Pursuing our investigations on 7-amino-2,3-polymethyleneindole derivatives, a set of 7-(dimethylaminomethylene)-amino-2,3-polymethyleneindoles, together with some other aryl or cycloalkyl substituted formamidines, were prepared and tested for analgesic and antiinflammatory activities. Several compounds resulted endowed with one or both of these activities; the indole derivatives 1 and 2 exhibited a good degree of both of them

Boatto, Gianpiero

Carrara, Maria

Cerri, Riccardo

Cima, Lorenzo

Pau, Amedeo

Satta, Margherita

Sparatore, Fabio

English

UnissResearch

IL FARMACO, 46 (2),369-378; 1991 
FORMAMIDINES WITH ANTINOCICEPTIVE AND 
ANTIINFLAMMATOR Y ACTIVITIES 
RICCARDO CERRI (*) - GIANPIERO BOATTO - AMEDEO PAU 
ISTlTUTO 01 ANALlTICA FARMACEUTlCA - UNIVERSITA DI SASSARI 
VIA MURONI , 23 - 07100 SASSARI, ITALV 
FABIO SPARATORE 
ISTITUTO 01 SCIENZE FARMA CE UTI C HE - UN1VERSITA 01 GENOVA 
VIALE BENEDETTO XV, 3 - 16132 GENOVA, ITALY 
LORENZO CIMA - MARIA CARRARA 
DIPARTlMENTO 01 FARMACOLOGIA " E. MENEGHETTI" - UNIVERSITA DI PADOVA 
LARGO E. MENEGHETTI , 2 - 35 131 PADOVA , ITALY 
MARGHERITA SATTA 
ISTITUTO 01 C HIMICA BIOLOGICA - FACOL TA 01 FARMACIA - UNIVERSITA 01 SASSA Rl 
VIA MURONI, 23 - 07100 SASSARI, ITALY 
SUMMARY Pursuing our investigations on 7-amino-2,3-polymethyleneindole 
derivatives, a set oj 7-(dimethy/aminomethy/ene)-amino-2,3-po/ymethy/eneindo/es, 
together with some other aryl or cye/oalkyl substitutedjormamidines, were prepared 
and tested jor analgesic and antiinjlammatory activities. Several compounds result-
ed endowed with one or both oj these activities; the indole derivatives I and 2 ex-
hibited a good degree oj both oj them. 
RIASSUNTO Proseguendo 10 studio dei derivati 7-ammino-2,3-
polimetilenindolici sono stati preparati alcuni 7-(dimetilamminometilen)ammino-2,3-
polimetilenindoli nonche a/cune altre amidine recanti spezzoni aromatici 0 alicie/i-
d. I composti sono stati studiati per quanto concerne le attivitii analgesica e antiin-
jiammatoria. Svariati prodotti sono risultati dotati dell'una e/o dell'altra attivitii; 
appaiono interessanti i derivati indolici 1 e 2 che presentano ad elevato live/lo le due 
. . , 
attlvlla. 
(*)To whom correspondence should be addressed 
370 R. CERRI et al. 
Introduction 
Several compounds bearing an amidine group seem to be involved in 
different biological and pharmacological areas (l), and recently a particu-
lar importance has been attained by compounds containing a formamidine 
moiety joined with an aromatic nucleus. 
Thus N' -(imidazolyl)phenyl-N" -alkylformamidines (2) show potent in-
hibition on hystamine receptors H-2 (bisfentidine), whereas, N'-(4-
arylmethylamino )-2,6-dichlorophenyl-N", N" -dimethylformamidines dis-
play different levels of antihypertensive and diuretic activities (3). 
On such a basis we deemed interesting to study the effects of the in-
troduction of a formamidine function on position 7 of certain 2,3-
polymethyleneindoles B, which since a long time are object of our investi-
gation. In fact, several 7-(N-acyl)amino derivatives of general formula A 
exhibited different levels, sometimes rather high, of antiinflammatory (4,5) 
and analgesic (6) activities. 
While our research was going on (7), Gall et al. (8) observed very high 
anti nociceptive activities, with virtually no hypotensive effects, in certain 
2,4- and 2,6-dimethylarylformamidines; this fact further validated our 
research project. 
In analogy with our former research (6), in order to evaluate the sig-
nificance of the polymethylene rings condensed with the indole nucleus, as 
well as the significance of the indole nucleus itself, we prepared also the 
7-formamidine derivative corresponding to 3-propylindole (C) and some aryl-
and cycloalkylformamidines (D - F). The structures of these compounds are 
