Synthesis and antifungal activity of substituted-10-methyl-1,2,3,4- tetrahydropyrazino[1,2-a]indoles

Article abstract of DOI:10.1016/j.bmc.2005.11.054

Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger (ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a-d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 μg/disc against A. fumigatus and 11.71 μg/disc against A. flavus and A. niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 μg/ml in case of A. fumigatus and 31.25 μg/ml with A. flavus and A. niger. The MIC₉₀ value of the most active compound by percent germination inhibition assay was found to be 15.62 μg/ml against A. fumigatus. The MIC₉₀ values of substituted-10-methyl-1,2,3,4- tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 μg/ml. The in vitro toxicity of the most active 1-(4-chlorophenyl)-10-methyl- 1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 μg/ml. The standard drug amphotericin B exhibited 100% lysis at a concentration of 37.5 μg/ml.

Full text of DOI:10.1016/j.bmc.2005.11.054

Bioorganic & Medicinal Chemistry 14 (2006) 2747–2752
Synthesis and antifungal activity of
substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles
Rakesh Kumar Tiwari,a Akhilesh K. Verma,a,* Anil K. Chhillar,b Devender Singh,a
Jaspal Singh,a V. Kasi Sankar,a Vibha Yadav,b G. L. Sharmab and Ramesh Chandraa,*
aSynthetic Organic Chemistry Research Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi,
Delhi 110007, India
bInstitute of Genomics and Integrative Biology, Mall Road, University Campus, Delhi 110007, India
Received 18 October 2005; revised 25 November 2005; accepted 29 November 2005
Available online 11 January 2006
Abstract—Series of substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles derivatives have been synthesized and examined
for their activity against pathogenic strains of Aspergillus fumigatus (ITCC 4517), Aspergillus flavus (ITCC 5192) Aspergillus niger
(ITCC 5405) and Candida albicans (ITCC No 4718). All synthesized compounds showed mild to moderate activity, except for
2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 6a–d. The most active 1-(4-chlorophenyl)-10-methyl-1,2,3,4-tetra-
hydropyrazino[1,2-a]indole 4c exhibited a MIC value of 5.85 lg/disc against A. fumigatus and 11.71 lg/disc against A. flavus and A.
niger in disc diffusion assay. Anti-Aspergillus activity of active compound 4c by microbroth dilution assay was found to be 15.62 lg/
ml in case of A. fumigatus and 31.25 lg/ml with A. flavus and A. niger. The MIC90 value of the most active compound by percent
germination inhibition assay was found to be 15.62 lg/ml against A. fumigatus. The MIC90 values of substituted-10-methyl-1,2,3,
4-tetrahydropyrazino[1,2-a]indoles against C. albicans ranged from 15.62 to 250 lg/ml. The in vitro toxicity of the most active 1-
(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was evaluated using haemolytic assay, in which the
compound was found to be non-toxic to human erythrocytes up to a concentration of 312.50 lg/ml. The standard drug amphoter-
icin B exhibited 100% lysis at a concentration of 37.5 lg/ml.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
spp., and increasing numbers with Cryptococcus spp.
or other fungi such as Fusarium spp.2,5
Invasive fungal infections, particularly in immunosup-
pressed patients, have continued to increase in incidence
during the past 20 years and are now significant causes
of morbidity and mortality.1 This is particularly true
in patients with haematological malignancies undergo-
ing induction or consolidation chemotherapy (especially
during the nadir of their granulocytopenia), in immuno-
suppressed organ transplant recipients and in patients
with acquired immunodeficiency secondary to infection
by human immunodeficiency viruses. These infections
also occur in some iatrogenic or nosocomial clinical set-
tings.2,3 Autopsy data indicate that more than half of
the patients who die with malignancies are infected with
Candida spp., approximately one-third with Aspergillus
Major factors which predispose patients to invasive
fungal diseases include: prolonged neutropenia (chemo-
therapy induced); defective T-lymphocyte function
(associated with organ transplantation and HIV infec-
tion); impaired macrophage function, particularly of
pulmonary macrophages (associated with high doses
and prolonged administration of corticosteroids); and
barrier defects (associated with invasive medical proce-
dures, vascular catheters, parenteral nutrition and hae-
modialysis and peritoneal dialysis) in compromised
patients.2–4,6–10
Although invasive fungal diseases are now more fre-
quent than during the first half of the century, they
are still difficult to diagnose clinically. During the latter
half of the century, particularly during the past two dec-
ades, a number of different classes of antifungal agents
have been discovered.2,3,6,10–13 Despite advances in anti-
fungal therapies, many problems remain to be solved for
Keywords: 1,2,3,4-Tetrahydropyrazino[1,2-a]indoles; Benzotriazole;
Antifungal agent; Aspergillus; Candida; Haemolytic activity.
