Structure - activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Article abstract of DOI:10.1021/jm900672t

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood - brain barrier (BBB) in an in vitro model using aMDR-MDCKmonolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3β (GSK-3β), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Full text of DOI:10.1021/jm900672t

J. Med. Chem. 2010, 53, 61–76 61
DOI: 10.1021/jm900672t
Structure-Activity Relationship and Mechanism of Action Studies of Manzamine Analogues for the
Control of Neuroinflammation and Cerebral Infections
Jiangnan Peng,† Sucheta Kudrimoti,† Sivaprakasam Prasanna,‡,O Srinivas Odde,‡ Robert J. Doerksen,‡,§ Hari K Pennaka,†
Yeun-Mun Choo,† Karumanchi V. Rao,† Babu L. Tekwan^i,§ Vamsi Madgula,§ Shabana I. Khan,†,§ Bin Wang,†
§
Alejandro M. S. Mayer, Melissa R. Jacob, Lan Chun Tu, Jurg Gertsch,#,3 and Mark T. Hamann*,†,§
€
†Department of Pharmacognosy, ‡Department of Medicinal Chemistry, and §National Center for Natural Products Research, School of
Pharmacy, The University of Mississippi, Oxford, Mississippi 38677, Department of Pharmacology, Chicago College of Osteopathic Medicine,
Midwestern University, Downers Grove, Illinois 60515, ^Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic
of China, #Department of Chemistry and Applied Biosciences, ETH Zurich, Ch-8093 Zurich, Switzerland, 3Institute of Biochemistry and Molecular
Medicine, University of Bern, CH-3012 Bern, Switzerland. OCurrent address: Computer-Aided Drug Design, Bristol-Myers Squibb Co., Wallingford, CT.
Received May 20, 2009
Structure-activity relationship studies were carried out by chemical modification of manzamine A (1),
8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongy-
lophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflam-
matory activities. Several modified products exhibited potent and improved in vitro antineuro-
inflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria
compared to chloroquine in both multi- and single-dose in vivo experiments. The significant anti-
malarial potential was revealed by a 100% cure rate of malaria in mice with one administration of
100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit
for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In
addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using
a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of
glycogen synthesis kinase-3β (GSK-3β), which is a putative target of manzamines. On the basis of the
results presented here, it will be possible to initiate rational drug design efforts around this natural
product scaffold for the treatment of several different diseases.
Introduction
their chemistry and pharmacology. To date, over 80 manza-
mine related alkaloids have been isolated from more than 16
species of marine sponges belonging to five families distrib-
uted from the Red Sea to Indonesia, which suggested a
potential microbial origin of manzamine alkaloids.2,3 The
manzamine alkaloids have shown a variety of bioactivities
including antimicrobial,4-7 antiparasitic,8 cytotoxic,1,9 anti-
neuroinflammatory,10-13 and pesticidal.14
Marine invertebrates have been recognized as an important
source of pharmacologically unique bioactive natural pro-
ducts. Apart from human medicines, the research involving
marine natural products in the last three decades has also
generated significant discoveries that are now utilized routi-
nely as pharmacological tools with unique cellular targets.
Some of them have become indispensable tools in biochemical
research and played significant roles in the recent advance-
ments of life sciences. In our search for bioactive compounds
against infectious and neurological diseases, we have focused
on marine alkaloids isolated from sponges and in particular
the manzamine alkaloids.
Manzamines are characterized by a unique 5-, 6-, 6-, 8-,
13-membered heterocyclic ring system coupled to a β-carbo-
line moiety. The first representative of this class, manzamine
A (1), was isolated from the marine sponge Haliclona
sp. collected near Manzamo Island by Higa and co-workers
in 1986 and its structure including absolute configuration was
established by X-ray diffraction.1 In recent years, the manza-
mines have been regarded as an interesting group of marine
alkaloids with extraordinary biological activities, and as a
result the molecules have received considerable attention for
The greatest potential for the manzamine alkaloids appears
to be against malaria and neuroinflammation, which is an
interesting combination and may make it possible to treat
both the infection and the symptoms with a single drug. The
most effective and widely used antimalarial drugs are those
derived from quinine and artemisinin, which are natural
products from terrestrial plants. Our previous studies showed
that the marine natural products 1 and 8-hydroxymanzamine
A (2) exhibited improved potency against the malarial para-
sites both in vitro and in vivo relative to chloroquine and
artemisinin.15 A remarkable aspect of 1 is its ability to prolong
the survival of highly parasitemic mice with significant recov-
ery after a single injection. Oral administration of 1 (2 ꢀ 100
μmol/kg) and 2 (2 ꢀ 100 μmol/kg) produced a significant
reduction (90%) in parasitemia. All mice treated with a single
dose (50 or 100 μmol/kg) of 1 or 2 showed significant
improvements in survival times over mice treated with chloro-
quine or artemisinin.16 Significant variation of antimalarial
*To whom correspondence should be addressed. Phone: 662-
9155730. Fax: 662-915697. E-Mail: mthamann@olemiss.edu.
r
2009 American Chemical Society
Published on Web 12/17/2009
pubs.acs.org/jmc

62 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
activity among naturally isolated manzamines such as
manzamine F (14) and ircinal A (19) provided some interest-
ing information regarding the structure-activity relationship
of these alkaloids.4,7,12,15-17
Manzamines have also been reported to have antimicrobial
activities against a number of bacteria and fungi.3 In parti-
cular, most naturally isolated manzamines were active against
Mycobacterium tuberculosis.15,16,18 Compounds 1 and 2
showed 98-99% inhibition of Mycobacterium tuberculosis,
with MIC end points of 1.56 and 0.91 μg/mL, respectively.7 1
showed antimicrobial activity against Staphylococcus aureus
and methicillin-resistant Staphylococcus aureus,7,12,17 with
IC50 values of 0.5 and 0.7 μg/mL, respectively.19
on different positions and functional groups of manzamines
to generate diverse analogues. For this reason, four major
manzamine alkaloids, 1, 2, 14, and 19, were chosen as start-
ing material for the synthesis of analogues, although 14 and
19 only show marginal biological activity. Large quantities
of these alkaloids had been produced from a common
Indonesian sponge Acanthostrongylophora sp. through an
optimized isolation procedure. Because of the number of
potential reactive functional groups and instability of large
and complex molecules such as manzamines, only mild and
highly efficient reactions were employed for modification of
manzamines. Thirty-nine analogues were synthesized in
total, using various synthetic strategies, including 16 analo-
gues of 1, four of 2, 11 of 14, and eight of 19.
