= 57 nM) and 4d with no aﬃnity. Also in

this case, if the additive eﬀects are assumed to be due

to activation of this subunit, 6d might behave on it like

antagonists.

These preliminary ﬁndings require further investigation

either through biological and electrophysiological stud-

ies or through the synthesis of new derivatives to con-

ﬁrm this binding hypothesis. In particular in this

paper, a new ester 6d endowed with inverse-agonist eﬃ-

cacy, opposite to reference compound 6 which showed

an anxiolytic proﬁle without side eﬀects, is identiﬁed.

This compound 6d causes no eﬀects on mice spontane-

ous motor activity (i.e., no sedative action), no protec-

tion from PTZ-induced convulsions, and no ethanol-

reinforcing actions. On the other hand, 6d antagonizes

the sedative eﬀect of diazepam and diazepam-induced

convulsion, suggesting an antagonist eﬀect on a₁sub-

type receptor. The anxiogenic eﬀect could be due to in-

verse agonism to the a₂subtype receptor. It could

become a new lead, and suitable chemical modiﬁcation

could possibly shift its inverse agonist eﬀect onto other

subtype receptors, that is, a₅subtype receptor, thus

yielding new ligands useful in therapy as cognition

enhancers in AlzheimerÕs disease and related

dementias.^36,37

5. Conclusion

The study here reported on new BZR ligands, A and B

series, 3-heteroarylester- and 3-heteroarylpyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide 8-substituted, has focused

the role of the substituent at the position 8, which ap-

pears to be in synergy with the substituent at position

3 but per se to have a crucial role in binding and in in vi-

vo activity. The lipophilic and steric features of the opti-

mum 8-substituent, to achieve high aﬃnity ligands, have

been determined improving the pharmacophore/recep-

tor model proposed for this class of ligands.¹²

6. Experimental

6.1. Chemistry

The replacement of the 8-chlorine of lead compounds 6

and 11 (see Chart 1) is favorable when the substituent is

endowed with a p value (lipophilic feature) in the range

0.38–1.19 (substituents as EtO-, Me-, MeS-, and Br-)

and the steric hindrance lower than iodine

(E_s= ꢀ1.40).¹⁵Interestingly, while these lipophilic and

steric hindrance limits seem to be the driving force to

achieve high binding ligands, the electronic parameter

(r) of 8-substituent seems to inﬂuence the subtype recep-

tor selectivity and pharmacological eﬃcacy. In fact

replacing the chlorine, electron-withdrawing atom

(r = 0.23)¹⁵with an electron-donor group, as

Melting points were determined with a Gallenkamp

apparatus and are uncorrected. Silica gel plates (Merk

F

₂₅₄) and silica gel 60 (Merk 70–230 mesh) were used

for analytical and column chromatography, respective-

ly. The structures of all compounds were supported by

their IR spectra (KBr pellets in nujol mulls, Perkin-El-

1

mer 1420 spectrophotometer) and H NMR data (mea-

sured with a Varian Gemini at 200 MHz). Chemical

shifts were expressed in d ppm, using DMSO-d₆or

CDCl₃as solvent. The coupling constant values (J_H6-

_H7,H7-H6; J_H7-H9,H9-H7;) were in agreement with the

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

767

assigned structure. The chemical and physical data of

new compounds are shown in Tables 7 and 8; Microa-

nalyses were performed with a Perkin-Elmer 260 analyz-

er for C, H, N, and the results were within 0.4% of the

theoretical value (Table 9).

After treatment with hydrochloric acid 6 M, the desired

acids, which were ﬁltered and puriﬁed by recrystalliza-

tion, were obtained.

6.1.3.1. 3-Carboxy- 8-bromopyrazolo[5,1-c][1,2,4]ben-

zotriazine 5-oxide 1a. Yellow crystals. TLC eluent: tolu-

ene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ11660,

1593; ¹H NMR (DMSO-d₆) d 12.5 (br s, exch. 1H,

OH); 8.60 (s, 1H, H-2); 8.54 (d, 1H, H-9); 8.35 (d, 1H,

H-6); 7.95 (dd, 1H, H-7). Anal. C, H, N.

6.1.1. General procedure for the synthesis of Ia and b. The

5-bromo-2-nitro-¹⁹

suitable

or

5-iodio-2-nitro-

phenylhydrazine²⁰was reacted with ethyl 2-cyano-3-eth-

oxypropeneate following a reported method.¹⁸

6.1.1.1. Ethyl 1-(2-nitro-5-bromophenyl)-5-amino-

pyrazol-4-carboxylate Ia. From 5-bromo-2-nitro-

phenylhydrazine.¹⁹Orange crystals. TLC eluent:

chloroform/ethanol 10:1 v/v; IR m^ꢀ13438, 3324, 1683;

¹H NMR (DMSO-d₆) d 8.06 (d, 1H, H-3⁰); 7.98 (m,

2H, H-4⁰and H-6⁰); 7.68 (s, 1H, H-3); 6.62 (br s, exch.

2H, NH₂); 4.20 (q, 2H, CH₂); 1.32 (t, 3H, CH₃). Anal.

C, H, N.

6.1.3.2. 3-Carboxy-8-iodiopyrazolo[5,1-c][1,2,4]benzo-

triazine 5-oxide 1b. Yellow crystals. TLC eluent: toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ13481, 1713,

1588; H NMR (DMSO-d₆) 12.8 (br s, exch. 1H, OH);

8.65 (d, 1H, H-9); 8.55 (s, 1H, H-2); 8.12 (m, 2H, H-6,

H-7). Anal. C, H, N.

1

6.1.4. 3-Carboxy-8-methoxypyrazolo[5,1-c][1,2,4]benzo-

triazine 5-oxide 1e. A suspension of 3-carboxy-8-

6.1.1.2.

pyrazol-4-carboxylate

Ethyl

1-(2-nitro-5-iodiophenyl)-5-amino-

Ib. From 5-iodio-2-nitro-

chloropyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide¹⁷

(0.5 mmol) was reacted with methanol/10% sodium

hydroxide 20 mL for 4 h. After treatment with hydro-

chloric acid 6 M, the desired acids, which were ﬁltered

and puriﬁed by recrystallization, were obtained. Yel-

low crystals. TLC eluent: toluene/ethyl acetate/acetic

acid 8:2:1 v/v/v; IR m^ꢀ13400, 1560; ¹H NMR

(DMSO-d₆) 8.57 (s, 1H, H-2); 8.39 (d, 1H, H-6);

7.72 (d, 1H, H-9); 7.35 (dd, 1H, H-7); 4.1 (s, 3H,

CH₃O). Anal. C, H, N.

phenylhydrazine.²⁰Orange crystals. TLC eluent:

chloroform/ethanol 10:1 v/v; IR m^ꢀ13387, 3324, 1682;

¹H NMR (DMSO-d₆) d 8.20 (m, 2H, H-4⁰and H-6⁰);

7.85 (d, 1H, H-3⁰); 7.68 (s, 1H, H-3); 6.62 (br s, exch.

2H, NH₂); 4.20 (q, 2H, CH₂); 1.32 (t, 3H, CH₃). Anal.

C, H, N.

6.1.2. General procedure for the synthesis of IIa–c, and

IIIc. The suitable 5-bromo-2-nitro-¹⁹or 5-iodio-2-ni-

tro-²⁰5-methyl-2-nitrophenylhydrazine¹⁸was reacted

with 2-(thien-3-yl)- and 2-(thien-2-yl)-3-oxapropanenit-

rile¹⁰following a reported methods¹⁰Derivatives IIa

and b were obtained after evaporation of solvent and

recrystallized. Compounds IIc and IIIc, 1-(2-nitro-5-

methylphenyl)-4-(thien-3-yl)-5-aminopyrazole and 1-(2-

nitro-5-methylphenyl)-4-(thien-2-yl)-5-aminopyrazole,

respectively, were not characterized. In this case only the

evaporation of reaction solvent (ethanol) was made: the

red-brown oils obtained were used for synthesis of 11c

and 12c, respectively.