illustrated in Scheme 1. 
H 
I 'A' 
Z 
Z= CO-R: CO-CF3 
S02-CFJ 
R 
• 
I.lN 
HCV 
H 
" N-CH 
, 3 
CH J 
'E' 
'B' 
R 
R= H 
OH 
I 
H 
'c 
'F' 
Formamidines with antinociceptive and antiinflammatory activities 371 
The arylformamidines D were already known, but no data on their phar-
macological activities were available. 
In the present paper we describe the results of the investigation on an-
tinociceptive and antiinflammatory activities, while those concerning pos-
sible antihypertensive and diuretic activities will be the object of a forth-
• 
commg paper. 
Chemistry 
The preparation of formamidines B - F was effected by refluxing the 
benzene solution of the suitable amino compounds with an excess of N,N-
dimethylformamide dimethylacetal: 
R-N 
The structure of the obtained compounds is supported by elemental ana-
lyses and IR and NMR spectra and, in the case of alicyclic compounds E 
and F, also by mass spectra (see Tables I and 11). 
The required 7-amino-2,3-polymethyleneindoles and 7-amino-3-
propylindole were prepared as already described by us (6,9). The alicyclic 
amines were prepared according to the literature: (+) trans-2-
aminocyclopentanol through ammonolysis of cyclopentene oxide (10, 11) 
and 3-aminocyclopentene through the reaction of cyclopentadiene with dry 
hydrogen chloride followed by the ammonolysis of the resulting chlorocy-
clopentene (12). 
Chemical Experimental Section 
Melting points were determined using a Kofler apparatus and were not corrected. 
Elemental analyses (C, H, N) were perfomed at the Microanalytical Laborato-
ry of the Department of Pharmaceutical Sciences, Padua University, and the ana-
lytical results for the elements indicated were within ± 0.3 070 of the calculated values. 
IR spectra were recorded with a Perkin-Elmer mod. 298 Spectrophotometer using 
liquid samples as films and including solid samples in KBr pellets; NMR spectra were 
taken on a Varian XL 200 speclrometer, using CDCl, as solvent with TMS as in-
ternal standard. Mass spectra were obtained on a HP 5970A apparatus, using a capil-
lary column SE 30 of 12 m length; programmed temperature was from 50°C to 270°C; 
detector temp. was 280°C; the carrier gas was helium at 99,9998070 purity. 
372 R. CERRI et al. 
7-(Dimethylamino)methylene amino-2,3-polymethyleneindoles (B) and 
7-(dimethylamino)methylene amino-3-propylindole (C). 
In a boiling solution of 5-7 mmoles of 7-amino-2,3-polymethyleneindoles or 
7-amino-3-propylindole in 15-25 ml of benzene were dropped rapidly 0.80 - 1.15 ml 
(stoichiometric quantity + 200/0 excess) of dimethylformamide dimethylacetal and the 
mixture was further refluxed for 0.5 to 1 hour. 
When the conversion of the amino compounds to formamidine derivatives [as 
detected through TLC on silica using benzene-aceton (4:1) as eluent] was practically 
completed, the reacting mixture was cooled and the precipitate was collected and 
crystallized from benzene B or aqueous ethanol C. 
All data concerning these compounds are collected in Tables I and n. 
N',N"-Dimethyl-N"-(3-R)aryljormamidines (D) and N',N'-dimethyl-N"-
cycloalkyljormamidines (E, F). 
These formamidines were prepared under the above mentioned conditions, but 
after 30 to 60 min of reflux the solvent was removed under reduced pressure and 
the residue was distilled in vacuo (bulb to bulb). All data concerning these compounds 
are collected in Tables I and n. The boiling points of aryl formamidines D are in 
agreement with those reported in the literature (13, 14, 15). 
TABLE I 
Formamidine derivatives 1-10 
Molecular m.p. or 
Comp. Structure Formula Analyses b.p. (mmHg) Yield 
weight °C 
% 
1 B; n= 1 C16H 19N3 241.3 C,H,N 205-207 75 
2 B; n=2 CI6H 21 N3 2553 C,H,N 181-183 73 
3 B; n=3 C17I-123N3 269.4 C)-I,N 144-145 83 
4 C C14H 19N 3 229.3 C,H,N 119-120 72 
5 (a) D' R=H , CgH12N2 148.2 C)I,N 78-80 (0.1) 70 
