Synthesis and Biological Evaluation of Tricyclic Guanidine Analogues of Batzelladine K for Antimalarial, Antileishmanial, Antibacterial, Antifungal, and Anti-HIV Activities

Article abstract of DOI:10.1111/cbdd.1427

Fifty analogues of batzelladine K were synthesized and evaluated for in vitro antimalarial (Plasmodium falciparum), antileishmanial (Leishmania donovani), antimicrobial (panel of bacteria and fungi), antiviral (HIV-1) activities. Analogues 14h and 20l exhibited potential antimalarial activity against chloroquine-sensitive D6 strain with IC₅₀ 1.25 and 0.88μm and chloroquine-resistant W2 strain with IC₅₀ 1.64 and 1.07μm, respectively. Analogues 12c and 14c having nonyl substitution showed the most potent antileishmanial activity with IC₅₀ 2.39 and 2.78μm and IC₉₀ 11.27 and 12.76μm, respectively. Three analogues 12c, 14c, and 14i were the most active against various pathogenic bacteria and fungi with IC₅₀<3.02μm and MIC/MBC/MFC <6μm. Analogue 20l having pentyl and methyl substituents on tricycle showed promising activities against all pathogens. However, none was found active against HIV-1. Our study demonstrated that the tricyclic guanidine compounds provide new structural class for broad spectrum activity. Fifty analogues of tricyclic guanidine derivative of batzelladine K were synthesized and tested for antimalarial, antileishmanial, antimicrobial, antifungal and anti-HIV activities. We have identified several active analogues.

Full text of DOI:10.1111/cbdd.1427

Chem Biol Drug Des 2013; 81: 491–498
Research Article
Synthesis and Biological Evaluation of Tricyclic
Guanidine Analogues of Batzelladine K for
Antimalarial, Antileishmanial, Antibacterial, Antifungal,
and Anti-HIV Activities
Nafees Ahmed1,6, Keyur G. Brahmbhatt1,
Shabana I. Khan2,3, Melissa Jacob2, Babu L.
Tekwani2,4, Sudeep Sabde5, Debashis Mitra5,
Inder P. Singh1, Ikhlas A. Khan2,3 and Kamlesh
K. Bhutani1,*
Received
3 December 2012, revised 7 April 2012 and
accepted for publication 5 June 2012
Among the parasitic diseases, leishmaniasis, and malaria
have a high mortality rate affecting millions of people
worldwide, particularly in developing countries. Malaria
ranks third among the major infectious diseases in causing
deaths after pneumococcal acute respiratory infections
and tuberculosis. Approximately, 1.5–2.5 million people die
of malaria every year, accounting for about 5% of all fatali-
ties in the world (1). Leishmaniasis caused by protozoa of
the genus Leishmania remains a significant health issue in
large part because of the lack of effective and affordable
drugs and increasing resistance against existing drugs.
More than 2 million new cases of leishmaniasis occur each
year, whereas 15 million people are infected (2).a
1Department of Natural Products, National Institute of
Pharmaceutical Education and Research (NIPER), Sector
67, S.A.S. Nagar, Mohali, Punjab 160062, India
2National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, Oxford, MS 38677,
USA
3Department of Pharmacognosy, School of Pharmacy,
University of Mississippi, Oxford, MS 38677, USA
4Department of Pharmacology, School of Pharmacy,
University of Mississippi, MS 38677, USA
5National Center for Cell Science (NCCS), Pune University
Campus, Ganeshkhind, Pune, Maharashtra 411007, India
6Jeffrey Cheah School of Medicine and Health Sciences,
MONASH University Sunway Campus, Bandar Sunway,
Selangor Darul Ehsan 46150, Malaysia
HIV and parasitic infections interact and affect each other
mutually. HIV infection may alter the natural history of
parasitic diseases, impede rapid diagnosis or reduce the
efficacy of antiparasitic treatment, whereas parasitoses may
facilitate the infection with HIV and the progression from
asymptomatic infection to AIDS. Recently, cause-effect
interactions between HIV and malaria were also shown to
occur in pregnant women (3). Leishmania/HIV co-infection
has emerged as a result of the increasing overlap of areas in
which HIV or leishmaniasis occur, particularly in eastern
Africa, India, Brazil, and Europe.a Multiple immunologic
mechanisms mediate the impact of HIV infection on visceral
leishmaniasis (VL) and vice versa. Both pathogens infect
*Corresponding author: Kamlesh K. Bhutani,
kkbhutani@niper.ac.in
Fifty analogues of batzelladine K were synthesized and
evaluated for in vitro antimalarial (Plasmodium falcipa-
rum), antileishmanial (Leishmania donovani), antimicro-
bial (panel of bacteria and fungi), antiviral (HIV-1)
activities. Analogues 14h and 20l exhibited potential
antimalarial activity against chloroquine-sensitive D6
strain with IC50 1.25 and 0.88 lM and chloroquine-
resistant W2 strain with IC50 1.64 and 1.07 lM,
respectively. Analogues 12c and 14c having nonyl sub-
stitution showed the most potent antileishmanial activ-
ity with IC50 2.39 and 2.78 lM and IC90 11.27 and
12.76 lM, respectively. Three analogues 12c, 14c, and
14i were the most active against various pathogenic
monocytes/macrophages and may establish
a latent
infection or may accelerate their intracellular multiplication.