6.1.5. General procedure for the synthesis of ester

derivatives 2a–b, 3c, 4a–f, 5c, 6a–f, 7c, 8c, 9c, and 10c.

The starting acids 1a–c (3-carboxy-8-methylpyrazolo-

[5,1-c][1,2,4]benzotriazine 5-oxide),¹⁸1d (3-carboxy-8-

ethoxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide),¹⁷1e,

and f¹⁸were transformed into ester derivatives by means

of two methods, A and B.

Method A: the ethyl esters 2a and b, the benzyl ester

3c, and the phenoxyester 10c were obtained from acids

1a–c¹⁸(0.32 mmol) by means of the corresponding 3-

carbonyl chlorides which were suspended in 2-methyl-

3-butene stabilized chloroform (5 mL) and then the

suitable alcohol was added. In the case of ester 10c,

addition of some pellets of sodium hydroxide was

needed.

6.1.2.1. 1-(2-Nitro-5-bromophenyl)-4-(thien-3-yl)-5-

aminopyrazole IIa. Brown crystals. TLC eluent: tolu-

ene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ13461,

1

3180; H NMR (DMSO-d₆) d 8.02 (d, 1H, H-3⁰); 7.96

(d, 1H, H-6⁰); 7.88 (dd, 1H, H-4⁰); 7.76 (s, 1H, H-3);

7.56 (dd, 1H, H-4⁰⁰4-thienyl); 7.52 (m, 1H, H-2⁰⁰4-thie-

nyl); 7.38 (dd, 1H, H-5⁰⁰4-thienyl); 5.54 (br s, exch. 2H,

NH₂). Anal. C, H, N.

Method B: all other esters (4a–f, 5c, 6a–f, 7c, 8c, and

9c) were obtained treating the corresponding acids

(100 mg) (1a–f) in tetrahydrofurane (THF) with triethyl

amine (1:3.5) in ice bath for 30⁰. To the suspension was

added ethylchlorocarbonate (1:2) and maintained un-

der stirring for 1 h, from 0 ꢁC to room temperature,

to permit the anhydride to form. The suitable alcohol

was added (1:2.5) and the mixture was heated at

60 ꢁC for 8–18 h and monitored by TLC. The ﬁnal sus-

pension was diluted with water and extracted with

chloroform which was in turn washed with sodium

hydrogen carbonate solution and, after the normal

workup, the residue was treated with isopropyl ether

or ethyl ether, ﬁltered, and recrystallized by a suitable

solvent.

6.1.2.2.

1-(2-Nitro-5-iodiophenyl)-4-(thien-3-yl)-5-

aminopyrazole IIb. Red crystals. TLC eluent: toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ13461, 3340;

¹H NMR (CDCl₃) d 8.08 (d, 1H, H-6⁰); 7.96 (dd, 1H,

H-4⁰); 7.76 (d, 1H, H-3⁰); 7.68 (s, 1H, H-3); 7.43 (dd,

1H, H-4⁰⁰4-thienyl); 7.22 (m, 2H, H-2⁰⁰, H-5⁰⁰4-thienyl);

3.80 (br s, exch. 2H, NH₂). Anal. C, H, N.

6.1.3. General procedure for the synthesis of 1a and b. A

suspension ofIa and b (0.5 mmol) in 25 mL of 10% sodi-

um hydroxide was kept at room temperature for 24 h.

768

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

Table 7. Chemical data for pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides

N

R₈

N

R₃

N

X

Compound

R₃

R₈

X

Yield (%)

Mp ꢁC (recryst. solvent)

1a

1b

COOH

Br

I

O

80

75

296ꢁ (methoxyethanol)

295–6ꢁ (methoxyethanol)

1c^a

1d^b

1e

COOH

Me

OEt

Ome

SMe

Br

COOH

O

—

55

288–90ꢁ (methoxyethanol)

1f^a

2a

COOEt

92

90

62

197–8ꢁ (ethanol 80%)

232–3ꢁ (ethanol 80%)

195–6ꢁ (ethanol)

2b

I

2bR

2c^a

2d^b

3c

COOEt

I

Me

OEt

Me

Br

COOCH₂Ph

COOCH₂-2-MeOPh

COOCH₂-2-NO₂Ph

COOCH₂-2-tienyl

COOCH₂-3-tienyl

COOCH₂-2-furyl

COOCH₂-3-furyl

COO-2-MeOPh

3-Tienyl

O

—

O

—

O

-

85

64

40

45

29

36

45

65

70

95

75

35

70

36

37

52

38

35

56

80

70

75

68

55

61

45

30

48

70

45

74

30

60

59

82

30

60

62

85

94

58

170–2ꢁ (2-propanol)

185–6ꢁ (2-propanol)

200–1ꢁ (ethanol)

189–90ꢁ (ethanol)

193–5ꢁ (ethanol)

3cR

4a

4b

4c

I

Me

OEt

Ome

SMe

Br

4d

4e

224–5ꢁ (ethanol)

200–2ꢁ (2-propanol)

225–6ꢁ (2-propanol)

179–80ꢁ (ethanol)

217–8ꢁ (methoxyethanol)

187–8ꢁ (CHX–EtOAc)^c

214–5ꢁ (ethanol)

174–5ꢁ (2-propanol)

184–5ꢁ (ethanol)

198–9ꢁ (2-propanol)

184–5ꢁ (methoxyethanol)

152–3ꢁ (ethanol)

4f

4aR

5c

Me

Br

6a

6b

I

6c

6d

Me

OEt

Ome

SMe

Me

Br

6e

6f

7c

8c

9c

163–4ꢁ (ethanol 80%)

165–6ꢁ (ethanol 80%)

223–4ꢁ (ethanol)

10c

11a

11b

11c

11cR

11d

11e

11f

11g

12c

12e

12f

13c

13d

13e

13f

14c

14d

15e

16c

16d

16e

16f

224–5ꢁ (methoxyethanol)

233–4ꢁ (methoxyethanol)

224–5ꢁ (methoxyethanol)

195–6ꢁ (methoxyethanol)

211–2ꢁ (methoxyethanol)

226–7ꢁ (methoxyethanol)

227–8ꢁ (methoxyethanol)

>300ꢁ (methoxyethanol)

207–8ꢁ (methoxyethanol)

218–9ꢁ (methoxyethanol)

195–7ꢁ (2-propanol)

192–3ꢁ (methoxyethanol)

189–9ꢁ(diisopropylether)^d

213–4ꢁ (methoxyethanol)

194–5ꢁ (methoxyethanol)

202–3ꢁ (methoxyethanol)

207–8ꢁ^d(diisopropylether)

218–9ꢁ (methoxyethanol)

228–9ꢁ (2-propanol)

244–5ꢁ (methoxyethanol)

234–5ꢁ (ethanol)

3-Tienyl

I

Me

OEt

Ome

SMe

OH

Me

Ome

SMe

Me

OEt

Ome

SMe

Me

OEt

Ome

Me

OEt

Ome

SMe

3-Tienyl

O

3-Tienyl

2-Tienyl

3-Furyl

2-Furyl

H

I

229–30ꢁ (ethanol)

^aSee Ref. 18.

^bSee Ref. 17.

^cCyclohexane/ethyl acetate.

^dSeparated by chromatography column is reported the eluent solvent.

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

Table 8. Chemical data for new 5-aminopyrazoles

769

N

R_5’: a= Br

b= I

R₄

N

R₅'

c= Me

NH₂

NO₂

0

Compound

R₄

R₅

MF (MW)

₁₂H₁₁O₄N₄Br (355.06)

C₁₂H₁₁O₄N₄I (402.15)

Yield (%)

Mp ꢁC (recryst. solvent)

Ia

COOEt

3-Tienyl

2-Tienyl

Br

I

C

78

60

139–40ꢁ (ethanol 80%)

169–70ꢁ (ethanol 80%)

Ib

Ic^a

I^b

Me

Cl

Br

I

IIa

IIb

IIc

II^c

IIIc

III^c

C

₁₃H₉O₂N₄SBr (365.11)

50

45

133–4ꢁ (ethanol 80%)

162–3ꢁ (ethanol)

Not separated

C₁₃H₉O₂N₄SI (412.11)

C₁₄H₁₂O₂N₄S (300.15)

Me

Cl

Me

Cl

C

₁₄H₁₂O₂N₄S (300.15)

Not separated

^aSee Ref. 18.