6 (b) 0; R=OCH3 CIOH 14N20 178.2 C,H,N 164 (12) 70 
7(c) D; R=CF3 CIOHII F3N 2 216.2 C,H,N 93-95 (12) 70 
8 E; R=H CgI-I J6l"l 2 140.2 C,H,N 59-60 (6) 70 
9 E; R=OH CgHl~20 156.2 CJlN 85 (7) 74 
10 F CgH14N2 138.2 C,H,N 60 (10) 30 
(a) known (13); (b) known (14); (c) known (15) 
IR, cm-! 
Comp.1 (in KBr or film) 
NH indole I -C=N-
1 3126 1630 
2 3120 1630 
3 3199 1626 
4 3130 1640 
8 1650 
9 1640 
10 1640 
TABLE II 
Spectral data of compounds 1-4 and 8-10 
IH-NMR,o 
(CDCI3) 
1.75 (m, 4H, 2CH2); 2.55 (m, 4H, 2CH2); 2.85 (5, 6H, 2CH3); 
6.85 (m, 3H arorn.); 755 (5, IH, -CH=); 9.05 (5, I H, NH indole) 
1.65 (m, 6H, 3CH2); 2.75 (m, 4H, 2CH2); 3.00 (5, 6H, :CH3); 
6.85 (m, 3H arorn.); 7.60 (5, IH, -CH=); 8.95 (5, IH, NH indole) 
150 (m, BH, 4CHZ); 2.70 (m, 4H, 2CH2); 3.00 (5, 6H, 2CH3); 
6.75 (m, 3H arom.); 7.60 (5, I H, -CH=); B.95 (5, I H, NH indole 
• 
1.00 (t, 3H, CHZ-CH3); 1.75 (m, 2H, CH2); 2.70 (t, ZH, CHZ); 
3.00 (5, 6H, 2CH3); 6.90 (m, 4H aram.); 7.65 (5, IH, -CH=); 
8.85 (5, I H, NH indole) 
1.60 (m, 8H, 4CH2); 2.80 (5, 6H, 2CH3); 3.4 (m, IH, CH-N>; 
7.25 (5, IH, -CH= ) 
1.75 (m, 6H, 3CH2); 2.85 (5, 6H, 2CH3); 3.2 (m, !H, CH-N); 
335 (5, IH, OH); 3.80 (m, lH, CH-OH); 7.25 (5, IH, -CH=) 
1.95 (m, 4H, :CH2); 2.80 (s, 6H, 2CH3); 4.25 (m, IH, CH-N); 
5.50-5.i5 (m, 2H, -CH=CH-); 7.20 (5, IH, -CH=) 
Retention 
time 
(min) 
5.93 
12_10 
GC-Mass 
Most important fragments 
M+/z 
44 [N(CH3)2!; 57 [CH-N(CH3)2!; 71 [N=CH-N(CH3)Z!; 
85 [CSH80HI; 99 [N-CSH80H!; 156 (M+! 
44 [N(CH3)2!; S7 [CH-N(CH3)21; 67 (CSH7!. 
71 [N=CH-N(CH3>z1; 138 [M+l 
"r1 
o 
~ 
3 
t>l 
3 
--0-_. 
::s (b 
'" ~ _. 
~ 
:r 
t>l 
::s 
~ _. 
::s 
o 
() _. 
() 
(b 
'0 
~ _.
<: 
(1) 
t>l 
::s 
0-
t>l 
::s 
~ _. 
_. 
::s 
:=l 
t>l 
3 
3 
t>l 
~ 
o 
~ 
'< 
t>l 
() 
~ _.
<: _. 
~ _.
(1) 
'" 
w 
-..l 
w 
374 
Pharmacology 
Materials and methods 
R. CERRI et al. 
For the detection of antinociceptive properties, female Swiss mice (Nossan) were 
used. The animals, weighing 18-22 g, were divided in groups of ten and stabulated 
at constant temperature and humidity (21 QC ± 1 QC; 60 ± 5070) with alternating 12-
hour periods of light and dark. 
Antiinflammatory activity was evaluated on male albino Wistar-Nossan rats 
(weight: 120-140 g). 
In all cases the animals were fed with Nossan feed in pellets and aqua jontis 
ad libitum. 
Antinociceptive activity against a chemical stimulus (peripheral analgesia). 
This type of analgesia, which involves receptors p. and x, was evaluated through 
the inhibition of writhings induced by the i.p. injection of phenylquinone (2 mg/ kg 
as a 0.02% solution in 5% ethanol). 
The test compounds were administered per os at the dose of 0.167 mmollkg 
suspended in a 10% arabic gum solution. 
The animals were dosed 15, 30, 60, and 120 minutes before the phenylquinone 
injection. 
For every point three groups of 10 mice each were used. For each experiment 
were employed two groups (one at the beginning and one at the end) of control animals 
receiving only the quinone i.p. and the arabic gum per os. 
After the injection of phenylquinone the animals were introduced into a glass 
cylinder and the writhings were recorded for 20 min starting from the fifth min af-
ter injection. 
The results are expressed as the percent variation in writhings number compared 
to that of the control animals. 
Antinociceptive activity against a thermic stimulus (central analgesia). 
To evaluate this kind of analgesia, typically overspinal and involving the p. and 
o receptors, we used the Eddy and Leimbach hot-plate test (16). 
The plate was kept at 55 ±0.5 Q C and the reaction time (in seconds) was deter-
mined on the basis of licking, raising of hind paws, jumping and escape reaction. 
The animals with a reaction time superior to 15 sec or inferior to 10 sec were dis-
