New fluoro derivatives of the pyrazolo[5,1- c ][1,2,4]benzotriazine 5-oxide system: Evaluation of fluorine binding properties in the benzodiazepine site on γ-aminobutyrric acid type A (GABAA) receptor. design, synthesis, biological, and molecul

DOI: 10.1021/jm1001887

Source and publish data:

Journal of Medicinal Chemistry p. 7532 - 7548 (2010)

Update date:2022-09-26

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Authors:

Guerrini, Gabriella

Ciciani, Giovanna

Bruni, Fabrizio

Selleri, Silvia

Guarino, Chiara

Melani, Fabrizio

Montali, Marina

Daniele, Simona

Martini, Claudia

Ghelardini, Carla

Norcini, Monica

Ciattini, Samuele

Costanzo, Annarella

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Article abstract of DOI:10.1021/jm1001887

In the search for potent ligands at the benzodiazepine site on the GABA_A receptor, new fluoro derivatives of the pyrazolo[5,1-c][1,2,4] benzotriazine system were synthesized to evaluate the importance of the introduction of a fluorine atom in t

Full text of DOI:10.1021/jm1001887

7532 J. Med. Chem. 2010, 53, 7532–7548

DOI: 10.1021/jm1001887

New Fluoro Derivatives of the Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide System: Evaluation

of Fluorine Binding Properties in the Benzodiazepine Site on γ-Aminobutyrric Acid Type A

(GABA_A) Receptor. Design, Synthesis, Biological, and Molecular Modeling Investigation^†

Gabriella Guerrini,*^,‡Giovanna Ciciani,^‡Fabrizio Bruni,^‡Silvia Selleri,^‡Chiara Guarino,^‡Fabrizio Melani,^§Marina Montali,

Simona Daniele, Claudia Martini, Carla Ghelardini,^{^}Monica Norcini,^{^}Samuele Ciattini,^#and Annarella Costanzo^‡

^‡Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi (HeteroBioLab),

Universitaꢀdegli Studi di Firenze, Via U. Schiff 6, 50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy, ^§Dipartimento di Scienze Farmaceutiche,

Laboratorio di Molecular Modeling, Cheminformatics and QSAR, Universitaꢀ degli Studi di Firenze, Via U. Schiff 6, 50019 Polo Scientifico,

Sesto Fiorentino, Firenze, Italy, Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitaꢀ degli Studi di Pisa,

via Bonanno 6, 56126 Pisa, Italy, ^{^}Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Universitaꢀ degli Studi di Firenze,

Viale Pieraccini 6, 50139 Firenze, Italy, and ^#Centro di Crystallografia, Universitaꢀ degli Studi di Firenze, Via della Lastruccia 3,

50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy

Received February 12, 2010

In the search for potent ligands at the benzodiazepine site on the GABA_Areceptor, new fluoro deriva-

tives of the pyrazolo[5,1-c][1,2,4]benzotriazine system were synthesized to evaluate the importance of

the introduction of a fluorine atom in this system. Biological and pharmacological studies indicate that

the substitution at position 8 with a trifluoromethyl group confers pharmacological activity due to

potential metabolic stability in comparison to inactive 8-methyl substituted analogues. In particular, the

compound 3-(2-methoxybenzyloxycarbonyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide

(21) emerges because of its selective anxiolytic profile without side effects. An analysis of all the newly

synthesized compounds in our pharmacophoric map confirms the essential interaction points for binding

recognition and the important areas for affinity modulation. The fluorine atom was able to form a hydrogen

bond interaction only when it is not in position 3.

Introduction

group has studied heterocyclic polyazotated polycondensed

systems as useful scaffolds for the synthesis of compounds

with a well-defined target.^4-7In regard to the benzodiazepine

site on the GABA_Areceptor (Bz site/GABA_A-R), various

ligands with a pyrazolobenzotriazine core were synthesized,

and in a more recent study, in comparison with the more

frequently used Cook’s model,^8,9a pharmacophoric model

was elaborated¹⁰as reported in Figure 1.

The notable results achieved prompted us to continue the

synthesis of other derivatives of this tricyclic system to obtain

useful compounds in the benzodiazepine receptor area. Here we

report the synthesis and binding studies of new derivatives of the

pyrazolo[5,1-c][1,2,4]benzotriazine system bearing one or more

fluorine atom. The synthesis of the 3-fluorine derivatives is

designed to complete the study on the 3-halogen series.^10,11The

insertion of the trifluoromethyl group in position 7 or 8 was used

to evaluate whether the negligible pharmacological activity of

7- and 8-methyl derivatives was due to metabolic instability even

though they showed high affinity binding.¹²Finally, the difluoro-

methoxy derivatives were synthesized to evaluate whether the

oxygen atom at position 8 influences receptor interaction as in

the 8-alkyloxyderivatives.¹¹We then carried out in vivo tests on

the most interesting ligands.

GABA (γ-aminobutyrric acid^a) is the major inhibitory neuro-

transmitter in the brain. The GABA_Areceptor is a ligand-gated

ion channel (LGIC) whose involvement in various diseases

(epilepsy, anxiety, panic disorder, cognitive impairment, pain)

is well-known, making the gabaergic system a useful target for

drug development.¹Even though various subunits (R(1-6),

β(1-3), γ(1-3), δ, ε, π, F, θ) have been identified, many

combinations should be possible, whereas only a few have been

shown to actually exist.²The most common form of GABA_A

receptor contains R, β, and γ subunits in different combinations.

The site of benzodiazepine ligands is located between the

R(1-3,5) and γ2 subunits. It is believed that the R-subunit is

the main determinant of the variability in the benzodiazepine

site’s affinity and efficacy.³For many years, our research

^†Crystal structural data are available from the Cambridge Crystal-

lographic Data Center CCDC 746814.

*To whom correspondence should be addressed. Phone: þ39-055-

4573766. Fax: þ39-055-4573671. E-mail: gabriella.guerrini@unifi.it.

^aAbbreviations: GABA_A, GABA type A receptor; LGCIs, ligand-

gated ion channels; Bz site/GABA_A-R, benzodiazepine site on GABA_A

receptor; SAR, structure-affinity relationships; NCS, N-chlorosucci-

nimmide; Pd(PPh₃)₄, tetrakis(triphenylphosphine)palladium (0); PTZ,

pentylenetetrazole; Lp, lipophilic point; HBp, hydrogen bond interac-

tion point; diazepam, 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiaze-

pin-2(1H)-one; CGS 9896, 2-(4-chlorophenyl)pyrazolo[4,3-c]quinolin-

3-one; CMC, carboxymethylcellulose; flu, flumazenil; PET, positron

emission tomography; SPECT, single photon emission computed tomog-

raphy.

Our decision to synthesize fluorine derivatives arises from

the increasing importance of fluorine in medicinal chemistry

in recent years. In fact, many compounds reported in litera-

ture are endowed with remarkable biological and medicinal

applications. The fluorine’s small size, high electronegativity,

pubs.acs.org/jmc

Published on Web 10/12/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7533

Table 1. Chemical Data for New Synthesized Compounds 1-6 and 33

Nꢀ

R₄

R₃

R_4’

R₅

MF (MW)

yield (%)

mp ꢀC (recryst solvent)

COOEt

COOH

CF₃

C₁₃H₁₁N₄O₄F₃(344.26)

C₁₃H₇N₄O₄F₃(316.26)

C₇H₆N₃O₃F₃(221.12)

C₁₃H₁₁N₄O₅F₃(360.4)

C₁₃H₁₁N₄O₄F₃(344.12)

132-134 (ethanol)

117-118 (water/ethanol)

168-170 (ethanol)

254-256 dec (ethanol)

70-72 (ethanol 80%)

155-157(ethanol)

CF₃

COOEt

OCF₃

250-252 (ethanol)

and low polarizability are intriguing characteristics that can

modify the chemical and physical parameters of the deriva-

tives. A single atom of fluorine or perfluorinated groups can

opportunely modulate the stability of compounds and affect

the metabolism.^13-19The development of procedures, meth-

ods or strategies for the synthesis of fluorine derivatives, and

commercially available intermediates have made possible

access to synthetic and design strategies to introduce fluorine

into target molecules.^15,20Moreover, fluorine derivatives

could be useful tools for potential development of radioli-

gands for brain imaging (positron emission tomography, PET

or single photon emission computed tomography, SPECT) of

neuropsychiatric diseases.

Compounds 3-carboxy-7/8-trifluoromethylpyrazolo[5,1-c]

[1,2,4]benzotriazine, 14 and 15, were respectively modified

(Scheme 3) by decarboxylation with HCl conc (compounds 16

and 17), by esterification (compounds 18-23), or by trans-

formation in ketones (compounds 24 and 25). The insertion of

a halogen atom (Scheme 4) at position 3 of compounds 16 and

17, by reaction with bromine, NCS, or ICl, gave respectively

the 3-halo derivatives 26-29. This latter, 29, the 3-iodo-8-

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

in turn useful to achieve, by Suzuki coupling, the corresponding

3-heteroaryl derivatives 30-32.

Finally, since in a our previous work,¹¹we evidenced the

importance of the alkoxy group at position 8 of pyrazolobenzo-

triazine system, the idea of obtaining derivatives bearing the

8-oxygen atom linked to the trifluoromethyl group was intrigu-

ing. To obtain the 8-trifluoromethoxypyrazolobenzotriazine

system, it was necessary to synthesize the new 2-nitro-5-

trifluoromethoxyphenylhydrazine, 5, following two synthetic

approaches as depicted in Scheme 5 (route a, route b).

Route a was the better synthetic pathway, using the 4-tri-

fluoromethoxyaniline as starting material. The halogenations

with NCS,²⁶followed by diazotation and subsequent decom-

position of the obtained diazonium salt with sodium nitrite

and cuprous oxide,²⁷gave the corresponding 2-chloro-4-

trifluoromethoxynitrobenzene.²⁸The next nucleophilic sub-

stitution of the chlorine atom by hydrazine hydrate afforded

the desired 2-nitro-5-trifluoromethoxyphenylhydrazine in

good yield.

In route b (Scheme 5), the 3-trifluoromethoxyaniline was used

as starting material. Nitration, diazotation, and the next reduc-

tion²⁹gave the desired compound 5, but this route b was not

exploited because of the low yields of the various reaction steps.

The next reaction of 5 with 3-ethoxy-2-cyanopropenate

gave compound 6, the ethyl 1-(2-nitro-5-trifluoromethoxy-

phenyl)-5-aminopyrazole-4-carboxylate. The attempt to obtain

the corresponding 3-carboxy-8-trifluoromethoxypyrazolo-

[5,1-c][1,2,4]benzotriazine 5-oxide in alkali medium was disap-

pointing. In fact, although the intramolecular condensation

between nitro and amino group occurred and the pyrazolobenzo-

triazine system was formed, liability of the CF₃-O bond was

evidenced and the 8-hydroxyderivative, previously synthesized

by another method,²¹was recovered.