^bSee Ref. 17.

^cSee Ref. 10.

6.1.5.1. 3-Ethoxycarbonyl-8-bromopyrazolo[5,1-c]-

[1,2,4]benzotriazine 5-oxide 2a. From acid 1a, method A.

Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic

NMR (CDCl₃) d 8.86 (d, 1H, H-9); 8.52 (s, 1H, H-2);

8.24 (d, 1H, H-6); 8.02 (dd, 2H, H-7); 7.56 (d, 1H, H-

3⁰Ph); 7.34 (t, 1H, H-5⁰Ph); 7.00 (t, 1H, H-4⁰Ph);

6.94 (d, 1H, H-6⁰Ph); 5.48 (s, 2H, CH₂); 3.87 (s, 3H,

OCH₃). Anal. C, H, N.

1

acid 8:2:1 v/v/v; IR m^ꢀ11719, 1572; H NMR (DMSO-

d₆) d 8.65 (s, 1H, H-2); 8.55 (d, 1H, H-9); 8.35 (d, 1H,

H-6); 7.95 (dd, 1H, H-7); 4.25 (q, 2H, CH₂); 1.35 (t,

3H, CH₃). Anal. C, H, N.

6.1.5.6. 3-(2-Methoxybenzyloxycarbonyl)-8-methylpy-

razolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4c. From acid

1c¹⁸method B. Yellow crystals. TLC eluent: chloro-

form/methanol 10:0.1 v/v; IR m^ꢀ11711, 1564; ¹H

NMR (CDCl₃) d 8.52 (s, 1H, H-2); 8.44 (d, 1H, H-6);

8.24 (d, 1H, H-9); 7.54 (m, 2H, H-7 and H-3⁰Ph);

7.34 (t, 1H, H-5⁰Ph); 7.00 (t, 1H, H-4⁰Ph); 6.94 (d,

1H, H-6⁰Ph); 5.49 (s, 2H, CH₂); 3.89 (s, 3H, OCH₃);

2.67 (s, 3H, CH₃). Anal. C, H, N.

6.1.5.2. 3-Ethoxycarbonyl-8-iodiopyrazolo[5,1-c]-

[1,2,4]benzotriazine 5-oxide 2b. From acid 1b, method

A. Yellow crystals. TLC eluent: toluene/ethyl acetate/

acetic acid 8:2:1 v/v/v; IR m^ꢀ11713, 1557; ¹H NMR

(CDCl₃) d 8.85 (d, 1H, H-9); 8.50 (s, 1H, H-2); 8.25

(d, 1H, H-6); 8.00 (dd, 1H, H-7); 4.40 (q, 2H, CH₂);

1.40 (t, 3H, CH₃). Anal. C, H, N.

6.1.5.3. 3-Benzyloxycarbonyl-8-methylpyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide 3c. From acid 1c,¹⁸meth-

od A. Yellow crystals. TLC eluent: toluene/ethyl ace-

tate/acetic acid 8:2:1 v/v/v; IR m^ꢀ11720, 1570; ¹H

NMR (CDCl₃) d 8.55 (s, 1H, H-2); 8.45 (d, 1H,

H-6); 8.25 (d, 1H, H-9); 7.45 (m, 6H, Ph and H-

7); 5.45 (s, 2H, CH₂); 2.65 (s, 3H, CH₃). Anal. C,

H, N.

6.1.5.7. 3-(2-Methoxybenzyloxycarbonyl)-8-ethoxypy-

razolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4d. From acid

1d¹⁷method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11711, 1564; ¹H

NMR (CDCl₃) d 8.52 (s, 1H, H-2); 8.45 (d, 1H, H-6);

7.65 (d, 1H, H-9); 7.60 (dd, 1H, H-7); 7.15 (m, 2H H-

3⁰and H-5⁰Ph); 7.00 (t, 1H, H-4⁰Ph); 6.94 (d, 1H, H-

6⁰Ph); 5.47 (s, 2H, CH₂); 4.30 (q, 2H, CH₂); 3.89 (s,

3H, OCH₃); 1.60 (t, 3H, CH₃). Anal. C, H, N.

6.1.5.4. 3-(2-Methoxybenzyloxycarbonyl)-8-bromopy-

razolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4a. From acid

1a, method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11711, 1566; ¹H

NMR (CDCl₃) d 8.62 (d, 1H, H-9); 8.48 (s, 1H, H-2);

8.40 (d, 1H, H-6); 7.80 (dd, 2H, H-7); 7.55 (d, 1H, H-

3⁰Ph); 7.32 (t, 1H, H-5⁰Ph); 6.98 (t, 1H, H-4⁰Ph);

6.92 (d, 1H, H-6⁰Ph); 5.48 (s, 2H, CH₂); 3.87 (s, 3H,

OCH₃). Anal. C, H, N.

6.1.5.8. 3-(2-Methoxybenzyloxycarbonyl)-8-methoxy-

pyrazolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4e. From acid

1e method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11711, 1564; ¹H

NMR (CDCl₃) d 8.55 (s, 1H, H-2); 8.48 (d, 1H, H-6);

7.74 (d, 1H, H-9); 7.60 (d, 1H, H-3⁰Ph); 7.32 (t, 1H,

H-5⁰Ph); 7.22 (dd, 1H, H-7); 7.01 (t, 1H, H-4⁰Ph);

6.93 (d, 1H, H-6⁰Ph); 5.50 (s, 2H, CH₂); 4.10 (s, 3H,

OCH₃); 3.59 (s, 3H, OCH₃). Anal. C, H, N.