N-Alkylations (Scheme 1) (6a-i) of the β-carboline moi-
ety with various alkyl halides were carried out starting with 1
in the presence of sodium hydride in dimethyl formamide at
0 ꢀC. The alkylated products were obtained in good yield
(81-87%). One- and two-dimensional NMR showed that
alkylation occurred at the 9-N position.
Reductions of 1 and 2 were performed in the presence of
hydrogen-10% Pd/C in ethanol at room temperature for 4 h.
Manzamine A afforded a major product, 15,16,32,33-tetra-
hydromanzamine A (3), and a minor product, 5, and 2 gave a
major product, 4, and a minor product, 12.
1 was nitrated by sodium nitrate using TFA as a solvent to
yield two nitrated products 7 and 8. The 6-nitromanzamine
A (8) was generated in excess when compared to 8-nitro-
manzamine (7). These two products could be readily purified
by flash column chromatography. Refluxing of 1 and 2 with
acetic anhydride and potassium carbonate in dry acetone
resulted in dehydration product 10 and 11. Oxidation of 1
with m-chloroperoxybenzoic acid in the presence of a cata-
lytic amount of sodium bicarbonate in dichloromethane
(DCM) solvent gave the corresponding N-oxides (13) with-
out disturbing the double bond.
Modification of 14 was focused on the carbonyl group in
the bottom eight-membered ring (Scheme 2). The carbonyl
functional group was first reduced when treated with sodium
borohydride to give a mixture of hydroxylated products and
only one isomer (16) was obtained after purification. 14
was treated with Grignard reagent in dry THF to give the
31-alkyl product (18). Manzamine F-31-hydrazone (15) was
obtained by heating 14 with hydrazine hydrate in ethanol.
Knovenagel condensation of 14 with different aromatic
aldehydes in the presence of a catalytic amount of piperidine
yielded R,β-unsaturated products (17a-h).
We next focused our efforts on converting 19 (Scheme 3)
to analogues of 1 with a modified β-carboline moiety, which
were otherwise difficult to synthesize directly from 1. 19 was
reacted with substituted tryptamines in the presence of
catalytic amounts of trifluoroacetic acid and activated
molecular sieves in DCM to yield products through Pictet-
Spengler cyclization. The Pictet-Spengler products33 were
further aromatized with DDQ to yield substituted analogues
of 1 (21a-d).
Several manzamines were reported to modulate the genera-
-
a
tion of O2 and TXB2 generated by activated rat neonatal
microglia.12,13 1 was the most potent inhibitor and did not
show in vitro toxicity to microglia, suggesting that it is a good
candidate for treatment of neuroinflammatory diseases.10,11
Recently, 1 and related derivatives have been identified as a
new class of GSK-3β inhibitors.20 Additionally, 1 was shown
to be effective in decreasing tau hyperphosphorylation in
human neuroblastoma cell lines, a demonstration of its ability
to enter cells and to interfere with tau pathology. Inhibition
studies of 1 against five different kinases related to GSK-3β
including CDK-1, PKA, CDK-5, MAPK, and GSK-3R
showed that manzamine A specifically inhibited GSK-3β
and CDK-5,20 the two kinases involved in tau pathological
hyperphosphorylation.21 These results suggest that the man-
zamines may represent a new class from which more potent
and selective GSK-3β/CDK5 inhibitors could be designed as
potential therapeutic agents for Alzheimer’s disease. In addi-
tion, these results suggest that a group of closely related
kinases may be responsible for the diverse activity seen for
this class and thattargetingMycobacterium tuberculosis(Mtb)
and plasmodial kinases may indeed be a viable approach to
drug discovery and development for Mtb and malaria.
The unique structure and extraordinary bioactivity of
manzamine alkaloids has attracted considerable interest from
synthetic chemists due to a challenging structure for total
synthesis.22-24 Methodology studies toward the synthesis of
manzamine structural units have also been reported,25-28
and a group of simplified analogues of 1 and manzamine C
were synthesized.29-31 The simplified manzamine C products
showed equal or more potent cytotoxicity than the natural
manzamine C, while simplified 1 products showed signifi-
cantly reduced antimalarial activity.
Modification of 1 using olefin metathesis has been carried
out to generate novel structures with important biological
properties.32 To better understand the structure-activity
relationship and to generate better leads as potential drug
candidates, we synthesized a number of new analogues of
manzamines and evaluated their biological activity. An eva-
luationof the activitywas made using molecular docking toan
ATP-noncompetitive binding site of human GSK-3β.
Results and Discussion
All of the above analogues were characterized by analysis
of spectral data, provided in the Experimental Section, and
were evaluated for antimalarial, antimicrobial, and anti-
inflammatory activities.