Chemistry

All chemical and physical data of the new compounds

are reported in Tables 1 and 2. The synthetic approach to

obtaining the desired fluorinated compounds is depicted

in Schemes 1-6.

The 3-fluoro-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide, 7, was obtained by reaction of the corresponding

3-unsubstituted compound²¹with an N-F fluorinating agent

(F-TEDA-BF₄, 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo-

[2.2.2]octanebis(tetra-fluoroborate)).²²The final complex mix-

ture was purified by column chromatography. Finally, 7 was

subjected to the nucleophilic substitution of the chlorine atom

at position 8 in PTC conditions¹¹to obtain the 3-fluoro-8-

alkyloxy-/arylmethoxy derivatives 8-13, see Scheme 1.

For synthesis of the trifluoromethyl derivatives (Scheme 2),

the intermediates ethyl 5-aminopyrazole-4-carboxylate 1-3

were synthesized from the corresponding 3-, 4-, 5-trifluoro-

methyl-2-nitro-phenylhydrazine^23-25and 2-cyano-3-ethoxy-

propenate, following a previously reported procedure.²¹The

next cyclization to the pyrazolobenzotriazine system was

obtained in standard conditions of 10% sodium hydroxide

solution²¹and then were acidifiedwith concentrated HCl. The

3-carboxy-7-trifluoromethyl- and the 3-carboxy-8-trifluoro-

methylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides, 14 and 15,

were obtained from 2and 3, respectively. The 5-aminopyrazole 1

in the same condition did not cyclize to the pyrazolobenzotriazine

system but afforded to the corresponding 5-amino-1-(2-nitro-3-

trifluoromethylphenyl)-1H-pyrazole-4 carboxylic acid 4.

7534 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Table 2. Chemical Data for New Synthesized Compounds 7-32, 34-42

Nꢀ

R₃

R_7/8

MF (MW)

yield (%)

mp ꢀC (recryst solvent)

8-Cl

C₉H₄N₄OClF (238.45)

C₁₂H₇N₄O₂F (258.40)

C₁₇H₁₃N₄O₃F (340.32)

C₁₇H₁₀N₄O₃F₄(394.30)

C₁₅H₁₀N₅O₂F₄(311.29)

C₁₄H₉N₄O₂SF (316.42)

C₁₄H₉N₄O₃F (300.25)

C₁₁H₅N₄O₃F₃(298.19)

C₁₀H₅N₄OF₃(254.18)

C₁₃H₉N₄O₃F₃(326.24)

C₁₉H₁₃N₄O₄F₃(418.34)

C₁₆H₉N₄O₃SF₃(394.34)

C₁₈H₁₃N₄O₄F₃(406.18)

C₁₅H₇N₄O₂SF₃(364.36)

C₁₀H₄N₄OF₃Br(254.18)

C₁₀H₄N₄OClF₃(289.71)

C₁₀H₄N₄OBrF₃(334.26)

C₁₀H₄N₄OIF₃(380.1)

C₁₄H₇N₄OSF₃(336.30)

C₁₄H₇N₄O₂F₃(320.23)

C₉H₅N₄OI (312.15)

204-205 (ethanol)

219-221 (ethanol)

205-206 (ethanol)

164-166 (ethanol)

264-265(ethanol)

8-O-CH₂CtCH

8-O-CH₂-2-OCH₃Ph

8-O-CH₂-2-OCF₃Ph

8-O-CH₂-4-Py

8-O-CH₂-2-thienyl

8-O-CH₂-2-furyl

7-CF₃

34^a

38^a

39^a

40^a

224-226 (ethanol)

190-192 (ethanol)

>300 dec (ethanol)

263-265 (ethanol)

282-283 (ethanol)

172-174 (ethanol)

223-224 (ethanol)

211-212 (ethanol)

205-206 (i-propyl alchol)

212-214 (i-propyl alcohol)

180-182 (i-propyl alcohol)

210-1ꢀ (i-propyl alcohol)

250-252 (ethanol)

230-231 (ethanol)

202-203 (ethanol)

203-205 (ethanol)

223-225(ethanol 80%)

194-196 (ethanol)

209-210 (i-propyl alcohol)

207-208 (ethanol)

222-224 (i-propyl alcohol)

225-226(ethanol 80%)

>300 (ethanol)

COOH

8-CF₃

7-CF₃

COOCH₂CH₃

8-CF₃

7-CF₃

COOCH₂CH₃

COOCH₂-2-OCH₃-Ph

8-CF₃

7-CF₃

COOCH₂-2-OCH₃-Ph

COOCH₂-2-thienyl

8-CF₃

7-CF₃

COOCH₂-2-thienyl

CO-2-OCH₃-Ph

8-CF₃

CO-2-thienyl

8-CF₃

7-CF₃

8-CF₃

2-thienyl

8-CF₃

3-thienyl

3-furyl

8-CF₃

COOH

8-OH

C₁₀H₆N₄O₃(230.20)

COOCH₂-2-thienyl

C₁₅H₁₀N₄O₃S (326.34)

C₁₀H₅N₄OF₂I (362.10)

C₁₃H₁₀N₄O₃F₂(308.25)

C₁₆H₁₀N₄O₃SF₂(376.35)

C₁₀H₅N₄O₂F₂I (378.10)

C₁₃H₁₀N₄O₄F₂(324.25)

>300 (ethanol)

8-OCF₂H

185-187 (ethanol)

148-150 (ethanol)

127-129 (ethanol)

170-172 (ethanol)

189-190 (ethanol)

COOCH₂CH₃

COOCH₂-2-thienyl

COOCH₂CH₃

^a5-Deoxide derivative.

However, to try to obtain the desired compound 3-carboxy-

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

another attempt was made using a different synthesis strat-

egy. A survey of the literature^30-32indicated that the pyrazolo-

[5,1-c][1,2,4]benzotriazine nucleus was also obtained by C-azo

coupling of aromatic diazonium salt. In particular, diazotized

aminopyrazole was reported to react with resorcinol to yield

a diazo derivative that in turn could cycle, with elimina-

tion of water, to a pyrazolobenzotriazine system.^31,32With

this in mind, the 3-trifluoromethoxyphenol was reacted

with the 4-ethoxycarbonylpyrazole-5-diazonium salt to obtain

the desired intermediate 5-(2-hydroxy-4-trifluoromethoxy-

phenyl)azopyrazole-4-carboxylate. Because the obtained

compound did not cycle under various condensation

conditions (acetic acid, ethylenglycol), we investigated

whether this lack of reactivity was due to a structural

feature and thus performed RX analysis. From this

study emerges unequivocally that the obtained compound

was not the desired one because the C-azo coupling

gave the regioisomer (ethyl 5-(2-trifluoromethoxy-4-

hydroxyphenyl)azopyrazole-4-carboxylate) 33 that can-

not eliminate water between the OH group and the NH-

pyrazole because they are not adjacent. (see Scheme 5 and

Figure 2).

Because the synthesis of 8-trifluoromethoxy compounds

was problematic, we attempted to obtain the 8-difluoro-

methoxy derivatives. The introduction of the difluoro-

methoxy functionality was made by exploiting the 8-hydroxy

group on the pyrazolobenzotriazine system. The reaction

with a new difluorocarbene reagent, the 2-chloro-2,2-

difluoroacetophenone³³(Scheme 6), was made on the 5-de-

oxide derivatives, the new 3-iodo-, 3-ethoxycarbonyl-,³²

and 3-(2-thienylmethoxycarbonyl)-8-hydroxypyrazolo-

[5,1-c][1,2,4]benzotriazine, 34, 35, and 37, respectively.

The reactions were complete, and the final compounds

38-40 were obtained.

Compound 37 was obtained by esterification of 36 in mild

conditions (using trichloroacetonitrile and triphenylphosphine

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7535

Scheme 1^a

^a(i) F-TEDA-BF₄, MeOH, room temperature, 25 h; (ii) ROH/NaOH 40% solution/NBu₄^þBr^-/CH₂Cl₂.

Scheme 2^a

^a(i) (a) NaOH 10% solution; (b) HCl conc.

to form the corresponding acid chloride³⁴with the addition of

the 2-thiophenmethanol). In turn, 36 was obtained by alkaline

hydrolysis of the ethyl 8-hydroxypyrazolo[5,1-c][1,2,4]benzo-

triazine 3-carboxylate 35.³²

Only compounds 38 and 39 were oxidized with acetic

anhydride/hydrogen peroxide to achieve the correspond-

ing 5-oxide derivatives 41 and 42. The attempt to oxidize

compound 40 failed because a mixture of byproduct was

recovered.

Most of the 3-fluoroderivatives (7-13) displayed affin-

ity in the range of 3.19 e K_ie 380.8 nM. The comparison

between compounds 9 and 10 with ortho-substituents in

the aromatic ring (OCH₃in 9 and OCF₃in 10) is inter-

esting. While compound 9 has the best affinity (K_i=

3.19 nM), compound 10 completely lost receptor recogni-

tion (I% = 27), thus we hypothesize a probable steric

hindrance of the 8-substituent. On the other hand, by

comparing these biological results with the corresponding

data of 3-iodine analogues (K_irange 0.42-6.6 nM),¹⁰it is

possible to assert that in the 3-fluorine series, the 8-sub-

stituent plays a critical role in the binding affinity.

Results and Discussion

Among the 8-trifluoromethyl derivatives (compounds 19,

21, 23-25, 27-32, range 5.8 e K_i(nM) e 316), compounds 21

and 23 (the 3-(2-methoxybenzyloxycarbonyl)- and the 3-(2-

thienylmethoxycarbonyl)-, respectively) had the best affinity

(K_i= 5.8 and 10 nM, respectively). The fact that these

compounds have a 2.5- and 10-fold lower binding affinity

than the corresponding 8-methyl derivatives¹²(K_i=2.3 nM

and 1.41 nM, respectively) could be due to the larger volume

of the trifluoromethyl than the methyl group.¹⁵

In Vitro and in Vivo Studies. The Bz site/GABA_A-R

binding affinity of newly synthesized compounds was eval-

uated by their ability to displace [³H]flumazenil (Ro15-1788)

from its specific binding in bovine brain membrane and was

expressed as K_ivalue only for those compounds inhibiting

radioligand binding by more than 80% at fixed concentra-

tions of 10 μM. The binding data (Table 3, compounds 7-13,

18-32, 38-42) show that all compounds were able to bind

to Bz site/GABA_A-R with good affinity, with only a few

exceptions.

