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5,6-Dihydro-1H, 4H-pyrazolo[4,3-f]pyrrolo[1,2-a][1,4]diazepines. Synthesis and pharmacological evaluation
233
Eur J Med Chem (1994) 29,233-239 0 Elsevier, Paris
Short communication
5,6-Dihydro-lH, 42%pyrazolo[4,3-fJpyrrolo[l,2-a][l,4]diazepines. Synthesis and pharmacological evaluation S Vega’, MS Gill, V Darias2, CC Sinchez Mateo2, MA Exp6sito2, MJ Oset-Gasques,M Pan-am&$, MP Gonzdezs ‘Institute de Quimica Me’dica, CSIC, Juan de la Cierva, 3,28006 Madrid; ZDepartamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, Tenerife; ‘Institute de Bioquimica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain (Received
14 May 1993; Final version received 28 October 1993; accepted 2 November
Summary - A series of new 5,6-dihydro- lH,4l+pyrazolo[4,3,fpyrrolo[ 1,2-a] [ 1,4]diazepines toxicity and CNS activity in mice. Some of these compounds were shown to possess anxiolytic with weak anticonvulsant and sedative action. pyrazolo[4,3-Jlpyrrolo[l,2-u][l,4]diazepine
/ anxiolytic
1993)
was synthesized and tested for acute activity similar to that of diazepam,
activity
Introduction In the last few years we have synthesized novel heterocyclic compounds with potential usefulness in the field of the psychoactive drugs. As part of this research program, we have already described 2 series of tricyclic derivatives belonging to the 4Hpyrrolo[l,2-a]thieno[3,2-fand 4H-pyrrolo[1,2-a]thieno[2,3--[ 1,4]diazepine ring systems [1,2], in which some degree of anxiolytic activity was found. In search for more active drugs, we planned to prepare other structures in which the thiophene ring of the pyrrolothienodiazepine moiety of the above derivatives was substituted by other heterocyclic systems, such as pyrazole, imidazole, pyridine or an additional pyrrole ring. In this context, we have prepared a number of new compounds 1 bearing the lH, 4H-pyrazolo[4,3--pyrrolo[ 1,2-a] [ 1,4]diazepine stmcture. The synthesis of these compounds and the results obtained in their preliminary pharmacological screening are the subject of the present report.
LiAlH4
THF
Ar 4
-
a /
HCI EtOH
NaOH, H@& N+H2CI
NH
/
Ar
Chemistry The 5,6-dihydro-lH, 4H-pyrazolo[4,3-flpyrrolo[l,2-a][ 1,4]diazepines la-i were synthesized by the sequence of reactions shown in scheme 1. The starting
m-i
Scheme
1.
Ar la-i
234 material, 5-amino-4-cyano-1-methylpyrazole 2 was readily prepared by reaction of ethoxymethylenemalononitrile with methylhydrazine according to the method described by Cheng and Robins [3]. Aminonitrile 2 was converted to 4-cyano-1-methyl-5-(1pyrryl)pyrazole 3 by condensation with 2,5dimethoxytetrahydrofuran in the presence of glacial acetic acid following the procedure reported by Clauson-Kaas and Tyle for the synthesis of pyrroles [4]. Reduction of compound 3 with lithium aluminium hydride in dry tetrahydrofuran furnished the desired aminomethylpyrazole 4 in 69% yield. Reaction of this aminomethyl derivative 4 with the appropriately substituted benzaldehydes in boiling ethanol afforded the intermediate Schiff bases 5a-i, which were subsequently cyclized into the target tricyclic diazepines la-i by treatment with gaseous hydrogen chloride in ethanol. Under these conditions, which have been successfully employed in preceding work [ 1, 2, 51, the expected hydrochloride salts 6a-i were obtained. Treatment with aqueous sodium hydroxide yielded the final pyrazolo-pyrrolo-diazepines la-i. This process was carried out in a 2-step synthetic sequence through the benzylidene intermediates 5 which, in general, were not isolated. An exception was compound 5e, which was a yellow crystalline solid. The rest of the Schiff bases prepared were oily products which were not characterized. As in the wellknown Pictet-Spengler reaction, this process can also be performed in one-pot procedure, but in this case the workup and isolation of the final compounds was more difficult and the overall yields were lower. The structures of the compounds synthesized were established by elemental and spectroscopic analyses. These characteristics and the yields and physical data of the products obtained are listed in tables I and II. Pharmacological
results and discussion
All of the compounds la-i presented in this paper were originally submitted for acute toxicity and behavioral studies in mice, using the method of Irwin [6]. Antagonism to pentylenetetrazole and strychnine, potentiation of pentobarbital sleeping time, exploratory behavior and effects on spontaneous motility, normal body temperature and muscle relaxation were studied in male mice at doses not greater than approximately 0.40 LD,,. The compounds were administered po and the results obtained in this study are recorded in tables III, IV and V. Some reference standards, such as chlordiazepoxide, diazepam, ketazolam, amphetamine and caffeine, were included in the tests for comparison purposes.
Table I. .5,6-Dihydro-6-aryl-l-methyl-lH,4H-pyrazolo[4,3-fpyrrolo[ 1,2-a][ 1,4]diazepin& la-i.
Ar Compound
Ar
ViaId (“4
Free base Pf. (“C) (ethanol)
Hydrochloride P.f. (“C) Formula (ethanol-water) (m.w.)
la
C6H5
28
113-115
246-248
ci 6HdIN4 (300.79)
lb’
‘%i-‘%H4
32
146-l 47
250 (dec)
C16H16C12N4 (335.23)
1c
mCI-CeH4
42
102-103
224 (dec)
C16h6’%N4 (335.23)
ld
pCI-CaH4
50
108-109
242 (die)
C16H16%‘N4 (335.23)
le
pN02-‘hH4
52
166-169
250 (dec)
C16H16Cb02 (345.79)
If
m-OCH3-CeH4
32
140-143
221 (dec)
‘h~wCN&J
1g
pOCH&$i4
52
oil
235 (dec)
‘W-hgCIN40
ih
(;;;)Z-C6H3
30
oil
240 (dec)
CiaH21 CIN402 (360.84)
li
OCH20-C6H3 (334)
40
oil
233 (dec)
CvHi7Ck02 (344.80)
(330.81)
(330.81)
It was found that, except for compounds la and Id, this series provided moderate to low toxicities. All of the screened compounds were tested by the Irwin test, and showed, at the highest doses assayed (500 mg/kg po for the less toxic compounds), depressant effects on‘ spontaneous activity, irritability, limb and abdominal tone, and righting reflex, as well as increased positional passivity and ataxia, which were increased after 2 h. These compounds showed only a slight hypothermic effect, except for compounds lc, le and lh ‘which produced a reduction of the body temperature higher than 2°C after 4 h. On the other hand, the tested compounds did not show any important relaxant activity in the well-known traction and chimney tests and, in contrast to the benzodiazepines used as reference standards, they were devoid of anticonvulsant activity in the pentylenetetrazole and strychnine seizures, with the exception of lb in which some degree of protection (20 and 60%, respectively) was detected at a dose of 250 mg/kg.