Biological Activity. In Vitro Antimalarial Activity. All
analogues were evaluated in vitro for antiprotozoal activity
against Plasmodium falciparum (D6 and W2 clones). In
addition, they were also tested for cytotoxicity against
Chemistry. To explore the structure-activity relationship,
diversity is the first thought. Modifications were carried out
a Abbreviations: TXB2, thromboxane B2; BBB, blood-brain barrier;
Mtb, Mycobacterium tuberculosis; DCM, dichloromethane; PO, oral
administration; GSK-3β, glycogen synthesis kinase-3β; HsGSK-3β,
Homo sapiens GSK-3β; PfGSK-3, Plasmodium falciparum parasite
GSK-3; AD, Alzheimer’s disease; LPS, lipopolysaccharide; HBSS,
Hanks’ balanced salt solution; PMA, phorbol 12-myristate 13-acetate.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 63
Scheme 1. Modification of Manzamine A
mammalian kidney fibroblasts (Vero cells) as shown in
Table 1. The activity profile exhibited varied results for each
analogue against Plasmodium strains. 1 still provided the
highest activity with IC50 of 4.5 ng/mL (D6 clone) and
8.0 ng/mL (W2 clone), which was comparable to the stan-
dard drugs chloroquine and artemisinin. Although toxicity
to Vero cells was observed for 1 (IC50 200 ng/mL), the selecti-
vity index (ratio of IC50 for cytotoxicity to IC50 for anti-
malarial activity) for antimalarial activity was high (44 and
25 for D6 and W2 clones, respectively). Introducing an alkyl
substituent on 9-N (6a-i) decreased the antimalarial activity
significantly in both D6 and W2 clones, showing that free
9-NH is required for antimalarial activity. Introducing lipo-
philic groups at 9N also diminished the antimalarial activity.
Substitution on other positions of the β-carboline moiety
also decreased antimalarial activity but not as much as did
the substitution at 9-NH. The hydroxyl group at position
8 (2) did not produce a significant change in antimala-
rial activity (IC50 6.0 ng/mL) but was less toxic (IC50
1100 ng/mL), yielding an improved selectivity index of 180
(D6 clone) and 140 (W2 clone). Nitration at the 6-position of
1 resulted in a slight decrease in the activity (IC50 18 ng/mL,
8), while nitration at the 8-position decreased the activity
significantly (IC50 310 ng/mL). Introduction of a 6-methoxyl
group (21a) also decreased the activity (IC50 28 ng/mL). The
methyl ester group in the 3-position in the β-carboline moiety
(21b) retained good activity (IC50 11 ng/mL). Reduction of
the pyridine ring in the β-carboline moiety (20a-d) also
decreased the activity. The above results indicated that
modification of the β-carboline ring is tolerated. Further
modification may result in a lead with a better therapeutic
index.
Reduction of the double bonds at 15 and 32 resulted in
differences in activity. When just the double bond at C-32 in
the bottom eight-membered ring of 1 or 2 was reduced (5 and
6), the activity was reduced significantly (IC50 200 ng/mL). A
second reduction of the double bond at C-15 gave 3 and 4 and
increased activities with IC50 values of 82 and 90 ng/mL,

64 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scheme 2. Modifications Beginning with Manzamine F
Peng et al.
Scheme 3. Modifications Beginning with Ircinal
respectively. These results indicate that the double bond at
C-32 is important for the activity (either bonding or con-
formation), while the double bond at 15 is not required.
Selectively reducing the double bond at C-15 in 1 and 2 may
result in more potent antimalarial products.
Table 1. In Vitro Antimalarial Activity against Plasmodium falciparum
and Cytotoxicity to Mammalian Cellsa
P. falciparum
(D6 clone)
IC50 (ng/mL)
P. falciparum
(W2 clone)
IC50 (ng/mL)
cytotoxicity (Vero)
IC50 ( μg/mL)
compd
Compound 14, differing from 2 by an additional carbonyl
functionality at position 31 and lack of a double bond at
position 32 in the bottom eight-membered ring, only showed
marginal antimalarial activity (IC50 780 ng/mL), a hundred-
fold less active than 2. Introduction of an R,β-unsaturated
system with carbonyl functionality intact (17a-h) did not
improve the antimalarial activity, nor did reduction of the
carbonyl group (16). Modification of the carbonyl func-
tional group with hydrazone (15) or alkylation (18) resulted
in significant improvement in antimalarial activity. Manza-
mine F-31-hydrazone (15) showed an IC50 of 29 ng/mL
1
2
4.5
6.0
11
8.0
8.0
15
0.2
1.1
21b
21a
0.2
0.5
28
29
58
38
15
18
NC
NC
NC
270
NC
NC
77
17
86
67
13
8
18
16
28
chloroquine
artemisinin
155
8
13
a For a full list of compounds and activity data, please see Supporting
Information. NC = no cytotoxicity up to 4.76 μg/mL.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 65
Table 2. In Vitro Antibacterial and Antifungal Activitiesa
Table 3. In Vitro Antineuroinflammatory Activitya
-
M. tuberculosis C. albicans C. neoformans M. intracellulare
IC50
O2
IC50 ( μM)
TXB2
IC50 ( μM)
LDH
LDH50 ( μM)
(H37Rv)
IC50
IC50
( μg/mL)
compd
compd MIC ( μg/mL) ( μg/mL)
( μg/mL)
1
0.1
<0.1
0.4
3.2
>10
>10
>10
>10
>10
>10
1
1.5
0.9
2.0
3.5
1.5
2.0
0.9
1.5
0.8
2.0
3.5
2.0
1.0
6.0
1.5
2.5
4.0
6.5
0.75
0.35
0.1
6d
6f
0.12
3.2
2
3
50
24
7.5
0.45
0.1
6g
21a
21b
0.07
0.1
0.07
0.11
0.11
4
20a
6
15
0.99
>64
>64
>64
1.9
0.1
0.2
0.03
a Antineuroinflammatory assay: effect of compounds on rat micro-
glia PMA [1 μM]-stimulated release of O2-, TXB2, and LDH. Data
shown corresponds to 2-7 independent experiments and is expressed as
20c
20d
>20
>20
>20
>20
2.5
0.06
<0.02
0.09
0.25
0.10
0.15
0.25
NT
21b
15
-
IC50 ( μM) for O2 and TXB2. LDH50 ( μM) is the compound’s
concentration, causing 50% percent of maximal LDH release triggered
by treating microglia with 0.1% Triton X-100. For a full list of
compounds and activity data, please see Supporting Information.