6.1.5.5. 3-(2-Methoxybenzyloxycarbonyl)-8-iodiopy-

razolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4b. From acid

1b, method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11709, 1564; ¹H

6.1.5.9. 3-(2-Methoxybenzyloxycarbonyl)-8-methylthio-

pyrazolo[5,1-c][1,2, 4]benzotriazine 5-oxide 4f. From acid

1f¹⁸method B. Yellow crystals. TLC eluent: isopropyl

770

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

Table 9. Elemental analyses

Compound

Formula

Anal. Calcd

Anal. Found

H

C

H

N

C

N

2a

2b

C

₁₂H₉O₃N₄Br

₁₂H₉O₃N₄I

42.75

37.52

39.15

64.66

67.91

50.37

45.40

62.63

58.11

60.00

57.57

52.32

56.99

44.46

39.84

56.46

55.13

53.93

51.60

56.46

59.26

61.71

44.97

39.61

59.56

63.14

57.68

56.37

53.49

54.92

56.56

56.37

53.49

63.15

60.81

59.57

56.37

63.15

60.81

55.55

63.83

37.10

35.11

33.53

2.69

2.36

2.46

4.22

4.43

3.05

2.75

4.43

4.22

4.24

4.07

3.17

3.45

2.24

2.01

3.55

3.81

3.39

3.25

3.55

3.73

4.03

2.03

1.79

3.57

3.78

3.87

3.38

3.21

2.84

3.57

3.38

3.21

3.79

4.08

3.57

3.38

3.79

4.08

3.73

2.16

2.55

2.06

1.97

16.62

14.59

15.22

16.76

17.60

13.05

11.76

15.38

14.65

14.73

14.13

13.56

18.46

13.83

12.39

16.46

15.13

15.72

15.04

16.46

17.28

15.99

16.14

14.21

19.84

21.04

17.94

18.78

17.82

19.71

19.84

18.78

17.82

21.04

18.91

19.85

18.78

21.04

18.91

25.91

17.18

15.73

16.38

15.64

42.96

37.88

38.90

64.89

67.63

50.64

45.72

62.31

57.84

59.84

57.78

52.69

56.78

44.79

40.05

56.82

55.44

53.64

51.84

56.78

59.58

59.35

61.99

44.62

39.78

59.85

62.89

57.98

56.72

53.74

54.61

56.19

56.48

53.74

63.54

61.04

59.84

56.68

63.29

61.04

55.84

63.54

37.36

35.36

35.85

2.81

2.72

2.15

4.58

4.15

3.35

2.95

4.26

3.97

3.93

4.42

3.37

3.13

2.57

2.26

3.76

4.18

3.62

3.46

3.74

4.03

3.85

4.35

1.98

1.85

3.90

3.57

4.03

3.58

3.51

2.65

3.20

3.64

3.51

3.99

4.35

3.67

3.57

3.98

4.27

3.98

1.89

2.86

2.38

2.13

16.88

14.79

14.99

16.78

17.25

13.28

12.03

15.18

14.35

14.57

14.36

13.85

18.20

13.99

12.59

16.74

15.36

15.99

15.38

16.80

17.54

17.35

16.28

16.01

14.35

19.99

19.85

18.02

19.15

18.00

19.54

19.74

18.98

18.04

21.38

19.06

20.10

18.95

21.34

19.21

26.15

16.99

15.99

16.65

15.84

C

2bR

3c

C₁₂H₉O₂N₄I

C₁₈H₁₄O₃N₄

C₁₈H₁₄O₂N₄

3cR

4a

C₁₈H₁₃O₄N₄Br

4b

4c

C₁₈H₁₃O₄N₄I

C₁₉H₁₆O₄N₄

C₂₀H₁₈O₅N₄

4d

4e

C₁₉H₁₆O₅N₄

C₁₉H₁₆O₄N₄S

C₁₈H₁₃O₃N₄Br

4f

4aR

5c

C₁₈H₁₃O₅N₅

6a

6b

C₁₅H₉O₃N₄SBr

C₁₅H₉O₃N₄SI

C₁₆H₁₂O₃N₄S

6c

6d

C

₁₇H₁₄O₄N₄S

₁₆H₁₂O₄N₄S

6e

C

6f

7c

C₁₆H₁₂O₃N₄S₂

C₁₆H₁₂O₃N₄S

C₁₆H₁₂O₄N₄

8c

9c

C₁₆H₁₂O₄N₄

10c

11a

11b

11c

11cR

11d

11e

11f

11g

12c

12e

12f

13c

13d

13e

13f

14c

14d

15e

16c

16d

16e

16f

C₁₈H₁₄O₄N₄

C₁₃H₇ON₄SBr

C₁₃H₇ON₄SI

C

₁₄H₁₀ON₄S

C₁₄H₁₀N₄S

₁₅H₁₂O₂N₄S

C₁₄H₁₀O₂N₄S

₁₄H₁₀ON₄S₂

C₁₃H₈O₂N₄S

₁₄H₁₀ON₄S

C₁₄H₁₀O₂N₄S

₁₄H₁₀ON₄S₂

C₁₄H₁₀O₂N₄

₁₅H₁₂O₃N₄

C

C₁₄H₁₀O₃N₄

C₁₄H₁₀O₂N₄S

C₁₄H₁₀O₂N₄

C₁₅H₁₂O₃N₄

C₁₀H₈O₂N₄

C₁₀H₇ON₄I

C

₁₁H₉O₂N₄I

C₁₀H₇O₂N₄I

₁₀H₇ON₄IS

C

ether/cyclohexane 8:3 v/v; IR m^ꢀ11710, 1560; ¹H

NMR (CDCl₃) d 8.54 (s, 1H, H-2); 8.40 (d, 1H, H-

6); 8.07 (d, 1H, H-9); 7.59 (d, 1H, H-3⁰Ph); 7.48

(dd, 1H, H-7); 7.32 (t, 1H, H-5⁰Ph); 7.01 (t, 1H,

H-4⁰Ph); 6.94 (d, 1H, H-6⁰Ph); 5.50 (s, 2H, CH₂);

3.90 (s, 3H, OCH₃); 2.70 (s, 3H, SCH₃). Anal. C,

H, N.

6.1.5.11. 3-(2-Thienylmethoxycarbonyl)-8-bromopy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 6a. From acid

1a method B. Yellow crystals. TLC eluent: chloro-

form/methanol/acetic acid 10:0.5:0.1 v/v/v; IR m^ꢀ1

1

1727, 1567; H NMR (DMSO-d₆) d 8.66 (s, 1H, H-2);

8.54 (d, 1H, H-9); 8.34 (d, 1H, H-6); 7.96 (dd, 1H, H-

7); 7.58 (m, 1H, H-3⁰2-thienyl); 7.28 (m, 1H, H-5⁰2-thi-

enyl); 7.04 (m, 1H, H-4⁰2-thienyl); 5.52 (s, 2H, CH₂).

Anal. C, H, N.

6.1.5.10. 3-(2-Nitrobenzyloxycarbonyl)-8-methylpy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 5c. From acid

1c¹⁸method B. Yellow crystals. TLC eluent: chloro-

6.1.5.12. 3-(2-Thienylmethoxycarbonyl)-8-iodiopyraz-

olo[5,1-c][1,2,4]benzotriazine 5-oxide 6b. From acid 1b

method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11723, 1565; ¹H

NMR (CDCl₃) d 8.85 (d, 1H, H-9); 8.52 (s, 1H, H-2);

8.25 (d, 1H, H-6); 8.05 (dd, 1H, H-7); 7.35 (m, 1H,

1

form/methanol 10:0.1 v/v; IR m^ꢀ11715, 1570; H NMR

(CDCl₃) d 8.59 (s, 1H, H-2); 8.47 (d, 1H, H-6); 8.28 (d,

1H, H-9); 8.19 (d, 1H, H-3⁰Ph); 8.07 (d, 1H, H-6⁰Ph);

7.75 (t, 1H, H-5⁰Ph); 7.52 (m, 2H, H-7 and H-4⁰Ph);

5.87 (s, 2H, CH₂); 2.69 (s, 3H, CH₃). Anal. C, H, N.

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

771

H-3⁰2-thienyl); 7.28 (m, 1H, H-5⁰2-thienyl); 7.02 (m,

1H, H-4⁰2-thienyl); 5.55 (s, 2H, CH₂). Anal. C, H, N.

methanol 10:0.1 v/v; IR m^ꢀ11730, 1570; ¹H NMR

(CDCl₃) d 8.50 (s, 1H, H-2); 8.45 (d, 1H, H-6); 8.24

(d, 1H, H-9); 7.62 (d, 1H, H-2⁰3-furyl); 7.51 (dd, 1H,

H-7); 7.42 (m, 1H, H-4⁰3-furyl); 6.57 (d, 1H, H-5⁰3-fur-

yl); 5.30 (s, 2H, CH₂); 2.67 (s, 3H, CH₃). Anal. C, H, N.

6.1.5.13. 3-(2-Thienylmethoxycarbonyl)-8-methylpy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 6c. From acid

1c¹⁸method B. Yellow crystals. TLC eluent: chloro-

form/methanol 10:0.1 v/v; IR m^ꢀ11700, 1565; ¹H

NMR (CDCl₃) d 8.50 (s, 1H, H-2); 8.44 (d, 1H, H-6);

8.23 (d, 1H, H-9); 7.52 (dd, 1H, H-7); 7.35 (m, 1H, H-

3⁰2-thienyl); 7.24 (m, 1H, H-5⁰2-thienyl); 7.02 (m,

1H, H-4⁰2-thienyl); 5.58 (s, 2H, CH₂); 2.67 (s, 3H,

CH₃). Anal. C, H, N.

6.1.5.20. 3-(2-Methoxyphenoxycarbonyl)-8-methylpy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 10c. From acid

1c¹⁸method A. Yellow crystals. TLC eluent: toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ11730, 1570;

¹H NMR (CDCl₃) d 8.68 (s, 1H, H-2); 8.45 (d, 1H, H-

6); 8.28 (d, 1H, H-9); 7.54 (dd, 1H, H-7); 7.24 (m, 2H,

H-4⁰and H-5⁰Ph); 7.02 (d, 2H, H-3⁰and H-6⁰Ph);

3.83 (s, 3H, OCH₃); 2.69 (s, 3H, CH₃). Anal. C, H, N.