235 Table II. 1H-NMRa data for compounds la-i. 6H3 64
6 CH2 CAB)
SH6 is)
6H7 (W
6H8 W)
6H9 (dd)
3.99
7.36
3.97 4.08
4.92
5.57
6.20
6.98
7.27-7.43 (m, H2’, 3’, 4’ and 5’)
1b
3.99
7.36
4.01 4.05
4.92
5.57
6.34
6.98
7.48-7.60 (m, H4’, 5’ and 6’) .8.30-8.35 (m, H2’)
1c
3.98
7.35
3.96 4.06
4.92
5.60
6.22
6.98
7.24-7.29 (m, H4’, 5’ and 6’) 7.43-7.46 (m, H2’)
1d
3.98
7.32
3.95 4.06
4.92
5.57
6.32
6.98
1.96
7.32 (d, JHT,H3’=JH6’,H5’=7.7, H2’ and 6’) 7.37 (d, H3’ and 5’)
1 e
3.97
7.36
3.98 4.07
5.08
5.59
6.24
7.00
1.98
7.60 (d, JHZ,H3’=JH6’,H5’=8 H2’ and 6’) 8.18 (d, H3’ and 5’)
11
3.99
7.36
3.98 4.02
4.90
5.64
6.20
6.96-7.20 Wd
1.97
3.78 (s, OCH3) 6.83 (ddd, JH4’,HS=8.2, JHe,H6’=2.5, JHe,H2’=1 .O, H4’) 6.96-7.26 (m, HS and 6’) 7.21-7.29 (m, H2’)
lcl
3.99
7.36
3.96 4.06
4.67
5.59
6.20
6.97
W
6CH3 wb
la
1 h
3.99
7.36
3.96 4.07
4.88
5.64
6.21
6.98
ii
3.99
7.36
3.96 4.07
4.84
5.60
6.18
6.95
6NH (WC
6Ar
3.81 (s, OCH3) 6.88 (d, JHZ,H3’=JHe,H5’=8 Hil’and 6’) 7.33 (d, H3’ and 5’) 2.03
3.87 3.88 6.83 6.93 7.02
9,
8,
(s, OCH3) (s, OCH3) (d, JHS,H6’=8.2, H5’) (dd, JH6’,H?=Z.O, H6’) (d, H2’)
5.80 and 5.82 (AB, JAk1.6, OCH20) 6.70 (d, JHS,HS=6.0, H5’) 6.74 (dd, JH6’,HZ’=1.5, H6’) 6.84 (d. H2’)
% CDCI, and at 200 MHz; bmultiplicity; Cexchange with D,O; doverlapped by protons signals of Ar. Coupling constants: JAB = 15.4, JH7,“* = 3.6, JH7,H9= 1.5, JH7,H6= 0.9, JH8,H9= 3.0 Hz.
As regards the effects of these tricyclic derivatives on potentiation of pentobarbital-induced sleep, it was observed without exception that all of the compounds not only increased the sleeping time of the animals but also reduced their sleeping induction period, lb being in this respect much more effective than diazepam, although at a higher dose. The possible involvement of an enzymatic inhibition in these effects was discarded by the use in the above assay of subhypnotic doses of sodium pentobarbital. As indicated in table IV, this experiment confirmed the potent depressant effect of compound lb which induced sleep in a 100% of the animals employed. Likewise, the behavioral effects of these products were investigated in the plus-maze test in combination with the holeboard assay (table V). The plus-maze test
uses exploratory activity in an elevated plus-maze in the mouse as a measure of anxiety [7, 81. In fact, exposure of animals to the open arms of the plus-maze produces significantly more anxiety-related behavior than exposure to the closed arms, caused by fear or anxiety induced by high and open spaces. Anxiolytic compounds (benzodiazepines, for example) selectively elevate the percentage of open arms entries into, and the time spent on, the open arms of the maze. In contrast, anxiogenic compounds (caffeine or amphetamine) diminish these 2 measures. The test therefore is equally capable of detecting anxiolytic and anxiogenic drug activities. Immediately before the plus-maze test, the animals were exposed for 5 min to a holeboard apparatus in order to increase maze exploration. As is well known, the holeboard test
236 Table III. Toxicity, caused by la-i.
spontaneous
motility
and variation
of body
temperature
Activity
Compound
Toxicity Approx. LD50 Owk P.0.)
Serum
la
lb lb 1C ld ie If 1g lh lh li Diazepam Ketazolam Amphetamine
125 >iOOO >lOOO 150 >lOOO 710 550 >lOOO >lOOO
-
Spontaneous motility Oral EDsovalues (95% confid. limits) DOW (mg/kg P.o.)
2h
Ih
Hypothermia (Variation ofrectaltemperature,
>50 262 (204.235)
>50 241 (164.295)
419 (330-587) >50 185 (131-268) >250 98.7 (66-141) 264.3 (219-318)
38;(315-448) >50 191 (98-291) >250 >250 105 (74.134)
318. (198.463) 9.3 (3.7-16.6) 8.7 (2519.2) 516.75' a
50 250 50 250 50 250
296(220-363) 8.6 (4.6-15.2) 8.9 (4.9-17.7) 88.87*' =
250 50 250 20 20 5
DTI XiE
-0.19f0.23 -0.24+0.51 1.42+0.26*' 1.10+0.30* 1.44+0.18" -0.58f0.20 2.88f0.37" 0.00~0.36 -0.64kO.58 1.24+0.25"' 0.80f0.12' 1.56f0.36" 1.76f0.54" 1.5480.22"
-
"C) DT4 XfE
DTZ XiE -0.50+0.24 -0.14iO.08 0.72+0.49* 0.60+0.25 1.42f0.27'* -0.34kO.26 2.44+0.56*' -0.42f0.13 -0.36f0.63 0.70+0.15* 0.30f0.20 0.44kO.49 2.24kO.89" 1.64+0.15*'
0.67fO.21 1.621t0.35 0.4OkO.42 0.32+0.35 2.28f0.34'* 1.58+0.43 2.94f0.38" 1.46+0.23 -0.40f0.33' 2.70+0.41'* 0.8OiO.50 1.26i0.48 2.63f0.71'" 3.12fl.44'" -
*p < 0.05; **p < 0.01; ?5 mg/kg po.
Table IV. Potentiation
Compound
Serum
la
lb lb 1c ld le If 1g lh lh li Chlordiazepoxide Diazepam Ketazolam Serum la lb lb 1C ld le If
1g
ih Ih li Diazepam Ketazolam
DO% O-mm P.o.) 50 250 2:: 50 250 50 50 250 50 250 15 15 15 50 250 50 250 2:: 50 50 250 50 250
of pentobarbital
Sodium pentobafbital (mg/kg P.o.)
:: 40 40 40 40 40 40 40 :i 40 4": 40 20 20 20 ii 20 20 20 20 20 si 20 20
sleeping
time caused by la-i.
24124 818 818 818 818 818 718 818 818 818 818 818 818 818 818 0116 O/8 818 318 418 i/8 218 O/8 O/8 l/8 O/8 t/8 18116 13/16
ta
t1 x f Emin
W'4
9.41 7.88 4.22 6.75 5.74 7.30 6.13 4.24 4.75 5.19 6.02 5.90 3.58 3.90 3.17
* i f f f f f + f + f f f f f
x f
1.03 10.6 0.22" 0.73 1.97 2.05 1.89 0.38" 0.38. 0.48 1.25 0.79 0.26" 0.52"' 0.27"'
26.58 41.68 147.63 58.32 61.52 49.72 61.78 41.41 57.65 33.58 50.23 69.05 42.30 113.49 74.19
? zk + ?? ? k i f f k f f + f
2.93 6.83 22.94" 5.02" 8.60'. 5.48*6.99" 4.03" 4.56'. 6.48 4.95'" 9.30" 6.85' 23.86'" 9.04**
5.20 4.70 0.90 0.90 1.01
58.50 28.45 39.04 19.73 33.38
i: t i i k
9.75 4.83 13.27 0.00 0.65
0.00
38.78
0.00 1.40 0.37
40.46 94.62 42.33
17.74 13.56 10.59 13.50 11.13 38.70 14.15 5.77 7.39
f IL + f f + f f f
Emin
*p < 0.05; **p < 0.01; N, = total number of mice; N, = number t, = sleep induction time (min); tz = sleeping time (min).
+ 0.00 + 0.00 r 20.77 i 6.88
of sleeping
mice;
237 Table V. Anxiolytic activity of compounds la-i.
Activity Plus-Maze Compound Serum la lb lb 1c Id le if 1g lh lh li Amphetamine Buspirone Caffeine Digzepam
DO% (mg/kg P.o.)
