Synthesis of new arylaminoquinoxalines and their antimalarial activity in mice

Article abstract of DOI:10.1211/0022357011777765

2-Arylaminoquinoxalines were prepared by the condensation of 2-chloroquinoxaline with the appropriate Mannich bases in the presence of HCI. To synthesize the Mannich bases, 4-acetamidophenol was reacted with formaldehyde and dialkylamine to yield 3-[(dial

Full text of DOI:10.1211/0022357011777765

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 1409–1413
# 2001 The Authors
Received January 26, 2001
Accepted July 7, 2001
ISSN 0022-3573
Synthesis of new arylaminoquinoxalines and their
antimalarial activity in mice
J. B. Rangisetty, C. N. V. H. B. Gupta, A. L. Prasad, P. Srinivas, N. Sridhar,
P. Parimoo* and A. Veeranjaneyulu
Abstract
2-Arylaminoquinoxalines were prepared by the condensation of 2-chloroquinoxaline with the
appropriate Mannich bases in the presence of HCl. To synthesize the Mannich bases, 4-
acetamidophenol was reacted with formaldehyde and dialkylamine to yield 3-[(dialkylamino)
methyl]-4-hydroxyacetanilide, followed by hydrolysis. Antimalarial activities of the new
arylaminoquinoxalines were evaluatedagainst the rodent malaria parasite Plasmodium yoelii at
a dose of 75 mg kg 1. Three compounds synthesized (2-[3-[(diethylamino) methyl]-4-hydroxy-
−
anilino]-quinoxaline dihydrochloride (2b), 2-[3-[(pyrrolidinyl) methyl]-4-hydroxyanilino]-quin-
oxaline dihydrochloride (2f), and 2-[3-[(piperidinyl) methyl]-4-hydroxyanilino]-quinoxaline
dihydrochloride (2g)) showed moderate antimalarial activity.
Introduction
Malaria remains a major health threat in the world as it has for the last century, and
a major cause of death, particularly among children and women, primarily in Africa
(Su et al 1999). Chloroquine was the most effective and widely used drug in malaria
therapy because of its rapid onset of action, good tolerability and low cost
(Hoffman 1996). However, emergence of strains of Plasmodium falciparum resistant
to chloroquine and many other drugs, including primaquine, pyrimethamine and
mefloquine, in succession has stimulated efforts to identify new antimalarial agents.
The emergence of multidrug-resistant strains of Plasmodia has created a near
desperate situation, where the need for new inexpensive antimalarials to circumvent
the parasite’s resistance mechanism has become vital. Recent efforts in modifying
presently available drugs has led to the discovery of potent molecules that
are active against resistant strains of P. falciparum (Barlin et al 1993; Ridley et
al 1996; De et al 1998; Chibale et al 2000). Amodiaquine (1, Figure 1), a Mannich
base derivative, has been shown to be a superior alternative to chloroquine in areas
of high chloroquine resistance (Churchill et al 1985). Subsequent research into the
synthesis of Mannich base compounds containing side chains afforded many
diverse Mannich base derivatives (Raynes et al 1999). Tebuquine, a hybrid
compound combining structural elements of amodiaquine and some 2-(dialkyl-
amino)-o-cresol derivatives have shown more potent activity than amodiaquine
(Werbel et al 1986).
Department of Pharmacy,
Birla Institute of Technology
and Science, Pilani 333031,
Rajasthan, India
J. B. Rangisetty,
C. N. V. H. B. Gupta,
A. L. Prasad, P. Srinivas,
N. Sridhar, P. Parimoo,
A. Veeranjaneyulu
Correspondence and present
address: J. B. Rangisetty,
Department of Medicinal
Chemistry, Medical College
of Virginia, Virginia
Commonwealth University,
Richmond, VA 23298-0540, USA.
E-mail: jrangise!hsc.vcu.edu
Funding: This work was
supported by a University Grants
Commission Award, New Delhi,
awarded to J. B. Rangisetty.
From time to time reports of antimalarial activity with quinoxaline derivatives
have appeared in the literature (Haworth & Robinson 1948; Growther et al 1949).