6.1.5.14. 3-(2-Thienylmethoxycarbonyl)-8-ethoxypy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 6d. From acid

1d¹⁷method B. Yellow crystals. TLC eluent: chloro-

form/methanol 10:0.1 v/v; IR m^ꢀ11700, 1565; ¹H

NMR (CDCl₃) d 8.51 (s, 1H, H-2); 8.46 (d, 1H, H-6);

7.70 (d, 1H, H-9); 7.34 (m, 1H, H-3⁰2-thienyl); 7.22

(m, 2H, H-7 and H-5⁰2-thienyl); 7.02 (m, 1H, H-4⁰2-

thienyl); 5.58 (s, 2H, CH₂); 4.30 (q, 2H, CH₂); 1.58 (t,

3H, CH₃). Anal. C, H, N.

6.1.6. General procedure for the synthesis of 3-heteroaryl

derivatives 11a–g, 12c, and 12e–f. The cyclization to pyr-

azolo[5,1-c][1,2,4]benzotriazine 5-oxide system was ob-

tained from the suitable 5-aminopyrazoles IIa, IIb, IIc,

and IIIc in alkaline medium, 10% sodium hydroxide

solution, according to a previously reported method.¹⁰

For compounds 11d–g the starting material was the pre-

viously described 1-(2-nitro-5-chlorophenyl)-4-(thien-3-

yl)-5-amino pyrazole¹⁰that was suspended in 10% sodi-

um hydroxide solution. In these conditions, the pyra-

zolobenzotriazine N-oxide system is formed and the

chlorine atom undergoes the nucleophilic substitution

yielding 11d as precipitate and 11g in solution, when

treated with ethanol/sodium hydroxide; yielding 11e

when treated with methanol/sodium hydroxide and 11f

when treated with sodium thiomethoxide/ethanol.

6.1.5.15. 3-(2-Thienylmethoxycarbonyl)-8-methoxypy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 6e. From acid 1e

method B. Yellow crystals. TLC eluent: isopropyl ether/

cyclohexane 8:3 v/v; IR m^ꢀ11718, 1560; ¹H NMR

(CDCl₃) d 8.53 (s, 1H, H-2); 8.48 (d, 1H, H-6); 7.74

(d, 1H, H-9); 7.36 (dd, 1H, H-7); 7.25 (dd, 1H, H-5⁰2-

thienyl); 7.22 (dd, 1H, H-3⁰2-thienyl); 7.02 (m, 1H, H-

4⁰2-thienyl); 4.10 (s, 3H, OCH₃). Anal. C, H, N.

6.1.5.16. 3-(2-Thienylmethoxycarbonyl)-8-methylthio-

pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 6f. From acid

1f¹⁸method B. Yellow crystals. TLC eluent: isopropyl

ether/cyclohexane 8:3 v/v; IR m^ꢀ11709, 1570; ¹H

NMR (CDCl₃) d 8.53 (s, 1H, H-2); 8.40 (d, 1H, H-6);

8.05 (d, 1H, H-9); 7.49 (dd, 1H, H-7); 7.36 (dd, 1H,

H-5⁰2-thienyl); 7.25 (dd, 1H, H-3⁰2-thienyl); 7.03 (m,

1H, H-4⁰2-thienyl); 5.60 (s, 2H, CH₂); 2.70 (s, 3H,

SCH₃). Anal. C, H, N.

6.1.6.1. 3-(Thien-3-yl)-8-bromopyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11a. From IIa. Red crystals. TLC

eluent: isopropyl ether/cyclohexane 8:3 v/v; IR m^ꢀ11590;

¹H NMR (CDCl₃) d 8.56 (d, 1H, H-9); 8.40 (d, 1H,

H-6); 8.32 (s, 1H, H-2); 7.90 (dd, 1H, H-2⁰3-thienyl);

7.72 (dd, 1H, H-7); 7.64 (dd, 1H, H-5⁰3-thienyl); 7.44

(dd, 1H, H-4⁰3-thienyl). Anal. C, H, N.

6.1.6.2.

3-(Thien-3-yl)-8-iodiopyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11b. From IIb. Red crystals. TLC

eluent: isopropyl ether/cyclohexane 8:3 v/v; IR m^ꢀ11578;

¹H NMR (CDCl₃) d 8.78 (d, 1H, H-9); 8.32 (s, 1H, H-2);

8.23 (d, 1H, H-6); 7.90 (m, 2H, H-7 and H-2⁰3-thienyl);

7.65 (dd, 1H, H-5⁰3-thienyl); 7.44 (dd, 1H, H-4⁰3-thie-

nyl). Anal. C, H, N.

6.1.5.17. 3-(3-Thienylmethoxycarbonyl)-8-methylpy-

razolo[5,1-c][1,2,4]benzotriazine 5-oxide 7c. From acid

1c¹⁸method B. Yellow crystals. TLC eluent: chloro-

1

form/methanol 10:0.1 v/v; IR m^ꢀ11740, 1570; H NMR

(CDCl₃) d 8.52 (s, 1H, H-2); 8.44 (d, 1H, H-6); 8.23 (d,

1H, H-9); 7.50 (m, 2H, H-7 and H-2⁰3-thienyl); 7.34 (m,

1H, H-4⁰3-thienyl); 7.22 (m, 1H, H-5⁰3-thienyl); 5.44

(s, 2H, CH₂); 2.67 (s, 3H, CH₃). Anal. C, H, N.

6.1.6.3. 3-(Thien-3-yl)-8-methylpyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11c. From IIc. Red crystals. TLC

eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR

1

6.1.5.18. 3-(2-Furylmethoxycarbonyl)-8-methylpyraz-

olo[5,1-c][1,2,4]benzotriazine 5-oxide 8c. From acid 1c¹⁸

method B. Yellow crystals. TLC eluent: chloroform/

methanol 10:0.1 v/v; IR m^ꢀ11710, 1570; ¹H NMR

(CDCl₃) d 8.51 (s, 1H, H-2); 8.44 (d, 1H, H-6); 8.24

(d, 1H, H-9); 7.52 (dd, 1H, H-7); 7.45 (d, 1H, H-3⁰2-fur-

yl); 6.55 (d, 1H, H-5⁰2-furyl); 6.40 (m, 1H, H-4⁰2-furyl);

5.38 (s, 2H, CH₂); 2.67 (s, 3H, CH₃). Anal. C, H, N.

m^ꢀ11540; H NMR (CDCl₃) d 8.45 (d, 1H, H-6); 8.30

(s, 1H, H-2); 8.17 (d, 1H, H-9); 7.90 (dd, 1H, H-2⁰3-thi-

enyl); 7.66 (dd, 1H, H-5⁰3-tienyl); 7.46 (m, 2H, H-7 and

H-4⁰3-thienyl). Anal. C, H, N.

6.1.6.4. 3-(Thien-3-yl)-8-ethoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11d. From 1-(2-nitro-5-chlorophe-

nyl)-4-(thien-3-yl)-5-aminopyrazole¹⁰in 10% sodium

hydroxide/ethanol at 60 ꢁC. Red crystals. TLC eluent:

toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ1

6.1.5.19. 3-(3-Furylmethoxycarbonyl)-8-methylpyraz-

olo[5,1-c][1,2,4]benzotriazine 5-oxide 9c. From acid 1c¹⁸

method B. Yellow crystals. TLC eluent: chloroform/

1

1540; H NMR (CDCl₃) d 8.46 (d, 1H, H-6); 8.30 (s,

1H, H-2); 7.90 (dd, 1H, H-2⁰3-thienyl); 7.68 (m, 2H,

772

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

H-9 and H-5⁰3-tienyl); 7.44 (dd, 1H, H-4⁰3-thienyl);

7.14 (dd, 1H, H-7). Anal. C, H, N.

the same procedure described for compounds 13c–f

and 14c–d.

6.1.6.5. 3-(Thien-3-yl)-8-methoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11e. From 1-(2-nitro-5-chlorophe-

nyl)-4-(thien-3-yl)-5-aminopyrazole¹⁰in 10% sodium

hydroxide solution/methanol at 60 ꢁC. Red crystals.

TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/

6.1.6.10. 3-(Thien-2-yl)-8-methylthiopyrazolo[5,1-c]

[1,2,4]benzotriazine 5-oxide 12f. From 1-(2-nitro-5-chlor-

ophenyl)-4-(thien-2-yl)-5-aminopyrazole¹⁰in sodium

thiomethoxide/ethanol under N₂. Red crystals. TLC

eluent: isopropyl ether/cyclohexane 8:3 v/v; IR m^ꢀ11540;

¹H NMR (CDCl₃) d 8.40 (d, 1H, H-6); 8.29 (s, 1H, H-

2); 8.02 (dd, 1H, H-9); 7.66 (dd, 1H, H-5⁰2-thienyl);

7.42 (dd, 1H, H-7); 7.35 (m, 1H, H-3⁰2-tienyl); 7.15 (m,

1H, H-4⁰2-thienyl); 2.70 (s, 3H, SCH₃). Anal. C, H, N.

1

v; IR m^ꢀ11540; H NMR (CDCl₃) d 8.50 (d, 1H, H-6);

8.34 (s, 1H, H-2); 7.92 (dd, 1H, H-2⁰3-thienyl); 7.71

(dd, 1H, H-9); 7.68 (m, 1H, H-4⁰3-tienyl); 7.45 (m,

1H, H-5⁰3-thienyl); 7.16 (dd, 1H, H-7); 4.10 (s, 3H,

OCH₃). Anal. C, H, N.

This compound is also obtained, with excellent yield, by

the Suzuki-coupling reaction using as starting material

8-methylthioypyrazolo[5,1-c][1,2,4]benzotriazine 5-ox-

ide¹⁸and 2-thiophenboronic acid following the same

procedure described for compounds 13c–f and 14c–d.

6.1.6.6. 3-(Thien-3-yl)-8-methylthiopyrazolo[5,1-c][1,2,

4]benzotriazine 5-oxide 11f. From 1-(2-nitro-5-chlor-

ophenyl)-4-(thien-3-yl)-5-aminopyrazole¹⁰in sodium

thiomethoxide/ethanol at RT ꢁ. Red crystals. TLC elu-

ent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m^ꢀ1

1

1540; H NMR (CDCl₃) d 8.41 (d, 1H, H-6); 8.32 (s,

6.1.7. General procedure for the synthesis of 3-furyl

derivatives 13c–f and 14c–d. Tetrakis-(triphenylphps-

phine)palladium(0) (30 mg, 0.026 mmol) and the suit-

able 3-iodio derivatives (0.30 mmol), 16c–f (see below),

were combined in anhydrous toluene (4.0 mL). The 3-

and 2-furanboronic acids (70 mg, 0.62 mmol) in abso-

lute ethanol (2.5 mL) and aqueous sodium carbonate

(2 M, 4 mL) were added and the reaction mixture was

heated to reﬂux for 12 h and monitored by TLC. The

red product was then extracted by methylene chloride

and the organic layer was washed with water and dried

over sodium sulfate. The evaporation of solvent gave the

crude product that was recrystallized by suitable solvent.

1H, H-2); 8.03 (dd, 1H, H-9); 7.92 (dd, 1H, H-2⁰3-thie-

nyl); 7.68 (m, 1H, H-4⁰3-tienyl); 7.45 (m, 1H, H-5⁰3-thi-

enyl); 7.42 (dd, 1H, H-7); 2.70 (s, 3H, SCH₃). Anal. C,

H, N.

6.1.6.7. 3-(Thien-3-yl)-8-hydroxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 11g. From 1-(2-nitro-5-chlorophe-

nyl)-4-(thien-3-yl)-5-aminopyrazole¹⁰in 10% sodium

hydroxide solution/ethanol at 60 ꢁC as by-product. This

compound was obtained after concentration of reaction

solvent and acidiﬁcation with hydrochloric acid. The

residue was treated with ethanol/water and ﬁltered.

Red crystals. TLC eluent: toluene/ethyl acetate/acetic

acid 8:2:1 v/v/v; IR m^ꢀ13400–2900, 1540; ¹H NMR

(DMSO-d₆) d 11.7 (br s, exch. 1H, OH); 8.65 (s, 1H,

H-2); 8.30 (d, 1H, H-6); 7.90 (dd, 1H, H-2⁰3-thienyl);

7.70 (m, 2H, H-4⁰and H-5⁰3-tienyl); 7.50 (d, 1H, H-

9); 7.14 (dd, 1H, H-7). Anal. C, H, N.

6.1.7.1. 3-(Fur-3-yl)-8-methylpyrazolo[5,1-c][1,2,4]ben-

zotriazine 5-oxide 13c. From 16c and 3-furanboronic

acid. Red crystals. TLC eluent: acetone/cyclohexane

1

1:4 v/v; IR m^ꢀ11530; H NMR (CDCl₃) d 8.44 (d, 1H,

H-6); 8.20 (s, 1H, H-2); 8.17 (d, 1H, H-9); 8.07 (d, 1H,

H-2⁰3-furyl); 7.53 (dd, 1H, H-4⁰3-furyl); 7.43 (dd,

1H, H7); 6.91 (dd, 1H, H-5⁰3-furyl); 2.64 (s, 3H,

CH₃). Anal. C, H, N.

6.1.6.8. 3-(Thien-2-yl)-8-methylpyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 12c. From IIIc. Red crystals.

TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/

1

v; IR m^ꢀ11550; H NMR (CDCl₃) d 8.44 (d, 1H, H-6);

6.1.7.2. 3-(Fur-3-yl)-8-ethoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 13d. From 16d and 3-furanboronic

8.28 (s, 1H, H-2); 8.18 (d, 1H, H-9); 7.64 (dd, 1H, H-

5⁰2-tienyl); 7.44 (dd, 1H, H-7); 7.34 (dd, 1H, H-3⁰2-tie-

nyl); 7.14 (dd, 1H, H-4⁰2-thienyl); 2.63 (s, 3H, CH₃).

Anal. C, H, N.

acid. Red crystals. TLC eluent: isopropylether; IR m^ꢀ1

1

1530; H NMR (CDCl₃) d 8.44 (d, 1H, H-6); 8.18 (s,

1H, H-2); 8.08 (d, 1H, H-2⁰3-furyl); 7.64 (d, 1H, H-9);

7.52 (dd, 1H, H-5⁰3-furyl); 7.10 (dd, 1H, H7); 6.90

(dd, 1H, H-4⁰3-furyl); 4.20 (q, 2H, CH₂); 1.40 (t, 3H,

CH₃). Anal. C, H, N.

6.1.6.9. 3-(Thien-2-yl)-8-methoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 12e. From 1-(2-nitro-5-chlorophe-

nyl)-4-(thien-2-yl)-5-aminopyrazole¹⁰in 10% sodium

hydroxide solution/methanol. Red crystals. TLC elu-

ent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR

m^ꢀ11540; ¹H NMR (CDCl₃) d 8.50 (d, 1H, H-6);

8.30 (s, 1H, H-2); 7.71 (dd, 1H, H-9); 7.66 (dd, 1H,

H-3⁰2-thienyl); 7.35 (dd, 1H, H-5⁰2-tienyl); 7.17 (dd,

1H, H-7); 7.15 (m, 1H, H-4⁰2-thienyl); 4.10 (s, 3H,

OCH₃). Anal. C, H, N.

6.1.7.3. 3-(Fur-3-yl)-8-methoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 13e. From 16e and 3-furanboronic

acid. Red crystals. TLC eluent: isopropyl ether/cyclo-

1

hexane 8:3 v/v; IR m^ꢀ11530; H NMR (CDCl₃) d 8.48

(d, 1H, H-6); 8.22 (s, 1H, H-2); 8.09 (d, 1H, H-2⁰3-fur-

yl); 7.70 (d, 1H, H-9); 7.54 (m, 1H, H-5⁰3-furyl); 7.15

(dd, 1H, H7); 6.93 (m, 1H, H-4⁰3-furyl); 4.10 (s, 3H,

OCH₃). Anal. C, H, N.