*
Corresponding authors. E-mail: akvacbr@rediffmail.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.054

2748
R. K. Tiwari et al. / Bioorg. Med. Chem. 14 (2006) 2747–2752
most antifungal drugs available. For example, the use of
amphotericin B, known as the Ôgold standardÕ, is limited
because of its infusion-related reactions and nephrotox-
icity.14,15 The use of azoles, such as fluconazole, keto-
conazole and miconazole, has resulted in clinically
resistant strains of Candida spp.16,17 A 3.6–7.2% of vag-
inal isolates of Candida albicans from women with can-
didal vaginitis is resistant to fluconazole.18 This
situation highlights the need for advent of safe, novel
and effective antifungal compounds.
2. Results and discussion
2.1. Chemistry
2-(3-Methyl-1H-indol-1-yl)ethylamine 2 was obtained
by the reaction of 3-methylindole 1 with 2-chloroethyl-
amine hydrochloride in CH3CN in the presence of
NaOH and tetrabutylammonium hydrogen sulfate
(TBAHS).32 Condensation of 2-(3-methyl-1H-indol-1-
yl)ethylamine 2 with 1 equiv of benzotriazole (BtH)
and 2 equiv of formaldehyde (37% aqueous solution)
in MeOH/H2O at 25 ꢁC gave 2-(1H-1,2,3-benzotriazol-
1-ylmethyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,
2-a]indole 5 in 96% yield as Bt1 (benzotriazol-1-yl) iso-
mers.32 Bt group attached at an a-carbon to a nitrogen
atom is easily replaced by nucleophiles. Treatment of 5
with 2 equiv of sodium borohydride in refluxing THF
replaced the benzotriazolyl group with hydrogen to give
2,10-dimethyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 8
in 90% yield. The Bt group in 5 can be substituted by
a cyano anion to afford 2-[10-methyl-3,4-dihydropyrazi-
Pyrazino[1,2-a]indoles have attracted a great deal of
attention due to their therapeutic uses as serotonin
antagonist,19 thrombolytic,20 cardiovascular diseases,21
antidepressant, anxiolitics,22 central nervous system
depressants,23 anticonvulsants,24 antihistaminic,25 pro-
27
27,28
tein kinase C inhibitors26 and 5-HT2A
,
5-HT2C,
and selective imidazoline I2 receptor ligands.29 One re-
port shows the activity of pyrazinoate towards resistant
Mycobacterium tuberculosis.30 Some triazino[5,6-b]-
indoles are reported to have antifungal properties.31
However, the potential of pyrazino[1,2-a]indoles as anti-
fungal agents has not been studied. Increases in the inci-
dence of fungal infections have prompted a search for
new antifungal agents with broad antifungal activities
and fungicidal actions, a low likelihood of resistance
development and minimal toxicity. Therefore, the
current study was undertaken to synthesize different
substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indole 4–8 derivatives and investigate their antifungal
potential using Aspergilli and Candida species as model
pathogens.
no[1,2-a]indol-2(1H)-yl]acetonitrile
7 in 96% yield.
Nucleophilic substitution of 5 with p-tolyl, 1-propynl,
n-pentyl and isopropylmagnesium bromides in dry
THF furnished the 2-substituted-10-methyl-1,2,3,4-tetr-
ahydropyrazino[1,2-a]indoles 6a–d in 90–96% yields.