% O.A.
% t.0 A.
Holeboard ("A variation)
T.N.E.
50 250 50 250 50 250
30.02 37.50 39.64 37.90 33.33 9.09 30.90
it + f f f A
3.8 5.1 2.9 2.5' 4.7 5.3" 2.2
28.21 19.10 34.34 36.52 16.61 1.33 22.59
f i k + i f
3.1' 1.9 4.9" 5.2" 3.3 4.1" 3.5
11.41 6.00 7.87 8.00 8.25 8.25 13.99
ii f + f f f
1.2 1.5' 0.9. 1.2' 3.1 1.2" 2.3
-15.80 ; 4.1' -56.99 i- 6.1' 28.78 f 5.2 1.61 YC 5.8 -41.94 + 6.0 -66.67 ? 7.2'*
z: 250 50 250 550
22.11 20.58 40.62 33.15 2.6.47 33.73 5.69
+i i f ii-+
2.7 4.1 3.5' 3.4 4.6 2.8 3.3"'
13.52 7.17 34:92 28.86 9.29 32.44 10.50
f + ? iik
2.2' 2.7'. 4.6" 3.6" 2.3" 5.1** 2.5"
8.50 7.37 8.00 8.50 8.50 10.50 9.37
I* I i i +k
0.9' 1.1' 1.4' 1.0' 2.1 1.8 2.5
-54.91 -17.83 -11.11 -10.85 -31.79 -48.32 37.25
i-+ 5.5 6.F k 4.3 i 4.7 Yc 3.9" +C 5.7" 4.2"
15 5
24.03 49.60
? 3.9 k 5.1
12.32 52.77
+ 2.7*' t 5.3'.
14.00 8.75
* 1.3 XL 0.9'
-15.62 -61.98
+ 3.6 i 6.5
*p < 0.05; **p < 0.01; % OA: percentage of entries into open arms; % tOA: percentage of time spent on open arms; TNE: total number of arm entries.
measures exploratory behavior (head-dipping) independently of locomotor activity. It was found that compounds la, lb, and lh were endowed with anxiolytic activity at the doses assayed since they increased both the percentage of entries and percentage of time on the open arms of the plus-maze compared with the control. In the case of lb, and to a lesser extent lh, this was accompanied by a moderate sedative action, while in the case of la there was no significant modification of the curiosity and motor activity of the animals. At 50 mg/kg the anxiolytic character of lb and lh was confirmed, although at this dose a less central depressant effect was observed. On the contrary, compounds Id, and to a lesser extent If, lg and li, seemed to have a rather anxiogenic character, because they reduced the 2 indices of anxiety used in this experiment. Nevertheless, an anxiogenic interpretation of the action of these compounds should be made with caution, since all the behavioral measures were depressed. These behavioral results led us to investigate the ability of the reported compounds to interact in vitro with central benzodiazepine receptors (mainly with the GABA,-BZD-Clchannel complex), since, despite their 5,6-dihydro character (reduced benzodiazepines do not elicit binding to this receptor), these compounds could exert their biological effects through that receptor. Therefore, we have tested their ability to displace the binding of [3H]-flunitrazepam (FNZ) to receptors from bovine cerebral cortex membranes. Among all of these compounds only la and lb were able to displace the specific binding of
the radioligand in a concentration-dependent form, although the affinity that characterizes the interaction with benzodiazepine receptors of both compounds was lower than that of FNZ. Thus, compound la, which possesses the best affinity with an IC,, of 420 nM (FNZ = 29 nM), was less potent in these tests than compound lb with an IC,, of 20 600 nM. In summary, and in spite of the difficulty of drawing firm conclusions from this preliminary pharmacological study, it may be stated that compound lb appears to have a pharmacological profile that is consistent with an anxiolytic activity similar to that of diazepam and the rest of the classical benzodiazepines, but showing weak anticonvulsant and moderate sedative activities. On the other hand, compounds la and lh have an anxiolytic profile with even low propensity to cause sedative effects. In contrast, compounds Id, lf, lg and li seem to be more akin to anxiogenic drugs, like amphetamines or caffeine. A more detailed investigation is now in progress to confirm these findings. Experimental
protocols
Chemistry All melting points were taken on a Gallenkamp capillary apparatus and are uncorrected. Infrared spectra were determined with a Shimadzu IR-435 instrument. ‘H-NMR spectra were obtained with Bruker AM-200 and Varian XL-300 spectrometers using TMS as an internal standard. Chemical shifts are expressed in 6 units and coupling constants
238 in Hz. Analyses indicated by symbols were within + 0.4% of the theoretical values. The purity of compounds was verified by thin-layer chromatography (TLC) which was run on silica gel GFZS, (Merck) or aluminium oxide 60F,, (Merck) with cyclohexane/ethyl acetate mixtures (2: 1 and 1: 1, v/v respectively) as eluents.
free base 1 was obtained by treatment of 6 with dilute sodium hydroxide and extraction with ether or chloroform. Table I lists the yields and the physical and analytical data of all the comuounds 6 and 1 svnthesized. The main ‘H-NMR suectroscop\c features of diazepines 1 are summarized in table II. Pharmacology
4-Cyano-I-methyl-5-(1-pyrryl)pyrazole 3 A mixture of 5-amino-4-cyano-1-methylpyrazole 2 [3] (122 g, 1.0 mol), 2,5-dimethoxytetrahydrofuran (132 g, 1.0 mol) and glacial acetic acid (1000 ml) was heated under reflux for 30 min. The mixture was concentrated in vacua and the oily residue was then distilled at 0.1 mmHg; a fraction (163.4 g, 95%) of bp = 125-130°C crystallized on cooling to give 4-cyano- 1-methyl-5-( 1-pyrryl)pyrazole 3, mp = 6667°C (ethanol). Anal C,H,N, (C, H, N). IR (KBr): 2240 cm-1 (CN). ‘H-NMR (DMSO-d,): 6 7.78 (s, lH, H3); 6.90 (t, 2H, JH2’,a3’= J H4’,H5’= 2.2 Hz, H2 and H5); 6.44 (t, 2H, H3’ and H4’); 3.80 (s, 3H, N-CH,). 4-Aminomethyl-methyl-T-(1 -pyrryl)pyrazole 4 To a stirred susnension of lithium aluminium hvdride (24 g. 0.6 mol) in dry tetrahydrofuran (750 ml) was slowly added”; solution of 3 (86 g, 0.5 mol) in dry tetrahydrofuran (750 ml). The mixture was stirred and heated under reflux for 3 h, then cooled and carefully treated with absolute ethanol. After filtration, the filtrate was evaporated and the residue was extracted with ether. The organic layer was washed with water, dried (magnesium sulfate) and evaporated. Distillation of the oily residue at 0.1 mm Hg gave the aminomethyl derivative 4, bp = 115-118°C (61 g, 69%). Picrate, mp > 194°C (dec) (ethanol). Anal C,,H,.&O, (Ci H, N). IR (film): 3380, 1306, 1590 cm-r (NH,). rH-NMR (CDCl,): 6 7.28 (s. 1H. H3): 6.75 (t, 2H, J,&j = yHt.,, = 2.2 Hz, Hf and H5’): 6.38 (t, 2H, H3’ and H4’); 3.62 (s, 3H, CH,); 3.59 (s, 2H, CH,); 1.58 (bs, 2H, exchangeable, NH,). MS: m/z 176 (M-t-, 100%). 4-Benzylideneaminomethyl-1
-methyl-T-(1
-pyrryl)pyrazoles
5