An increasing number of multidrug-resistant microbial
pathogens have become a serious problem, particularly
during the last decade (4,5). Consequently, these conditions
demand the rapid search and discovery of new broad spec-
trum antiparasitic agents with novel structural backbone.
bacteria and fungi with IC50
<
3.02 lM and MIC/
MBC/MFC <6 lM. Analogue 20l having pentyl and
methyl substituents on tricycle showed promising
activities against all pathogens. However, none was
found active against HIV-1. Our study demonstrated
that the tricyclic guanidine compounds provide new
structural class for broad spectrum activity.
The batzelladines, a class of polycyclic marine alkaloids
containing a guanidine group, have been isolated from var-
ious Batzella species. Batzelladines A-N, isolated from var-
ious sponges of the genus Batzella are of biological
interests (6–10). Batzelladines A and B were found to inhi-
Key words: anti-HIV, antileishmanial, antimalarial, antimicro-
bial, batzelladine, tricyclic guanidine
ª 2012 John Wiley & Sons A/S. doi: 10.1111/j.1747-0285.2012.01427.x
491

Ahmed et al.
roquine-resistant (W2) clones of Plasmodium falciparum
based on the determination of plasmodial LDH activity
(13). All the analogues were evaluated for in vitro cytotoxic-
ity against mammalian kidney cell line (Vero) up to a high-
est concentration of 4.76 lg/mL by neutral red assay
(14,15). Selectivity index was calculated for all analogues
(Data not shown). S.I. is calculated as the ratio of IC50 for
cytotoxicity and IC50 for antimalarial activity and measures
the therapeutic index of the compound under investigation
to malaria parasites in comparison to its toxicity to the
mammalian cells (if there is any).
Antileishmanial activity
The antileishmanial activity of all analogues was evaluated
in vitro against Leishmania donovani promastigotes by Ala-
mar Blue assay (16,17). The activity is reported in terms of
IC50 and IC90 values. Pentamidine and Amphotericin B are
used as standards.
Figure 1: Selected batzelladine alkaloids.
bit the binding of HIV glycoprotein gp-120 to CD4 recep-
tors (6). Batzelladines F, G, and a mixture of H and I were
active in the p56lck-CD4 dissociation assay (8). Few
selected batzelladine alkaloids are shown in Figure 1. It
has been identified that the tricyclic core of batzelladines
is essential for anti-HIV activity (11). However, antiparasitic
potential of this class of compounds has never been
explored which led us to synthesize a series of com-
pounds with tricyclic core of batzelladines. Total synthesis
of complex batzelladines with many numbers of stereocen-
tres is one of the challenging endeavors for synthetic
chemists. We have previously reported the total synthesis
of batzelladine K using a biomimetic approach (12). We
report herein, the synthesis of tricyclic guanidine analogues
of batzelladines and evaluation of their in vitro antimalarial,
antileishmanial, antimicrobial, and anti-HIV activities.
Antimicrobial activity
Synthesized analogues were evaluated for their antibacterial
properties against Staphylococcus aureus ATCC 29213,
Methicillin-resistant S. aureus ATCC 33591 (MRSA), Escher-
ichia coli ATCC 35218, Pseudomonas aeruginosa ATCC
27853, and Mycobacterium intracellulare ATCC 23068.
Susceptibility testing was performed using a modified ver-
sion of the CLSI (formerly NCCLS) methods (18–22). Myco-
bacterium intracellulare was tested using a modified method
of Franzblau et al. (23). Ciprofloxacin was used as standard.
Antifungal activity
The antifungal activities of all analogues against the oppor-
tunistic fungi Candida albicans ATCC 90028 (Ca), Crypto-
coccus neoformans ATCC 90113 (Cn), Aspergillus
fumigatus ATCC 204305 (Afu), Candida glabrata ATCC
90030 (Cg), and Candida krusei ATCC 6258 (Ck) were
determined. Amphotericin B was used as positive control.
Materials and Methods
General
All commercial chemicals and solvents were reagent grade
and were used without further treatment, unless otherwise
noted. Nuclear magnetic resonance spectra were recorded
on Brukers avance (400 MHz) with tetramethyl silane
(TMS) as internal standard. Chemical shifts were recorded
in parts per million (ppm, d) and were reported relative to
TMS. Mass spectra were recorded on GCMS-QS Shimadzu
(QP-500) and LCMS waters ((Micromass ZQ, Milford, MA,
USA). IR spectra were recorded on Nicolet spectrometer.
HRMS was recorded on LCMS (Bruker MAxis, Fremont,
CA, USA). TLC was performed on Merck 0.25 mm Kiesel-
gel 60 F254 plates. Column chromatography was per-
formed using either silica gel-60 (60–120 mesh).
Anti-HIV activity
Selected synthesized analogues were tested for anti-HIV
potential. They were first evaluated for their cytotoxicity in
MTT based cell viability assay in CEM-GFP cells (24). Further
non-cytotoxic concentration of each analogue was used for
determination of anti-HIV activity. They were evaluated for
anti-HIV potential in human CD4+ T cell line CEM-GFP,
infected with HIV-1NL4.3 virus by p24 antigen capture ELISA
assay (25–27). Zidovudine (AZT) was used as standard.
