Leishmanicidal potential of N-substituted morpholine derivatives: Synthesis and structure-activity relationships

Article abstract of DOI:10.1080/14786410802090359

A series of N-substituted morpholines 2-20 was synthesised by reacting various acid chlorides and alkyl halides with morpholine (1). All of the synthesised compounds 2-20 were screened for their leishmanicidal effects using amphotericin B (IC50 = 0.24 μg L-1) and pentamidine (IC50 = 2.56 μg mL-1) as standards and a structure-activity relationship (SAR) study was established. The compounds 2 (IC50 = 48 μg mL-1), 3 (IC50 = 30.0 μg mL-1), 10 (IC50 = 41.0 μg mL-1), 15 (IC50 = 33.0 μg mL-1), 16 (IC50 = 35.0 μg mL-1) and 20 (IC50 = 47.0 μg mL-1) showed weak leishmanicidal activities.

Full text of DOI:10.1080/14786410802090359

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Natural Product Research: Formerly
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Leishmanicidal potential of N-
substituted morpholine derivatives:
Synthesis and structure–activity
relationships
a
a
Khalid Mohammed Khan , Muhammad Zarrar Khan , Muhammad
a
a
a
Taha , Ghulam Murtaza Maharvi , Zafar Saeed Saify , Shahnaz
b
a
Parveen & Muhammad Iqbal Choudhary
a
HEJ Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi , Karachi,
Pakistan
b
PCSIR Laboratories Complex , Karachi, Pakistan
Published online: 29 Oct 2009.
To cite this article: Khalid Mohammed Khan , Muhammad Zarrar Khan , Muhammad Taha , Ghulam
Murtaza Maharvi , Zafar Saeed Saify , Shahnaz Parveen & Muhammad Iqbal Choudhary (2009)
Leishmanicidal potential of N-substituted morpholine derivatives: Synthesis and structure–activity
relationships, Natural Product Research: Formerly Natural Product Letters, 23:5, 479-484, DOI:
10.1080/14786410802090359
To link to this article: http://dx.doi.org/10.1080/14786410802090359
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Natural Product Research
Vol. 23, No. 5, 20 March 2009, 479–484
Leishmanicidal potential of N-substituted morpholine derivatives: Synthesis
and structure–activity relationships
a
a
a
Khalid Mohammed Khan *, Muhammad Zarrar Khan , Muhammad Taha ,
a
Ghulam Murtaza Maharvi , Zafar Saeed Saify , Shahnaz Parveen and
Muhammad Iqbal Choudhary
a
b
a
a
HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan; PCSIR Laboratories Complex, Karachi, Pakistan
b
(
Received 26 June 2006; final version received 5 October 2007)
A series of N-substituted morpholines 2–20 was synthesised by reacting various
acid chlorides and alkyl halides with morpholine (1). All of the synthesised
compounds 2–20 were screened for their leishmanicidal effects using amphotericin
ꢀ
1
ꢀ1
B (IC50 ¼ 0.24 mg L ) and pentamidine (IC50 ¼ 2.56 mg mL ) as standards and
a structure–activity relationship (SAR) study was established. The compounds
2
ꢀ
1
1
ꢀ1
ꢀ1
(IC50 ¼ 48 mg mL ), 3 (IC50 ¼ 30.0 mg mL ), 10 (IC50 ¼ 41.0 mg mL ), 15
ꢀ ꢀ1 ꢀ1
(
showed weak leishmanicidal activities.
IC50 ¼ 33.0 mg mL ), 16 (IC50 ¼ 35.0 mg mL ) and 20 (IC50 ¼ 47.0 mg mL
)
Keywords: N-substituted morpholine; synthesis; anti-Leishmanial activity; SAR
1
. Introduction
Leishamaniasis is one of the major parasitic diseases, causes enormous suffering in
many parts of the tropical and subtropical regions of the world, and contributes to serious
health problems (World Health Organisation (WHO), 2001). This disease manifests in
different clinical forms, including cutaneous, mucocutaneous and visceral leishmaniasis.
The disease is caused by a parasitic protozoa (Kinetoplastida: Trypanosomatideae) (Olliaro
&
Bryceson, 1993; WHO, 2001), which is transmitted as metacyclic flagellated
promastigote forms from host to host by the bite of infected sand flies. This species has
been isolated from patients with visceral disease or with post-Kala Azar dermal
leishmaniasis (Barral et al., 1991; Leon, Machado, Carvalho-Paes, & Grimaldi, 1990).
Therapy of patients with leishmaniasis remains a serious problem. The drugs for the
treatment of all clinical forms of leishmaniasis are sodium stibogluconate (pentostam) and
meglumine antimonate (glucantime), although they exhibit renal and cardiac toxicity
(
Raht et al., 2003). Alternative drugs such as pentamidine, amphotericin B and some
azo-derivatives are also very toxic, with serious side effects (Mcgregor, 1998). Miltefosine,
a phosphocholine analogue, originally developed as an anticancer agent, has been found to
be highly effective against leishmaniasis in vitro and in vivo. Presently, this compound is the
*
Corresponding author. Email: hassaan2@super.net.pk
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802090359
http://www.informaworld.com