General method. A mixture of 4 (0.02 mol), the appropriate benzaldehyde (0.02 mol) and ethanol (40 ml) was heated under reflux for 15 min. The solvent was evaporated in vacua to give the corresponding Schiff base 5, which, in general, could-not be uurified bv distillation or crvstallization from the common solGents and for this reason they were used as such in the next reaction. Compound Se precipitated on cooling the reaction mixture, and so it was isolated by filtration and identified as follows. 4-(p-Nitrobenzylideneaminomethyl)-I -methyl-T-(1 -pyrryl)uvrazole 5e. This comvound was obtained in 78% vield from &itrobenzaldehyde as Lyellow crystals of mp = f48-151°C (ethanol). Anal C,6H,SN,0, (C, H, N). IR (nujol): 1640, 1570 cm-l (C=N). rH-NMR (CDCl,): 8 8.19 (d, 2H, JH3.‘H2.’= J H4”FTC- 8.9 Hz, H3” and H5”); 8.03 (s, lH, CH): 7.75 (d, 2H. H2”?&d H6”); 7.46 (s, lH, H3); 6.66‘(t, 2H, J,;,.,Y = Jis.H4. = 2.1 Hz. H2’ and H5’): 6.26 It. 2H. H3’ and H4’): 4.49 fd. 2H. J = 1.5 Hz; CH,); 3.52 (s, 3H, &I,). ’ 5,6-Dihydro-6-aryl-l-methyl-IH,4H-pyrazolo[4,3-f]pyrrolo[l, 2-a][l,4]diazepines 1 The crude Schiff bases 5 obtained above were dissolved in absolute ethanol saturated with dry hydrogen chloride (50 ml) and the solution stirred at room temperature for 45 min. The precipitated hydrochloride salt 6 was filtered off, washed with dry ether and recrystallized from the appropriate solvent. The
Effects on behavior and LD,, in mice According to the method of Irwin [6], the behavior of the mice was observed 1 and 2 h after po administration of the test drugs. The LD,, values were calculated from the lethality within 3 d after po administration of the drugs by the method of Litchfield and Wilcoxon [9]. Effect on spontaneous motor activity Locomotor activity was recorded with a photocell activity meter for 15 min, beginning 60 and 120 min after po administration of each test drug. Effect on normal body temperature The rectal temperature of the mice was measured with a thermistor thermometer (Panlab 0331) prior to the experiment and 1, 2 and 4 h after po administration of each test drug. Muscle relaxant
activity
Traction test. Experiments were performed by the method of Courvoisier et al [lo]. Mice were forced to hang with their forelegs on a wire of 1 mm in diameter, which was stretched horizontally at a height of 35 cm. When they fell off the wire within 5 s or they failed to grasp the wire with their hind legs 3 times successively, muscle relaxation was judged to be positive. Chimney test. In a Pyrex tube (30 cm long and 28 mm diameter) marked at 20 cm from the base, a mouse was introduced at the end near the mark. When the animal reached the other end of the tube, the tube was moved to the vertical position and immediately the mouse tried to climb the tube backwards. Only those mice which reached the mark within 30 s were selected for further testing. The operation was repeated 60 and 120 min after po administration of the test drugs [ll]. Exploratory
behavior
pattern
Holeboard test and plus-maze test. The holeboard apparatus consisted of a wooden board (40 x 40 cm) which had 16 equally spaced holes. The holeboard testing involved placing the mouse 55 min after po injection with the control vehicle or test material in the center of the floor and counting the number of head-dips during 5-min trials ] 121. The plus-maze apparatus was made of Plexiglas and consisted of 2 open arms (30 x 5 cm) and two enclosed (30 x 5 x 15 cm) arms connected by a central platform (5 x 5 cm). The open arms, the central platform and the floor of the closed arms were made of black nlexinlas. and the sides of the closed arms were made of clear Plexiglas. The apparatus was mounted on a base raising it 38.5 cm above the floor. The test consisted of injecting po mice with the drug or its vehicle 55 min before being tested individually in the holeboard for 5 min. Immediately after the holeboard test each animal was placed in the center of the plus-maze facing an open arm and allowed to freely explore for 5 min. The number of entries made on the open and closed arms and the time spent in each type of arm I
YI
239 were recorded. Three measures were obtained from the test: the total number of arm entries; the percentage of arms entries made on the open arms; and the time spent on the open arms expressed as a percentage of the time spent on both the open and closed arms. The last 2 measures are used as indices of anxiety: they increase following the administration of anxiolytics and decrease following the administration of anxiogenic drugs [7, 81. Interaction with barbiturate-induced sleep in mice Sodium pentobarbital at a subhypnotic dose of 20 mg/kg or at a hypnotic dose of 40 mg/kg dissolved in 0.9% saline was injected ip to groups of 8 mice 60 min after po injection of the drug. The number of mice which lose righting reflex (in the case of subhypnotic dose) and the latency and the duration of sleep (loss and recovery of the righting reflex) were recorded. Anticonvulsant Convulsions (120 mg/kg) 0.9% saline. administered anticonvulsive tonic seizures
activity: effect on chemically induced seizures were induced by an ip injection of pentetrazol or strychnine sulfate (2.5 mg/kg) dissolved in Test drugs, suspended in 1% Tween 80, were orally 60 min before the convulsant injection, and activity was determined by loss of clonic and and mortality.
Binding of [3H]-junitrazepam to GABAlbenzodiazepine receptor complex The binding of [sH]-flunitrazepam to the GABA/benzodiazepine receptor complex of bovine cerebral cortex was determined by the method described by Sigel et al [13], with minor modifications. Preparation of membranes. Bovine brain was obtained fresh from a slaughterhouse, the cortex was rapidly removed, chopped, frozen immediately and stored at -80°C until use. Cortex (199 g) was thawed, chopped and homogenized in 10 mM HEPES (pH 7.5), 1 mM EDTA, 300 mM sucrose, 0.5 mM dithiothreitol, 1 mM benzamidine/HCl, 0.3 mM phenylmethylsulfonyl fluoride (1 1). All operations were made at 0-4”C. The homogenate was centrifuged at 1000 x g,, for 12 min. The supernatant was centrifuged at 27 000 x g,, for 35 min. The pellet was resuspended, using a glass-teflon homogenizer, in 500 ml of the same medium except that sucrose and phenylmethylsulfonyl fluoride were omitted. After recentrifugation, the pellet was resuspended in the same medium (final volume 150 ml; 15 mg protein/ml).
Binding assay in solution. Membranes were diluted at 1 mg protein/ml in 20 mM potassium phosphate (pH 7.4) 0.1 mM EDTA, 0.1% (w/v) Triton X-100. Diluted membranes (140 ml) were incubated with [sH]-flunitrazepam (10 nM final concentration) with and without unlabelled ligands at different concentrations with incubation at 0°C for I h. Duplicates of incubations were filtered on GF/C glass fiber filters (Whatman) under suction. The filters were washed 3 times with 4 mi 20 mM uotassium ohosnhate (nH 7.4). 0.1 mM EDTA. dried and counted in a l&id &intil&ion fluid. Non-specific bmding was determined in the presence of cold 10 mM flunitrazepam and represented 5 5% of the total binding. The results were estimated by considering the maximal specific binding obtained with 10 nM flunitrazepam as 100%.
Acknowledgments We are indebted to the Comisidn fnterministerial de Ciencia y Tecnologia, and the Vicerrectorado de Investigation, Universidad de La Laguna, Spain, for the financial support of this work.