Results and Discussion
Antimalarial activity
Chemistry
In vitro antimalarial activity of all synthesized compounds
was evaluated against chloroquine-sensitive (D6) and chlo-
Four series of tricyclic guanidine analogues were synthe-
sized using the protocol developed in our laboratory (12).
492
Chem Biol Drug Des 2013; 81: 491–498

Synthesis and Evaluation of Batzelladine is Analogues
Scheme 1: Reagents and conditions: (a) HCl, Na2CO3, 2 h (repeated 5 times), 2: 70%; (b) DCM, 24 h, 4: 60%, 7: 65%; (c) Guanidine,
DMF/0 ꢀC -RT/5 h, 3:1:3 DMF/H2O/MeOH, NaBH4/16 h, 5: 30%, 8: 25%.
Scheme 2: Reagents and conditions: (a) BuLi/À78 ꢀC, RT/16 h, 95–98%; (b) DCM/24 h, 60–70%; (c) DMF, 0 ꢀC -RT/5 h, 3:1:3
DMF/H2O/MeOH, NaBH4/16 h, 25–40%.
Scheme 3: Reagents and conditions: (a) BuLi/À78 ꢀC, RT/16 h, 95–98%; (b) DCM/24 h, 60–70%; (c) DCM/24 h, 70–80%; (d)
DMF/0 ꢀC -RT/5 h, 3:1:3 DMF/H2O/MeOH, NaBH4/16 h, 25–45%.
Chem Biol Drug Des 2013; 81: 491–498
493

Ahmed et al.
Table 1: In vitro antimalarial activity and cytotoxicity of tricyclic guanidine derivatives
Plasmodium
falciparum
(D6 Clone)IC50
(lM)
P. falciparum
(W2 Clone)IC50
(lM)
Cytotoxicity
(Vero)IC50
(lM)
P. falciparum
(D6 Clone)IC50
(lM)
P. falciparum
(W2 Clone)IC50
(lM)
Compd.
No.
Cytotoxicity
(Vero)IC50 (lM)
Compd. No.
12a
12b
12c
12d
12e
12f
6.49
1.98
1.48
6.16
10.62
7.29
9.66
6.34
4.23
5.62
3.01
2.99
1.52
1.39
6.20
6.27
1.69
14.13
1.25
1.37
7.22
2.70
1.43
8.04
10.98
7.59
5.57
7.71
4.98
7.33
3.60
4.81
1.96
1.76
8.26
7.66
2.18
–
NC
NC
6.71
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
6.96
NC
NC
NC
NC
NC
NC
14l
2.25
3.04
4.59
7.62
16.05
20.60
10.09
9.39
15.76
14.85
3.21
4.69
3.44
9.24
19.27
6.54
17.14
2.41
5.78
0.88
2.82
3.53
6.20
9.93
–
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
17b
17c
17d
17f
17g
17h
17i
20.60
7.25
8.05
13.24
13.65
5.62
7.22
3.76
12.45
19.27
9.45
19.24
3.02
7.71
1.07
12g
12h
12i
17j
17l
12j
12l
20a
20b
20c
20d
20e
20f
14a
14b
14c
14d
14e
14f
20g
20h
20i
14g
14h
14i
1.64
2.26
20l*
IC50, sample concentration that causes 50% cell growth inhibition compared to vehicle control; NC, no cytotoxicity upto 4.76 lg/mL; ‘–’,not
active; *S.I., selectivity index [TC50 (Vero)/IC50 (P. falciparum)] >17; Chloroquine: IC50 0.052 lM (D6 clone); IC50 0.46 lM, (W2
clone). Artemisinin: IC50 0.018 lM (D6 clone); IC50 0.015 lM (W2 clone).
=
=
=
=
As shown in Scheme 1, succinaldehyde (2) was freshly
prepared by hydrolysis of commercially available 2,5-dim-
ethoxytetrahydrofuran (1) with 0.6 N HCl (28). A Wittig
reaction of phosphorane 3 with succinaldehyde (0.5 equiv.)
at room temperature for 24 h afforded E-isomer of ke-
tone 4 in 60% yield. The Michael addition of guanidine to
4 at 0 ꢀC followed by a reductive imination with sodium
borohydride gave 5.
Table 2: In vitro antileishmanial activity (Leishmania donovani) of
tricyclic guanidine derivatives
L. donovani
L. donovani
Compd.
No.
IC50 (lM) IC90 (lM) Compd. No IC50 (lM) IC90 (lM)
12a
12b
12c
12d
12e
12f
64.98
12.31
2.39
126
17b
17c
17d
17e
17f
17h
17i
14.44
10.13
59.60
85.69
61.06
63.09
63.75
62.91
48.19
29.31
57.76
10.34
57.23
133.6
50.90
12.08
48.23
9.52
29.65
23.85
112.5
>13
33.03
11.27
91.15
>130
91.18
72.56
79.80
78.24
55.73
106.5
19.59
12.76
93.02
128.9
20.40
14.91
15.90
21.94
48.25
73.26
28.87
29.47
32.41
29.34
13.33
34.02
9.51
To identify the role of tricyclic guanidinium core in biologi-
cal activity, the pyrrolidine ring of tricycle was replaced by
piperidine ring. Glutaraldehyde was employed to con-
struct the piperidine ring in previously described synthetic
route. As shown in Scheme 2, Wittig reaction of 3 with
glutaraldehyde (0.5 equiv.) gave ketone 7. Condensation
of 7 with guanidine in DMF via sequential double Michael
addition, followed by sodium borohydride reduction affor-
ded the 8.