Compounds 24 and 25, bearing an acyl group (2-methoxy-

benzoyl-, thien-2-carbonyl-) at position 3, have low affinity

7536 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Scheme 3^a

^a(i) HCl conc for compounds 16, 17; SOCl₂/EtOH for compounds 18, 19; NEt₃/ClCOOEt/THF, ROH for compounds 20-23; toluene/tetrakis/

Cs₂CO₃(2M), 2-methoxyphenylboronic acid/EtOH for compound 24; SOCl₂, CH₂Cl₂/SnCl₄/thiophene for compound 25.

Scheme 4^a

^a(i) NCS/CHCl₃or Br₂/CHCl₃or ICl/CHCl₃; (ii) toluene/tetrakis/Na₂CO₃(2M); 2-thienyl-, 3-thienyl-, 3-furylboronic acid/EtOH.

(K_i= 1543 and 164 nM) according to previous reports.³⁵In

the case of the 3-halogen series (27, 3-Cl; 28, 3-Br; 29, 3-I), the

affinity increased up to 10-fold from chlorine to iodine: 27,

K_i=175 nM, 28, K_i=139 nM, 29 K_i=18 nM. Thus, the

best substituent was the more lipophilic and the larger

halogen atom, allowing us to hypothesize that these features

were necessary for binding. The binding affinity for the

3-heteroaryl series (30, 2-thienyl; 31, 3-thienyl; 32, 3-furyl)

was in the nanomolar range (30, K_i=57 nM, 31, K_i=192 nM,

and 32, K_i=112 nM).

The 8-difluoromethoxy derivatives, 38-42, were synthe-

sized to evaluate if the oxygen atom at position 8 influences

the receptor interaction. The affinity data indicate that

the 8-difluoromethoxy substitution leads to higher affinity

(K_irange 1.04-51.68 nM) than the 8-alkyloxy derivatives

bearing the same substituents at position 3.^11,12The presence

of the N-oxide group slightly improves the binding affinity as

seen by comparing compounds 41 vs 38 (K_i=2.47 nM vs

5.01 nM) and 42 vs 39 (K_i=5.75 nM vs 51.68 nM).

The difference in affinity between compounds 33 and 34

(3-ethoxycarbonyl- and the 3-(2-thienylmethoxycarbonyl)-

derivative, K_i= 51.68 and 1.04 nM, respectively) is probably

due to the better ability of the 3-(2-thienylmethoxycarbonyl)

The lack of binding affinity of the 7-trifluoromethyl

derivatives (18, 20, 22, 26; I% range 3-38%) shows that

position 7 must not be occupied.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7537

Scheme 5^a

^aRoute a: (1) NCS/CH₃CN;²⁶(2) HCl/NaNO₂, NaNO₂/Cu₂O;^27,28(3) N₂H₄hydrate/ethanol. Route b: (1) acetic anhydride, HNO₃conc, NaOH

10% solution; (2) AcOH/HCl/NaNO₂; (3) SnCl₂/HCl.²⁹(i) 3-Ethoxy-2-cyanopropenate/EtOH; (ii) NaOH 10%; (iii) EtOH/sodium acetate.

group to engage in a strong lipophilic interaction, e.g. π-π

stacking, with the receptor protein as previously evidenced.³⁶

Compounds 21, 23, and 41 were chosen for in vivo tests in

mice. Six potential benzodiazepine actions were considered.

Murine motor coordination and muscle relaxant effects

were screened with the rota-rod test and the grip strength

meter test; the anticonvulsant action was evaluated using the

new drugs against pentylenetetrazole-induced convulsions

(Table 4). The drugs were also tested for their ethanol-

potentiating action (Table 5). Potential anxiolytic-like effects

were screened using the light-dark choice test (Figure 3).

Mouse learning and memory modulation were evaluated by

the passive avoidance test (Figure 4); diazepam was used as

the positive reference molecule (see Tables 4 and 5 and

Figures 3 and 4).

corresponding 8-methyl derivatives¹²was due to the meta-

bolic transformation of the methyl group. Of the 8-difluoro-

methoxyderivatives, compound 41 was chosen for in vivo

evaluation.

According to the mouse rota-rod test and the grip strength

meter test, neither 21 nor 23 induced any pharmacological

effect on motor coordination or myorelaxation (Table 4),

whereas the reference compound (diazepam 3 mg/kg ip)

increased the number of falls from the rotating rod and

induced muscle relaxant effect in a statistically significant way.

Protection from convulsions was evaluated in mice using

pentylentetrazole (PTZ) as a chemical convulsant agent.

Compounds 21, 23 (10 and 30 mg/kg po) and 41 (10 mg/kg

po) were devoid of any effect on PTZ-shock, whereas diaze-

pam (1 mg/kg ip) completely protected against PTZ-induced

shocks and convulsions (Table 4).

The behavioral effects of the 8-trifluoromethyl derivatives

21 and 23 were tested in comparison with diazepam with the

aim of evaluating whether the in vivo inactivity of the

The effects of the newly synthesized molecules, 21, 23, and

41, in comparison with diazepam on mouse anxiety, were

7538 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Scheme 6^a

Figure 2. X-ray structure of compound 33.

Table 3. BzR Ligand Affinity of New Compounds

^bAcetic acid/acetic anhydride/H₂O₂on 38 and 39. ^ab(i) NaOH 40%

solution refluxing conditions, HCl; (ii) CCl₃CN/CH₂Cl₂/Ph₃P, thio-

phenmethanol; (iii) 2-chloro-2,2-difluoroacetophenone, water/CH₃CN/

K₂CO₃.

Nꢀ

R₃

R₇

R₈

I% or K_i(nM)^a

OCH₂CtCH

50% ( 0.18

268.3 ( 8.32

3.19 ( 0.79

27% ( 2.30

380.8 ( 35.0

93.3 ( 0.10

129.34 ( 9.76

3% ( 0.31

316 ( 25.60

38% ( 3.50

5.8 ( 0.60

34% ( 3.40

10 ( 1.00

1543 ( 120

164 ( 16.0

38% ( 3.50

175 ( 15.0

139 ( 14.2

18 ( 2.0

57 ( 3.70

192 ( 20

112 ( 10

5.01 ( 0.20

51.68 ( 5.89

1.04 ( 0.03

2.47 ( 0.20

5.75 ( 0.40

10 ( 1.1

OCH₂-2-OCH₃Ph

OCH₂-2-OCF₃Ph

OCH₂-4-Py

38^b

OCH₂-2-thienyl

OCH₂-2-furyl

COOCH₂CH₃

CF₃

COOCH₂-2-OCH₃-Ph CF₃

COOCH₂-2-OCH₃-Ph

COOCH₂-2-thienyl

CF₃

CO-2-OCH₃-Ph

CO-2-thienyl

CF₃

2-thienyl

3-thienyl

3-furyl

CF₃

OCF₂H

39^bCOOCH₂CH₃

40^bCOOCH₂-2-thienyl

Diaz^c

COOCH₂CH₃

Figure 1. (A) Overlappingofligand conformations consideredinthis

study (82 compounds): in the figure are reported the essential inter-

action points for binding recognition (yellow circles) and the impor-

tant areas for affinity modulation (blue circles). (B) One of the likely

orientations that our system can adopt. It is evidenced that the

lipophilic points Lp-1 and Lp-2 and hydrogen bond interaction points

HBp-1 and HBp-2 coincide, respectively, with the L_Di, L-1/L-2, H1,

and H2 sites of the Cook model.⁸Instead, the HBp-3 (corresponding

to the acceptor hydrogen bond site A2 in Cook’s model) probably is

an acceptor/donor hydrogen bond area which is not essential for

receptor recognition but modulates the affinity ligands.

^aPercent of inhibition of specific [³H]Ro15-1788 binding at 10 μM

concentration; K_iare means ( SEM of five determination. ^b5-Deoxide

derivative. ^cSee ref 12.

studied using a light-dark box apparatus. Compound 21

(10 mg/kg po), had good anxiolytic activity, comparable to

diazepam, while 23 (3, 10, and 30 mg/kg po) and 41 (10 mg/

kg po) exhibited a slight anxiogenic effect (Figure 3). The

anxiolytic-like effects of 21 (10 mg/kg po) and the anxiogenic

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7539

Table 4. Effects of the New Compounds in Comparison with Diazepam on Motor Coordination, Muscle Relaxant Effect, and Convulsion

motor coordination

rota-rod test (16 rpm)

myorelaxant activity

grip-strength meter

anticonvulsant activity

Nꢀ of falls

in 30 min

Nꢀ of mice

(number of dead mice)

against PTZ-induced

attacks (%)

treatment^a

mg/kg po

test 35 min (g)

CMC 1%^b

diazepam

0.1 mL

0.3 (ip)

0.3 ( 0.1

58.3 ( 1.0

28(9)

7.1

71.4**

100**

0.3 ( 0.2

0.9 ( 0.3*

0.2 ( 0.1

0.1 ( 0.1

0.2 ( 0.1

55.8 ( 3.7

0.5 ( 0.5**

51.7 ( 1.2

48.5 ( 1.7

52.6 ( 1.9

57.9 ( 1.6

10(3)

10(4)

8(1)

^aNew compounds were administered 30 min and diazepam (ip) 20 min before the test. *P < 0.01, **P < 0.001 versus control mice.

^bCarboxymethylcellulose 1%. Each value represents the mean ( SEM of treated-mice.

Table 5. Effect on Ethanol-Induced Sleeping Time

whereas compounds 21 (10 mg/kg po) and 41 (3, 10, 30 mg/kg po)

treatment^a

CMC 1%^b

diazepam

mg/kg po

sleep time (s)

did not influence mouse performance. In the same experimental

test, flumazenil (100 mg/kg po) showed a statistically significant

increase in retention latency (Figure 4).

In the test of ethanol-induced sleeping time (Table 5),

compounds 21 (3, 10 mg/kg po) and 23 (3, 10 mg/kg po) did

not modify the duration of loss of righting reflex. As

expected, the reference drug diazepam (diaz, ip) enhanced

sedation in a statistically significant manner.

Pharmacophoric Model. Our previously reported pharmaco-

phoric model¹⁰that illustrated the essential interaction points

for binding recognition (HBp-1, HBp-2, and Lp-1) and the

important areas for affinity modulation (HBp-3 and Lp-2) of

a pyrazolobenzotriazine system (Figure 1) is used here to

rationalize the affinity data. The compound orientations

(one or more), reported in Figures 5-9, are the most likely

and emerged from the method reported in the Experimental

Protocols and Supporting Information.

0.1 mL

0.3 (ip)

59.2 ( 8.7

134.5 ( 7.9*

140.5 ( 4.0*

36.2 ( 11.6

54.3 ( 21.0

48.6 ( 7.0

40.8 ( 11.7

^aNew compounds were administered 30 min and diazepam (ip) 20

min before the test. *P < 0.001 versus control mice. ^bCarboxymethyl-

cellulose 1%. Each value represents the mean ( SEM of treated-mice.