4
80
K.M. Khan et al.
only oral agent effective against both cutaneous (Soto et al., 2004) and visceral (Prasad,
Kumar, Jaiswal, & Singh, 2004) leishmaniasis, although causing severe gastrointestinal
problems (Sangraula, Sharma, Rijal, Dwivedi, & Koirala, 2003). Since the chemotherapy
against leishmaniasis is still inefficient and with toxic side effects, there is an urgent need
for the development of new, efficient and non-toxic drugs for the treatment of this disease
(
Carvalho, Arribas, & Ferreira, 2000).
In a continuation of research work on bioactive molecules, and as a part of a
programme to study potential leishmanicidal agents (Khan et al., 2003, 2008),
9 morpholine derivatives 2–20 (Scheme 1) were synthesised and randomly screened for
1
their leishmanicidal potential. These studies may provide lead compounds for future drug
development.
2
. Results and discussion
2
.1. Chemistry
A series of morpholine derivatives 2–20 (Scheme 1) were synthesised by treating various
acid chlorides with morpholine (1) in tetrahydrofuran at room temperature. The structures
of the resultant products 2–20 were determined spectroscopically, including UV, IR,
NMR, and MS. CHN analyses were found in good agreement with the calculated values.
2
.2. Biology
Several reports (Ram & Nath, 1996) describing the leishmanicidal activity of nitrogen-
containing compounds suggested random evaluation of the leishmanicidal potential of the
synthetic N-substituted morpholines in the present series. The testing was performed at
ꢀ
1
the 100 mg mL level according to a literature protocol (Atta-ur-Rahman, Choudhary, &
Thomsen, 2001) in duplicate. Amphotericin B and pentamidine were used as the positive
ꢀ
1
controls, having an IC50 of 0.24 and 2.56 mg mL , respectively.
All of the synthetic derivatives 2–20 of morpholine (1) were tested for their effects
against Lesihmania major and the results of the leishmanicidal activity are collected in
Table 1. The compounds 3 and 16 were found to be the most active compounds in the
ꢀ
1
present series, having IC50 values of 30.0 and 33.0 mg mL , respectively. Compounds 3,
1
3
0, 16, and 20, which were long, straight chain aliphatic amides, showed IC values of
50
ꢀ1
0.0, 41.0, 47.0, and 35.0 mg mL , respectively. However, the aliphatic amide 4 exhibited
1
ꢀ
an IC50 value of 80.0 mg mL . The difference in activities of compounds may be explained
on the basis of their structures and suggested that the IC50 values of these compounds
depends on the length of the carbon chain attached to the amide moiety. A nine-carbon
chain attached to the amide residue, as in compound 3, was found to be the most suitable
for leishmanicidal activity. The slight decline in activity of compound 20, having an IC
5
0
ꢀ
1
value of 47.0 mg mL , may be due to the reduction of one or two carbons in the carbon
chain. Unfortunately, a compound with 10 carbons could not be prepared, but compound
ꢀ
1
1
6 (IC ¼ 35.0 mg mL ), with an 11-carbon chain, showed a slight decline in activity,
5
0
demonstrating that either an increase or a decrease in carbon chain affects the
leishmanicidal activity. This hypothesis also establishes that the relative IC value of
5
0
ꢀ
1
compound 4 (IC ¼ 80.0 mg mL ), which has only one carbon attached to the amide
5
0
ꢀ
1
residue, with compound 15 (IC ¼ 35.0 mg mL ) may be explained on the basis of its
5
0
branched carbon chain. The aromatic amides 5, 6 and 9 showed very weak activity, which