1-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indoles 4a–j were obtained as a racemic mixture in
high yields by the reaction of 2-(3-methyl-1H-indol-1-
yl)ethylamine 2 with benzotriazole and aldehydes 3a–j
in the presence of catalytic amount of AlCl3 in CH2Cl2
(Scheme 1).32 The structures of all the synthesized
CH3
CH3
CH3
R
b
R
a
RCHO
N
N
C6H5
NH
4a
4b
4c
4d
4e
4f
4g
4h
4i
N
H
NH2
p-BrC6H4
p-ClC6H4
p-FC6H4
p-CH3C6H4
p-NO2C6H4
m-CH3OC6H4
2-pyridyl
4a-j
2
3a-i
1
c
CH3
2-thienyl
m-NO2C6H4
CH3
f
CH3
d
4j
N
N
N
N
N
CH3
N
R1
Bt
6a-d
5
8
R1
e
p-CH3C6H4
n-C5H11
iso-C3H7
H3C
6a
6b
6c
6d
N
BtH =
CH3
N
N
H
N
N
CN
7
Scheme 1. Reagents and conditions: (a) ClCH2CH2NH2HCl, NaOH, TBAHS, CH3CN, reflux, 36 h; (b) BtH, DCM, catalytic AlCl3, 25–28 ꢁC,
1–6 h; (c) BtH, HCHO (2 equiv), 25–28 ꢁC stirring; (d) R1MgX, THF, reflux, 4–12 h; (e) NaCN, DMSO, 25 ꢁC, 36 h; (f) NaBH4, THF, reflux, 12 h.

R. K. Tiwari et al. / Bioorg. Med. Chem. 14 (2006) 2747–2752
2749
compound were clearly supported by their spectral
analyses.32 The structures of new compounds are clearly
pound 4f showed moderate activity against A. flavus
and A. niger in the DDA assay. All compounds
showed mild to moderate activity against the patho-
genic strains used in the study, except for 6a–d.
However, pyrazino[1,2-a]indoles have not been investi-
gated. We, for the first time, synthesized substituted-
10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles for
their antimycobial properties. The activity of the com-
pound appeared to be associated with the 1 or 2
substitution on the 1,2,3,4-tetrahydropyrazino[1,2-a]in-
doles. The presence of NH group with substitution on
1-position of the compound 4a–j showed better activ-
ity than compound 5–8 with substituents present on
2-position. The compound 4b–d showed potential
activity due the presence of 4-fluorophenyl, 4-chlor-
ophenyl and 4-bromophenyl at 1-position. Enhanced
activity of l-substituted-10-methyl-1,2,3,4-tetrahydro-
pyrazino[1,2-a]indoles 4a–j as compared to that of
2-substituted-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indoles, 5–8, was noticed due to the presence of
hydrogen atom on the pyrazino nitrogen atom which
might be important to exhibit inhibitory activity.
1
supported by their H, 13C NMR spectra and micro-
analysis. The H NMR spectra showed NCH-pyrazino
1
singlet signal for 4a–j at ꢀ5.3 ppm and the presence of
exchangeable NH pyrazino was confirmed by deuterium
exchange.
2.2. Antifungal activity
Series of substituted-10-methyl-1,2,3,4-tetrahydropyraz-
ino[1,2-a]indoles 4a–j and 5–8 have been examined for
activity against pathogenic strains of A. fumigatus, A.
flavus, A. niger and C. albicans. The anti-Aspergillus
activity of all the synthesized compounds was evaluated
by disc diffusion (DDA), microbroth dilution (MDA)
and percentage spore germination inhibition (PSGI) as-
says;33 the results are given in Table 1. The anti-Candida
activity of all the synthesized compounds 4a–j and 5–8
has been investigated by microbroth dilution assay36
and the results are given in Table 1.
2.2.1. Anti-Aspergillus activity. The results of anti-
Aspergillus activity evaluation revealed that one of
the 1,2,3,4-tetrahydropyrazino[1,2-a]indole, that is, 1-
(4-chlorophenyl)-10-methyl-1,2,3,4-tetrahydropyrazino-
[1,2-a]indole (4c) is a potential inhibitor of the growth
of A. fumigatus, A. flavus and A. niger. The com-
pound 4c exhibited appreciable activity even in the
range of 5.85–11.71 lg/disc in disc diffusion and
15.62–31.25 lg/ml in microbroth dilution assays (Table
1). The MIC90 value of 4c by percentage spore germi-
nation inhibition assay was found to be in the range
of 15.62–31.25 lg/ml (Table 1). Significant activity
was observed with compounds 4b and d which exhib-
ited MIC value in the range of 23.43–46.87 lg/disc in
DDA and 62.50–125.05 lg/ml by MDA and PSGI as-
say, respectively. Compound 4h show good activity
against A. fumigatus with MIC 93.75 lg/disc and com-
2.2.2. Anti-Candidal activity. The results of anti-Candi-
da activity showed that all the synthesized compounds
had variable activity against the pathogenic strains of
C. albicans but no activity was found in compounds
6a–d. The most active compound 1-(4-chlorophenyl)-
10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole
4c
was a potential inhibitor of the growth of C. albicans
with a MIC90 value of 15.62 lg/disc by microbroth
dilution assay. The significant activity was found for
compounds 4b and d, which exhibited activity at
62.50 and 125.0 lg/ml, respectively, in microbroth
dilution assay. The only difference in the structures
of compounds 4b–d is the presence of a halogen group
at C-4 position of the 1-substituted phenyl group.