The 3-fluoro derivatives (7-13) present three different

orientations in our pharmacophoric model. For clarity,

Parts A, B, and C of Figure 5 illustrate the three orientations

of compound 9, of which 5C is the most probable. In the first

two orientations (Figure 5A,B), the HBp-1 or HBp-3 points

engage respectively a hydrogen bond with a fluorine atom at

position 3, confirming its ability to form this interaction.^18,37,38

The lipophilic areas, Lp-2 in orientation 5A and Lp-1/Lp-2 in ori-

entation 5B (essential interaction points for binding recognition/

modulation) are not involved.

Instead, in the third orientation (Figure 5C), all essential

pharmacophoric points, HBp-1, HBp-2, and Lp-1, interact

efficiently through the N-oxide group, the N1-pyrazole, and

the 8-substituent, respectively. The 3-fluorine atom is located

in the Lp-2 area, but because it is unable to form efficient

lipophilic interactions (F, π = 0.14), the driving force for the

binding affinity could be due to physical and chemical

features of the 8-substituent that occupies the Lp-1 area. In

fact, by comparing compounds 9 and 10 (9, R₈= OCH₂-2-

OCH₃Ph, and 10, R₈= OCH₂-2-OCF₃Ph), we can explain

the different binding affinity in terms of steric hindrance

(9 K_i= 3.19 nM vs 10 I% = 27). The ortho-OCF₃-phenyl

ring interacts with a steric repulsive area near Lp-1

(Figure 5C,D).

Figure 3. Effect of compounds 21, 23, and 41 in comparison with

diazepam (diaz, ip) on mouse light-dark box test. The first column

represents the number of transfers in 5 min, and the second column

represents the time spent in light. Compounds and diazepam were

administered 30 min and flumazenil (flu, ip) 40 min before the test.

Each column represents the means ( SEM of 9-10 mice. *P < 0.05,

**P < 0.01, ***P < 0.001 versus control. ^∧P < 0.05, ^∧∧P < 0.01

versus 21-treated and 23-treated mice.

effect of 23 (10 mg/kg po) were completely antagonized by

flumazenil (100 mg/kg ip, dose at which flumazenil was able

to antagonize the anxiolytic effect of diazepam).

To evaluate the effect of the newly synthesized compounds

on learning and memory processes, mouse performance of

passive avoidance test was investigated. In this assay, the

parameters taken into consideration are training and retention

latencies expressed in seconds. While the training latencies did

not differ among the various groups, some retention latencies

were significantly different from others. As can be observed in

Figure 4, only compound 23 (3 and 10 mg/kg po) improved, in

a statistically significant manner, mouse memory processes,

Compounds having the CF₃group in position 8 (19, 21,

23-25, 27-32) show all pharmacophoric groups correctly

set to form efficient hydrogen bonds and lipophilic interac-

tions, thus explaining the good/fair affinity of the 8-CF₃

derivatives compared to the 7-CF₃(18, 20, 22, and 26). The

7540 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Figure 4. Effects of compounds 21, 23, and 41 in comparison with diazepam (diaz, ip) and flumazenil (flu, ip) on mouse passive avoidance test.

The first column represents the training test, and the second column represents the retention latency. Mice were treated 30 min before

the training test. The retention test was performed 24 h later. Each column represents the mean ( SEM of 9-11 mice. *P < 0.05, **P < 0.01,

***P < 0.001 versus control.

Figure 5. (A-C) Three orientations of compound 9. Orientation depicted in 5C is the most probable. (D) Compound 10 has a steric hindrance

evidenced with red arrow. The essential interaction points are represented by yellow circles and the areas for affinity modulation with blue

circles.

different affinity of 20 and 21 (I% = 38 and K_i= 5.8 nM)

can be explained because the CF₃group of 20 interacts with

a steric hindrance near previously individualized Lp-1

(Figure 6A,B).¹⁰

The position of a trifluoromethyl group at the pyrazolo-

benzotriazine core is important even when a halogen atom

(26-29) is present in position 3. Parts A and B of Figure 7

illustrate compounds 26 and 28 that fit into our pharmaco-

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7541

Figure 6. (A) The inactivity of compound 20 is due to the steric hindrance evidenced with the red arrow due to CF₃group in position 7.

(B) Compound 21 fits in the pharmacophoric model, with the 8-CF₃group in the suitable position. The essential interaction points are

represented by yellow circles and the areas for affinity modulation with blue circles.

Figure 7. (A) The inactivity of compound 26 is due to the steric hindrance evidenced with the red arrow. (B) Compound 28 fits in the

pharmacophoric model, with the 8-CF₃group in the suitable position to engage the hydrogen bond with HBp-1 area. The essential interaction

points are represented by yellow circles and the areas for affinity modulation with blue circles.

Figure 8. Compound 38 and 41 in the pharmacophoric model. (A) The N-deoxide derivative 38 interacts with all hydrogen bond areas HBp

involving also the fluorine atom of difluoromethoxy group. The corresponding N-oxide derivative 41 (B) fits in the pharmacophoric model in

two possible orientations. The essential interaction points are represented by yellow circles and the areas for affinity modulation with blue

circles.

phore model. They present only one possible orientation in

which the fluorine atom(s) of the trifluoromethyl group

interact with the hydrogen bond point HBp-1 and the

3-halogen atom fits into the lipophilic area Lp-1. When

the trifluoromethyl is in position 8, a right alignment for

the hydrogen bond occurs, permitting the halogen atom

to fit into the lipophilic area more efficiently. In the case

of the 7-trifluoromethyl derivative, if an efficient hydro-

gen bond interaction between fluorine and HBp-1 occurs,

the 3-halogen atom interacts with the probable steric

repulsive area near Lp-1 and the receptor recognition

fails.

7542 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Figure 9. (A) The 5-N-deoxide derivative 39, and in (B) compound 42, the 5-N-oxide derivative in the pharmacophoric model. The essential

interaction points are represented by yellow circles and the areas for affinity modulation with blue circles.

Concerning the 8-difluoromethoxy derivatives, molecular

modeling suggests that the fluorine atoms (of 8-OCF₂H group)

contribute significantly to receptor interaction for compounds

38 and 41 (3-iodo derivatives), while the oxygen atom of the

same 8-substituent plays an important role in the binding for

compounds 39 and 42 (3-ethoxycarbonyl derivatives). Com-

pound 38 presents one possible orientation in which the

fluorine atom(s) of the difluoromethoxy group cause hydrogen

bond interaction with the HBp-1 area. Compound 41 presents,

instead, two possible orientations in which the fluorine atom(s)

of the difluoromethoxy group alternatively interact with the

HBp-1 or HBp-3 and the iodine atom at position 3 fits with Lp-1

or Lp-2 (Figure 8A,B). The same hydrogen bond interaction

is evidenced, also, for the oxygen atom of the 8-OCF₂H

group in compounds 39 and 42, in the assumed orientations

(Figure 9A,B). The difference in the affinity shown by 39 and

42 (39, 5-N-deoxide, K_i= 51.68 nM, and 42, 5-N-oxide K_i=

5.75 nM) may be due to the better alignment of 42 in the

pharmacophoric map (Figure 9A,B). Thus, it emerges that

the substituent at position 3 determines the interaction of the

fluorine or oxygen atom of the -OCF₂H group, within the

HBp areas.

We have updated this pharmacophoric model, previously

validated¹⁰by inserting two ligands (CGS 9698 and diazepam,

already used by Cook⁹), and further confirmed the essential

interaction points for binding recognition.

Experimental Protocols

Chemistry. Melting points were determined with a Gallenkamp

apparatus and were uncorrected. Silica gel plates (Merk F₂₅₄) and

Silica Gel 60 (Merk 70-230 mesh) were used for analytical and

column chromatography, respectively. The structures of all com-

pounds were supported by their IR spectra (KBr pellets in nujol

mulls, Perkin-Elmer 1420 spectrophotometer) and ¹H NMR data

(measured with a Bruker 400 MHz). Chemical shifts were ex-

pressed in δ 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 assigned structure. The chemical and physical

data of new compounds are shown in Tables 1 and 2. All new

compounds possess a purity g95%;microanalyses were performed

with a Perkin-Elmer 260 analyzer for C, H, N. Mass spectra (m/z)

were recorded on a LTQ mass spectrometer (ThermoFisher, San

Jose, CA, USA). The crystal structure of compound 33 was solved

by means of single-crystal X-ray diffraction.

2-Nitro-5-trifluoromethoxyphenylhydrazine (5). A solution

in THF (20 mL) of 2-chloro-4-trifluoromethoxynitrobenzene

(270 mg, 1.12 mmol), synthesized by diazotation of the 2-chloro-

4-trifluoromethoxyaniline²⁶and subsequent decomposition of the

obtained diazonium salt with sodium nitrite and cuprous oxide,²⁷

was added of hydrazine hydrate (1 mL) and maintained at reflux-

ing temperature. The reaction was monitored by TLC (toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v), and when the starting material

disappeared, the red solution was evaporated to dryness. The

residue was recovered by ethanol 80% and recrystallized by the

same solvent. Red crystals. ¹H NMR (CDCl₃) δ 9.00 (bs, 1H, NH

exch), 8.15 (d, 1H, H-3), 7.52 (s, 1H, H-6), 6.51 (d, 1H, H-4), 3.85

(bs, 2H, NH₂exch). Anal. C, H, N.

General Procedure for the Synthesis of 5-Aminopyrazoles 1-3,

6. A suspension of the suitable 3-, 4-, 5-trifluoromethyl-2-nitro-

phenylhydrazine^23-25or 2-nitro-5-trifluoromethoxy phenylhydra-

zine, 5 (1 mmol), and 2-cyano-3-ethoxypropenate (1 mmol) in

ethanol (50 mL) with HCl as acid catalyst, were refluxed until the

starting material disappeared in TLC (toluene/ethyl acetate/acetic

acid 8:2:1 v/v/v). The workup of the final solution gave the ethyl

1-(2-nitro-3-, 4-, 5-trifluoromethylphenyl)-5-aminopyrazol-4-car-

boxylate, 1-3, and the ethyl 1-(2-nitro-5-trifluoromethoxy-

phenyl)-5-aminopyrazol-4-carboxylate 6, that were recrystallized

by suitable solvent.

Ethyl 1-(2-Nitro-3-trifluoromethylphenyl)-5-aminopyrazol-4-

carboxylate (1). Yellow crystals. IR ν cm^-13300, 3200, 1687.

¹H NMR (CDCl₃) δ 7.90 (d, 1H, H-4 phenyl), 7.88 (s, 1H, H-3),

7.70 (d, 1H, H-6 phenyl), 7.58 (t, 1H, H-5 phenyl), 5.10 (bs, 2H,

NH₂exch), 4.35 (q, 2H, CH₂), 1.40 (t, 3H, CH₃). Anal. C, H, N.