18
9
1.9
NT
1.6
1.5
8
>20
>20
15
7
14
NT
NT
NT
study. Compounds 3, 20a, and 15 showed potent activity
(IC50 0.9, 0.8, and 1.0 μg/mL, respectively), comparable with
that of amphotericin B (IC50 0.75 μg/mL), and can be
considered as leads. The remaining compounds are less
active than 1.
1 showedmoderateactivityagainstCandida albicans. Modi-
fication resulted in decreasing of activity for all products.
Antineuroinflammatory Activity. The in vitro antineuro-
inflammatory activity of natural and synthetic analogues of
ampho-
tericin B
ciproflo-
xacin
rifampin
0.20
NT
NT
NT
NT
0.30
0.09
NT
NT
NT
a For a full list of compounds and activity data, please see Supporting
Information. NT = not tested.
against P. falciparum (D6 clone), which is a ca. 27-fold
increase in activity over the mother compound. 18 showed
an IC50 of 77 ng/mL, a 1 order of magnitude increase.
Neither compound showed toxicity against Vero cells at
the highest tested concentration of 4.6 μg/mL. The improved
activity of 14 analogues clearly showed that the carbonyl
functional group is a liability for the bioactivity, and a
lipophilic functionality is preferred for the antimalarial
activity.
manzamines was investigated on phorbol 12-myristate
-
13-acetate-stimulated generation of superoxide anion (O2
)
and thromboxane B2 (TXB2) from activated rat neonatal
microglia as previously described.13,34 Lactate dehydrogen-
ase (LDH) was used as a marker for cell cytotoxicity.35 As
-
shown in Table 3, 21b potently inhibited O2 (IC50 =0.03
μM) and TXB2 (IC50=0.11 μM) release from brain micro-
glia, followed by 6g, 21a, and 6d. Furthermore, minimal
cytotoxicity, determined as lactate dehydrogenase release,
was observed for these compounds and for 1 at the highest
concentration tested in vitro (10 μM). This data thus
supports the hypothesis that these manzamine compounds
may be potential leads for the development of novel
agents to modulate activated microglia cells in neuroin-
flammatory disorders such as Alzheimer’s disease and
malaria.10,11,36-38 All other N-alkylation products (com-
pounds 6a,b,c,d,e,f) exhibited decreased activity on micro-
Antibacterial and Antifungal Activity. In addition to the
antiprotozoal assays, in vitro antimicrobial activities of the
manzamine analogues against Mycobacterium tuberculosis,
Candida albicans, Cryptococcus neoformans, and Mycobac-
terium intracellulare were investigated. Manzamines showed
moderate activity against Mycobacterium tuberculosis
(H37Rv), of which 2 exhibited the best activity with an
MIC of 0.9 μg/mL (see Table 2). In contrast, manzamine
derivatives exhibited highly potent activity against M. intra-
cellulare. Compounds 21b and 20d showed the most potent
activity, with IC50 values of <0.02 and 0.06 μg/mL, which
are about 1 order of magnitude more potent than 1 and the
positive control ciprofloxacin. 2, tetrahydro-8-hydroxy-
manzamine A (4), manzamine D (20a), 8-acetoxymanza-
mine A (9), 6-methoxymanzamine D (20c), 6-nitromanza-
mine A (8), and 8-nitromanzamine A (7) showed slightly
improved activity compared to 1 and ciprofloxacin with
IC50 values of 0.1-0.25 μg/mL, indicating that the β-carbo-
line ring can be modified to yield improved activity against
M. intracellulare. The decreased activity of the 9-N alkyla-
tion products of 1 (6a-g) revealed that the free NH is
required for the activity. Modification of the carbonyl
group of 14 gave varied results. While the R,β-unsaturated
derivatives of 14 (17a-g) diminished the activity, 15 and 18
exhibited potent activity, with IC50 values of 0.09 and
0.25 μg/mL, respectively, more than 1 order of magnitude
more potent than 14.
-
glia O2 and TXB2 generation. Hydrogenation (3, 4) also
-
appeared to decrease the inhibitory activity on O2 and
TXB2 generation. 20a derivatives showed both less activity
in the antineuroinflammatory assay and increased cyto-
toxicity to microglia cells.
Blood-Brain Barrier Permeability. Because of the efficacy
of 1 and its analogues in neuroinflammation and against
infectious agents that could cause cerebral infections (e.g.,
Cryptococcus and Plasmodium), the transport of 1 was
evaluated across MDR-MDCK monolayer-a model for in
vitro BBB transport study. The permeability of the com-
pound was observed in both absorptive and secretory direc-
tions but the efflux ratio was <1, indicating no net efflux.
The transport was linear with time (Figure 1A,B) in both
directions but drug permeated at a higher rate from the
apical side to the basolateral side (absorptive direction) of
the monolayer. The permeability of 1 HCl salt in the
absorptive direction (Papp 1.9 ꢀ 10-6 cm/s) was much higher
than that of its free base form (Papp 0.87 ꢀ 10-6 cm/s) as
shown in Figure 2. However, the permeability of 1 was much
lower in comparison to caffeine (Papp 17 ꢀ 10-6 cm/s),
which is a highly permeable drug. That 1 can permeate across
All the analogues were evaluated for activity against
Cryptococcus neoformans, and improved activities were ob-
served with several of the analogues synthesized for this

66 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
in Basel and Northwick Park Institute for Medical Research,
Harrow, UK, through a WHO contract. 1 has shown
ED50/ED90 values of 0.57/1.7 mg/kg po in Plasmodium yoelii
infected mice, which were significantly better than sodium
artesunate, which had ED50/ED90 value of 4.1/17.0 mg/kg.
Administration of 3 ꢀ 5 mg/kg of 1 subcutaneously or orally
to mice inoculated with P. berghei resulted in prolonged
mean survival times of 24.3 and 19.7 days in comparison with
14.3 days for chloroquine at 3 ꢀ 10 mg/kg po.