125
>100
>100
>100
124.4
116
119
22.25
114
>100
>100
45.31
106
12h
12i
17j
17l
12j
12l
20a
20b
20c
20d
20e
20f
20h
20i
14a
14b
14c
14d
14e
14f
2.78
46.51
62.71
10.20
7.23
7.71
10.97
A series of compounds 12a–l were synthesized following
a strategy shown in Scheme 2 (Series 1). The synthesis
was started with alkylation of phosphorane 3 with alkyl
halides (9a–l) employing butyllithium as a base to obtain
phosphorane 10a–l. Wittig reaction of 10a–l with succinal-
dehyde afforded 11a–l which was identified by NMR
experiment. The NMR data showed coupling constants in
range of 15–18 Hz for trans protons indicating synthesis
of E-isomeric form of 11a–l. Condensing guanidine with
11a–l at 0 ꢀC via Michael addition, followed by a reduc-
14h
14i
20l
20.95
14l
IC50 and IC90, sample concentrations that kill 50% and 90% cells
compared to the solvent controls; Pentamidine:
IC50 2.94 lM, IC90 5.88 lM; Amphotericin B: IC50
0.173 lM, IC90 0.358 lM.
=
=
=
=
494
Chem Biol Drug Des 2013; 81: 491–498

Synthesis and Evaluation of Batzelladine is Analogues
Table 3: In vitro antibacterial activity of tricyclic guanidine derivatives
Staphylococcus
aureus
Pseudomonas
aeruginosa
Mycobacterium
intracellulare
MRSA
Escherichia coli
Compd. IC50
MIC
MBC
IC50
MIC
MBC
IC50
MIC
MBC
IC50
MIC
MBC IC50
MIC
MBC
No.
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
12b
12c
12f
12h
12i
8.13
3.02
15.01 30
5.99
6.48
23.98 2.44
15
–
39.94
7.02
–
–
–
–
–
–
–
–
–
–
–
–
–
–
28.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.74 15
7.41 12
20.3 30
11.1 22.67 45.35
12 12.46 24.93
16.8 24.45 24.45
13.5 32.78 65.57
8.12 14.40 14.40
9.81 11.6
7.05 14.5
30
23.9
60
5.99 12
15.80 30.39 60.79 11.67 30.3
–
–
–
–
–
–
3.26
3.44
6.89
8.19
6.25
3.01
4.60
3.18
3.20
5.66
6.23
45.35 3.28
49.87 3.39
5.66 22.67
6.23 25
–
16.3
14.4 28.8
5.8 11.6
14.5 14.5
5.48 10.96 5.96
12
15.6 15.6
19.0
–
15.7
–
12j
12l
12.22 48.90 11.63
16.39 65.57 8.59
14.40 57.63 4.26
–
–
14b
14c
14f
14h
14i
14.26 28.8
6.33
29.82 58.3
5.80
7.28
5.48
6.02
5.80
3.10
–
45
–
23.20
29
14.57 5.07
21.93 2.89
24.09 5.54
58.3
10.96 10.96 10.96 21.9
37.88 48.2
8.26 10.96 43.86
5.95 12
12
12.38 24.09
31.91 62.69 62.69 30.31
69.12
57.22
–
–
–
–
–
12
31.2
38
14l
12.32 31.34 31.34 8.27
8.21 38.02 38.02 7.90
17.28 32.68 65.35 27.38
16.40 31.54
19.0 33.11
64.09 80.32
24.33 40.16
36.49 72.20
8.74
8.52
4.44
–
–
–
–
–
–
–
–
–
–
–
11
13
15.6
19
17b
17c
17h
17j
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
35.3 65.35 65.35
34.1 63.09 31.54
34.9 66.22 66.22
–
–
–
–
–
8.51
56.85
56.06 80.3
26.78 40.1
29.09 36.1
17l
79.5
–
–
20a
20b
20c
20h
20l
34.4 80.32 80.32
44.3 72.20 72.20
25.9 62.6
15.8 30.2
–
–
15.67 31.34 11.28 31.3 31.3
15.10 30.21 8.30 15.1 30.2
7.93 15.87 7.07 15.8
62.6
30.2
47.88
23.27 63.49
–
–
48.19
6.73 15.87 31.74
IC50, concentration (lM) that affords 50% growth inhibition; MIC, minimum inhibitory concentration (the lowest concentration in lM that allows
no detectable growth); MBC, minimum bactericidal concentration (the lowest concentration in lM that kills the organism); ‘–’, not active at
the highest test concentration of 20 lg/mL; Ciprofloxacin: IC50
0.3 lM, MIC 0.75 lM, MBC 1.5 lM (MRSA); IC50
0.311 lM, MIC lM, MBC ‘–’ (Pa); IC50 0.69 lM, MIC
=
0.3 lM, MIC
0.009 lM, MIC
1.5 lM, MBC
=
1.5 lM, MBC
0.024 lM, MBC
lM (Mi).