Conclusion

We have evaluated the binding properties of the fluorine

atom(s) in the Bz site on GABA_Areceptors and their ability to

render compounds more metabolically stable. These findings

indicate that the fluorine atom is very important for recognition,

engaging hydrogen bond interaction, when it is in position 8

inserted in the -CF₃or -OCF₂H groups. When the fluorine

atom is in position 3, it does not cause any interaction and the

driving force for recognition is the substituent in position 8.

The in vivo results on compounds 21, the 3-(2-methoxy-

benzyloxycarbonyl)- and 23, the 3-(2-thienylmethoxycar-

bonyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide, confirm the potential metabolic stability due to the

CF₃group versus the CH₃group. In fact, these ligands,

although having about 2-10-fold less affinity than corre-

sponding 8-methyl derivatives,¹²present interesting pharma-

cological activity. Compound 21 has a selective anxiolytic-like

profile (10 mg/kg) without impairing memory or modifying

the duration of righting reflex loss. Compound 23 has an

inverse-agonist profile because it shows anxiogenic and

pro-mnemonic activity (3 and 10 mg/kg). These activities

are mediated by acting on the benzodiazepine site on

GABA_Areceptors because they are completely antagonized

by flumazenil.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7543

Ethyl 1-(2-Nitro-4-trifluoromethylphenyl)-5-aminopyrazol-4-

3-Fluoro-8-(thien-2-ylmethoxy)pyrazolo[5,1-c][1,2,4]benzo-

triazine 5-Oxide (12). From 7 and thiophen-2-methanol.

Yellow crystals. ¹H NMR (DMSO-d₆) δ 8.44 (d, 1H, H-2,

carboxylate (2). Yellow crystals. IR ν cm^-13400, 3300, 1687.

¹H NMR (CDCl₃) δ 8.25 (s, 1H, H-3 phenyl), 8.10 (d, 1H,

H-5 phenyl), 7.80 (m, 2H, H-6 phenyl and H-3), 5.30 (bs,

2H, NH₂exch), 4.35 (q, 2H, CH₂), 1.40 (t, 3H, CH₃). Anal.

C, H, N.

_H-F= 3.84 Hz), 8.36 (d, 1H, H-6), 7.81 (d, 1H, H-9), 7.62 (d,

1H, H-5⁰thienyl), 7.35 (m, 2H, H-7 and H-3⁰thienyl), 7.08 (t,

1H, H-4⁰thienyl), 5.65 (s, 2H, CH₂O). Anal. C, H, N.

3-Fluoro-8-(fur-2-ylmethoxy)pyrazolo[5,1-c][1,2,4]benzotri-

azine 5-Oxide (13). From 7 and furan-2-methanol (furfuryl

alcohol). Yellow crystals. ¹H NMR (DMSO-d₆) δ 8.42 (d, 1H,

H-2, J_H-F= 3.84 Hz), 8.36 (d, 1H, H-6), 7.82 (d, 1H, H-9), 7.75

(d, 1H, H-5⁰furyl), 7.35 (dd, 1H, H-7), 6.70 (d, 1H, H-3⁰furyl),

6.51 (m, 1H, H-4⁰furyl), 5.45 (s, 2H, CH₂O). Anal. C, H, N.

General Procedure for the Synthesis of Compounds 14 and 15.

A suspension of 1.0 mmol of suitable 5-aminopyrazole 2, 3

in 10% solution of sodium hydroxide (15 mL) was stirred at

30-40 ꢀC. The reaction was monitored by TLC (eluent toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v), the final suspension was

acidified with concentrated hydrochloric acid; crude acids, 14

and 15, were recovered by filtration and recrystallized by

ethanol.

Ethyl 1-(2-Nitro-4-trifluoromethylphenyl)-5-aminopyrazol-4-

carboxylate (3). Yellow crystals. IR ν cm^-13380, 3300, 1687.

¹H NMR (CDCl₃) δ 8.15 (dd, 1H, H-3 phenyl), 7.95 (m, 2H, H-4

and H-6 phenyl), 7.82 (s, 1H, H-3), 5.30 (bs, 2H, NH₂exch), 4.35

(q, 2H, CH₂), 1.40 (t, 3H, CH₃). Anal. C, H, N.

Ethyl 1-(2-Nitro-4-trifluoromethyoxyphenyl)-5-aminopyrazol-

4-carboxylate (6). Yellow crystals. IR ν cm^-13380, 3300,

1687. H NMR (CDCl₃) δ 8.13 (dd, 1H, H-3 phenyl), 7.82 (s,

1H, H-3), 7.49 (m, 2H, H-4 and H-6 phenyl), 5.25 (bs, 2H, NH₂

exch), 4.40 (q, 2H, CH₂), 1.40 (t, 3H, CH₃). Anal. C, H, N.

1-(2-Nitro-3-trifluoromethylphenyl)-5-aminopyrazol-4-carboxylic

Acid (4). A suspension of compound 1 (0.2 mmol) in a 10%

solution of sodium hydroxide (15 mL) was warmed at 60 ꢀC for

3 days. The final suspension was made slightly acid, and the final

compound was recovered. The recrystallization with ethanol gave

the pure compounds. Yellow crystals. IR ν cm^-13380, 3300, 1687.

¹H NMR (DMSO-d₆) δ 12.00 (s, 1H, COOH), 8.05 (d, 1H, H-4),

7.85 (d, 1H, H-6 phenyl), 7.65 (t, 1H, H-5 phenyl), 7.68 (s, 1H, H-3),

6.38 (s, 2H, NH₂exch). Anal. C, H, N.

3-Carboxy-7-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-Oxide (14). From the 5-aminopyrazole 2. Yellow crystals. IR

ν cm^-12700-2600, 1680, 1550. ¹H NMR(DMSO-d₆) δ 13.00 (bs,

1H, COOH), 8.72 (d, 1H, H-6), 8.68 (s, 1H, H-2), 8.60 (d, 1H,

H-9), 8.46 (dd, 1H, H-8). Anal. C, H, N.

3-Fluoro-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide

(7). From 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide²¹

(100 mg, 0.45 mmol) and Selectfluor (F-TEDA-BF₄, 1-chloro-

methyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetra-fluoro-

borate) (2 equiv)) in methanol (6 mL) for 24 h at room tempera-

ture. The final suspension was evaporated and washed with water,

and the raw precipitate was purified by column chromatography

(eluent toluene/ethyl acetate/acetic acid 8:2:1 v/v/v). Yellow crys-

tals. ¹H NMR (CDCl₃) δ 8.49 (d, 1H, H-6), 8.36 (d, 1H, H-9), 8.02

(d, 1H, H-2, J_H-F= 3.84 Hz), 7.60 (dd, 1H, H-7). MS(ESI) m/z:

239.09 ([M þ H]^þ). Anal. C, H, N.

3-Carboxy-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-Oxide (15). From the 5-aminopyrazole 3. Yellow crystals. IR

ν cm^-12700-2600, 1680, 1550. ¹H NMR(DMSO-d₆) δ 13.00 (bs,

1H, COOH), 8.65 (m, 3H, H-2, H-6 and H-9), 8.12 (dd, 1H, H-7).

Anal. C, H, N.

General Procedure for the Synthesis of Compounds 16-17.

Compounds 14 or 15 (0.5 mmol) were suspended in concen-

trated hydrochloric acid (15 mL) at refluxing temperature. After

5 h,, the reaction was stopped, the suspension was extracted with

chloroform, and the organic layer was dried with anhydrous

sodium sulfate. Evaporation under vacuum gave yellow-

orange residue recuperated by ethanol.

General Procedure for the Synthesis of Compounds 8-13. A

mixture of compound 7 (0.45 mmol), 10 mL of dichloro-

methane, 5 mL of 40% sodium hydroxide solution, 0.1 mol of

tetrabutylammonium bromide, and suitable alcohol in large

excess (5 mL) was vigorously stirred at 30-50 ꢀC and monitored

by TLC (eluent toluene/ethyl acetate/acetic acid 8:2:1 v/v/v).

The organic layer was then separated and the aqueous layer

extracted twice with 10 mL of dichloromethane. The combined

organic extracts were evaporated, and the residue was recovered

with 1-2 mL of ethanol and recrystallized by suitable solvent.

3-Fluoro-8-(2-propynyloxy)pyrazolo[5,1-c][1,2,4]benzotriazine

5-Oxide (8). From 7 and propargyl alcohol. Yellow crystals.

¹H NMR (DMSO-d₆) δ 8.43 (d, 1H, H-2, J_H-F= 3.84 Hz), 8.38

(d, 1H, H-6), 7.78 (d, 1H, H-9), 7.32 (dd, 1H, H-7), 5.17 (d, 2H,

CH₂O), 3.78 (t, 1H, CH). Anal. C, H, N.

7-Trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide

(16). From 14; TLC eluent: toluene/ethyl acetate/acetic acid

8:2:1 v/v/v. IR ν cm^-11550. H NMR (CDCl₃) δ 8.85 (d, 1H,

H-6), 8.55 (d, 1H, H-9), 8.20 (m, 2H, H-2 and H-8), 6.80 (d, 1H,

H-3). Anal. C, H, N.

8-Trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxide

(17). From 15; TLC eluent: toluene/ethyl acetate/acetic acid

8:2:1 v/v/v. IR ν cm^-11550. H NMR (CDCl₃) δ 8.72 (m, 2H,

H-6 and H-9), 8.18 (d, 1H, H-2), 7.88 (dd, 1H, H-7), 6.82 (d, 1H,

H-3). Anal. C, H, N.

General Procedure for the Synthesis of Compounds 18-23.

The starting acids 14 and 15 were transformed into ester

derivatives (18-23) by means of two methods, A and B.

Method A. The ethyl esters 18 and 19 were obtained from

corresponding acids 14 and 15 (0.5 mmol) by means of synthesis

of the corresponding 3-carbonyl chloride, which was, in turn,

suspended in anhydrous ethyl alcohol (15 mL). The precipitate

was filtered and purified by recrystallization.

Method B. All other esters (20-23) were obtained by treating

the corresponding acids (0.5 mmol) (14, 15) in tetrahydrofurane

(THF), with triethyl amine (1:3.5) in an ice bath for 30 min. The

suspension was supplemented with ethylchlorocarbonate (1:2)

and maintained from 0 ꢀC to room temperature under stirring

for 1 h to permit the anhydride to form. Alcohol was added

(1:2.5), and the mixture was heated at 60 ꢀC for 8-18 h and

monitored by TLC. The final suspension was diluted with water

and extracted with chloroform that 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 using a suitable solvent.