This compound is also obtained, with excellent yield, by

the Suzuki-coupling reaction using as starting material

8-methoxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide,

15e (see below), and 2-thiophen boronic acid following

6.1.7.4. 3-(Fur-3-yl)-8-methylthiopyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 13f. From 16f and 3-furanboronic

acid. Red crystals. TLC eluent: isopropyl ether/cyclo-

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

773

hexane 8:3 v/v; IR m^ꢀ11530; ¹H NMR (CDCl₃) d

8.40 (d, 1H, H-6); 8.21 (s, 1H, H-2); 8.08 (d, 1H,

H-2⁰3-furyl); 8.02 (d, 1H, H-9); 7.54 (m, 1H, H-5⁰

3-furyl); 7.41 (dd, 1H, H7); 6.93 (m, 1H, H-4⁰3-fur-

yl); 2.70 (s, 3H, SCH₃). Anal. C, H, N.

1H, H-6); 8.10 (s, 1H, H-2); 7.65 (d, 1H, H-9); 7.15

(dd, 1H, H-7); 4.30 (q, 2H, CH₂); 1.50 (t, 3H, CH₃).

Anal. C, H, N.

6.1.8.3. 3-Iodio-8-methoxypyrazolo[5,1-c][1,2,4]benzo-

triazine 5-oxide 16e. From 15e and ICl. Dark yellow

crystals. TLC eluent: toluene/ethyl acetate/acetic

acid 8:2:1 v/v/v; IR m^ꢀ11570; ¹H NMR (CDCl₃) d

8.48 (d, 1H, H-6); 8.11 (s, 1H, H-2); 7.69 (d, 1H,

H-9); 7.17 (dd, 1H, H-7); 4.10 (s, 3H, OCH₃). Anal.

C, H, N.

6.1.7.5. 3-(Fur-2-yl)-8-methylpyrazolo[5,1-c][1,2,4]ben-

zotriazine 5-oxide 14c. From 16c and 2-furanboronic

acid. Red crystals. TLC eluent: ethyl acetate/cyclohex-

1

ane 1:3 v/v; IR m^ꢀ11530; H NMR (CDCl₃) d 8.43 (d,

1H, H-6); 8.36 (s, 1H, H-2); 8.17 (d, 1H, H-9); 7.51 (d,

1H, H-3⁰2-furyl); 7.43 (dd, 1H, H-7); 6.97 (d, 1H, H-

5⁰2-furyl); 6.54 (dd, 1H, H-4⁰2-furyl); 2.64 (s, 3H,

CH₃). Anal. C, H, N.

6.1.8.4. 3-Iodio-8-methylthiopyrazolo[5,1-c][1,2,4]ben-

zotriazine 5-oxide 16f. From 8-methylthiopyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide¹⁸and ICl. Dark yellow

crystals. TLC eluent: toluene/ethyl acetate/acetic acid

6.1.7.6. 3-(Fur-2-yl)-8-ethoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide 14d. From 16d and 2-furanboronic

1

8:2:1 v/v/v; IR m^ꢀ11560; H NMR (CDCl₃) d 8.40 (d,

acid. Red crystals. TLC eluent: isopropylether; IR m^ꢀ1

1H, H-6); 8.11 (s, 1H, H-2); 8.01 (d, 1H, H-9); 7.43

(dd, 1H, H-7); 2.65 (s, 3H, SCH₃). Anal. C, H, N.

1

1530; H NMR (CDCl₃) d 8.44 (d, 1H, H-6); 8.38 (s,

1H, H-2); 7.64 (d, 1H, H-9); 7.48 (d, 1H, H-3⁰2-furyl);

7.12 (dd, 1H, H-7); 6.96 (m, 1H, H-5⁰2-furyl); 6.54

(m, 1H, H-4⁰2-furyl); 4.20 (q, 2H CH₂); 1.40 (t, 3H,

CH₃). Anal. C, H, N.

6.1.9. General procedure for synthesis of compounds, 3cR,

4aR and 11cR. These compounds were obtained by fol-

lowing a previously described procedure of reduction

with triethyl phosphite (1.5 mL) in toluene¹⁸(5.0 mL)

at reﬂuxing temperature for 6–8 h, starting from com-

pounds 3c, 4a, and 11c.

6.1.7.7. 8-methoxypyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide 15e. Compound 1e, 3-carboxy-8-methoxypyraz-

olo[5,1-c][1,2,4]benzotriazine 5-oxide, was treated with

concd hydrochloric acid until the starting material dis-

appeared by evaluation with TLC. The ﬁnal solution

was treated with ice and the precipitate was ﬁltered

and crystallized by a suitable solvent.

6.1.9.1. 3-Benzyloxycarbonyl-8-methylpyrazolo[5,1-

c][1,2,4]benzotriazine 3cR. From 3c. Yellow crystals.

TLC eluent: toluene/ethyl acetate 8:3 v/v; IR m^ꢀ1

1

1711; H NMR (CDCl₃) d 8.66 (s, 1H, H-2); 8.63 (d,

1H, H-6); 8.32 (d, 1H, H-9); 7.66 (dd, 1H, H-7); 7.58

(dd, 2H, H-2⁰and H-6⁰Ph); 7.40 (m, 3H, H-3⁰, H-4⁰

and H-5⁰Ph); 5.51 (s, 2H, CH₂); 2.72 (s, 3H, CH₃).

Anal. C, H, N.

Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic

1

acid 8:2:1 v/v/v; IR m^ꢀ11560; H NMR (DMSO-d₆) d

8.50 (d, 1H, H-6); 8.10 (s, 1H, H-2); 7.73 (d, 1H, H-9);

7.17 (dd, 1H, H7); 6.47 (d, 1H, H-3); 4.08 (s, 3H,

OCH₃). Anal. C, H, N.

6.1.9.2. 3-(2-Methoxybenzyloxycarbonyl)-8-bromopy-

razolo[5,1-c][1,2,4]benzotriazine 4aR. From 4a. Yellow

crystals. TLC eluent: isopropyl ether/cyclohexane 8:3

6.1.8. General procedure for the synthesis of compounds

16c–f. Compounds 8-methylpyrazolo[5,1-c][1,2,4]benzo-

triazine 5-oxide,¹⁸8-ethoxy pyrazolo[5,1-c][1,2,4]benzo-

1

v/v; IR m^ꢀ11720; H NMR (CDCl₃) d 8.72 (d, 1H, H-

9); 8.68 (s, 1H, H-2); 8.62 (d, 1H, H-6); 7.960 (dd, 2H,

H-7); 7.62 (d, 1H, H-3⁰Ph); 7.34 (t, 1H, H-5⁰Ph); 7.02

(t, 1H, H-4⁰Ph); 6.92 (d, 1H, H-6⁰Ph); 5.60 (s, 2H,

CH₂); 3.87 (s, 3H, OCH₃). Anal. C, H, N.

triazine

5-oxide,¹⁷

8-methoxypyrazolo[5,1-c][1,2,4]

benzotriazine 5-oxide, 15e, and 8-methylthiopyrazol-

o[5,1-c][1,2,4]benzotriazine 5-oxide¹⁸were dissolved in

chloroform and treated with an excess of iodine

monochlorine (ICl) in chloroform. The reaction was

monitored by TLC; the ﬁnal solution was evaporated

and the residue was treated with ethanol 80%, ﬁltered,

and recrystallized by suitable solvent.

6.1.9.3. 3-(Thien-3-yl)-8-methylpyrazolo[5,1-c][1,2,4]-

benzotriazine 11cR. From 11c. Dark orange crystals.

TLC eluent: acetone/cyclohexane 1:4 v/v; ¹H NMR

(CDCl₃) d 8.52 (d, 1H, H-6); 8.42 (s, 1H, H-2); 8.26

(d, 1H, H-9); 8.22 (dd, 1H, H-2⁰3-thienyl); 7.88 (dd,

1H, H-5⁰3-thienyl); 7.58 (dd, 1H, H-7); 7.50 (dd, 1H,

H-4⁰3-thienyl). Anal. C, H, N.