=
=
1.5 lM (Sa); IC50
0.377 lM (Ec); IC50
=
=
=
=
=
=
=
=
3
=
=
= 3
tion with sodium borohydride accomplished synthesis of
compounds 12a–l. Using the similar synthetic route, com-
pounds 14a–l (Series 2) were synthesized where glutaral-
dehyde was employed instead of succinaldehyde as
depicted in Scheme 2. In Series 1 and 2, compounds
were substituted with identical alkyl chains on both side of
tricycle.
derivatives was established by NOE experiments as shown
previously (12).
Antimalarial activity
The antimalarial activities of all the compounds are
reported as IC50 values against D6 and W2 strains of P. fal-
ciparum. Activity data are shown only for active com-
pounds in Table 1. Compounds having methyl
substituents, 5 and 8 did not show any antimalarial activ-
ity. In Series 1 and 2 (compounds 12a–l and 14a–l), all
analogues were active against the P. falcifarum. Amongst,
compound 14h was the most active and showed IC50 of
1.25 and 1.64 lM against D6 and W2 strains, respec-
tively. Analogue 14b exhibited IC50 of 1.52 and 1.96 lM
against D6 and W2 strains, respectively, whereas 14i dis-
played IC50 of 1.37 and 2.26 lM against D6 and W2
strains, respectively. Whereas in Series 3 and 4 (com-
pounds 17a–l and 20a–l), the most potent analogue, 20l
displayed IC50 of 0.88 lM for D6 clone and 1.07 lM for
W2 clone. The activity of this compound was much higher
in comparison to the natural product, batzelladine K (17l)
which showed IC50 of 14.85 lM for D6 clone and
13.65 lM for W2 clone.
Compounds of Series 3 (17a–l) were synthesized as
shown in Scheme 3, with two different alkyl groups on
each side of tricyclic core. Synthesis of phosphorane
10a–l was carried out as shown in Scheme 2. Wittig
reaction of 10a–l with excess of succinaldehyde afforded
ketone 15a–l. It is noteworthy that excess of succinalde-
hyde (>3 equiv.) is essential to obtain the ketone (15a–l),
to avoid the reaction of the phosphorane (3) with free alde-
hyde group of 15a–l to form di-a, b-unsaturated ketone
(symmetrical). Furthermore, 15a–l were treated with 3 at
room temperature to provide 16a–l, which were reacted
with guanidine followed by a reduction with sodium boro-
hydride to accomplish synthesis of compounds 17a–l as
single diastereo-isomers. Similarly, compounds 20a–l (Ser-
ies 4) were synthesized using glutaraldehyde in the syn-
thetic Scheme 3. The relative stereochemistry of all
Chem Biol Drug Des 2013; 81: 491–498
495

Ahmed et al.
Table 4: In vitro antifungal activity of tricyclic guanidine derivatives
Cryptococcus
neoformans
Candida albicans
Candida glabrata
Candida krusei
Aspergillus fumigatus
IC50
Compd. No. lM
MIC
lM
MFC
lM
IC50
MIC
lM
MFC
lM
IC50
MIC
lM
MFC
lM
IC50
MIC
lM
MFC
lM
IC50
MIC
lM
MFC
lM
lM
lM
lM
lM
12b
12c
12f
12h
12j
18
3.02
–
–
–
–
7.03
3.89
60
5.99
–
–
–
60
5.99
–
–
–
–
2.18
–
–
–
–
–
2.99
–
–
–
–
–
5.80
–
2.99
–
–
–
–
–
5.80
19.76 60
60
2.99
–
8.73
2.11
15
2.99
30
2.99
33.54
5.46
–
–
–
–
–
–
2.47
2.99
11.99 11.99
33.22
–
29.54 60.79 30.39
11.33 11.33
23.71 48.9
–
–
–
–
–
–
–
–
–
–
35.89 45.35 45.35 6.71
–
–
–
–
–
48.9
12l
–
–
39.21
28.68 32.78 32.78
14b
14c
14f
14h
14i
28.81 57.63 35.96
5.80 5.80 3.75
13.91 28.81 28.81 4.20
1.87 2.90 2.90 2.39
11.39 29.15 29.15 12.47 14.57 14.57 35.27
12.34 21.93 43.86 0.46
16.84 24.09 24.09 1.03
3.60
5.80
3.60
2.90
35.64
10.11 23.20 23.20
16.44 29.15 58.30 34.66 58.30
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
34.93
25.90 48.19
9.09
22.09 38.0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.68
1.51
7.83
0.68
1.51
7.83
38
–
47.78
59.34
47.37
–
–
–
–
–
14l
31.34 62.69 34.16 62.69 62.69 12.94 31.34 31.34 4.29
17b
17c
17h
17j
76
–
–
–
–
–
–
–
–
–
–
–
24.37 76
38
17.60 38
–
–
–
–
–
–
–
–
17.32 32.68 65.36 9.57
–
–
–
–
–
19.0
–
16.34 16.34 2.61
8.17
8.17
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
16.49 31.54 31.54
35.49 66.22 66.22
76.18
19.96 40.16 40.16
15.34 18.0
17l
–
–
20a
20b
20c
20h
20l
18.0
15.67
7.55
3.96
–
–
–
31.34 62.69 6.99
–
15.67 15.67 3.66
3.56
15.87 15.87 2.12
7.83
7.55
3.96
–
–
–
–
16.79 31.74 31.74 19.30 31.74 63.49 8.98
34.25
IC50, concentration (lM) that affords 50% growth inhibition; MIC, minimum inhibitory concentration (the lowest concentration in lM that
allows no detectable growth); MFC, minimum fungicidal concentration (the lowest concentration in lM that kills the organism); ‘–’, no activ-
ity at the highest test concentration of 20 lg/mL. Amphotericin B: IC50
=
=
0.30 lM, MIC
0.56 lM, MIC =
=
1.35 lM, MFC
1.35 lM, MFC
=
=
1.35 lM (Ca);
1.35 lM (Ck);
IC50
IC50
=
=
0.39 lM, MIC
0.86 lM, MIC
=
=
0.68 lM, MFC
1.35 lM, MFC
=
=
1.35 lM (Cg); IC50
2.57 lM (Afu); IC50
=
0.26 lM, MIC
=
0.68 lM, MFC
=
0.68 lML (Cn).