3-Ethoxycarbonyl-7-trifluoromethylpyrazolo[5,1-c][1,2,4]-

benzotriazine 5-Oxide (18). From 14 and ethanol (method A).

Yellow crystals. TLC eluent: toluene/ethyl acetate 8:2 v/v.

3-Fluoro-8-(2-methoxybenzyloxy)pyrazolo[5,1-c][1,2,4]benzo-

triazine 5-Oxide (9). From 7 and 2-methoxybenzyl alcohol.

Yellow crystals. H NMR (CDCl₃) δ 8.45 (d, 1H, H-6), 8.00

(d, 1H, H-2, J_H-F= 3.84 Hz), 7.85 (d, 1H, H-9), 7.49 (d, 1H,

H-6⁰phenyl), 7.38 (t, 1H, H-4⁰phenyl), 7.23 (dd, 1H, H-7), 7.05 (t,

1H, H-5⁰phenyl), 6.98 (d, 1H, H-3⁰phenyl), 5.35 (s, 2H, CH₂O),

3.90 (s, 3H, OCH₃). Anal. C, H, N.

3-Fluoro-8-(2-trifluoromethoxybenzyloxy)pyrazolo[5,1-c][1,2,4]-

benzotriazine 5-Oxide (10). From 7 and 2-trifluoromethoxybenzyl

alcohol. Yellow crystals. ¹H NMR (DMSO-d₆) δ 8.44 (d, 1H, H-2,

J_H-F= 3.84 Hz), 8.38 (d, 1H, H-6), 7.81 (d, 1H, H-9), 7.76 (d, 1H,

H-3⁰phenyl), 7.58 (t, 1H, H-5⁰phenyl), 7.48 (m, 1H, H-6⁰and H-4⁰

phenyl), 7.36 (dd, 1H, H-7), 5.48 (s, 2H, CH₂O). Anal. C, H, N.

3-Fluoro-8-(pyridine-4-ylmethoxy)pyrazolo[5,1-c][1,2,4]benzo-

triazine 5-Oxide (11). From 7 and pyridine-4-methanol. Yellow

crystals. ¹H NMR (DMSO-d₆) δ 8.65 (d, 2H, H-3⁰and H-5⁰Py),

8.44(d, 1H, H-2, J_H-F= 3.84 Hz), 8.38(d, 1H, H-6), 7.79(d, 1H,

H-9), 7.57 (d, 2H, H-2⁰and H-6⁰Py), 7.42 (dd, 1H, H-7), 5.52 (s,

2H, CH₂O). Anal. C, H, N.

7544 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

IR ν cm^-11720, 1550. ¹H NMR (CDCl₃) δ 8.85 (d, 1H, H-6),

8.60 (m, 2H, H-2 and H-9), 8.22 (dd, 1H, H-8), 4.45 (q, 2H,

CH₂), 1.45 (t, 3H, CH₃). Anal. C, H, N.

3-Ethoxycarbonyl-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzo-

triazine 5-Oxide (19). From 15 and ethanol (method A). Yellow

crystals. TLC eluent: toluene/ethyl acetate 8:2 v/v. IR ν cm^-1

1720, 1550. ¹H NMR (CDCl₃) δ 8.78 (d, 1H, H-9), 8.72 (d, 1H,

H-6), 8.58 (s, 1H, H-2), 7.94 (dd, 1H, H-7), 4.45 (q, 2H, CH₂), 1.45

(t, 3H, CH₃). Anal. C, H, N.

3-(2-Methoxybenzyloxycarbonyl)-7-trifluoromethylpyrazolo-

[5,1-c][1,2,4]benzotriazine 5-Oxide (20). From 14 and 2-meth-

oxybenzyl alcohol (method B). Yellow crystals. TLC eluent:

toluene/ethyl acetate 8:2 v/v. IR ν cm^-11720, 1550. ¹H NMR

(CDCl₃) δ 8.85 (d, 1H, H-6), 8.60 (m, 2H, H-2 and H-9), 8.22

(dd, 1H, H-8), 7.56 (d, 1H, H-6⁰phenyl), 7.34 (t, 1H, H-4⁰

phenyl), 7.02 (t, 1H, H-5⁰phenyl), 6.92 (d, 1H, H-3⁰), 5.45 (s,

2H, CH₂), 3.85 (s, 3H, OCH₃). Anal. C, H, N.

3-(2-Methoxybenzyloxycarbonyl)-8-trifluoromethylpyrazolo-

[5,1-c][1,2,4]benzotriazine 5-Oxide (21). From 15 and 2-meth-

oxybenzyl alcohol (method B). Yellow crystals. TLC eluent:

isopropyl ether/cyclohexane 8:3 v/v. IR ν cm^-11720, 1550.

¹H NMR (CDCl₃) δ 8.78 (d, 1H, H-9), 8.70 (d, 1H, H-6), 8.58 (s,

1H, H-2), 7.94 (dd, 1H, H-7), 7.58 (d, 1H, H-6⁰phenyl), 7.35 (t,

1H, H-4⁰phenyl), 7.02 (t, 1H, H-5⁰phenyl), 6.95 (d, 1H, H-3⁰

phenyl), 5.50 (s, 2H, CH₂), 3.80 (s, 3H, OCH₃). Anal. C, H, N.

3-(2-Thienylmethoxycarbonyl)-7-trifluoromethylpyrazolo[5,1-

c][1,2,4]benzotriazine 5-Oxide (22). From 14 and thiophen-2-

methanol (method B). Yellow crystals. TLC eluent: toluene/

ethyl acetate 8:2 v/v. IR ν cm^-11720, 1550. ¹H NMR (CDCl₃)

δ 8.87 (d, 1H, H-6), 8.59 (m, 2H, H-2 and H-9), 8.24 (dd, 1H,

H-8), 7.36 (dd, 1H, H-5⁰thienyl), 7.24 (d, 1H, H-3⁰thienyl), 7.02

(m, 1H, H-4⁰thienyl), 5.60 (s, 2H, CH₂). Anal. C, H, N.

3-(2-Thienylmethoxycarbonyl)-8-trifluoromethylpyrazolo[5,1-

c][1,2,4]benzotriazine 5-Oxide (23). From 15 and thiophen-2-

methanol (method B). Yellow crystals. TLC eluent: isopropyl ether/

cyclohexane 8:3 v/v. IR νcm^-11720, 1550. ¹HNMR(CDCl₃) δ8.78

(d, 1H, H-9), 8.70(d, 1H, H-6), 8.58(s, 1H, H-2), 7.94(dd, 1H, H-7),

7.38 (dd, 1H, H-5⁰thienyl), 7.24 (d, 1H, H-3⁰thienyl), 7.02 (m, 1H,

H-4⁰thienyl), 5.60 (s, 2H, CH₂). Anal. C, H, N.

3-(2-Methoxybenzoyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]-

benzotriazine 5-Oxide (24). Compound 15 (0.4 mmol) was

+reacted with 2.0 mL of thionyl chloride and maintained at

60 ꢀC for 30 min. The final solution was evaporated in vacuum,

and the intermediate 3-carbonylchloride was suspended in 8 mL

of absolute toluene. 2-Methoxyphenylboronic acid (0.2 mmol),

cesium carbonate (0.5 mmol), and an excess of triphenylphos-

phine palladium (0) (tetrakis, 10 mg) were added. The mixture

was maintained at refluxing temperature under nitrogen for

2 days. The final suspension was diluted with ethyl acetate and

washed with water and a saturated solution of sodium bicarbon-

ate. The ethyl acetate solution was then dried over sodium sul-

fate and evaporated under pressure. The residue was recrystal-

lized by ethanol. Yellow crystals. TLC eluent: isopropyl ether/

cyclohexane 8:3 v/v. IR ν cm^-11680, 1550. ¹H NMR (CDCl₃) δ

8.78 (d, 1H, H-9), 8.68 (d, 1H, H-6), 8.50 (s, 1H, H-2), 7.92 (dd,

1H, H-7), 7.58 (m, 2H, H-6⁰and H-4⁰phenyl), 7.12 (t, 1H, H-5⁰

phenyl), 6.95 (d, 1H, H-3⁰phenyl), 3.80 (s, 3H, OCH₃). Anal.

C, H, N.

separated. This layer was washed with a saturated solution of

sodium bicarbonate, dried over anhydrous sodium sulfate, and

evaporated. The residue was recovered with isopropyl ether and

recystallized. Yellow crystals. IR ν cm^-11680, 1550. H NMR

(CDCl₃) δ 8.82 (d, 1H, H-9), 8.72 (d, 1H, H-6), 8.68 (s, 1H, H-2),

8.02 (dd, 1H, H-3⁰thienyl), 7.94 (dd, 1H, H-7), 7.78 (d, 1H, H-5⁰

thienyl), 7.25 (m, 1H, H-4⁰thienyl). Anal. C, H, N.

General Procedure for the Synthesis of Compounds 26-29. A

solution in chloroform (10 mL) of starting material 16 or 17 (0.5

mmol) was supplemented with an excess of bromine (1.0 mL) to

obtain compounds 26 and 28, with N-chlorosuccinimide (NCS,

100 mg) and a catalytic amount of benzoyl peroxide to obtain

compound 27, and with iodine monochloride (1:2) chloroform

solution to obtain compound 29. The final solution was evapo-

rated to dryness and the residue purified by recrystallization.

3-Bromo-7-trifluoromethylpyrazolo[5,1-c][1,2,4]pyrazolo-

benzotriazine 5-Oxide (26). From 16 and bromine. TLC

eluent: toluene/ethyl acetate 8:2:1 v/v/v. IR ν cm^-11550.

¹H NMR (CDCl₃) δ 8.85 (d, 1H, H-6), 8.50 (d, 1H, H-9), 8.18

(dd, 1H, H-8), 8.10 (s, 1H, H-2). Anal. C, H, N.

3-Bromo-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-Oxide (27). From 17 and bromine. TLC eluent: toluene/ethyl

acetate/aceticacid 8:2:1 v/v/v. IR ν cm^-11550. ¹H NMR(CDCl₃)

δ 8.70 (m, 2H, H-6 and H-9), 8.15 (d, 1H, H-2), 7.89 (dd, 1H,

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

3-Chloro-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide (28). From 17 and NCS. TLC eluent: isopropyl ether/

cyclohexane 8:3 v/v. IR ν cm^-11550. ¹H NMR (CDCl₃) δ 8.70

(m, 2H, H-6 and H-9), 8.12 (d, 1H, H-2), 7.89 (dd, 1H, H-7).

Anal. C, H, N.

3-Iodo-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotriazine

5-oxide (29). From 17 and ICl. TLC eluent: toluene/ethyl

acetate/acetic acid 8:2:1 v/v/v. IR ν cm^-11550. ¹H NMR

(CDCl₃) δ 8.70 (m, 2H, H-6 and H-9), 8.18 (d, 1H, H-2),

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

General Procedure for the Synthesis of Compounds 30-32.