* Deceased March 7, 1998
1409

1410
J. B. Rangisetty et al
2-Chloroquinoxaline (4)
Phosphorous oxychloride (75 mL) was added slowly
dropwise to 2-hydroxyquinoxaline (3) (14.6 g, 0.10 mol)
at 0mC. A few drops of DMF were added to the reaction
mixture and heated to reflux for 45 min. Excess of
phosphorous oxychloride was removed under reduced
pressure and the residue was then poured into crushed
ice and extracted with Et2O (5i50 mL), washed with
H2O, dried (MgSO4) and evaporated to give 2-chloro-
quinoxaline and was recrystallized from n-pentane to
give a pure compound. Yield 14.5 g, 88%; mp 46–48mC
(Gowenlock et al (1945): 46mC)
Figure 1 The structures of amodiaquine 1 and the new arylamino-
quinoxalines 2.
In light of these observations, we have attempted to
combine a quinoxaline system with a series of Mannich
base derivatives to develop new arylaminoquinoxalines
(2, Figure 1) for antimalarial evaluation.
General procedure for the preparation of 2-[3-
[(dialkylamino) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2a–2j)
A mixture of 4-acetamidophenol (3.8 g, 25 mmol), 37%
formaldehyde (2.0 mL) and dialkylamine (25 mmol)
suspended in alcohol (50 mL) was heated on a steam
bath for 3 h. The volatile materials were removed under
reduced pressure, 20% HCl (20 mL) was added and
heated at refluxing temperature for 3 h. The reaction
mixture was cooled. The pH was adjusted to alkaline
with ammonia solution, extracted with CH2Cl2 (4i
30 mL), washed with H2O, dried (MgSO4) and evapo-
rated to give the free amine. The mixture of the above
amine, 2-chloroquinoxaline (25 mmol) was dissolved in
ethanol (20 mL), pH was adjusted to pH 4 with hy-
drochloric acid and was heated to reflux for 20 h. The
reaction mixture was cooled and pH was adjusted to
alkaline with NH4OH and extracted with CH2Cl2 (4i
50 mL), dried (MgSO4), evaporated under pressure and
purified by column chromatography (CH2Cl2\CH3OH,
20:1) to yield freebase and the freebase was converted to
their respective hydrochloride salts.
Materials and Methods
Chemical procedures
All melting points were determined on a Buchi model
530 melting point apparatus and were uncorrected.
Infrared spectra were recorded on a JASCO IR report-
1
−
100 infrared spectrophotometer and were given in cm .
1H NMR spectra were recorded using a Bruker AC
300F NMR spectrometer. Chemical shifts were reported
in parts per million (δ) relative to tetramethylsilane as
internal standard. All the HCl salts were characterized
by elemental analyses. Elemental analyses are indicated
by the symbols of the elements and were withinp0.4 of
the theoretical values. Hydrochloride salts were ob-
tained by the dropwise addition of a saturated, an-
hydrous solution of ethereal HCl into a cold solution of
the free base in anhydrous ether until further addition of
ethereal HCl did not produce any more precipitate.
Reactionswere performed under a nitrogen atmosphere.
2-[3-[(Dimethylamino) methyl]-4-
hydroxyanilino]-quinoxaline dihydrochloride
(2a)
1
−
Yield 49%; mp 86–88mC. IR (cm ): 3522 (OH), 3216
(NH), 1507, 1375 (aromatic). H NMR (DMSO-d6,
1
2-Hydroxyquinoxaline (3)
freebase): δ 2.4 (s, 6H, 2iCH3), 3.7 (s, 2H, CH2),
6.9–8.4 (m, 8H, ArH), 11.6 (bs, 1H, OH). Anal.
(C17H18N4O. 2HCl) C, H, N.
A mixture of n-butylgyloxylate (12.75 g, 0.10 mol) and
o-phenylenediamine (10.8 g, 0.10 mol) in ethanol
(100 mL) was heated at reflux for 2 h. The reaction
mixture was cooled to give 2-hydroxyquinoxaline. The
product was purified by dissolving in 10% NaOH
solution (125 mL), treated with charcoal and then
2-[3-[(Diethylamino) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2b)
1
−
precipitated by addition of dilute HCl to give the Yield 58%; mp 249–251mC. IR (cm ): 3390 (OH),
1
title compound. Yield 11.4 g, 78%, mp 268–269mC 3198 (NH), 1546, 1418 (aromatic). H NMR (DMSO-
(Atkenson et al (1956): 267–269mC).
d6, freebase): δ 1.1 (t, 6H, 2iCH3), 2.4 (m, 4H, 2iCH2),

1411
4.1 (s, 2H, CH2), 6.7–8.3 (m, 8H, ArH), 11.8 (bs, 1H, 2iCH2), 3.73 (s, 2H, CH2), 6.5–8.1 (m, 8H, ArH),
Synthesis and antimalarial activity of new arylaminoquinoxalines
OH). Anal. (C19H22N4O. 2HCl.0.25H2O) C, H, N.