References
6 7 8 9 10 11 12 13
Gil S (1989) An R Acad Farm 55,339-352 Vega S, Gil MS (1991) J Heterocycl Chem 28, 945-950 Cheng CC, Robins RK (1956) JOrg Chem 21, 1240-1257 Clauson-Kaas N, Tyle N (1952) Acta Chem Stand 6, 567-878 Massa S, Stefancich G, Artico M, Corelli F (1984) J Hererocycl Chem 21. 1877-1880 Irwin S (1968) Psychopharmacology 13,222-250 Pellow S, File SE (1986) Pharmacol Biochem Behav 11, 99-106 Pellow S, Chopin P, File SE, Briley J (1985) J Neurosci Methods 14. 149-167 Litchfield JT, Wilcoxon F (1949) JPharmacol Exp Ther 96,99-l 13 Courvoisier S, Ducrot R, Julou L (1957) In: Psy&zotropic Drugs (Garattini S, Ghetti V, eds) Elsevier, Amsterdam, p 373 Boissier JR, Tardy J, Divemes JC (1960) Med Exp 3, 81-84 B&&r JR, Simon P (1962) Therapie 17, 1225-1232 Sigel E, Stephenson FA, Mamalaki C, Barnard EA (1983) J Biol Chem 258, 6969-6971
Eur J Med Chem (1994) 29,233-239 0 Elsevier, Paris
Short communication
5,6-Dihydro-lH, 42%pyrazolo[4,3-fJpyrrolo[l,2-a][l,4]diazepines. Synthesis and pharmacological evaluation S Vega’, MS Gill, V Darias2, CC Sinchez Mateo2, MA Exp6sito2, MJ Oset-Gasques,M Pan-am&$, MP Gonzdezs ‘Institute de Quimica Me’dica, CSIC, Juan de la Cierva, 3,28006 Madrid; ZDepartamento de Farmacologia, Facultad de Farmacia, Universidad de La Laguna, Tenerife; ‘Institute de Bioquimica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain (Received
14 May 1993; Final version received 28 October 1993; accepted 2 November
Summary - A series of new 5,6-dihydro- lH,4l+pyrazolo[4,3,fpyrrolo[ 1,2-a] [ 1,4]diazepines toxicity and CNS activity in mice. Some of these compounds were shown to possess anxiolytic with weak anticonvulsant and sedative action. pyrazolo[4,3-Jlpyrrolo[l,2-u][l,4]diazepine
/ anxiolytic
1993)
was synthesized and tested for acute activity similar to that of diazepam,
activity
Introduction In the last few years we have synthesized novel heterocyclic compounds with potential usefulness in the field of the psychoactive drugs. As part of this research program, we have already described 2 series of tricyclic derivatives belonging to the 4Hpyrrolo[l,2-a]thieno[3,2-fand 4H-pyrrolo[1,2-a]thieno[2,3--[ 1,4]diazepine ring systems [1,2], in which some degree of anxiolytic activity was found. In search for more active drugs, we planned to prepare other structures in which the thiophene ring of the pyrrolothienodiazepine moiety of the above derivatives was substituted by other heterocyclic systems, such as pyrazole, imidazole, pyridine or an additional pyrrole ring. In this context, we have prepared a number of new compounds 1 bearing the lH, 4H-pyrazolo[4,3--pyrrolo[ 1,2-a] [ 1,4]diazepine stmcture. The synthesis of these compounds and the results obtained in their preliminary pharmacological screening are the subject of the present report.
LiAlH4
THF
Ar 4
-
a /
HCI EtOH
NaOH, H@& N+H2CI
NH
/
Ar
Chemistry The 5,6-dihydro-lH, 4H-pyrazolo[4,3-flpyrrolo[l,2-a][ 1,4]diazepines la-i were synthesized by the sequence of reactions shown in scheme 1. The starting
m-i
Scheme
1.
Ar la-i
234 material, 5-amino-4-cyano-1-methylpyrazole 2 was readily prepared by reaction of ethoxymethylenemalononitrile with methylhydrazine according to the method described by Cheng and Robins [3]. Aminonitrile 2 was converted to 4-cyano-1-methyl-5-(1pyrryl)pyrazole 3 by condensation with 2,5dimethoxytetrahydrofuran in the presence of glacial acetic acid following the procedure reported by Clauson-Kaas and Tyle for the synthesis of pyrroles [4]. Reduction of compound 3 with lithium aluminium hydride in dry tetrahydrofuran furnished the desired aminomethylpyrazole 4 in 69% yield. Reaction of this aminomethyl derivative 4 with the appropriately substituted benzaldehydes in boiling ethanol afforded the intermediate Schiff bases 5a-i, which were subsequently cyclized into the target tricyclic diazepines la-i by treatment with gaseous hydrogen chloride in ethanol. Under these conditions, which have been successfully employed in preceding work [ 1, 2, 51, the expected hydrochloride salts 6a-i were obtained. Treatment with aqueous sodium hydroxide yielded the final pyrazolo-pyrrolo-diazepines la-i. This process was carried out in a 2-step synthetic sequence through the benzylidene intermediates 5 which, in general, were not isolated. An exception was compound 5e, which was a yellow crystalline solid. The rest of the Schiff bases prepared were oily products which were not characterized. As in the wellknown Pictet-Spengler reaction, this process can also be performed in one-pot procedure, but in this case the workup and isolation of the final compounds was more difficult and the overall yields were lower. The structures of the compounds synthesized were established by elemental and spectroscopic analyses. These characteristics and the yields and physical data of the products obtained are listed in tables I and II. Pharmacological
results and discussion
All of the compounds la-i presented in this paper were originally submitted for acute toxicity and behavioral studies in mice, using the method of Irwin [6]. Antagonism to pentylenetetrazole and strychnine, potentiation of pentobarbital sleeping time, exploratory behavior and effects on spontaneous motility, normal body temperature and muscle relaxation were studied in male mice at doses not greater than approximately 0.40 LD,,. The compounds were administered po and the results obtained in this study are recorded in tables III, IV and V. Some reference standards, such as chlordiazepoxide, diazepam, ketazolam, amphetamine and caffeine, were included in the tests for comparison purposes.
Table I. .5,6-Dihydro-6-aryl-l-methyl-lH,4H-pyrazolo[4,3-fpyrrolo[ 1,2-a][ 1,4]diazepin& la-i.
Ar Compound
Ar
ViaId (“4
Free base Pf. (“C) (ethanol)
Hydrochloride P.f. (“C) Formula (ethanol-water) (m.w.)
la
C6H5
28
113-115
246-248
ci 6HdIN4 (300.79)
lb’
‘%i-‘%H4
32
146-l 47
250 (dec)
C16H16C12N4 (335.23)
1c
mCI-CeH4
42
102-103
224 (dec)
C16h6’%N4 (335.23)
ld
pCI-CaH4
50
108-109
242 (die)
C16H16%‘N4 (335.23)
le
pN02-‘hH4
52
166-169
250 (dec)
C16H16Cb02 (345.79)
If
m-OCH3-CeH4
32
140-143
221 (dec)
‘h~wCN&J
1g
pOCH&$i4
52
oil
235 (dec)
‘W-hgCIN40
ih
(;;;)Z-C6H3
30
oil
240 (dec)
CiaH21 CIN402 (360.84)
li
OCH20-C6H3 (334)
40
oil
233 (dec)
CvHi7Ck02 (344.80)
(330.81)
(330.81)
It was found that, except for compounds la and Id, this series provided moderate to low toxicities. All of the screened compounds were tested by the Irwin test, and showed, at the highest doses assayed (500 mg/kg po for the less toxic compounds), depressant effects on‘ spontaneous activity, irritability, limb and abdominal tone, and righting reflex, as well as increased positional passivity and ataxia, which were increased after 2 h. These compounds showed only a slight hypothermic effect, except for compounds lc, le and lh ‘which produced a reduction of the body temperature higher than 2°C after 4 h. On the other hand, the tested compounds did not show any important relaxant activity in the well-known traction and chimney tests and, in contrast to the benzodiazepines used as reference standards, they were devoid of anticonvulsant activity in the pentylenetetrazole and strychnine seizures, with the exception of lb in which some degree of protection (20 and 60%, respectively) was detected at a dose of 250 mg/kg.