However, 4-chlorophenyl derivative 4c was found to
be the most active compound among all the halogen
Table 1. In vitro antifungal activity against Aspergilli and Candida albicans
Compound
DDA MIC (lg/disc)
MDA MIC (lg/ml)
PSGI MIC90 (lg/ml)
MDA MIC90
(lg/ml)
A. flavus A. fumigatus A. niger A. flavus A. fumigatus A. niger A. flavus A. fumigatus A. niger C. albicans
4a
4b
4c
4d
4e
4f
—
—
—
500.0
125.0
31.25
125.0
500.0
500.0
500.0
500.0
500.0
500.0
500.0
—
500.0
125.0
15.62
62.50
500.0
250.0
500.0
250.0
500.0
500.0
500.0
—
1000.0
500.0
62.50 125.0
500.0
62.50
15.62
62.50
500.0
250.0
500.0
125.0
500.0
250.0
500.0
—
500.0
62.50
15.62
62.50
500.0
250.0
500.0
250.0
500.0
500.0
250.0
—
125.0
62.50
15.62
125.0
250.0
250.0
250.0
125.0
250.0
250.0
250.0
—
46.87
11.71
46.87
23.43
5.85
46.87
46.87
11.71
46.87
31.25
125.0
1000.0
500.0
500.0
500.0
500.0
500.0
500.0
—
31.25
125.0
500.0
500.0
500.0
250.0
500.0
500.0
500.0
—
—
187.5
—
—
187.5
—
—
—
4g
4h
4i
—
187.5
—
—
93.75
—
—
187.5
—
—
4j
5
—
—
—
—
—
—
6a
6b
6c
6d
7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
187.5
—
—
—
—
—
—
—
—
—
—
250.0
—
500.0
500.0
1.95
250.0
500.0
1.95
500.0
500.0
1.95
500.0
500.0
1.95
250.0
250.0
1.95
250.0
500.0
1.95
8
AmpB
—
—
0.73
0.73
0.73
0.97
The Ô—Õ means no Activity.

2750
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R. K. Tiwari et al. / Bioorg. Med. Chem. 14 (2006) 2747–2752
4. Experimental
100
80
60
40
20
0
4.1. General
4c
AmpB
All reagents used were of AR grade. THF was dis-
tilled from sodium/benzophenone prior to use. Melt-
ing points were determined with a Thomas Hoover
melting point apparatus and are uncorrected. 1H
(300 MHz) and 13C NMR (75 MHz) spectra were
recorded on a Bruker 300 NMR spectrometer in
CDCl3 (with TMS for 1H and chloroform-d for
13C as internal references) unless otherwise stated.
Mass spectrum was recorded on a Hybrid Quadru-
pole-TOF LCnMSnMS mass spectrometer (Q. Star
XL). Infrared spectra (mmax) were recorded on a Per-
kin Elmer FTIR spectrophotometer as thin films on
KBr plates (for oils) or KBr discs (for solids). Col-
umn chromatography was performed on silica gel
(230–400 mesh). Microanalyses were obtained with
an Elemental Analysensysteme GmbH VarioEL
V3.00 element analyser. The reactions were moni-
tored by thin-layer chromatography (TLC) using
aluminium sheets with silica gel 60 F254 (Merck).
All the reactions were carried out under nitrogen
atmosphere.
0
1
2
3
4
5
Log concentration (µg/ml)
Figure 1. The cytotoxicity of 1-(4-chlorophenyl)-10-methyl-1,2,3,4-
tetrahydropyrazino[1,2-a]indole (4c) analysed by haemolytic assay.
derivatives 4b–d. Mild to moderate activity was shown
by the 2-substituted-10-methyl-1,2,3,4-tetrahydropyraz-
ino[1,2-a]indoles 5–8 against the C. albicans strain,
except for compounds 6a–d. (10-Methyl-3,4-dihydro-
1H-pyrazino[1,2-a]indol-2-yl)-acetonitrile 7 and 2-ben-
zotriazol-1-ylmethyl-10-methyl-1,2,3,4-tetrahydropyraz-
ino[1,2-a]indole 5 showed moderate activity. Activity
data of all the compounds listed in Table 1 revealed
that the presence of a hydrogen atom on the pyrazino
nitrogen atom showed potent antifungal activity. Pres-
ence of alkyl, alkynyl and p-tolyl groups in com-
pounds 6a–d at 2-position of compounds does not
cause any significant effect on the antifungal activity.