Tetrakis (triphenylphosphinepalladium(0), 30 mg, 0.026 mmol)

and compound 29 (0.30 mmol) were combined in anhydrous

toluene (4.0 mL). The suitable heteroarylboronic acid (2-, 3-thio-

phen boronic acid and 3-furanboronic acid, 0.60 mmol) in

absolute ethanol (2.5 mL) and aqueous sodium carbonate (2M,

4 mL) were added, and the reaction mixture was heated to reflux

for 12 h. The red product was then extracted by dichloromethane,

and the organic layer was washed with water and dried over

anhydrous sodium sulfate. The evaporation of the solvent gave

the crude product that was recrystallized by suitable solvent.

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

triazine 5-Oxide (30). Dark-red crystals. IR ν cm^-11550. H

NMR (CDCl₃) δ 8.68 (m, 2H, H-6 and H-9), 8.35 (s, 1H, H-2),

7.85 (dd, 1H, H-7), 7.68 (dd, 1H, H-3⁰thienyl), 7.40 (d, 1H,

H-5⁰thienyl), 7.18 (m, 1H, H-4⁰thienyl). Anal. C, H, N.

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

azine 5-Oxide (31). Dark-red crystals; IR ν cm^-11550. ¹H NMR

(CDCl₃) δ 8.70 (m, 2H, H-6 and H-9), 8.39 (s, 1H, H-2), 7.94 (dd,

1H, H-2⁰thienyl), 7.85 (dd, 1H, H-7), 7.68 (dd, 1H, H-4⁰thienyl),

7.48 (d, 1H, H-5⁰thienyl). Anal. C, H, N.

3-(Fur-3-yl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]benzotri-

azine 5-Oxide (32). Dark-red crystals. IR ν cm^-11550. ¹H

NMR (CDCl₃) δ 8.70 (m, 2H, H-6 and H-9), 8.28 (s, 1H, H-2),

8.10 (dd, 1H, H-2⁰furyl), 7.85 (dd, 1H, H-7), 7.58 (dd, 1H, H-5⁰

furyl), 6.92 (d, 1H, H-4⁰furyl). Anal. C, H, N.

3-(Thien-2-ylcarbonyl)-8-trifluoromethylpyrazolo[5,1-c][1,2,4]-

benzotriazine 5-Oxide (25). Compound 15 (0.3 mmol) was re-

acted with 2.0 mL of thionyl chloride and maintained at 60 ꢀC for

30 min. The final solution was evaporated in vacuum, and the

intermediate 3-carbonylchloride was suspended in a solution of

15 mL of methylene chloride and 0.9 mmol of anhydrous tin

tetrachloride; the dark-yellow solution was supplemented, after

5 min, with thiophene (1.2 mmol) and the reaction monitored by

TLC (eluent toluene/ethyl acetate/acetic acid 8:2:1 v/v/v). The

obtained suspension became red, and when the starting material

disappeared, the reaction was quenched by treatment with HCl

1:1, diluted with methylene chloride, and the organic layer

Ethyl 5-(2-Trifluoromethoxy-4-hydroxyphenyl)azopyrazole-

4-carboxylate (33). A solution of diazotized ethyl 3(5)-amino-1H-

pyrazole-4-carboxylate (commercially available) (2.0 mmol) was

slowly added to a solution in ethanol (10 mL) of 3-trifluoromethox-

yphenol (1.0 mmol) containing sodium acetate (500 mg) at 0 ꢀCwith

stirring. After complete addition, the reaction mixture was stirred for

a further hour and then 100 g of ice were added. An orange-

red precipitate was formed that was collected by filtration and

recrystallized by ethanol. IR ν cm^-13178, 1693, 1572. H NMR

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7545

(DMSO-d₆) δ 13.70 (bs, 1H, OH, exch), 11.00 (bs, 1H. NH, exch),

8.37 (s, 1H, H-3 pyrazole), 7.72 (d, 1H, H-6 phenyl), 6.98 (m, 2H,

H-3 and H-5 phenyl), 4.20 (q, 2H, CH₂),1.20(t, 3H,CH₃). MS(ESI)

m/z: 343.0, 344.1 ([M þ H]^þ). Crystallographic data for compound

33 are reported in the Supporting Information. Anal. C, H, N.

3-Iodo-8-hydroxypyrazolo[5,1-c][1,2,4]benzotriazine (34). From

starting material 3-iodo-8-hydroxypyrazolo[5,1,-c][1,2,4]benzotri-

azine 5-oxide,¹¹following a previously described procedure, was

reduced with triethylphosphite (TEP, 1.0 mL) in toluene (10 mL)⁷at

refluxing temperature for 8 h. Yellow crystals. TLC eluent: toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v. IR νcm^-13700-3600. ¹HNMR

(DMSO-d₆) δ 11.90 (bs, 1H, OH, exch), 8.50 (m, 2H, H-6 and H-2),

7.59 (d, 1H, H-9), 7.30 (dd, 1H, H-7). Anal. C, H, N.

General Procedure for the Synthesis of Compounds 41-42. A

solution of compound 38 or 39 (0.150 mmol) in acetic acid (7.0 mL)

and acetic anhydride (4.0 mL) was added, with caution, to hydrogen

peroxide (35 wt % in water, 4.0 mL). The mixture was refluxed and

stirred for 2-3 h and monitored by TLC (eluent: toluene/ethyl

acetate/acetic acid 8:2:1 v/v/v). The final solution was treated with

ice, and the suspension formed was extracted by ethyl acetate; this

was dried, evaporated to dryness, and the residue was recovered with

a few milliliters of ethanol and purified by recrystallization.

3-Iodo-8-difluoromethoxypyrazolo[5,1-c][1,2,4]benzotriazine

5-Oxide (41). From starting material 38. Orange crystals.

¹H NMR (DMSO-d₆) δ 8.49 (d, 1H, H-6), 8.41 (s, 1H, H-2),

8.00 (d, 1H, H-9), 7.72 (t, 1H, CHF₂, J_H-F73 Hz), 7.50 (dd, 1H,

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

Ethyl 8-Hydroxypyrazolo[5,1-c][1,2,4]benzotriazine-3-carboxyl-

ate (35). This compound was synthesized following a described

procedure.³²Melting point and ¹H NMR data are according to the

literature.

3-Ethoxycarbonyl-8-difluoromethoxypyrazolo[5,1-c][1,2,4]-

benzotriazine 5-oxide (42). From starting material 39. Yellow

crystals. H NMR (DMSO-d₆) δ 8.68 (s, 1H, H-2), 8.54 (d, 1H,

8-Hydroxypyrazolo[5,1-c][1,2,4]benzotriazine-3-carboxylic Acid

(36). Compound 35 (0.70 mmol) was reacted with 20% sodium

hydroxide solution at refluxing temperature. The final acid was

recovered by filtration after cooling and acidification with con-

centrated hydrochloric acid. Yellow crystals. TLC eluent: toluene/

ethyl acetate/acetic acid 8:2:1 v/v/v. IR ν cm^-13700-3600, 1680.

¹H NMR (DMSO-d₆) δ 12.90 (bs, 1H, COOH, exch), 12.40 (s, 1H,

OH, exch), 8.68 (s, 1H, H-2), 8.55 (d, 1H, H-6), 7.77 (d, 1H, H-9),

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

H-6), 8.06 (d, 1H, H-9), 7.76 (t, 1H, CHF₂, J_H-F73 Hz), 7.58 (dd,

1H, H-7), 4.34 (q, 2H, CH₂), 1.34 (t, 3H, CH₃). Anal. C, H, N.

Radioligand Binding Assay. [³H]Ro15-1788 (specific activity

78.8 Ci/mmol) was obtained from Perkin-Elmer. All the other

chemicals, which were of reagent grade, were obtained from

commercial suppliers.

Bovine cerebral cortex membranes were prepared as previously

described.^39,40The membrane preparations were diluted with 50

mM tris-citrate buffer pH 7.4 and used in the binding assay.

Protein concentration was assayed using the method of Lowry

et al.⁴¹[³H]Ro 15-1788 binding studies were performed as previously

reported.⁴²At 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, USA), which provided

IC₅₀, K_i, and SEM values for tested compounds, the K_ivalues being

calculated from the Cheng and Prusoff equation.⁴³

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 h/12 h

light/dark cycles (lights on at 7:00). The cages were brought

into the experimental room the day before the experiment

for acclimatization purposes. All experiments were performed

between 10:00 and 15:00.

3-(2-Thienylmethoxycarbonyl)-8-hydroxypyrazolo[5,1-c][1,2,4]-

benzotriazine (37). A solution of compound 36 (0.40 mmol) in di-

chloromethane (5 mL) was supplemented with a suspension of tri-

chloroacetonitrile (TCA, 0.80 mmol) and triphenylphospine (0.80

mmol) in dichloromethane (5 mL). The reaction was stirred until the

acid disappeared, and 2-thiophenemethanol (0.2 mL, large excess)

was added and maintained at refluxing temperature, with stirring,

monitored by TLC (eluent: tolurne/ethyl acetate/acetic acid 8:2:1

v/v/v). The mixture was washed with water, and the organic layer was

then dried over anhydrous sodium sulfate. The evaporation gave a

brown residue that crystallized after being exposed to a few milliliters

of ethanol. The raw product was recrystallized by ethanol. Yellow

crystals. IR ν cm^-13700-3600, 1720. ¹H NMR (DMSO-d₆) δ 12.02

(s, 1H, OH, exch), 8.78 (s, 1H, H-2), 8.58 (d, 1H, H-6), 7.65 (d, 1H,

H-9), 7.60 (dd, 1H, H-5⁰thienyl), 7.40 (dd, 1H, H-7), 7.32 (d, 1H, H-3⁰

thienyl), 7.08 (m, 1H, H-4⁰thienyl), 5.60 (s, 2H, CH₂). Anal. C, H, N.

General Procedure for the Synthesis of Compounds 38-40.

Chlorodifluoro-acetophenone (1.0 mmol) was added to a mixture of

compounds 34, 35, or 36 (0.20 mmol), potassium carbonate (2.0

mmol), acetonitrile (2 mL), and water (2.0 mL) and maintained at

room temperature. The reaction was warmed to 70-80 ꢀC and

monitored by TLC. Then the mixture was extracted with ethyl

acetate, and the organic layer was dried over anhydrous sodium

sulfate and evaporated to dryness. The crude product was purified

by chromatography column or by recrystallization.

Rota-Rod Test. The integrity of the animals’ motor coordination

was assessed using a rota-rod apparatus (Ugo Basile, Varese, Italy)

at a rotating speed of 24 rpm The number of falls from the rod over

30 s, 25 min after drug administration, were counted.