In our in vivo experiments, 1 was orally administered to
mice from day one after the infection at three doses, 3 ꢀ
1.1 mg/kg, 3 ꢀ 3.3 mg/kg, and 3 ꢀ 10 mg/kg. 1 was able to
produce a 75% reduction of the parasitemia level and
significantly increase the survival time of mice infected with
P. berghei with a dose of as little as 1.1 mg/kg (Table 4). A
75% cure rate of infected mice was achieved by a treatment
with 3 ꢀ 10 mg/kg of 1, while 25% cure of the infected
animals was seen at 3 ꢀ 3.3 mg/kg. This antimalarial activity
of 1 in mice was better than chloroquine at a similar dose.
We have shown that 1 has good oral bioavailability, a long
half-life of 54 h, and low plasma clearance.14 This facilitates
the possible clearance of malaria with a single treatment of 1.
For this reason, single dose schedules of 1 in mice infected
with malaria were evaluated. A single dose of 50 mg/kg
showed complete clearance of parasitemia until day 14 post
infection and was able to produce a 60% cure rate. Dosing
with 100 mg/kg or higher was found to cure all the mice
infected with P. berghei (Table 5). In contrast, treatment with
quinine (200 mg/kg) failed to cure any mice while chloro-
quine (200 mg/kg) only produced an 80% cure rate. The
untreated infected mice showed some initial weight gain
followed by marked weight loss about 10 days after the
inoculation, which correlated with the development of para-
sitemia. Treatment with 1 caused mild weight loss initially
until day 5 followed by normal weight gain thereafter. Mice
treated with 100 mg/kg of 1 did not show any behavioral
signs of toxicity. Mice treated with higher doses of 200 or
300 mg/kg began to exhibit signs of toxicity beyond weight
loss. A successful single dose treatment for malaria would be
highly significant because of its potential to reduce the
onset of drug-resistance associated with noncompliance of
multiple dose schedules.
Figure 1. Time dependent transport of 1 salt and its free base (50
μM) across MDR-MDCK monolayer.
Intraperitoneal administration of 1 and 2 (10 ꢀ 3 mg/kg)
in mice infected with malarial parasites was also evaluated
(see Table 6). 1 possesses strong antimalarial activity in vivo
with 100% suppression of the parasitemia at day 5, but it is
toxic at 10 mg/kg. No immediate adverse/toxic reactions
were noticed until day 3 post treatment with 1. 2 produced a
remarkable 80% cure with mild reversible weight loss. This
result is consistent with the in vitro data, which indicated
lower toxicity of 2 relative to 1 in Vero cells.
GSK-3β Docking. Glycogen synthase kinase 3 (GSK-3)
was originally identified as a protein kinase involved in the
regulation of glycogen metabolism and now is known to
function in a wide range of cellular processes including in the
cell life cycle, apoptosis, and development. Aberrant regula-
tion of GSK-3 has been implicated in a range of human
pathologies such as Alzheimer’s disease, type-2 diabetes,
bipolar disorder, and cancer.39-42 GSK-3 was originally
identified in mammals, and homologues have been found
in all eukaryotes. Because several of the manzamines were
recently discovered to be inhibitors of Homo sapiens GSK-3β
(HsGSK-3β),20,43,44 we performed molecular docking stu-
dies44 for the representative compound 2 with HsGSK-3β.
Figure 2. Permeability coefficient of 1 salt and its free base in
absorptive and secretory directions of MDR-MDCK monolayer.
the BBB strengthens the therapeutic value of this class of
compounds, which already have promising anti-infectious
and antineuroinflammatory activities and significant oral
bioavailability.
In Vivo Antimalarial Evaluation of 1 and 82. Initial in vivo
evaluations of 1 were carried out with the assistance of
Tropical Disease Research at the Swiss Tropical Institute

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 67
Table 4. Multiple Dose Treatments of Manzamine A in Plasmodium berghei Infected Mice
average % parasitemia (days post infection)
inhibition of
parasitemia
(%)a
compd
(dose)
7
10
14
21
28
MST days (%)b
cure (%)c
control
1 (3 ꢀ 1.1 mg/kg)
16.7
5.5
2.8
0.2
4.5
22.1
11.5
4.3
11.8 (0)
26.5 (50)
0
0
75
88
98
88
21.7
11.9
0.9
39.6
21.6
1.9
58.6
53.9
3.3
1 (3 ꢀ 3.3 mg/kg)
>30 (100)
>30 (100)
>30 (100)
25
75
0
1 (3 ꢀ 10 mg/kg)
0.4
7.8
chloroquine (3 ꢀ 5 mg/kg)
13.5
21.7
39.3
a % Decrease in level of parasitemia in mice on day 5 post infection, as compared to untreated controls. b MST: mean survival time in days (% of mice
remaining alive until day 28 post infection). c Mice without parasitemia until 28 days after the infection were considered as cure.
Table 5. Oral Single Dose Treatment of Manzamine A on Plasmodium berghei Infection in Mice
average parasitemia (%) days post infection
compd
(dose)
activity
(%)a
cure
(%)b
5
7
10
14
21
28
control
1 (50 mg/kg)
9.8
0
15.9
0
21.7
0.4
0
39.8
1.0
0
100
100
100
100
26
5.2
0
6.25
0
60
100
100
80
1 (100 mg/kg)
1 (200 mg/kg)
0
0
0
0
0
0
0
4.0
0
8.0
chloroquine (200 mg/kg)
quinine (200 mg/kg)
0
7.3
0
11.6
0
14
0.2
32
45
0
a % Decrease in level of parasitemia in mice on day 5 post infection, as compared to untreated controls. b Percentage of mice without parasitemia until
day 28 post infection.