carded. The reaction time was measured thrice before the administration of the test 
compounds and 15, 30, 60, 90, 120, 150,210 and 270 minutes after the dosing. The 
test compounds were administered p.o. at the dose of 0.167 mmollkg, suspended 
in a 10% solution of arabic gum. 
Antiinflammatory activity 
Antiinflammatory activity was evaluated by means of the already described (5) 
inhibition of the carrageenan-induced hind paw edema in rats. 
One hour before the injection of carrageenan the test compounds were ad-
ministered per os at the dose of 1 mmollkg suspended in about 10 mllkg of a 5% 
arabic gum solution. The control animals received only arabic gum. The rats had 
been fasting from 12 hours, but with free access (0 water. The activity was evaluat-
ed by the edema volume at the 1", 2nd , 3"1, and 4 th hour after treatment with car-
rageenan and results are expressed as mean percent variation of edema volume com-
Formamidines with antinociceptive and anliinflammatory activities 375 
pared to that in control animals. Each value corresponds to the mean of six animals. 
Indomethacin at the dose of 0.028 mmollkg (10 mg/ kg) was used for comparison. 
All animals were observed for 48 hours after administration of test compounds. 
Results and Discussion 
Depending on the quantity available for each compound the tests were 
not performed for all of them. The anti nociceptive activity against a chemi-
cal stimulus was determined on all compounds except one (4). The an-
tinociceptive activity against a thermic stimulus was investigated only with 
the four compounds 1,2, 7 and 10, wich resulted the most active in the fore-
said test. The inhibitory activity against the carrageenan edema was tested 
on all compounds except compounds 9 and 10. The results are illustrated 
in Tables Ill-IV. 
Even if the compounds have been tested at a single dose, some indica-
tions have been provided by these preliminary assays. 
It is worthy of note that all the three 2,3-polymethyleneindole deriva-
tives (I, 2, 3) exhibited analgesic activity against a chemical stimulus, 
although of different degree and duration. For the remaining aromatic and 
alicyclic compounds the activity appeared to be erratic, depending on the 
nature of substituents (compounds 7 and 10). 
Compound 1, 15 min after administration, exhibited strong antinocicep-
tive activity which reached maximal intensity (-80070) after 30 min and then 
decreased slowly. Compound 2 presented a slow onset of activity which, 
however, growed steadily for the whole duration of the experiment; thus, 
2 h after administration it produced 87070 inhibition of writhings. The differ-
ent behaviours may be attributed to a remarkable difference in the kinetics 
of absorption and/ or of metabolic activation. Compound 3 gave rise to an 
alternating response. In fact, after only 30 min it exhibited a strong inhibi-
tory activity that was of short duration; actually after 60 min the activity 
was over but it was then progressively restored reaching 38070 inhibition of 
writhings 2 h after administration. 
Concerning the arylformamidines, a short lived analgesic activity was 
observed in the un substituted compound and only a late activity in the 3-
methoxyderivative; on the contrary, compound 7, bearing a strong electron-
withdrawing substituent, exhibited a strong activity with rapid onset and 
long duration, which was comparable with that of compound 1. Even if 
with some reservations due to the different methodologies used, the activi-
ty of N', N' -dimethyl-N" -(3-trifluoromethyl)phenylformamidine appeared 
to be comparable with that of all the variously substituted arylformamidines 
described by Gall et at (8), with the exception of the 2,4- and 2,6-dimethyl 
derivatives. 
Among the alicyclic derivatives a short lasting hyperalgesic activity was 