Natural Product Research
481
THF
r.t.
O
NH
+
RCl
O
NR
1
2−20
Compounds
R
Yield (%)
87
Physical data
O
S
Off white solid; m.p. 160−162°C; Rf = 0.47
1:1, Ethyl acetate/n-hexane).
2
CH3
(
O
O
C
Colourless liquid; R = 0.57 (1:1, Ethyl
acetate/n-hexane).
CH3
72
81
f
3
4
O
Yellow liquid; R = 0.47 (1:1, Ethyl
f
C
CH3
CH3
OCH3
acetate/n-hexane).
O
C
Brown liquid; R = 0.47 (1:1, Ethyl
f
5
6
89
88
acetate/n-hexane).
O
C
Yellow liquid; R = 0.47 (1:1, Ethyl
OCH3
OCH3
f
acetate/n-hexane).
Colourless crystals; m.p. 150−152°C;
Rf = 0.42 (1:1, Ethyl acetate/n-hexane).
CH3
7
8
81
74
Yellow liquid; R = 0.47 (1:1, Ethyl
f
CH3
acetate/n-hexane).
O
C
Yellow liquid; R = 0.52 (1:1, Ethyl
f
9
80
77
86
acetate/n-hexane).
O
C
Yellow liquid; R = 0.56 (1:1, Ethyl
f
1
1
0
1
CH3
acetate/n-hexane).
Light yellow crystals; m.p. 170−172°C;
Rf = 0.48 (1:1, Ethyl acetate/n-hexane).
CH2
O
S
White powder; m.p. 155−157°C; R = 0.53
f
1
2
3
CH3
79
79
(1:1, Ethyl acetate/n-hexane).
O
CH3
CH3
Colourless liquid; R = 0.51 (1:1, Ethyl
CH2
f
1
acetate/n-hexane).
H C
3
O
Pale yellow crystals; m.p. 180−182°C;
Rf = 0.57 (1:1, Ethyl acetate/n-hexane).
S
CH3
86
79
1
4
5
O
3
H C
O
C
CH3
CH3
CH3
Brown liquid; R = 0.45 (1:1, Ethyl
f
1
acetate/n-hexane).
O
C
Colourless crystals; m.p. 163−165°C;
Rf = 0.45 (1:1, Ethyl acetate/n-hexane).
Colourless crystals; m.p. 187−189°C;
Rf = 0.54 (1:1, Ethyl acetate/n-hexane).
1
1
1
6
7
8
CH3
81
80
89
O
C
CH2
OC H
2 5
O
C
Yellow liquid; R = 0.46 (1:1, Ethyl
f
OCH3
CH3
acetate/n-hexane).
Yellow liquid; R = 0.46 (1:1, Ethyl
f
1
2
9
0
88
81
acetate/n-hexane).
Colourless crystals; m.p. 172−174°C;
Rf = 0.46 (1:1, Ethyl acetate/n-hexane).
O
C
CH3
Scheme 1. Synthetic route and physical data of compounds 2–20.
indicate that an aromatic linkage with an amide residue in this type of compound is not
desirable for leishmanicidal activity.
The sulphonamides 2, 12 and 14 were also evaluated for leishmanicidal activity,
ꢀ1
having IC50 values 48.0, 51.0 and 100.0 mg mL , respectively. The difference in activities