Table 6. Antimalarial Activity of Manzamine A and 8-Hydroxyman-
zamine A by ip
8-hydroxyl group interacted with a donor NH from Arg96,
whereas the 8-hydroxyl donor hydrogen interacted with both
the backbone carbonyl of Ala204 and with an intermediary
water molecule, which in turn was hydrogen bonded to the
NH of Arg96. The tertiary nitrogen of the β-carboline ring of
2 showed water mediated hydrogen bonding with the NH of
Asp90. This may partly explain why 20a, which has a reduced
β-carboline ring, is less active than 2. A key hydrophobic
interaction between the 8-membered ring of 2 and Phe93 was
found. This may partly explain why 5, which has a reduced
8-membered ring, is less active than 2. The β-carboline ring
also showed hydrophobic interactions with the backbone of
Arg96. Thus the combination of three polar interactions and
several nonpolar interactions lead to a favorable docking
pose for 2 in the ATP-noncompetitive binding site of
HsGSK-3β.
% parasitemia suppressiona
survivalb
day of the death
treatments
day 5
day 7
(MST)c
cured
0/5
vehicle control
0/5
12/16/12/16/12
(14)
0/5
5e/6e/5e/6e/6e
5.6e
5/5 (>28)
1 10 ꢀ 3 mg/kg
2 10 ꢀ 3 mg/kg
100
100
0/5
100
4/5
a % Suppression in parasitemia is calculated by considering the mean
parasitemia in control group as 100%. b Number of animal survived day
28/total animal in group (the day of the death-post infection). c MST:
mean survival time (days). d Number of mice without parasitemia
(cured) through day 28 post infection. e Death related to toxicity.
To date, the mechanism for the diverse bioactivities of
manzamine alkaloids is still unclear. The above docking
results and our recent discovery of 1 as a selective inhibitor
of GSK-3β and CDK-5 suggest that GSK-3β and closely
related kinases may represent common targets for the diver-
sity of biological activity observed including the antimalarial
activity. Recently, Plasmodium falciparum parasite GSK-3
(PfGSK-3) was identified by Droucheau et al. and was
suggested to be a possible antimalarial target. The whole
protein sequence of PfGSK-3 is 43.1% identical and 58.9%
similar to HsGSK-3β (central catalytic domain is 58.2%
identical and 76.0% similar).50 Hence it is possible that the
antimalarial activity exhibited by manzamine and its analo-
gues is due to inhibition of Pf-kinases. However, further
experimental studies are required to prove that the antima-
larial activity of manzamines is through the inhibition of Pf-
kinases. Although PfGSK-3 shares similarity with human
GSK-3, divergent sensitivity to a series of GSK-3β inhibitors
was observed, suggesting that selective inhibitors to either
PfGSK-3 or HsGSK-3β might be identified.50 The high
activity of 1 against P. falciparum parasites and the moderate
inhibitory activity of HsGSK-3β by 1 may indicate that 1 is a
Building on the recently published evidence that 2 was an
ATP-noncompetitive inhibitor of HsGSK-3β,20,43,45 we ex-
plored the ability of 2 to bind in a pocket that is located in the
vicinity of the activation pocket formed by three basic
residues, Arg96, Arg180, and Lys205. In the first X-ray
crystal structures of HsGSK-3β46,47 a charged phosphate,
sulfate, or sulfonate-containing moiety was found to be
bound in that vicinity. This pocket was proposed to be the
binding site for thiadiazolidinones by Martinez et al. in their
reports on the first ATP-noncompetitive inhibitor.48,49 For
this work, we used an X-ray crystal structure of HsGSK-3β
without a cocrystallized ligand in the ATP-binding site but
which had a sulfate coordinated to Arg96, Arg180, and
Lys205, with Val204 and Asn203 making fairly close inter-
actions as well (pdb code: 1gng).47
The docking results showed that 2 fits well into the ATP-
noncompetitive pocket (Figures 3 and 4). The three polar
interactions exhibited by 2 are shown in Figure 4. The 8-
hydroxyl group of 2 formed significant electrostatic interac-
tions with Arg96 and Ala204. The acceptor oxygen of the

68 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
highly selective inhibitor of PfGSK-3. A new function has
been found recently for GSK-3 as a vital factor in the
inflammation process.51,52 Thus, the antineuroinflammatory
activity of manzamines may be also caused by inhibition of
GSK-3β. There is no published information about GSK-3 as
a target for antibacterial agents, but broad-spectrum kinase
inhibitors like staurosporine do indeed show antibiotic
activity.53,54
Conclusion
This structure-activity relationship study has allowed us to
refine the structural requirements for the unique manzamine
alkaloids to act against neuroinflammation and infectious
disease including malaria. A number of derivatives exhibited
highly promising activity and can be considered as potential
drug leads.
Both in vitro and in vivo data clearly revealed significant
potential for 1 and 2 analogues in the treatment of malaria.
Although toxicity has been seen in high doses and daily dosing
schedules of 1, the successful development of commonly
utilized antimalarial agents from quinine and quinolines,
despite their significant toxicity at higher doses, bodes well
for the development of a novel class of antimalarial drugs
from manzamines by optimization of both structure and drug
administration. This was evidenced by the significantly re-
duced toxicity of 2 compared to 1 in intraperitoneal admin-
istration to mice (Table 6). Highly encouraging results have
been found by modification of 14. Two derivatives with
modification of the carbonyl group of 14 (15 and 18) showed
10- to 27-fold increase in antimalarial activity without show-
ing toxicity to Vero cells. This suggests that further modifica-
tion along this direction may result in an optimized candidate
with suitably potent antimalarial activity and a better ther-
apeutic window.
One of the most deadly complications of malaria is cerebral
malaria, which is fatal in about 30-50% of the cases.36
Although the immunopathogenesis of cerebral malaria re-
mains incompletely understood,55 it appears that one of the
earliest events is an increase in the permeability of the blood-
brain barrier, probably mediated by CD8þ T cell damage to
the microvascular endothelium, an event that is followed by
leakage of cytokines, malaria antigens, and other potentially
harmful molecules into the cerebral parenchyma, with con-
comitant activation of microglia cells.36 Thus a significant
pathophysiological alteration observed in human cerebral
malaria is widespread activation and proliferation of micro-
glial cells.56,57 The antineuroinflammatory and immunosup-
pressive activity of manzamines13,58 along with their BBB
permeation is exceptional for the development of novel treat-
ments of cerebral malaria because currently there is no specific
treatment for cerebral malaria.59
Figure 3. Binding mode of docked 2 (green C, red O, blue N, cyan
H) within the ATP-noncompetitive pocket found in the vicinity of
Arg96, Arg180, and Lys205. Key amino acids in the binding pocket
for ATP-competitive inhibitors (left side of the enzyme) are labeled.