4.2. Materials
2.2.3. Cytotoxicity study of 1-(4-chlorophenyl)-10-meth-
yl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c. The in vitro
cell cytotoxicity of 1-(4-chlorophenyl)-10-methyl-
1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c was investi-
gated using haemolytic assay.35 In a dose-dependent
study, compound 4c was found to be non-toxic up
to a concentration of 312.50 lg/ml and lysed only
4.5% of human erythrocytes (Fig. 1). The standard
drug amphotericin B (AmpB) exhibited 100% lysis at
a concentration of 37.5 lg/ml. However, it was found
to be much less toxic than the standard drug ampho-
tericin B up to the tested concentration, that is,
10,000.0 lg/ml and lysed only 43.75% of human
erythrocytes.
SabouraudÕs dextrose agar and SabouraudÕs dextrose
broth were purchased from Hi Media, Mumbai, India.
Amphotericin B and DMSO were purchased from
Sigma Chemical Company, USA.
4.3. Pathogens
Pathogenic strain of A. fumigatus, A. flavus and A. niger
were obtained from the Microbiology Department of
Vallabhbhai Patel Chest Institute; Delhi, India and C.
albicans was obtained from IARI, New Delhi, India.
A. fumigatus, A. flavus, A niger and C. albicans were
grown on SabouraudÕs dextrose agar at 37 ꢁC.
4.4. General method of synthesis for the 1-substituted-10-
methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 4a–j
3. Conclusion
To a mixture of 2-(3-methyl-1H-indol-1-yl) ethylamine
(2, 0.5 g, 2.9 mmol), an aldehyde (3a–j) (2.9 mmol),
benzotriazole (0.342 g, 2.9 mmol), in the presence of
catalytic amount of Lewis acid (AlCl3) in dichloro-
methane (20 ml) was stirred at 25 ꢁC for 0.5–8.0 h
(completion of the reaction was monitored by
TLC).32 The reaction mixture was quenched with
water and extracted with ethyl acetate (3· 100 ml).
The combined extract was washed with 1 N NaOH,
brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to give solid compounds 4a–j
in 70–90% yields. For microanalysis, compounds were
recrystallized from ethyl acetate/hexanes. The struc-
tures of 4a–f and h,i were unambiguously established
on the basis of their spectral analysis (IR, 1H, 13C
and mass) and comparison of their melting points
and/or spectral data with those reported in the
literature.32
Series of substituted-10-methyl-1,2,3,4-tetrahydropy-
razino[1,2-a]indoles has been synthesized and the
antifungal activity evaluation studies on 1-substitut-
ed-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles
4a–j and 2-substituted-10-methyl-1,2,3,4-tetrahydropy-
razino[1,2-a]indoles 5–8 have revealed that 1-(4-chloro-
phenyl)-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-a]in-
dole 4c is a potent antifungal agent. Although the
potency of compound 4c was less than that of
the standard antifungal compound amphotericin B,
the toxicity of this active pyrazino[1,2-a]indole deriv-
ative was much less than that of the standard drug
amphotericin B. This shows that pyrazino[1,2-a]in-
dole derivatives are safe drug candidates and are
taken up for the development of safer antifungal
drugs through generation of a library of analogues
of 4c and for further studies.

R. K. Tiwari et al. / Bioorg. Med. Chem. 14 (2006) 2747–2752
2751
4.4.1. 1-(3-Methoxyphenyl)-10-methyl-1,2,3,4-tetrahyd-
5. Antifungal activity assay
ropyrazino[1,2-a]indole 4g. Colourless needless; mp
108–111 ꢁC; Found: C, 77.94; H, 6.98; N, 9.75.