All the analogues were evaluated for in vitro cytotoxicity
and Selectivity Index was calculated. S.I. ꢀ 10 is generally
considered significant, indicating that antimalarial activity is
not because of cytotoxicity. None of the compounds were
found cytotoxic with the exception of 12c and 14c, indi-
cating their selectivity for antimalarial activity.
the range of 2.44–10 lM and MIC in the range of
5.99–20 lM. Of these, 12c having a nonyl substituent
was the most potent having IC50 of 3.02 and 2.44 lM,
and bactericidal activity at 23.98 and 12 lM against
S. aureus and MRSA, respectively. The analogues 14c
and 14h exhibited IC50 values of 3.01 and 3.18 lM, and
were bactericidal at 5.8 and 5.48 lM respectively against
S. aureus. The 14h inhibited the growth of E. coli with
IC50 value of 5.96 lM and showed bactericidal activity at
10.96 lM. Only four compounds from Series 2 were
active against P. aeruginosa (14c, 14h, 14i, and 14l) with
14h being most active with IC50 of 10.96 lM and MIC of
21.9 lM. The 14i was found to be the most effective
against M. intracellulare with IC50 value of 5.95 lM and
MBC of 12 lM.
Antileishmanial activity
Activity data are shown only for active compounds in
Table 2. From Series 1 and 2, analogues 12c and 14c
were the most potent with IC50 of 2.39 and 2.78 lM and
IC90 of 11.27 and 12.76 lM, respectively. Remaining
compounds also displayed antileishmanial activity to vari-
ous extents with the exception of 12g and 14g. Among
Series 3 and 4, 20l was found be to the most active with
IC50 of 9.52 lM and IC90 of 20.95 lM. Natural product,
The analogues from Series 3 and 4 were also active
against all strains; ten compounds have shown moderate
to potent antibacterial activity against both the strains of
S. aureus with IC50 values in the range of 4.44–64 lM
and all were bactericidal at <80 lM. The most potent
activity was shown by 20l having IC50 of 4.44 and
7.07 lM, MIC of 7.93 and 15.8 lM and bactericidal at
15.87 lM against S. aureus and MRSA, respectively.
Four analogues showed moderate activity against E. coli;
batzelladine
K (17l) was weakly active with IC50 of
48.19 lM and IC90 of 124 lM.
Antimicrobial activity
Activity data are shown only for active compounds in
Table 3. In Series 1 and 2, thirteen analogues have shown
inhibitory activity against S. aureus and MRSA with IC50 in
496
Chem Biol Drug Des 2013; 81: 491–498

Synthesis and Evaluation of Batzelladine is Analogues
20l having IC50 value of 23.27 lM, and MIC of
63.49 lM. None of analogues in Series 3 and 4 were
active against P. aeruginosa. Ten analogues exhibited the
promising activity against M. intracellulare; 20l being the
most active with IC50 of 6.73 lM, MIC of 15.87 lM, and
bactericidal at 31.74 lM. In comparison, batzelladine K
(17l) showed a weak activity against S. aureus, MRSA,
and M. intracellulare (IC50 64.09, 56, and 79.5 lM,
respectively).
The most potent analogues having nonyl substituent, 12c
and 14c, not only exhibited antileishmanial activities, but
also showed potent antibacterial and antifungal activities.
Analogue 20l having pentyl and methyl substitutions
showed promising activities against all pathogens. How-
ever, none was active against HIV. The combination of
broad spectrum of activities makes these tricyclic guani-
dines a promising new structural class of compounds. Fur-
ther exploration of these tricyclic guanidines can provide
lead compounds for further development as antimicrobial
agents.
Antifungal activity
Activity data are shown only for active compounds Table 4.