235 Table II. 1H-NMRa data for compounds la-i. 6H3 64
6 CH2 CAB)
SH6 is)
6H7 (W
6H8 W)
6H9 (dd)
3.99
7.36
3.97 4.08
4.92
5.57
6.20
6.98
7.27-7.43 (m, H2’, 3’, 4’ and 5’)
1b
3.99
7.36
4.01 4.05
4.92
5.57
6.34
6.98
7.48-7.60 (m, H4’, 5’ and 6’) .8.30-8.35 (m, H2’)
1c
3.98
7.35
3.96 4.06
4.92
5.60
6.22
6.98
7.24-7.29 (m, H4’, 5’ and 6’) 7.43-7.46 (m, H2’)
1d
3.98
7.32
3.95 4.06
4.92
5.57
6.32
6.98
1.96
7.32 (d, JHT,H3’=JH6’,H5’=7.7, H2’ and 6’) 7.37 (d, H3’ and 5’)
1 e
3.97
7.36
3.98 4.07
5.08
5.59
6.24
7.00
1.98
7.60 (d, JHZ,H3’=JH6’,H5’=8 H2’ and 6’) 8.18 (d, H3’ and 5’)
11
3.99
7.36
3.98 4.02
4.90
5.64
6.20
6.96-7.20 Wd
1.97
3.78 (s, OCH3) 6.83 (ddd, JH4’,HS=8.2, JHe,H6’=2.5, JHe,H2’=1 .O, H4’) 6.96-7.26 (m, HS and 6’) 7.21-7.29 (m, H2’)
lcl
3.99
7.36
3.96 4.06
4.67
5.59
6.20
6.97
W
6CH3 wb
la
1 h
3.99
7.36
3.96 4.07
4.88
5.64
6.21
6.98
ii
3.99
7.36
3.96 4.07
4.84
5.60
6.18
6.95
6NH (WC
6Ar
3.81 (s, OCH3) 6.88 (d, JHZ,H3’=JHe,H5’=8 Hil’and 6’) 7.33 (d, H3’ and 5’) 2.03
3.87 3.88 6.83 6.93 7.02
9,
8,
(s, OCH3) (s, OCH3) (d, JHS,H6’=8.2, H5’) (dd, JH6’,H?=Z.O, H6’) (d, H2’)
5.80 and 5.82 (AB, JAk1.6, OCH20) 6.70 (d, JHS,HS=6.0, H5’) 6.74 (dd, JH6’,HZ’=1.5, H6’) 6.84 (d. H2’)
% CDCI, and at 200 MHz; bmultiplicity; Cexchange with D,O; doverlapped by protons signals of Ar. Coupling constants: JAB = 15.4, JH7,“* = 3.6, JH7,H9= 1.5, JH7,H6= 0.9, JH8,H9= 3.0 Hz.
As regards the effects of these tricyclic derivatives on potentiation of pentobarbital-induced sleep, it was observed without exception that all of the compounds not only increased the sleeping time of the animals but also reduced their sleeping induction period, lb being in this respect much more effective than diazepam, although at a higher dose. The possible involvement of an enzymatic inhibition in these effects was discarded by the use in the above assay of subhypnotic doses of sodium pentobarbital. As indicated in table IV, this experiment confirmed the potent depressant effect of compound lb which induced sleep in a 100% of the animals employed. Likewise, the behavioral effects of these products were investigated in the plus-maze test in combination with the holeboard assay (table V). The plus-maze test
uses exploratory activity in an elevated plus-maze in the mouse as a measure of anxiety [7, 81. In fact, exposure of animals to the open arms of the plus-maze produces significantly more anxiety-related behavior than exposure to the closed arms, caused by fear or anxiety induced by high and open spaces. Anxiolytic compounds (benzodiazepines, for example) selectively elevate the percentage of open arms entries into, and the time spent on, the open arms of the maze. In contrast, anxiogenic compounds (caffeine or amphetamine) diminish these 2 measures. The test therefore is equally capable of detecting anxiolytic and anxiogenic drug activities. Immediately before the plus-maze test, the animals were exposed for 5 min to a holeboard apparatus in order to increase maze exploration. As is well known, the holeboard test
236 Table III. Toxicity, caused by la-i.
spontaneous
motility
and variation
of body
temperature
Activity
Compound
Toxicity Approx. LD50 Owk P.0.)
Serum
la
lb lb 1C ld ie If 1g lh lh li Diazepam Ketazolam Amphetamine
125 >iOOO >lOOO 150 >lOOO 710 550 >lOOO >lOOO
-
Spontaneous motility Oral EDsovalues (95% confid. limits) DOW (mg/kg P.o.)
2h
Ih
Hypothermia (Variation ofrectaltemperature,
>50 262 (204.235)
>50 241 (164.295)
419 (330-587) >50 185 (131-268) >250 98.7 (66-141) 264.3 (219-318)
38;(315-448) >50 191 (98-291) >250 >250 105 (74.134)
318. (198.463) 9.3 (3.7-16.6) 8.7 (2519.2) 516.75' a
50 250 50 250 50 250
296(220-363) 8.6 (4.6-15.2) 8.9 (4.9-17.7) 88.87*' =
250 50 250 20 20 5
DTI XiE
-0.19f0.23 -0.24+0.51 1.42+0.26*' 1.10+0.30* 1.44+0.18" -0.58f0.20 2.88f0.37" 0.00~0.36 -0.64kO.58 1.24+0.25"' 0.80f0.12' 1.56f0.36" 1.76f0.54" 1.5480.22"
-
"C) DT4 XfE
DTZ XiE -0.50+0.24 -0.14iO.08 0.72+0.49* 0.60+0.25 1.42f0.27'* -0.34kO.26 2.44+0.56*' -0.42f0.13 -0.36f0.63 0.70+0.15* 0.30f0.20 0.44kO.49 2.24kO.89" 1.64+0.15*'
0.67fO.21 1.621t0.35 0.4OkO.42 0.32+0.35 2.28f0.34'* 1.58+0.43 2.94f0.38" 1.46+0.23 -0.40f0.33' 2.70+0.41'* 0.8OiO.50 1.26i0.48 2.63f0.71'" 3.12fl.44'" -
*p < 0.05; **p < 0.01; ?5 mg/kg po.
Table IV. Potentiation
Compound
Serum
la
lb lb 1c ld le If 1g lh lh li Chlordiazepoxide Diazepam Ketazolam Serum la lb lb 1C ld le If
1g
ih Ih li Diazepam Ketazolam
DO% O-mm P.o.) 50 250 2:: 50 250 50 50 250 50 250 15 15 15 50 250 50 250 2:: 50 50 250 50 250
of pentobarbital
Sodium pentobafbital (mg/kg P.o.)
:: 40 40 40 40 40 40 40 :i 40 4": 40 20 20 20 ii 20 20 20 20 20 si 20 20
sleeping
time caused by la-i.
24124 818 818 818 818 818 718 818 818 818 818 818 818 818 818 0116 O/8 818 318 418 i/8 218 O/8 O/8 l/8 O/8 t/8 18116 13/16
ta
t1 x f Emin
W'4
9.41 7.88 4.22 6.75 5.74 7.30 6.13 4.24 4.75 5.19 6.02 5.90 3.58 3.90 3.17
* i f f f f f + f + f f f f f
x f
1.03 10.6 0.22" 0.73 1.97 2.05 1.89 0.38" 0.38. 0.48 1.25 0.79 0.26" 0.52"' 0.27"'
26.58 41.68 147.63 58.32 61.52 49.72 61.78 41.41 57.65 33.58 50.23 69.05 42.30 113.49 74.19
? zk + ?? ? k i f f k f f + f
2.93 6.83 22.94" 5.02" 8.60'. 5.48*6.99" 4.03" 4.56'. 6.48 4.95'" 9.30" 6.85' 23.86'" 9.04**
5.20 4.70 0.90 0.90 1.01
58.50 28.45 39.04 19.73 33.38
i: t i i k
9.75 4.83 13.27 0.00 0.65
0.00
38.78
0.00 1.40 0.37
40.46 94.62 42.33
17.74 13.56 10.59 13.50 11.13 38.70 14.15 5.77 7.39
f IL + f f + f f f
Emin
*p < 0.05; **p < 0.01; N, = total number of mice; N, = number t, = sleep induction time (min); tz = sleeping time (min).