observed for the unsubstituted compound and a fleeting analgesic activity 
376 R. CERRI et al. 
TABLE III 
Antinociceptive activity against a c;hemical stimulus (phenylquinone) 
Dose Percent variation of writhings during 20 min compared with 
Comp. mg/Kg control animals (b) . Reading was started 5 min after phenylquino-
t?.o~ ne injection; the irritant was introduced after the indicated time 
(a) from compounds administration. 
15' 30' 
1 40.2 -60.3 -80.1 
2 42.5 -15.3 -30.0 
3 44.9 +7.4 -52.3 
5 24.7 -9.3 -38.8 
6 29.7 -3.9 -1.1 
7 36.0 -32.7 -71.9 
B 23.4 +37.8 +1.2 
9 26.0 -3.5 -22.2 
10 23.0 -25.4 -18.8 
(a) corresponding to - 0.167 nmoles/Kg 
(b) inhibition (-); increase (+) 
60' 120' 
-64.7 6 -51.2 
-65.3 6 -87.3 
-6.0 -38.2 
+21.3 +9.4 
+1.1 -35.1 
. 
-56.1 -53.4 
+9.6 +5.3 
+ 13 .6 + 12.5 
-45.7 -7.5 
for the hydroxyderivative 9. It is worthy of note that the activity of the un-
saturated compound 10 was quite long lasting though at only moderate lev-
el (-46070 at 60 min): this could indicate that the presence of an aromatic 
moiety is not necessary for the expression of analgesic activity. 
Compounds 1, 2, 7 and 10 which proved to be the most active in the 
peripheral analgesia test, were also tested for central analgesia. None of them 
resulted active in the hot plate test, at least at the used dose of 0.167 
mmollkg; on the contrary, some contraction of the reaction time was ob-
served, which normally lasted for several hours. 
For what concerns the anti inflammatory activity, we noted that, at the 
oral dose of 1 mmollkg, all the tested compounds exhibited a significant 
inhibition of edema (~30%) at the 3rd hour after the carrageenan injec-
tion. However, only the indole derivatives 1, 2, 3, 4 exhibited a rather strong 
, 
activity, spanning the whole experimentation time. At the indicated dose, 
these compounds showed an activity which was equal to or higher than that 
of indomethacin at the dose of 10 mg/ kg; and was also comparable to that 
of phenylbutazone (17) at the same dose of 1 mmollkg (308 mg/ kg). 
Formamidines with anti nociceptive and antiinflammatory activities 377 
TABLE IV 
AntiinJlammatory activity in the carrageenan induced rat paw edema 
Dose 
Compound mgKg Percent variation of edema volume at the following 
J1.~. times (hours) after carrageenan injection (b) 
(a) 
I 2 3 4 
1 241 -40 -58 -69 -79 
2 255 -18 -SI -65 
-63 
3 269 -18 -60 -53 N.D. 
4 229 N.D. -77 -74 -72 
5 148 -4 -13 -33 -21 
6 178 -7 -21 -34 -25 
7 216 -3 -6 -33 -27 
8 140 -6 -25 -29 N.D. 
indomethacin 10 -9 -58 -64 -43 
(a) Corresponding to 1 mmole/Kg 
(b) Inhibition (-) or increase (+) compared to the edema volume of control animals 
N.D. Not determined 
On doubling the dose of polymethyleneindole derivatives any sign of 
toxicity did not appear during the whole time (48 h) of observation, while 
antiinflammatory activity was only slightly increased. 
Concluding, we can remark that compounds 1 and 2 appeared to be 
the most interesting, having shown a good degree of both peripheral an-
tinociceptive and antiinflammatory activity, therefore their pharmacologi-
cal profile will be deeply examined. Moreover, these results support our in-
terest on the 7-aminoindole derivatives, whose pharmacogenic potentiali-
ties will be further investigated. 
Acknowledgement 
The financial support from the Ministero della Pubblica Istruzione and 
from C.N.R. are gratefully acknowledged. 
378 R. CERRl et al. 
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Received August 2, 1990; accepted November 9, 1990. 


Chimica Farmaceutica della S.C.!.; Alghero,

Patai &quot;The Chemistry of amidines and imidates&quot;,

Formamidines with antinociceptive and antiinflammatory activities

Formamidines with antinociceptive and antiinflammatory activities

Abstract

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