(A) Connolly molecular surface of GSK-3β, with surface red and
blue regions indicating the presence of hydrogen bond donors and
acceptors, respectively, in the protein. (B) Tube model (colors) of
the two active sites of GSK-3β (magenta, yellow and cyan indicate
R-helices, β-sheets and loops, respectively; protein side-chain gray
C, red O, blue N, cyan H).
Manzamine analogues exhibited selective activity against
Mycobacterium intracellulare. Eleven derivatives (2, 4, 7, 8, 9,
15, 18, 20a, 20c, 20d, and 21b) exhibited improved activity
compared to manzamine A and the positive control cipro-
floxacin. The two derivatives with a 3-methyl carboxylate, 21b
and 20d, were 5- to 15-fold more potent than ciprofloxacin.
Compounds 20a, 3, and 15 showed potent activity against
Cryptococcus neoformans, which are comparable with am-
photericin B. These manzamine analogues clearly have great
potential as antibiotics or antifungals, which are selectively
active against M. intracellulare and C. neoformans.
With regard to antineuroinflammatory activity, the most
effective manzamine derivatives were 21b and 6g, with 21b the
Figure 4. Key interactions of 2 within the ATP-noncompetitive
pocket of GSK-3β (gray C, red O, blue N, cyan H).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 69
best for O2- and 6g best for TXB2 release from brain micro-
glia. 21a and 6d were also highly active. The four compounds
showed no toxicity at 10 μM, suggesting they are potentially
good leads for the development of novel agents for the control
of neuroinflammatory diseases.
column, eluted with gradient acetonitrile:H2O:0.1% formic
acid, detected at 235 and 282 nm).
General Preparation of Compounds 6a-i. 1 (54.8 mg, 0.1
mmol) was dissolved in 5 mL of dry N,N-dimethylformamide
and stirred for 15 min in a Dewar flask at 0 ꢀC. To this 95% NaH
(2.33 mg, 0.12 mmol) was added with care and stirred for an
additional 5 min; methyl iodide (MeI, 15 μL, 0.24 mmol) was
added to the reaction mixture at 0 ꢀC. After 1 h, the mixture was
allowed to warm to room temperature and was stirred continu-
ously for 2-3 h. The completion of the reaction was monitored
by TLC, and the reaction mixture was slowly added to a mixture
of ice-cold water and stirred for 15 min.
The aqueous layer was extracted with Et2O (2 ꢀ 50 mL), and
the combined organic layers were washed with brine and dried
over anhydrous Na2SO4, with the solvent being removed under
reduced pressure. The crude product was chromatographed on a
250 mm ꢀ 10 mm, 5 m Luna C8 Phenomenex HPLC column
using a water and acetonitrile gradient with the flow rate of 8
mL/min. The remainder of the manzamine A analogues (6b-i)
were synthesized using the same reaction conditions.
Evidence shows that GSK-3 plays a key role in the neuro-
degenerative diseases including Alzheimer’s (AD), Parkin-
son’s, and related neurodegenerative diseases, and GSK-3
has been considered as one of the most important potential
targets for neurodegenerative disease.40-42 It is also well
established that inflammation occurs in AD and contributes
to the pathological progression of AD.60-62 Leads like man-
zamines, which have both antineuroinflammatory and GSK-
3β inhibitory activity, would have increased benefits for the
treatment of neurodegenerative diseases. Moreover, because
GSK-3 is involved in so many pathways for cell function,
selective and moderate inhibition is the best way to reach the
therapeutic goal and avoid or decrease potential side effects.
Manzamines exhibited selective, moderate inhibition to
GSK-3β, which for 1 was shown to be in a specific ATP-
noncompetitive manner,34 suggesting that they are appropri-
ate candidates for GSK-3β inhibition.
9N-Methylmanzamine A (6a): Yield 84%. 1H NMR (CDCl3)
δ 8.40 (d, J=5.2), 7.78 (d, J=5.2), 7.51 (d, J=7.8), 7.36 (t, J=
8.0), 7.56 (d, J=7.4), 7.59 (d, J=8.0), 5.78 (s), 1.59 (m), 2.23 (m),
2.42 (m), 2.79 (m), 5.54 (brs), 5.30 (brs), 1.55 (m) 1.81(m), 1.30
(m), 1.56 (m), 1.42 (m), 1.50 (m), 2.31 (m), 2.82 (m), 2.07 (m),
2.94 (m), 2.45 (m), 2.71 (m), 2.29 (m) 3.76 (s), 2.79 (m), 3.19 (m),
1.70 (m), 1.79 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m), 5.90
(brs), 5.66 (brs), 3.05 (m), 2.30 (m), 2.43 (m), 2.64 (d, J=11.3),
2.21 (d, J=11.3), 3.87 (s). 13C NMR (CDCl3) δ 142.9, 142.8,
139.5, 138.1, 136.5, 134.8, 130.5, 130.0, 129.2, 128.4, 128.1,
127.4, 123.2, 120.7, 120.5, 113.8, 113.4, 75.0, 70.1, 69.5, 63.8,
58.9, 53.6, 52.3, 51.8, 44.1, 37.9, 37.6, 33.6, 32.7, 31.7, 29.6, 28.0,
27.1, 26.7, 24.6, 20.3. HRESIMS m/z calcd for C37H47N4O [M þ
H]þ 563.3750, found 563.3764.
Molecular docking of 2 to HsGSK-3β was used to find a
binding pose in the previously identified ATP-noncompetitive
binding site, which was stabilized by a combination of three
polar interactions and several nonpolar interactions. This
opens up the exciting possibility of rational structure-based
optimization of manzamines as GSK-3β inhibitors.