+ 0.00 + 0.00 r 20.77 i 6.88
of sleeping
mice;
237 Table V. Anxiolytic activity of compounds la-i.
Activity Plus-Maze Compound Serum la lb lb 1c Id le if 1g lh lh li Amphetamine Buspirone Caffeine Digzepam
DO% (mg/kg P.o.)
% O.A.
% t.0 A.
Holeboard ("A variation)
T.N.E.
50 250 50 250 50 250
30.02 37.50 39.64 37.90 33.33 9.09 30.90
it + f f f A
3.8 5.1 2.9 2.5' 4.7 5.3" 2.2
28.21 19.10 34.34 36.52 16.61 1.33 22.59
f i k + i f
3.1' 1.9 4.9" 5.2" 3.3 4.1" 3.5
11.41 6.00 7.87 8.00 8.25 8.25 13.99
ii f + f f f
1.2 1.5' 0.9. 1.2' 3.1 1.2" 2.3
-15.80 ; 4.1' -56.99 i- 6.1' 28.78 f 5.2 1.61 YC 5.8 -41.94 + 6.0 -66.67 ? 7.2'*
z: 250 50 250 550
22.11 20.58 40.62 33.15 2.6.47 33.73 5.69
+i i f ii-+
2.7 4.1 3.5' 3.4 4.6 2.8 3.3"'
13.52 7.17 34:92 28.86 9.29 32.44 10.50
f + ? iik
2.2' 2.7'. 4.6" 3.6" 2.3" 5.1** 2.5"
8.50 7.37 8.00 8.50 8.50 10.50 9.37
I* I i i +k
0.9' 1.1' 1.4' 1.0' 2.1 1.8 2.5
-54.91 -17.83 -11.11 -10.85 -31.79 -48.32 37.25
i-+ 5.5 6.F k 4.3 i 4.7 Yc 3.9" +C 5.7" 4.2"
15 5
24.03 49.60
? 3.9 k 5.1
12.32 52.77
+ 2.7*' t 5.3'.
14.00 8.75
* 1.3 XL 0.9'
-15.62 -61.98
+ 3.6 i 6.5
*p < 0.05; **p < 0.01; % OA: percentage of entries into open arms; % tOA: percentage of time spent on open arms; TNE: total number of arm entries.
measures exploratory behavior (head-dipping) independently of locomotor activity. It was found that compounds la, lb, and lh were endowed with anxiolytic activity at the doses assayed since they increased both the percentage of entries and percentage of time on the open arms of the plus-maze compared with the control. In the case of lb, and to a lesser extent lh, this was accompanied by a moderate sedative action, while in the case of la there was no significant modification of the curiosity and motor activity of the animals. At 50 mg/kg the anxiolytic character of lb and lh was confirmed, although at this dose a less central depressant effect was observed. On the contrary, compounds Id, and to a lesser extent If, lg and li, seemed to have a rather anxiogenic character, because they reduced the 2 indices of anxiety used in this experiment. Nevertheless, an anxiogenic interpretation of the action of these compounds should be made with caution, since all the behavioral measures were depressed. These behavioral results led us to investigate the ability of the reported compounds to interact in vitro with central benzodiazepine receptors (mainly with the GABA,-BZD-Clchannel complex), since, despite their 5,6-dihydro character (reduced benzodiazepines do not elicit binding to this receptor), these compounds could exert their biological effects through that receptor. Therefore, we have tested their ability to displace the binding of [3H]-flunitrazepam (FNZ) to receptors from bovine cerebral cortex membranes. Among all of these compounds only la and lb were able to displace the specific binding of
the radioligand in a concentration-dependent form, although the affinity that characterizes the interaction with benzodiazepine receptors of both compounds was lower than that of FNZ. Thus, compound la, which possesses the best affinity with an IC,, of 420 nM (FNZ = 29 nM), was less potent in these tests than compound lb with an IC,, of 20 600 nM. In summary, and in spite of the difficulty of drawing firm conclusions from this preliminary pharmacological study, it may be stated that compound lb appears to have a pharmacological profile that is consistent with an anxiolytic activity similar to that of diazepam and the rest of the classical benzodiazepines, but showing weak anticonvulsant and moderate sedative activities. On the other hand, compounds la and lh have an anxiolytic profile with even low propensity to cause sedative effects. In contrast, compounds Id, lf, lg and li seem to be more akin to anxiogenic drugs, like amphetamines or caffeine. A more detailed investigation is now in progress to confirm these findings. Experimental
protocols
Chemistry All melting points were taken on a Gallenkamp capillary apparatus and are uncorrected. Infrared spectra were determined with a Shimadzu IR-435 instrument. ‘H-NMR spectra were obtained with Bruker AM-200 and Varian XL-300 spectrometers using TMS as an internal standard. Chemical shifts are expressed in 6 units and coupling constants
238 in Hz. Analyses indicated by symbols were within + 0.4% of the theoretical values. The purity of compounds was verified by thin-layer chromatography (TLC) which was run on silica gel GFZS, (Merck) or aluminium oxide 60F,, (Merck) with cyclohexane/ethyl acetate mixtures (2: 1 and 1: 1, v/v respectively) as eluents.
free base 1 was obtained by treatment of 6 with dilute sodium hydroxide and extraction with ether or chloroform. Table I lists the yields and the physical and analytical data of all the comuounds 6 and 1 svnthesized. The main ‘H-NMR suectroscop\c features of diazepines 1 are summarized in table II. Pharmacology
4-Cyano-I-methyl-5-(1-pyrryl)pyrazole 3 A mixture of 5-amino-4-cyano-1-methylpyrazole 2 [3] (122 g, 1.0 mol), 2,5-dimethoxytetrahydrofuran (132 g, 1.0 mol) and glacial acetic acid (1000 ml) was heated under reflux for 30 min. The mixture was concentrated in vacua and the oily residue was then distilled at 0.1 mmHg; a fraction (163.4 g, 95%) of bp = 125-130°C crystallized on cooling to give 4-cyano- 1-methyl-5-( 1-pyrryl)pyrazole 3, mp = 6667°C (ethanol). Anal C,H,N, (C, H, N). IR (KBr): 2240 cm-1 (CN). ‘H-NMR (DMSO-d,): 6 7.78 (s, lH, H3); 6.90 (t, 2H, JH2’,a3’= J H4’,H5’= 2.2 Hz, H2 and H5); 6.44 (t, 2H, H3’ and H4’); 3.80 (s, 3H, N-CH,). 4-Aminomethyl-methyl-T-(1 -pyrryl)pyrazole 4 To a stirred susnension of lithium aluminium hvdride (24 g. 0.6 mol) in dry tetrahydrofuran (750 ml) was slowly added”; solution of 3 (86 g, 0.5 mol) in dry tetrahydrofuran (750 ml). The mixture was stirred and heated under reflux for 3 h, then cooled and carefully treated with absolute ethanol. After filtration, the filtrate was evaporated and the residue was extracted with ether. The organic layer was washed with water, dried (magnesium sulfate) and evaporated. Distillation of the oily residue at 0.1 mm Hg gave the aminomethyl derivative 4, bp = 115-118°C (61 g, 69%). Picrate, mp > 194°C (dec) (ethanol). Anal C,,H,.&O, (Ci H, N). IR (film): 3380, 1306, 1590 cm-r (NH,). rH-NMR (CDCl,): 6 7.28 (s. 1H. H3): 6.75 (t, 2H, J,&j = yHt.,, = 2.2 Hz, Hf and H5’): 6.38 (t, 2H, H3’ and H4’); 3.62 (s, 3H, CH,); 3.59 (s, 2H, CH,); 1.58 (bs, 2H, exchangeable, NH,). MS: m/z 176 (M-t-, 100%). 4-Benzylideneaminomethyl-1
-methyl-T-(1
-pyrryl)pyrazoles
5