4
82
K.M. Khan et al.
Table 1. Leishmanicidal
compounds 2–20.
activity
of
ꢀ
1
Compounds
IC50 (mg mL )
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
48.0
30.0
80.0
83.0
100.0
100.0
80.0
100.0
41.0
100.0
51.0
100.0
100.0
35.0
1
1
1
1
1
1
1
1
1
1
2
35.0
100.0
100.0
100.0
47.0
Amphotericin B
Pentamidine
0.24
2.56
may again be explained on the basis of their structures. Compound 2, which was a
-methyl phenyl sulphonamide, was found to be the most active sulphonamide, whereas
4
methane sulphonamide showed a slight decline in activity, suggesting that aromatic
sulphonamides with suitable substitution may have better potential for leishmanicidal
activity as compared to aliphatic sulphonamides. The marked reduction in activity of
2,4,6-trimethylphenyl sulphonamide (14) may be explained on the basis of the steric
hindrances of the sulphonamide.
The compounds 7, 8, 11, 13 and 17–19, which were tertiary amine derivatives of
morpholine, showed only weak potential for leishmanicidal activity. Only compound 8
ꢀ1
ꢀ1
showed activity of 580.0 mg mL , while all other compounds showed 100.0 mg mL
The enhanced activity of compound 8 may be due to the presence of a small carbon
.
chain (ethyl) on the nitrogen atom of morpholine (1), while compounds 11, 13 and
17–19 showed a decrease in activity due to an unsuitable carbon chain attached to the
nitrogen atom.
In conclusion, aliphatic, straight carbon chain amides enhance the leishmanicidal
activity of morpholine (1), while an aliphatic, branched chain amide linkage at the
nitrogen atom of morpholine (1) also increased the leishmanicidal potential of
morpholine (1). For the sulphonamides, it was concluded that a suitable substitution
on an aromatic sulphonamide may enhance the activity of morpholine (1) and that a
small carbon chain sulphonamide of morpholine (1) might play an important role in
increasing leishmanicidal activity. Further research on the tertiary amides of compound
1 may also produce good results, either using aliphatic, straight carbon chains with some
functional groups like amidine, or hydrazino residues, which may enhance the activity,

Natural Product Research
483
as in the case of pentamidine and acyl hydrazides. This research suggested that
morpholine may act as possible lead compound for further research for potential
leishmanicidal agents.
3
. Experimental
3
.1. General
¨
Melting points were determined using a Buchi 434 melting point apparatus and are
uncorrected. NMR spectroscopy was performed on a Bruker AVANCE 400 MHz.
Elemental analyses were carried out on a Carlo Erba Strumentazion-Mod-1106, Italy.
UV and IR spectra were recorded on a Perkin-Elmer Lambda-5 UV/VIS and a JASCO
IR-A-302 spectrophotometer, respectively. A Finnigan MAT-311A (Germany) spectro-
meter was used to record mass spectra. Thin layer chromatography (TLC) was performed
on pre-coated silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany) and
chromatograms were visualised by UV light at 254 and 365 nm or iodine vapours.
3
.2. General procedure for the preparation of compounds 2–20
To a mixture of morpholine (1, 1 mmol) dissolved THF (10 mL) was added a mixture of
potassium carbonate (2 mmol), and the resultant mixture was stirred at room
temperature for 15 min and then various acid chlorides (1 mmol) were added. The
mixture was stirred for 48 h and the reaction was monitored by TLC analysis. On
completion, the solid was filtered with a sintered glass funnel. The filtrate was evaporated
on a rotary evaporator, the residue was dried on a two-stage pump (high vacuum) and
the resultant product, if solid, was recrystalised and dried in a desiccator, while the liquid
products were purified by vacuum distillation. The compounds were characterised using
different spectroscopic techniques, including UV, IR, NMR and MS, elemental analyses,
to determine the purity.
3
.3. Leishmanicidal bioassay (in vitro)
Leishmania major were grown in bulk in modified NNN biphasic medium by using normal
physiological saline. Leishmania promastigotes were cultured with RPMI 1640 medium,
supplemented with 10% heat-inactivated foetal bovine serum (FBS). Parasites at log phase
were centrifuged at 2000 rpm for 10 min, and washed three times with saline at the same
6
speed and time. Parasites were diluted with fresh culture medium to a final density of 10
cells per millilitre.
In a 96-well microtiter plate, 180 mL of medium was added in the first row and 100 mL
of medium was added in others wells. A total of 20 mL of the experimental compound was
added in medium and serially diluted. A total of 100 mL of the parasite culture was added
in all wells. Two rows were left for negative and positive controls. Negative controls
received only medium while the positive control contained varying concentrations of
standard anti-leishmanial compounds amphotericin B and petamidine. The plate was
ꢁ
incubated 21–22 C for 72 h. The culture was examined microscopically on an improved
Neubauer counting chamber and IC50 values of compounds were calculated by software
EZfit 5.03 (Perella Scientific). All assays were performed in duplicate (Atta-ur-Rahman,
Choudhary, & Thomsen, 2001).