The tricyclic guanidine analogues showed activities to a
variable extent against various strains. In Series 1, 12c
having nonyl substitution produced the most potent activity
with IC50 of 3.02, 2.18, 2.11, and 5.46 lM, MIC and
MFC of 5.99, 2.99, 2.99, and 11.99 lM against C. albi-
cans, C. glabrata, C. neoformans, and A. fumigatus,
respectively; whereas in Series 2, analogue 14c showed
IC50 of 3.83, 3.75, and 2.39 lM, and fungicidal at 5.80,
5.80, and 2.90 lM against C. albicans, C. glabrata, and
C. neoformans, respectively. 14c was the most potent in
Acknowledgments
Dr Nafees Ahmed and Dr Keyur Brambhatt thank the
Council of Scientific and Industrial Research (CSIR) and
Department of Biotechnology (DBT), New Delhi for the
award of Senior Research Fellowships. Antimicrobial
assays were supported by NIH grant No. AI 27094. Anti-
malarial and antileishmanial screening assays are partly
supported by USDA-ARS cooperative scientific agreement
number 58-6408-2-0009 (NCNPR) and US Department of
Defense CDMRP grant number W81XWH-09-20093 (BLT).
We thank John Trott, Marsha Wright, and Surendra Jain
for technical assistance in antimalarial, antibacterial/anti-
fungal, and antileishmanial assays, respectively. Anti-HIV
screening was supported by Department of Biotechnology
project (Grant No. BT/PR7020/Med/14/930/2005 Dt. 23/
05/2006).
Series 2 against C. krusei (IC50
MFC 2.90 lM). The most potent activity against
C. neoformans was observed with analogues 14h and 14i
(IC50 0.46 and 1.03 lM, MIC and MFC 0.68
and 1.51 lM, respectively).
=
1.87 lM, MIC and
=
=
=
In Series 3 and 4, 17b was moderately active against
C. albicans and A. fumigatus (IC50 22 and
=
46.73 lM) and 17c, 20c, and 20l were active against
C. glabrata (IC50 of 17.32–19.30 lM) whereas, they
showed promising activity against C. krusei (IC50 values of
9.57, 6.99, and 8.98 lM, respectively, and MIC and MFC
value of 15 lM), and C. neoformans (IC50 of 2.61, 3.66,
and 2.12 lM, MIC of 8.17, 7.83, and 3.96 lM, and
MFC of 8.17, 15.67, and 3.96 lM, respectively). Batzella-
dine K (17l) was inactive against all pathogens except
C. neoformans (IC50 76.18 lM).
References
1. Kaur K., Jain M., Khan S.I., Jacob M.R., Tekwani B.L.,
Singh S., Singh P.P., Jain R. (2011) Synthesis, antipro-
tozoal, antimicrobial, beta-hematin inhibition, cytotoxic-
ity and methemoglobin (MetHb) formation activities of
bis(8-aminoquinolines). Bioorg Med Chem;19:197–210.
2. Bharate S.B., Khan S.I., Yunus N.A.M., Chauthe S.K.,
Jacob M.R., Tekwani B.L., Khan I.A., Singh I.P. (2007)
Antiprotozoal and antimicrobial activities of O-alkylated
and formylated acylphloroglucinols. Bioorg Med
Chem;15:87–96.
Anti-HIV activity
None of tested analogues were found to inhibit the growth
of HIV-1 (Table S1). Batzelladine K also demonstrated no
anti-HIV activity.
3. Harms G., Feldmeier H. (2002) HIV infection and tropi-
cal parasitic diseases – deleterious interactions in both
directions? Trop Med Int Health;7:479–488.
Conclusion
4. Mazu T.K., Etukala J.R., Zhu X.Y., Jacob M.R., Khan
S.I., Walker L.A., Ablordeppey S.Y. (2011) Identification
of 3-phenylaminoquinolinium and 3-phenylaminopyridi-
nium salts as new agents against opportunistic fungal
pathogens. Bioorg Med Chem;19:524–533.
5. Groll A.H., Tragiannidis A. (2009) Recent advances in
antifungal prevention and treatment. Semin Hema-
tol;46:212–229.
We have synthesized four series of tricyclic guanidine ana-
logues based on batzelladine K structural framework. The
analogues were evaluated for antimalarial, antileishmanial,
antibacterial, antifungal, and anti-HIV activities. The 14h
and 20l produced the most potent antimalarial activity
against chloroquine-sensitive D6 (IC50 of 1.25 and
0.88 lM, respectively) and chloroquine-resistant W2 (IC50
of 1.64 and 1.07 lM, respectively) strains of P. falciparum.
6. Patil A.D., Kumar N.V., Kokke W.C., Bean M.F., Freyer
A.J., Brosse C.D., Mai S., Truneh A., Carte B. (1995)
Chem Biol Drug Des 2013; 81: 491–498
497

Ahmed et al.
Novel alkaloids from the sponge Batzella sp. – inhibi-
tors of HIV GP120-human CD4 binding. Org
Chem;60:1182–1188.
7. Patil A.D., Freyer A.J., Offen P., Bean M.F., Johnson
R.K. (1997) Three new tricyclic guanidine alkaloids from
the sponge Batzella sp. J Nat Prod;60:704–709.
standard M27-A2. National Committee on Clinical Lab-
oratory Standards;22:15.
21. NCCLS (2002) Reference method for broth dilution
antifungal susceptibility testing of filamentous fungi;
approved standard, M38-A. National Committee on
Clinical Laboratory Standards;22:16.