6.1.8.1. 3-Iodio-8-methylpyrazolo[5,1-c][1,2,4]benzotri-

azine 5-oxide 16c. From 8-methylpyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide¹⁸and ICl. Dark yellow

crystals. TLC eluent: toluene/ethyl acetate/acetic acid

6.2. Radioligand binding assay

1

8:2:1 v/v/v; IR m^ꢀ11570; H NMR (CDCl₃) d 8.43 (d,

1H, H-6); 8.17 (d, 1H, H-9); 8.09 (s, 1H, H-2); 7.44

(dd, 1H, H-7); 2.64 (s, 3H, CH₃). Anal. C, H, N.

6.2.1. Binding Studies. [³H]Ro15-1788 (speciﬁc activity

70.8 Ci/mmol) was obtained from NEN Life Sciences

products. All the other chemicals, which were of reagent

grade, were obtained from commercial suppliers.

6.1.8.2. 3-Iodio-8-ethoxypyrazolo[5,1-c][1,2,4]benzotri-

azine 5-oxide 16d. From 8-ethoxypyrazolo[5,1-

c][1,2,4]benzotriazine 5-oxide¹⁷and ICl. Dark yellow

crystals. TLC eluent: toluene/ethyl acetate/acetic acid

Bovine cerebral cortex membranes were prepared as

previously described.^38,39The membrane preparations

were diluted with 50 mM Tris–citrate buﬀer, pH 7.4,

1

8:2:1 v/v/v; IR m^ꢀ11560; H NMR (CDCl₃) d 8.45 (d,

774

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

and used in the binding assay. Protein concentration

was assayed using the method of Lowry et al.⁴⁰

[³H]Ro15-1788 binding studies were performed as pre-

viously reported.¹⁰Clonal mammalian cell lines,

expressing relatively high levels of GABA_Areceptor

subtypes (a₁b₂c₂, a₁b₂c₂, a₃b₂c₂, and a₅b₃c₂), were

maintained as previously described⁴¹in minimum

essential medium EagleÕs with EBSS, supplemented

with 10% fetal calf serum, ^L-glutamine (2 mM), penicil-

lin (100 U/ml), and streptomycin (100 lg/ml) in a

humidiﬁed atmosphere of 5% CO₂/95% air at 37 ꢁC.

After removal, the cells were harvested by centrifuga-

tion at 500g. The crude membranes were prepared after

homogenization in 10 mM potassium phosphate, pH

7.4, and diﬀerential centrifugation at 48,000g for

30 min at 4 ꢁC. The pellets were washed twice in this

manner before ﬁnal resuspension in 10 mM potassium

phosphate, pH 7.4, that contained 100 mM potassium

chloride.⁴¹[³H]Ro15-1788 binding assays to transfected

cell membranes were carried out as previously de-

scribed.⁴¹In brief, the cell line membranes were incu-

a grasping trapeze. Mouse instinctively grabs the tra-

peze, when raised by trail, trying to stop this involun-

tary backward movement until the pulling force

overcomes animalÕs grip strength. After the animal los-

es its grip, the peak preampliﬁer automatically stores

the peak pull force and shows it on a liquid crystal

display.

6.3.3. Hole-board test. The hole-board test was used to

evaluate the eﬀects of drugs on a mouseÕs explorative

capacity and curiosity. Mice were placed individually

on the board and left free to explore both panel and

holes for 5 min, 30 min after drug administration.

6.3.4. Mouse light/dark box test. The apparatus (50 cm

long, 20 cm wide, and 20 cm high) consisted of two

equal acrylic compartments, one dark and one light,

illuminated by a 60 W bulb lamp and separated by a

divider with a 10 · 3-cm opening at ﬂoor level. Each

mouse was tested by placing it in the center of the

lighted area, facing away from the dark one, and

allowing it to explore the novel environment for

5 min. The number of transfers from one compartment

to the other and the time spent in the illuminated side

were measured. This test exploited the conﬂict between

the animalÕs tendency to explore a new environment

and its fear of bright light.

bated in

a volume of 500 ll, which contained

[³H]Ro15-1788 at a concentration of 1–2 nM and test

compound in the 10^ꢀ9–10^ꢀ5M range. Nonspeciﬁc

binding was deﬁned by 10^ꢀ5M diazepam. Assays were

incubated to equilibrium for 1 h at 4 ꢁC. The com-

pounds were dissolved in DMSO, the level of which

did not exceed 1% and which was maintained constant

in all tubes. At least six diﬀerent concentrations of each

compound were used. The data of n = 5 experiments

carried out in triplicate were analyzed by means of

an iterative curve-ﬁtting procedure (program Prism,

GraphPad, San Diego, CA), which provided IC₅₀, K_i,

and SEM values for tested compounds, the K_ivalues

being calculated from the Cheng and Prusoﬀ

equation.⁴²

6.3.5. Pentylenetetrazole-induced seizure. PTZ (90 mg/kg

sc) was injected 30 min after the administration of drugs.

The frequency of the occurrence of clonic generalized

convulsions was noted over a period of 30 min.

6.3.6. Ethanol-induced sleeping time test. Ethanol (4 g/kg

ip) was injected 30 min after drug administration. The

duration of a loss of the righting reﬂex was measured

as the sleep time. If the mice slept more than 210 min,

the end-point was recorded as 210 min.

6.3. Pharmacological methods

The experiments were carried out in accordance with the

Animal Protection Law of the Republic of Italy, DL

No. 116/1992, based on the European Communities

Council Directive of 24 November 1986 (86/609/EEC).

All eﬀorts were made to minimize animal suﬀering and

to reduce the number of animals involved. Male CD-1

Albino mice (22–24 g) and male Wistar rats (180–

200 g) (Harlan, Italy) were used. Twelve mice and three

rats were housed per cage and fed a standard laboratory

diet, with tap water ad libitum for 12/12 h light/dark cy-

cles (lights on at 7:00). The cages were brought into the

experimental room the day before the experiment, for

acclimatization purposes. All experiments were per-

formed between 10:00 and 15:00.

Drugs: Diazepam (Valium 10—Roche), Flumazenil

(Roche), Pentylenetetrazole (Sigma), and Zolpidem

(Tocris) were the drugs used. All drugs, except for

PTZ, were suspended in 1% carboxymethylcellulose

sodium salt and sonicated immediately before use.

PTZ was dissolved in isotonic (NaCl 0.9%) saline solu-

tion and injected sc. All benzodiazepine receptor

ligands were administered by the po route, except for

ﬂumazenil, which was administered ip. Drug concen-

trations were prepared in such a way that the necessary

dose could be administered in a 10 ml/kg volume of

carboxymethylcellulose (CMC) 1% by the po, ip or sc

routes.

Statistical analysis: Results are given means SEM.

Statistical analysis was performed by means of ANO-

VA, followed by ScheﬀeÕs post hoc test. StudentÕs two-

tailed t test was used to verify signiﬁcance between

two means. Data were analyzed using a computer pro-

gram (Number Cruncher Statistical System, Version

5.03 9/92). For percentage values, chi-square analysis

was used in accordance with Tallarida and Murray.

6.3.1. Rota-rod test. The integrity of the animalsÕ motor

coordination was assessed using a rota-rod apparatus

(Ugo Basile, Varese, Italy) at a rotating speed of

24 rpm. The numbers of falls from the rod in 30 s,

25 min after drug administration, were counted.

6.3.2. Grip-strength meter test. The grip-strength meter

measures forelimb grip-strength in rodents. The appa-

ratus is formed by a Perspex basis on which is located

P

values of less than 0.05 were considered

signiﬁcant.⁴³

G. Guerrini et al. / Bioorg. Med. Chem. 14 (2006) 758–775

775

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Article Doi

Benzodiazepine receptor ligands. 8: Synthesis and pharmacological evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide 3- and 8-disubstituted: High affinity ligands endowed with inverse-agonist pharmacological efficacy

DOI: 10.1016/j.bmc.2005.08.058

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Full text of DOI:10.1016/j.bmc.2005.08.058

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