4
84
K.M. Khan et al.
Acknowledgement
This work was financially supported by the Pakistan Telecommunication Company Limited (PTCL)
through a research project entitled, ‘‘Synthesis of Leishmanicidal Chemotherapeutic Agents’’.
References
Atta-ur-Rahman, Choudhary, M.I., & Thomsen, W.J. (2001). Bioassay techniques for drug
developement (pp. 60–64). The Netherlands: Harwood Academic Publisher.
Barral, A., Pedral-Sampaio, D., Grimaldi, G. Jr, Momen, H., McMahon-Pratt, D.,
Ribeiro de Jesus, A., et al. (1991). Leishmaniasis in Bahia, Brazil: Evidence that
Leishmania amazonensis produces a wide spectrum of clinical disease. The American Journal
of Tropical Medicine and Hygiene, 44, 536–546.
Carvalho, P.B., Arribas, M.A.G., & Ferreira, E.I. (2000). Leishmaniasis: What do we know about its
chemotherapy? Brazilian Journal of Pharmaceutical Science, 36, 69–96.
Khan, K.M., Ahmed, S., Khan, Z.A., Zia-Ullah, Rani, M.,Choudhary, M.I., et al. (2008). In vitro
leishmanicidal activity of 3-substituted isocoumarins: Synthesis and structure-activity
relationship. Medicinal Chemistry (Shariqah, United Arab Emirates), 4, 163–169.
Khan, K.M., Rasheed, M., Zia-Ullah, Hayat, S., Kaukab, F., Choudhary, M.I., et al. (2003).
Synthesis and in vitro leishmanicidal activity of some hydrazides and their analogues.
Bioorganic & Medicinal Chemistry, 11, 1381–1387.
Leon, L.L., Machado, G.M.C., Carvalho-Paes, L.E., & Grimaldi, G. Jr (1990). Antigenic differences
of Leishmania amazonensis isolates causing diffuse cutaneous leishmaniasis. Transactions of
the Royal Society of Tropical Medicine and Hygiene, 84, 678–681.
Mcgregor, A. (1998). WHO warns of epidemic leishmania. Lancet, 351, 575.
Olliaro, P.L., & Bryceson, A.D.M. (1993). Practical progress and new drugs for changing patterns of
leishmaniasis. Parasitol Today, 9, 323–328.
Prasad, R., Kumar, R., Jaiswal, B.P., & Singh, U.K. (2004). Miltefosine: An oral drug for visceral
leishmaniasis. Indian Journal of Pediatrics, 71, 143–144.
Raht, S., Trivellin, A., Imbrunito, T.R., Tomazela, D.M., Jesus, M.N., Marzal, P.C., et al. (2003).
Antimoniais empregados no tratamento da Leishmaniose: Estado de arte. Quı ´m ica Nova, 26,
550–557.
Ram, V.J., & Nath, M. (1996). For a review see: Progress in chemotherapy of leishmaniasis. Current
Medicinal Chemistry, 3, 303–316.
Sangraula, H., Sharma, K.K., Rijal, S., Dwivedi, S., & Koirala, S. (2003). Orally effective drugs for
kala-azar (visceral leishmaniasis): Focus on miltefosine and sitamaquine. The Journal of the
Association of Physicians of India, 51, 686–690.
Soto, J., Arana, B.A., Toledo, J., Rizzo, N., Vega, J.C., Diaz, A., et al. (2004). Miltefosine for new
world cutaneous leishmaniasis. Clinical Infectious Diseases: An Official Publication of the
Infectious Diseases Society of America, 38, 1266–1272.
World Health Organisation. (2001). Tropical Disease Research: Progress 1999–2000; Fifteenth
Programme Report of the UNDP/World Bank/WHO Special Programme for Research &
Training in Tropical Diseases. (TDR/GEN/01.5). Geneva: World Health Organization.