J
8. Patil A.D., Freyer A.J., Taylor P.B., Carte B., Zuber G.,
Johnson R.K., Faulkner D.J. (1997) Batzelladines F-I,
novel alkaloids from the sponge Batzella sp.: inducers of
p56(lck)-CD4 dissociation. J Org Chem;6:1814–1819.
9. Hua H.M., Peng J., Dunbar D.C., Schinazi R.F.,
Andrews A.G.D.C., Cuevas C., Garcia-Fernandez L.F.,
Kelly M., Hamann M.T. (2007) Batzelladine alkaloids
from the caribbean sponge Monanchora unguifera
and the significant activities against HIV-1 and AIDS
22. NCCLS (2000) Susceptibility testing of mycobacteria,
nocardia, and other aerobic actinomycetes; tentative
standard-second edition, M24-T2. National Committee
on Clinical Laboratory Standards;20:26.
23. Franzblau S.G., Witzig R.S., McLaughlin J.C., Torres
P.M., Hernandez G.A., Degnan M.T., Cook M.B.,
Quenzer V.K., Ferguson R.M., Gilman R.H. (1998)
Rapid, low-technology MIC determination with clini-
cal Mycobacterium tuberculosis isolates by using the
opportunistic
dron;63:11179–11188.
10. Gallimore W.A., Kelly M., Scheuer P.J. (2005) Alkaloids
infectious
pathogens.
Tetrahe-
microplate Alamar Blue assay.
biol;36:362–366.
24. Denizot F., Lang R.J. (1986) Rapid colorimetric assay
for cell growth and survival. Modifications to the tetra-
zolium dye procedure giving improved sensitivity and
reliability. Immunol Methods;89:271–277.
25. Li F., Goila-Gaur R., Salzweddel K., Kilgore N.R., Red-
dick M., Matallana C., Castillo A., Zoumplis D., Martin
D.E., Orenstein J.M., Allaway G.P., Freed E.O., Wild
C.T. (2003) A potent HIV inhibitor that disrupts core
condensation by targeting a late step in Gag process-
ing. Proc Natl Acad Sci USA;100:13555–13560.
J
Clin Micro-
from the sponge Monanchora unguifera.
J Nat
Prod;68:1420–1423.
11. Bewley C.A., Ray S., Cohen F., Collins S.K., Overman
L.E. (2004) Inhibition of HIV-1 envelope-mediated
fusion by synthetic batzelladine analogues.
Prod;67:1319–1324.
J Nat
12. Ahmed N., Brahmbhatt K.G., Singh I.P., Bhutani K.K.
(2010) Total synthesis of (+/À)-batzelladine K: a biomi-
metic approach. Synthesis;15:2567–2570.
13. Makler M.T., Hinrichs D.J. (1993) Measurement of the
lactate-dehydrogenase activity of plasmodium-falcipa-
rum as an assessment of parasitemia. Am J Trop Med
Hyg;48:205–210.
26. Ladha J.S., Tripathy M.K., Mitra D. (2005) Mitochon-
drial complex
induced T-cell
fer;12:1417–1428.
I
activity is impaired during HIV-1-
apoptosis. Cell Death Dif-
14. Borenfreund E., Babich H., Martin-Alguacil N. (1990)
Rapid chemosensitivity assay with human normal
and tumor cells in vitro. In Vitro Cell Dev
Biol;26:1030–1034.
27. Gervaix A.W.D., Leoni L.M., Richman D.D., Wong-Sta-
al F., Corbeil J. (1997) A new reporter cell line to moni-
tor HIV infection and drug susceptibility in vitro. Proc
Natl Acad Sci USA;94:4653–4658.
15. Mustafa J., Khan S.I., Ma G., Walker L.A., Khan I.A.
(2004) Synthesis and anticancer activities of fatty acid
analogs of podophyllotoxin. Lipids;39:167–172.
16. Harrison J.H., Jollow D.J. (1987) Contribution of aniline
metabolites to aniline-induced methemoglobinemia.
Mol Pharmacol;32:423–431.
17. Mikus J., Steverding D. (2000) A simple colorimetric
method to screen drug cytotoxicity against Leishmania
using the dye Alamar Blue. Parasitol Int;48:265–269.
18. Ma G., Khan S.I., Jacob M.R., Tekwani B.L., Li Z.,
Pasco D.S., Walker L.A., Khan I.A. (2004) Antimicrobial
and antileishmanial activities of hypocrellins A and B.
Antimicrob Agents Chemother;48:4450–4452.
19. NCCLS (2000) Methods for dilution antimicrobial sus-
ceptibility tests for bacteria that grow aerobically M7-
A5. National Committee on Clinical Laboratory Stan-
dards;20:2.
28. House H.O., Cronin T.H. (1965) A study of intramolec-
ular Diels–Alder reaction. J Org Chem;30:1061–1070.
Note
ahttp://www.who.int/leishmaniasis/resources/en/index.
html.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Anti HIV activity of compounds.
Appendix S1. Experimental procedures and spectral data
of intermediates and final compounds.
20. NCCLS (2002) Reference method for broth dilution
antifungal susceptibility testing of yeasts; approved
498
Chem Biol Drug Des 2013; 81: 491–498