11.92 (bs, 1H, OH). Anal. (C20H22N4O. 2HCl.0.25H2O)
C, H, N.
2-[3-[(Dipropylamino) methyl]-4-
hydroxyanilino]-quinoxaline dihydrochloride
(2c)
2-[3-[(N-2-Methyl-piperdinyl) methyl]-4-
hydroxyanilino]-quinoxaline dihydrochloride
(2h)
1
−
Yield 73%; mp 271–273mC. IR (cm ): 3342 (OH),
3163 (NH), 1561, 1459 (aromatic). H NMR (DMSO-
1
−
1
Yield 43%; mp 178–180mC. IR (cm ): 3428 (OH),
3188 (NH), 1561, 1493 (aromatic). H NMR (DMSO-
1
d6, freebase): δ 0.98 (t, 6H, 2iCH3), 1.4 ( m, 4H,
2iCH2), 2.6 (m, 4H, 2iCH2), 3.6 (s, 2H, CH2), 6.7–8.2
(m, 8H, ArH), 12.0 (bs, 1H, OH). Anal. (C21H26N4O.
2HCl) C, H, N.
d6, freebase): δ 0.97 (s, 3H, CH3), 1.1–1.7 (m, 6H,
3iCH2), 2.42 (t, 2H, CH2), 2.71 (m,1H, CH), 3.92 (s,
2H, CH2), 6.9–8.3 (m, 8H, ArH), 11.82 (bs, 1H, OH).
Anal. (C21H24N4O. 2HCl) C, H, N.
2-[3-[(Diisopropylamino) methyl]-4-
hydroxyanilino]-quinoxaline dihydrochloride
(2d)
2-[3-[(N-Methylpiperazin-1-yl) methyl]-4-
hydroxyanilino]-quinoxaline dihydrochloride
(2i)
1
−
Yield 64%; mp 209–211mC. IR (cm ): 3436 (OH),
3184 (NH), 1459, 1376 (aromatic). H NMR (DMSO-
1
−
Yield 41%; mp 174–176mC. IR (cm ): 3386 (OH),
1
3212 ( NH), 1558, 1458 (aromatic). 1H NMR (DMSO-
d6, freebase): δ 1.12 (s, 3H, CH3), 2.6–2.95 (m, 8H,
4iCH2), 3.69 (s, 2H, CH2), 6.7–8.1 (m, 8H, ArH), 11.87
(bs, 1H, OH). Anal. (C20H23N5O. 2HCl) C, H, N.
d6, freebase): δ 1.1 (m, 12H, 4iCH3), 1.37 (s, 1H, CH),
2.61 (q, 2H, CH2), 3.97 (s, 2H, CH2), 6.62–9.42 (m, 8H,
ArH), 11.98 (bs, 1H, OH). Anal. (C21H26N4O. 2HCl) C,
H, N.
2-[3-[(Morpholinyl) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2j)
2-[3-[(Dibutylamino) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2e)
1
−
Yield 48%; mp 279–281mC. IR (cm ): 3386 (OH),
3212 (NH), 1558, 1458 (aromatic). H NMR (DMSO-
1
−
Yield 66%; mp 280–282mC. IR (cm ): 3361 (OH),
3114 (NH), 1512, 1459 (aromatic). H NMR (DMSO-
1
1
d6, freebase): δ 2.34–2.46 (m 4H, 2iCH2), 3.31 (m, 4H,
2iCH2), 4.20 (s, 2H, CH2), 6.1–8.5 (m, 8H, ArH), 11.98
(bs, 1H, OH). Anal. (C19H20N4O2. 2HCl) C, H, N.
d6, freebase): δ 0.9 (t, 6H, 2iCH3), 1.89 (m, 8H,
4iCH2), 2.89 (m, 4H, 2iCH2), 3.93 (s, 2H, CH2),
6.47–8.49 (m, 8H, ArH), 12.2 (bs, 1H, OH). Anal.