3-Iodo-8-difluoromethoxypyrazolo[5,1-c][1,2,4]benzotriazine (38).

From starting material 34 and purified by chromatography (eluent:

toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; fast band running).

Yellow crystals. ¹H NMR (DMSO-d₆) δ 8.78 (d, 1H, H-6), 8.61 (s,

1H, H-2), 8.10 (d, 1H, H-9), 7.77 (t, 1H, CHF₂, J_H-F73 Hz), 7.66

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

Ethyl 8-Difluoromethoxypyrazolo[5,1-c][1,2,4]benzotriazine-

3-carboxylate (39). From starting material 35, purified by

recrystallization. Yellow crystals. ¹H NMR (DMSO-d₆) δ 8.86

(m, 2H, H-2 and H-6), 8.18 (d, 1H, H-9), 7.80 (t, 1H, CHF₂, J_H-F

73 Hz), 7.75 (dd, 1H, H-7), 4.40 (q, 2H, CH₂), 1.40 (t, 3H, CH₃).

MS(ESI) m/z: 262.9, 308.9, 310.0 ([M þ H]^þ). Anal. C, H, N.

3-(2-Thienylmethoxycarbonyl)-8-difluoromethoxypyrazolo[5,1-c]-

[1,2,4]benzotriazine (40). From starting material 37 and purified by

recrystallization. Yellow crystals. IR ν cm^-1, 1720. ¹H NMR

(DMSO-d₆) δ 8.80 (s, 1H, H-2), 8.77 (d, 1H, H-6), 8.09 (d, 1H,

H-9), 7.71 (t, 1H, CHF₂, J_H-F73 Hz) 7.67 (dd, 1H, H-7), 7.51 (dd,

1H, H-5⁰thienyl), 7.25 (d, 1H, H-3⁰thienyl), 6.98 (m, 1H, H-4⁰

thienyl), 5.60 (s, 2H, CH₂). Anal. C, H, N.

Grip-Strength Meter Test. The grip strength meter measures

forelimb grip-strength in rodents. The apparatus is formed by a

Perspex base on which is located a grasping trapeze. The mouse

instinctively grabs the trapeze, when raised by trail, trying to

stop this involuntary backward movement until the pulling

force overcomes the animal’s grip strength. After the animal

loses its grip, the peak preamplifer automatically stores the peak

pull force and shows it on a liquid crystal display.

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 cm ꢀ 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 animal’s tendency to explore

a new environment and its fear of bright light.

7546 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

Pentylenetetrazole (PTZ)-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.

one point (T) located 1.8 A from oxygen or nitrogen forming

an angle of 120ꢀ between ROT or R⁰OT or R⁰NT. In the case

of -N < , one point (T) was considered located 1.8 A from

the nitrogen atom orthogonal to the plane formed by R, R⁰,

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.

Drugs. Diazepam (Valium 10, Roche), flumazenil (Roche),

pentylenetetrazole (PTZ) (Sigma), and zolpidem (Tocris) were

used. All drugs except PTZ were suspended in 1% carboxy-

methylcellulose sodium salt and sonicated immediately before

use. PTZ was dissolved in isotonic (NaCl 0.9%) saline solution

and injected sc. All benzodiazepine receptor ligands were ad-

ministered by po route, except for flumazenil, which was

administered ip. Drug concentrations were prepared in such a

way that the necessary dose could be administered in a volume of

10 mL/kg by the po, ip, or sc routes.

and R⁰⁰. If the acceptor atom is the fluorine, the point T is

located 1.8 A from fluorine and the angle CFT is 180ꢀ.

Statistical Analysis. Results are given as the mean ( SEM.

Statistical analysis was performed by means of ANOVA, fol-

lowed by Scheffe’s post hoc test. Student’s two-tailed t test was

used to verify the significance between two means. Data were

analyzed using a computer program (Number Cruncher Statis-

tical System, version 5.03 9/92). For percentage values, χ-square

analysis was used in accordance with Tallarida and Murray.

P < 0.05 were considered significant.

Geometry Optimization of Structures. Geometry optimiza-

tions were achieved with the cff91 force field (consistent force

field) of the DISCOVER module of the Insight II⁴⁴program by

applying the Conjugate Gradients algorithm with a convergence

criterion of 0.001 kcal/mol.

(d) Donor (D): the point in which it is necessary to have the

target atom (N, O) to form an efficient hydrogen bond

with hydrogen of the donor atom (NH or OH). The Pp is

located 2.8 A from the hydrogen bond donor atom (N or

O), with a DHT (D generic donor) angle of 180ꢀ.

Conformational Research (Simulated Annealing). We carried

out a stochastic process by a simulated annealing procedure, in

vacuum.

The calculations were carried out with the DISCOVER module

of the Insight II program using the cff91 force field. A multiple-step

procedure was used. The molecule was energetically minimized. The

minimized system was used as the initial structure for the subsequent

molecular dynamics (MD) simulation. The structures were heated

gradually to 900 K and cooled gradually, after 5 ps, until 300 K.

Finally, the structure was energetically minimized. This procedure

was repeated 200 times for every molecule (therefore 200 conforma-

tions were collected for every molecule).

Procedure for Obtaining the Pharmacophoric Model. We have

developed a procedure capable of predicting a combination of

pharmacophoric points by taking into account the number of

conformers needed to describe the conformational space which

represents all the reasonable 3D conformational structures for all

considered molecules. We call this original “homemade” procedure

ADLR (Acceptor, Donor, Lipophilic and Ring). This procedure

groups the molecules in clusters based on common combinations of

pharmacophoric points (same number, type, and position).

We consider the chemical groups present in the molecules and we

define a “pharmacophoric point” (Pp) the mapping of a chemical

group to its corresponding chemical interaction type. ADLR con-

siders the hydrogen bond (HBAcceptor and HBDonor) lipophilic

and aromatic (to simplify, ring) interaction and the four Pps are

defined as follows:

Conformations for each molecule are collected in clusters

based on the similarity of the Pp position. For each cluster, the

mean and the standard deviation of the distances among the Pps

of the conformers is calculated. All of these distance means

generate the “matrix of the distances”. The combination of the

mean of the distances of Pps, for each cluster of conformers, is

called “structure”. Each molecule is represented by one or more

“structures”.

The similarity among the “structures” is evaluated on the

bases of the similarity of Pp couples. Each Pp couple of one

structure is compared with all other Pp couples of the other

structures.

The couples are similar if they have the followings character-

istics:

(1) Similar features of the Pps (acceptor, donor, lipophilic,

ring).

(2) The mean of distance of the ij Pp couple of structure 2

(d²) is in the range of the mean of distance of the Pp

couple ij of structure 1 (d¹), that is:if d²_ij> d¹, the couple

ij of structure 2 is “similar” to Pp couple ij of structure 1 if

(d²_ij- SD²_ij- Tol)/2 < (d¹_ijþ SD¹_ijþ Tol)/2or:if d²

d¹, the Pp couple ij of structure 2 is “similar” to couple ij

of structure 1 if (d²_ijþ SD²_ijþ Tol)/2 > (d¹_ij- SD¹_ij- Tol)/2.

(a) Ring (R): the geometric center of atoms forming the

aromatic moiety.

SD¹represent the standard deviation of distance of the Pp

couple ij of structure 1, and SD²is the standard deviation of

distance of the Pp couple ij of structure 2. “Tol” represent a

(b) Lipophilic (L): the geometric center calculated among the

clusters of lipophilic atoms. Lipophilic atoms are carbon

atoms sp²and sp³, sulfur, halogen as bromine and iodine.

respective target atom (hydrogen) to form an efficient hydro-

gen bond with the acceptor atoms (N, O, and F). It is possible

to have various types of acceptor atoms. In the case of

R-CdO, two points (T1 and T2) were considered, with a

tolerance value (the default value is 0 A).

ADLR searches common combinations of 3, 4, 5, 6, or 7 Pps

among all structures. For example, two structures have in

common a combination of n (3 e n e 7) Pps, if they have a

n!/k! (n - k)! number of similar couples in common (n is the

number of Pps, k is 2 (the two element of the couple)). So for

three Pps (n = 3), three “similar” couples in common are

needed, for four Pps (n = 4), six “similar” couples in common

are needed, and so on.

distance of 1.8 A from the oxygen atom and the angle (COT)

of 120ꢀ. In the case of R-O-R⁰, R-NdR⁰was considered

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7547

Chart 1

The similarity among combinations of Pps of different struc-

The whole procedure for obtaining the pharmacophoric model is

schematically reported (Chart 1):

tures is calculated as the sum of square of mean distances

difference, among the couples of Pps of both structures

(distance difference sum square, DDSS). The DDSS and simi-

larity are inversely proportional.

Crystallographic Analyses. The data were collected at 293

(2)K on Xcalibur3 4-circle diffractometer using a graphite

monochromator, Mo KR radiation. A reference frame was

monitored every 50 frames to control the stability of the crystal

and the system revealed no intensity decay. The data set was

corrected for Lorentz, polarization effects, and absorption

correction was made by multiscan method using the CrysAlis

Software package. The structure was solved using direct method

with SIR97 software, and the refinement was carried out using

the SHELXL-97 software package. All non-hydrogen atoms

were located from the initial solution or from subsequent

electron density difference maps during the initial course of

the refinement. After locating the non-hydrogen atoms, the

models were refined against F2, first using isotropic and finally

anisotropic thermal displacement parameters. The hydrogen

atoms were treated with the riding coordinate method.

In general,

DDSS ¼

ðd_i¹_j- d_i²_jÞ =n

where N represents the number of pairs for combination, d¹

represents the distance between the Pp_iand the Pp_jof the

structure 1, and d²represents the distance between the Pp_i

and the Pp_jof the structure 2, etc.

For each cluster of conformers, the conformer that is closest

to the average conformation is selected. All the selected con-

formers are aligned with the conformer of the reference mole-

cule.

Alignment is optimized using the Simplex method that mini-

mizes the sum of the squares of the distance differences among

the Pp positions compared to the Pp positions of the reference

structure. Alignment with the Simplex method originates from a

number of 56 positions (vertexes) identified by six variables that

represent three rotations (around axes x, y, and z) and three

translations (along the axes x, y, and z). These initial positions

are calculated following an experimental design.

Acknowledgment. We thank Dr. Giovanni Cambi, who

contributed to the synthesis of some compounds during the

doctoral thesis.

Alignment by the Simplex method is preceded, for all the

selected conformers, by a first alignment positioning the first Pp

in the origin of the Cartesian axes and the second along the x axis

(with coordinates y and z equal to 0).

Supporting Information Available: Crystallographic data for

compound 33 and a thermal ellipsoid figure. An example of

research of Pps common combinations. This material is avail-

able free of charge via the Internet at http://pubs.acs.org.

7548 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Guerrini et al.

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