General method. A mixture of 4 (0.02 mol), the appropriate benzaldehyde (0.02 mol) and ethanol (40 ml) was heated under reflux for 15 min. The solvent was evaporated in vacua to give the corresponding Schiff base 5, which, in general, could-not be uurified bv distillation or crvstallization from the common solGents and for this reason they were used as such in the next reaction. Compound Se precipitated on cooling the reaction mixture, and so it was isolated by filtration and identified as follows. 4-(p-Nitrobenzylideneaminomethyl)-I -methyl-T-(1 -pyrryl)uvrazole 5e. This comvound was obtained in 78% vield from &itrobenzaldehyde as Lyellow crystals of mp = f48-151°C (ethanol). Anal C,6H,SN,0, (C, H, N). IR (nujol): 1640, 1570 cm-l (C=N). rH-NMR (CDCl,): 8 8.19 (d, 2H, JH3.‘H2.’= J H4”FTC- 8.9 Hz, H3” and H5”); 8.03 (s, lH, CH): 7.75 (d, 2H. H2”?&d H6”); 7.46 (s, lH, H3); 6.66‘(t, 2H, J,;,.,Y = Jis.H4. = 2.1 Hz. H2’ and H5’): 6.26 It. 2H. H3’ and H4’): 4.49 fd. 2H. J = 1.5 Hz; CH,); 3.52 (s, 3H, &I,). ’ 5,6-Dihydro-6-aryl-l-methyl-IH,4H-pyrazolo[4,3-f]pyrrolo[l, 2-a][l,4]diazepines 1 The crude Schiff bases 5 obtained above were dissolved in absolute ethanol saturated with dry hydrogen chloride (50 ml) and the solution stirred at room temperature for 45 min. The precipitated hydrochloride salt 6 was filtered off, washed with dry ether and recrystallized from the appropriate solvent. The
Effects on behavior and LD,, in mice According to the method of Irwin [6], the behavior of the mice was observed 1 and 2 h after po administration of the test drugs. The LD,, values were calculated from the lethality within 3 d after po administration of the drugs by the method of Litchfield and Wilcoxon [9]. Effect on spontaneous motor activity Locomotor activity was recorded with a photocell activity meter for 15 min, beginning 60 and 120 min after po administration of each test drug. Effect on normal body temperature The rectal temperature of the mice was measured with a thermistor thermometer (Panlab 0331) prior to the experiment and 1, 2 and 4 h after po administration of each test drug. Muscle relaxant
activity
Traction test. Experiments were performed by the method of Courvoisier et al [lo]. Mice were forced to hang with their forelegs on a wire of 1 mm in diameter, which was stretched horizontally at a height of 35 cm. When they fell off the wire within 5 s or they failed to grasp the wire with their hind legs 3 times successively, muscle relaxation was judged to be positive. Chimney test. In a Pyrex tube (30 cm long and 28 mm diameter) marked at 20 cm from the base, a mouse was introduced at the end near the mark. When the animal reached the other end of the tube, the tube was moved to the vertical position and immediately the mouse tried to climb the tube backwards. Only those mice which reached the mark within 30 s were selected for further testing. The operation was repeated 60 and 120 min after po administration of the test drugs [ll]. Exploratory
behavior
pattern
Holeboard test and plus-maze test. The holeboard apparatus consisted of a wooden board (40 x 40 cm) which had 16 equally spaced holes. The holeboard testing involved placing the mouse 55 min after po injection with the control vehicle or test material in the center of the floor and counting the number of head-dips during 5-min trials ] 121. The plus-maze apparatus was made of Plexiglas and consisted of 2 open arms (30 x 5 cm) and two enclosed (30 x 5 x 15 cm) arms connected by a central platform (5 x 5 cm). The open arms, the central platform and the floor of the closed arms were made of black nlexinlas. and the sides of the closed arms were made of clear Plexiglas. The apparatus was mounted on a base raising it 38.5 cm above the floor. The test consisted of injecting po mice with the drug or its vehicle 55 min before being tested individually in the holeboard for 5 min. Immediately after the holeboard test each animal was placed in the center of the plus-maze facing an open arm and allowed to freely explore for 5 min. The number of entries made on the open and closed arms and the time spent in each type of arm I
YI
239 were recorded. Three measures were obtained from the test: the total number of arm entries; the percentage of arms entries made on the open arms; and the time spent on the open arms expressed as a percentage of the time spent on both the open and closed arms. The last 2 measures are used as indices of anxiety: they increase following the administration of anxiolytics and decrease following the administration of anxiogenic drugs [7, 81. Interaction with barbiturate-induced sleep in mice Sodium pentobarbital at a subhypnotic dose of 20 mg/kg or at a hypnotic dose of 40 mg/kg dissolved in 0.9% saline was injected ip to groups of 8 mice 60 min after po injection of the drug. The number of mice which lose righting reflex (in the case of subhypnotic dose) and the latency and the duration of sleep (loss and recovery of the righting reflex) were recorded. Anticonvulsant Convulsions (120 mg/kg) 0.9% saline. administered anticonvulsive tonic seizures
activity: effect on chemically induced seizures were induced by an ip injection of pentetrazol or strychnine sulfate (2.5 mg/kg) dissolved in Test drugs, suspended in 1% Tween 80, were orally 60 min before the convulsant injection, and activity was determined by loss of clonic and and mortality.
Binding of [3H]-junitrazepam to GABAlbenzodiazepine receptor complex The binding of [sH]-flunitrazepam to the GABA/benzodiazepine receptor complex of bovine cerebral cortex was determined by the method described by Sigel et al [13], with minor modifications. Preparation of membranes. Bovine brain was obtained fresh from a slaughterhouse, the cortex was rapidly removed, chopped, frozen immediately and stored at -80°C until use. Cortex (199 g) was thawed, chopped and homogenized in 10 mM HEPES (pH 7.5), 1 mM EDTA, 300 mM sucrose, 0.5 mM dithiothreitol, 1 mM benzamidine/HCl, 0.3 mM phenylmethylsulfonyl fluoride (1 1). All operations were made at 0-4”C. The homogenate was centrifuged at 1000 x g,, for 12 min. The supernatant was centrifuged at 27 000 x g,, for 35 min. The pellet was resuspended, using a glass-teflon homogenizer, in 500 ml of the same medium except that sucrose and phenylmethylsulfonyl fluoride were omitted. After recentrifugation, the pellet was resuspended in the same medium (final volume 150 ml; 15 mg protein/ml).
Binding assay in solution. Membranes were diluted at 1 mg protein/ml in 20 mM potassium phosphate (pH 7.4) 0.1 mM EDTA, 0.1% (w/v) Triton X-100. Diluted membranes (140 ml) were incubated with [sH]-flunitrazepam (10 nM final concentration) with and without unlabelled ligands at different concentrations with incubation at 0°C for I h. Duplicates of incubations were filtered on GF/C glass fiber filters (Whatman) under suction. The filters were washed 3 times with 4 mi 20 mM uotassium ohosnhate (nH 7.4). 0.1 mM EDTA. dried and counted in a l&id &intil&ion fluid. Non-specific bmding was determined in the presence of cold 10 mM flunitrazepam and represented 5 5% of the total binding. The results were estimated by considering the maximal specific binding obtained with 10 nM flunitrazepam as 100%.
Acknowledgments We are indebted to the Comisidn fnterministerial de Ciencia y Tecnologia, and the Vicerrectorado de Investigation, Universidad de La Laguna, Spain, for the financial support of this work.
References
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