(C23H30N4O. 2HCl) C, H, N.
Antimalarial activity
The institutional ethics committee of the Birla Institute
of Technology and Science, Pilani, India, approved the
animal experiments.
2-[3-[(Pyrrolidinyl) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2f)
1
−
Yield 54%; mp 276–278mC. IR (cm ): 3384 (OH),
3207 (NH), 1558, 1543 (aromatic). H NMR (DMSO-
The in-vivo studies were carried out following the
method described by Puri & Dutta (1989) for anti-
malarial evaluation of new arylamine derivatives.
Plasmodium yoelii nigerensis was obtained from the
National Malaria Research Center, New Delhi, India.
Swiss albino mice (27p2 g) were allowed free access to
water and food. For the evaluation of blood schizon-
tocidal activity against rodent malaria, the mice, in
groups of five, were inoculated with 1i106 parasites.
Inoculumfor infectionwas preparedfrom the previously
1
d6, freebase): δ 1.74 (m, 4H, 2iCH2), 2.49 (m, 4H,
2iCH2), 3.78 (s, 2H, CH2), 6.4–8.6 (m, 8H, ArH),
11.84 (bs, 1H, OH). Anal. (C19H20N4O. 2HCl.0.5H2O)
C, H, N.
2-[3-[(Piperidinyl) methyl]-4-hydroxyanilino]-
quinoxaline dihydrochloride (2g)
1
−
Yield 49%; mp 261–263mC. IR (cm ): 3405 (OH), infected donor mouse with rising parasitaemia (20%).
3190 ( NH), 1524, 1477 (aromatic). 1H NMR (DMSO- Blood was drawn from the heart of an infected mouse
d6, freebase): δ 1.68 (m, 6H, 3iCH2), 2.52 (m, 4H, under anaesthesia in a sterile, heparinized disposable

1412
J. B. Rangisetty et al
Table 1 Antimalarial activity of 2-arylaminoquinoxalines against blood-induced Plasmodium yoelli in
mice.
Compound
NR
Mean day four
parasitaemia
Mean survival
time (meanp
s.e.m.)
(meanps.e.m.)
2a
Dimethylamino
Diethylamino
20.19p3.6
10.23p0.96*
26.64p2.48
24.48p4.22
9.42p4.02*
4.96p1.08*
21.79p2.12
20.08p3.16
16.38p1.12
kve
5.50p1.6
10.54p1.92*
5.94p0.88
6.12p2.20
11.20p0.48*
13.24p2.24*
6.40p1.48
7.12p2.18
8.96p2.96
" 15
2b
2d
Diisopropylamino
Dibutylamino
2e
2f
Pyrrolidinyl
2g
Piperidinyl
2h
N-(2-methyl) piperidinyl
N-methylpiperazinyl
Morpholinyl
2i
2j
Chloroquine
Control
23.12p3.2
6.18p0.46
*P ! 0.05 compared with the control group.
syringe. The test compounds, 2-arylaminoquinoxalines
1
at 75 mg kg , were administered orally. The compounds
−
were triturated with 1% w\v tragacanth solution for
uniform mixing and finally reconstituted with water.
The drug treatment was administered for four days
from day 0 to day 3. Blood smears were prepared on day
4, fixed in methanol and stained with giemsa stain. The
slides were observed under oil immersion lens (1000i)
to obtain the percentage of parasitaemia. A group of
five infected mice served as negative control, while a
similar test with mice treated with chloroquine 20 mg
1
−
kg for four days served as a positive control. The
percentage parasitaemia on day 4 and extension of mean
survival time of treated mice was interpreted as evidence
of antimalarial activity. Table 1 shows the antimalarial
activity of the new 2-arylaminoquinoxaline derivatives.
Statistical analysis
Figure 2 Synthesis of 2-arylaminoquinoxalines (2a–2j). Reagents
are : a, n-butylgyloxalate, ethanol, reflux; b, POCl3, reflux; c, HCHO,
dialkylamine (Mannich Reaction), ethanol, reflux; d, 6  HCl, reflux;
e, 2-chloroquinoxaline (4), pH 4, ethanol.