C19H20N2O requires C, 78.05; H, 6.89; N, 9.58; mmax
(KBr) 3311, 3048, 3041, 2960, 2921, 2870, 753,
677 cmꢁ1; dH (300 MHz, CDCl3) 7.52–6.50 (8H, m,
Ph), 5.34 (1H, s), 4.08–3.91 (2H, m), 3.85 (3H, s,
OCH3), 3.21–3.12 (2H, m), 2.42 (1H, s, NH); 2.39
(3H, s, CH3); dC (75 MHz, CDCl3) 8.6, 40.2, 42.4,
56.2, 58.5, 106.2, 108.7, 118.5, 120.0, 121.2, 125.2,
129.0, 129.2, 130.2, 132.5, 135.4, 141.2, 143.5, 147.5;
LCMS m/z 291 (100%, Mꢁ1), 293.4 (20%, M+1),
292.2 (8%, M).
The anti-Aspergillus activity of all the compounds was
studied by disc diffusion, microbroth dilution and per-
centage spore germination inhibition assays.33 The
activity against C. albicans was carried out by the meth-
ods of Iijima et al.36 and Chhillar et al.34
6. Anti-Aspergillus activity assay
6.1. Disc diffusion
The disc diffusion assay was performed in radiation-
sterilized petri plates of 10.0 cm diameter (Tarsons) as
described.33 Different concentrations in the range of
750–1.46 lg of the test compounds were impregnated
on the sterilized discs (5.0 mm in diameter). The discs
were placed on the surface of the agar plates already
inoculated with A. fumigatus spores. The plates were
incubated at 37 ꢁC and examined at 24, 48 and 96 h
for zone of inhibition, if any, around the discs.
4.4.2. 10-Methyl-1-(3-nitrophenyl)-1,2,3,4-tetrahydropy-
razino[1,2-a]indole 4j. (Yellow crystals from EtOAc/
hexanes); mp 121–123 ꢁC; Found: C, 70.31; H, 5.56;
N, 13.45. C18H17N3O2 requires C, 70.34; H, 5.58; N,
13.67; mmax (KBr) 3321, 3050, 3042, 2955, 2922,
2876, 1520, 1360, 750, 680 cmꢁ1
. dH (300 MHz,
CDCl3) 7.61–6.93 (8H, m, Ph); 5.30 (1H, s), 4.18–
3.94 (2H, m), 3.25–3.12 (2H, m), 2.65 (3H, s, CH3),
1.72 (1H, s, NH); dC (75 MHz, CDCl3) 8.6, 40.8,
42.5, 56.5, 109.2, 111.5, 118.0, 118.9, 120.0, 127.5,
128.5, 129.5, 132.5, 132.8, 139.5, 137.0, 138.5, 142.5;
LCMS m/z 307 (80%, M), 308 (20%, M+1), 185.2
(14%, Mꢁ122).
6.2. Microbroth dilution
The test was performed in 96-well culture plates (Nunc,
Nunclon). Various concentrations of synthetic com-
pounds in the range of 1000–7.81 lg/ml were prepared
in the wells by twofold dilution method. Assay was per-
formed as per the standard method described earlier.33
4.5. General method for the synthesis of 2-substituted–10-
methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indoles 5–8
The 2-substituted-10-methyl-1,2,3,4-tetrahydropyrazi-
no[1,2-a]indoles 5–8 were synthesized by the literature
procedure32 and the structures of 5–8 were unambigu-
ously established on the basis of their spectral analysis
(IR, 1H, 13C and mass) and comparison of their melting
points and/or spectral data with those reported in the
literature.32
6.3. Percentage spore germination inhibition
Different concentrations of the test compounds in
90.0 ll of culture medium were prepared in 96-well flat
bottomed microculture plates (Nunc, Nunclon) by dou-
ble dilution method. The wells were prepared in tripli-
cate for each concentration. Each well was then
inoculated with 10.0 ll of spore suspension containing
100 5 spores. The plates were incubated at 37 ꢁC for
16 h and then examined for spore germination with an
inverted microscope (Nikon, Diphot). The number of
germinated and non-germinated spores was counted.
The percentage spore germination inhibition was calcu-
lated.33 All the tests were repeated at least three times.
The lowest concentration of the compound, which
resulted in >90% inhibition of germination of spores
in the wells, was considered as MIC90.