The data were expressed as meanps.e.m. Statistical
tests were performed by one-way analysis of variance
followed by Dunnett’s test for multiple comparisons of
test groups with control group. The significance level
was P ! 0.05.
sized according to the method of Gowenlock et al (1945)
by chlorinating 2-hydroxyquinoxaline (3) with POCl3 in
high yield. 2-Hydroxyquinoxaline was prepared accord-
ing to the method of Atkenson et al (1956) by reacting o-
Results and Discussion
The arylaminoquinoxalines were prepared by the con- phenylenediamine and n-butylgyloxylate. The pro-
densation of 2-chloroquinoxaline with Mannich aryl- cedure of Burckhalter et al (1948) was used for the
amines (Figure 2). 2-Chloroquinoxaline (4) was synthe- synthesis of 4-hydroxy-3-[(dialkylamino) methyl] ani-

1413
Synthesis and antimalarial activity of new arylaminoquinoxalines
Chibale, K., Moss, J. R., Blackie, M., Schalkwyk, V. D., Smith, J. P.
(2000) New amine and urea analogues of ferrochloroquine: syn-
thesis, antimalarial activity in vitro and electrochemical studies.
Tetrahedron. Lett. 41: 6231–6235
lines. Reactions of equimolar quantities of 4-hydroxy
acetanilide, formaldehyde and requisite amine were
refluxed in ethanol to yield 4-hydroxy-3-[(dialkylamino)
methyl] acetanilide. Acetanilide derivatives were con-
verted to free amines using 20% hydrochloric acid.
After hydrolysis, the pH of the reaction mixture was
adjusted to pH 4 and refluxed with 2-chloroquinoxaline
for 20 h in ethanol to bring about the condensation of
the in-situ free amine (Banks 1944).
Churchill, F. C., Patchen, L. C., Campbell, C. C., Schwartz, L. K.,
Nguyen-Dinh, P., Dickinson, C. M. (1985) Amodiaquine as a
prodrug: importance of metabolites in the antimalarial effect of
amodiaquine in humans. Life Sci. 36: 53–62
De, D., Krogstad, M. F., Byers, D. L., Krogstad, J. D. (1998) Struc-
ture-activity relationships for antiplasmodial activity among 7-
substituted 4-aminoquinolines. J. Med. Chem. 41: 4918–4926
Gowenlock, A. H., Newbold, G. T., Spring, F. S. (1945) Syntheses of
2-monosubstituted and 2:3-disubstituted quinoxalines. J. Chem.
Soc. 622–625
All the compounds were tested for their antimalarial
activity in mice infected with P. yoelii. The antimalarial
activity of the 2-arylaminoquinoxalines is shown in
Table 1. From the ten compounds tested, only com-
pounds 2b, 2f and 2g showed moderate antimalarial
Growther, A. F., Curd, F. H. S., Davey, D. G., Stacey, G. J. (1949)
Synthetic antimalarials. Dialkylaminoalkylaminoquinoxalines. J.
Chem. Soc. 1260–1271
1
−
activity at 75 mg kg . The mean survival time of the
Hoffman, S. L. (1996) Artemether in severe malaria – still too many
deaths. N. Engl. J. Med. 335: 124–126
mice treated with chloroquine was more than 15 days,
where as the mean survival time of control mice was 6.18
days. Arylaminoquinoxalines containing diethylamine,
pyrrolidine and piperidine moieties were found to have
better antimalarial activity than the other amines. How-
ever, none of the tested compounds had significant
antimalarial activity when compared with chloroquine.
Haworth, R. D., Robinson, S. (1948) Synthetic antimalarials. Some
derivatives of phthalazine, quinoxalines and isoquinolines. J. Chem.
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of a new macrolide antibiotic midecamycin in mice and monkeys.
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Raynes, J. K., Stocks, A. P., O’Neill, M. P., Park, K. B., Ward, A. S.
(1999) New 4-aminoquinoline Mannich base antimalarials. 1. Effect
of an alkyl substituent in the 5h-position of the 4h-hydroxyanilino
side chain. J. Med. Chem. 42: 2747–2751
Ridley, G. R., Hofheinz, W., Matile, H., Jaquet, C., Dorn, A.,
Masciadri, R., Jolidon, S., Richter, F. W., Guenzi, A., Girometta,
M., Urwyler, H., Huber, W., Thaithong, S., Peters, W. (1996) 4-
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