4.5.1. 2-Hexyl-10-methyl-1,2,3,4-tetrahydropyrazino[1,2-
a]indole 6b. Colourless oil; mmax (KBr) 3324, 3053,
3046, 2952, 2910, 2865, 759, 680 cmꢁ1
;
dH
(300 MHz, CDCl3) 7.41 (1H, d, J = 7.2 Hz), 7.13
(1H, d, J = 7.4 Hz), 7.07–6.98 (2H, m), 3.95 (2H, t,
J = 5.3 Hz), 3.65 (2H, s), 2.83 (2H, t, J = 5.3 Hz),
2.48 (2H, t, J = 7.4 Hz), 2.12 (3H, s, CH3), 1.52–
1.18 (8H, m), 0.82 (3H, t, J = 6.6 Hz, Me); dC
(75 MHz, CDCl3) 8.0, 14.2, 22.5, 28.0, 31.0, 31.5,
42.0, 46.5, 53.2, 54.5, 105.2, 108.1, 118.2, 119.2,
120.7, 128.9, 131.2, 136.2; TOF-MS ES m/z: 271.41
(M+1).
7. Anti-Candidal activity assay
4.5.2.
2-Isobutyl-10-methyl-1,2,3,4-tetrahydropyrazi-
The activity against C. albicans was carried out by the
methods of Iijima et al.36 and Chhillar et al.34 C. albi-
cans cells in the exponential phase of growth were sus-
pended in SabouraudÕs dextrose medium at a density
of 1.6 · 103 cells/ml and a volume of 100.0 ll of the
suspension was inoculated into each well of a 96-well
microtitre plate with 100.0 ll of test compound solu-
tion. After incubation at 37 ꢁC for 12–16 h, the OD
was measured at A650 nm of the suspension to assess
the inhibition of cell growth due to treatment with
compounds.
no[1,2-a]indole 6c. Colourless oil; mmax (KBr) 3324,
3059, 3034, 2951, 2920, 2846, 757, 689 cmꢁ1; dH
(300 MHz, CDCl3) 7.39 (1H, d, J = 7.1 Hz), 7.16 (1H,
d, J = 7.6 Hz), 7.09–7.01 (2H, m), 4.10 (2H, t,
J = 5.3 Hz), 3.78 (2H, s), 2.92 (2H, t, J = 6.2 Hz), 2.52
(2H, d, J = 7.8 Hz), 2.14 (3H, s, CH3), 2.04–2.01 (1H,
m), 1.21 (6H, d, J = 6.9 Hz); dC (75 MHz, CDCl3) 8.0,
22.0 · 2, 30.1, 46.5, 53.2, 54.2, 65.5, 105.6, 109.2,
118.7, 119.5, 122.1, 128.7, 130.9, 136.1; TOF-MS ES
m/z: 243.4 (M+1).

2752
R. K. Tiwari et al. / Bioorg. Med. Chem. 14 (2006) 2747–2752
13. Current, W. L.; Tang, J.; Boylan, C.; Watson, P.; Zeckner,
8. Haemolytic assay
W.; Turner, W. In Antifungal Agents: Discovery and Mode;
Dixon, G. K., Copping, L. G., Hollomon, D. W., Eds.;
BIOS: Oxford, 1995; pp 143–160.
The toxicity of pyrazino indole derivatives having an anti-
fungal potential was investigated by using haemolytic as-
say.35 A slight modification37 was employed to determine
the haemolytic effect of antifungal 1-(4-chlorophenyl)-10-
methyl-1,2,3,4-tetrahydropyrazino[1,2-a]indole 4c deriv-
ative. Human erythrocytes collected from apparently
healthy volunteers were washed thrice with PBS and
2.0% (v/v) suspension of erythrocytes was prepared in
phosphate-buffered saline at pH 7.2. Half millilitre of
(2.0%) human erythrocyte suspension in 16 duplicate sets
of tubes was treated with compound 4c at a concentration
of 1.22 lg/ml for 1 h at 37 ꢁC. After incubation, tubes
were centrifuged at 5000 rpm for 10 min. The supernatant
was collected and the OD was measured at A415 nm using
a spectrophotometer (UV Vis Spect Lambda Bio 20 Per-
kin Elmer). Results were expressed as percentage haemol-
ysis by the compound. Only a buffer of pH 7.2 was used
for background lysis in negative control sets, whereas in
positive controls, lysis buffer was used for completely lys-
ing the erythrocytes.
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Acknowledgments
We gratefully acknowledge the financial support from
the Department of Science and Technology, New Delhi.
R.K. thanks Jean and Ashit Ganguly Trust for JRF and
J.S. is thankful to CSIR for JRF.
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