Despite the diverse bioactivities which have been shown for
manzamine alkaloids, our results clearly show that promising
drug leads for treatment of malarial, bacterial, neuroinflam-
matory, and neurodegenerative diseases are achievable by
modification and optimization of derivatives of 1, which also
shows moderate BBB permeation in an in vitro model. 1 has
been shown to be highly stable at room temperature in
aqueous solution. No significant decomposition of the com-
pound was observed for over 24 months during exposure to
light at room temperature in a 0.05 N HCl aqueous solution
(20 mg/mL). A putative Micromonospora (Actinomycetes sp.)
isolated from the manzamine producing sponge has been
patented for manzamine production, but validation through
incorporation experiments remains a challenge.63 Although
considerable effort remains to optimize the culture condition
to reach a high yield of manzamines, access to a reliable
microbial source for the production of the manzamines may
provide potential cost-effective production of manzamines
without disruption to fragile marine ecosystem by excessive
sponge harvest. As a result, there is significant potential for
analogues of the manzamines class as antiparasitic-antibiotics
and in the control of neuroinflammation. This unique combi-
nation of activities could be exploited to control both the
pathogen as well as the associated inflammation.
9N-Ethylmanzamine A (6b): Yield 87%. 1H NMR (CDCl3) δ
8.38 (d, J=5.2) 7.72 (d, J=5.2), 7.57 (d, J=7.8), 7.38 (t, J=8.0)
7.51 (d, J=7.4), 7.59 (d, J=8.0), 5.73 (s), 1.56 (m), 2.24 (m), 2.47
(m), 2.76 (m) 5.55 (brs), 5.23 (brs), 1.52 (m), 1.78 (m), 1.54 (m),
1.67 (m), 1.76 (m), 1.56 (m), 2.67 (m), 2.78 (m), 2.02 (m), 2.95
(m), 2.44 (m), 2.77 (m), 2.26 (m), 3.75 (s),2.74 (m), 3.23 (m), 1.67
(m), 1.76 (m), 1.26 (m), 1.75 (m), 1.54 (m), 1.75 (m), 5.94 (brs),
5.67 (brs), 3.04 (m), 2.36 (m), 2.46 (m), 2.63 (d, J=11.3), 2.27 (d,
J=11.3), 3.84 (m), 1.53 (m). 13C NMR (CDCl3) δ 142.7, 142.6,
139.7, 138.4, 136.5, 134.6, 130.3, 130.1, 129.1, 128.2, 128.2,
127.5, 123.3, 120.7, 120.4, 113.6, 113.2, 75.2, 70.4, 69.4, 63.7,
58.9, 53.5, 52.4, 51.7, 47.8, 44.3, 37.6, 37.5, 32.5, 31.5, 29.7, 28.1,
27.3, 26.4, 24.5, 20.2, 14.1. HRESIMS m/z calcd for C38H49N4O
[M þ H]þ 577.3906, found 577.3911.
9N-Propylmanzamine A (6c): Yield 82%. 1H NMR (CDCl3) δ
8.42 (d, J=5.2), 7.81 (d J=5.2), 7.69 (d, J=7.8), 7.34 (t, J=8.0),
7.53 (d, J=7.4), 7.57 (d, J=8.0), 5.83 (s), 1.42 (m), 2.13 (m),
2.30(m), 2.61 (m), 5.59 (brs), 5.34 (brs), 1.57 (m), 1.77 (m), 1.37
(m), 1.62 (m), 1.48 (m), 1.51 (m), 2.38 (m), 2.73 (m), 2.01 (m),
2.99 (m), 2.30 (m), 2.62 (m), 2.14 (m), 3.78 (s), 2.68 (m), 3.15 (m),
1.82 (m), 1.95 (m), 1.35 (m), 1.67 (m), 1.51 (m), 1.66 (m),
5.92(brs), 5.63 (brs), 3.11 (m), 2.25 (m), 2.56 (m), 2.62 (d, J=
11.3), 2.30 (d, J=11.3), 3.83 (m), 1.81 (m), 1.27 (m). 13C NMR
(CDCl3) δ 142.7, 142.6, 139.4, 138.3, 136.8, 134.8, 130.3, 130.2,
129.6, 128.4, 128.1, 127.3, 123.1, 120.8, 120.6, 113.7, 113.5, 75.0,
70.6, 69.5, 63.4, 58.6, 54.2, 53.3, 52.7, 51.4, 44.4, 37.5, 37.3, 32.8,
31.4, 29.6, 28.4, 27.5, 26.6, 24.3, 21.3, 20.7, 13.8. HRESIMS m/z
calcd for C39H51N4O [M þ H]þ 591.4063, found 591.4067.
9N-butylmanzamine A (6d): Yield 87%. 1H NMR (CDCl3) δ
8.48 (d, J=5.2), 7.78 (d, J=5.2), 7.51 (d, J=7.8), 7.34 (t, J=8.0),
7.58 (d, J=7.4), 7.59 (d, J=8.0), 5.72 (s), 1.56 (m), 2.23 (m), 2.47
(m), 2.79 (m), 5.54 (brs), 5.30 (brs), 1.59 (m), 1.81 (m), 1.30 (m),
1.56 (m), 1.43(m), 1.50 (m), 2.31 (m), 2.86 (m), 2.08 (m), 2.94 (m),
2.45 (m), 2.71 (m), 2.25 (m), 3.77(s), 2.77 (m), 3.19 (m), 1.70(m),
1.79 (m), 1.29 (m), 1.70(m), 1.54 (m), 1.75 (m), 5.90 (brs), 5.66
(brs), 3.09 (m), 2.30 (m), 2.43 (m), 2.64 (d, J=11.3), 2.21 (d,
Experimental Section
General Experimental Procedures. The NMR data were re-
corded in CDCl3 on a Bruker DRX NMR spectrometer operat-
ing at 400 MHz for 1H and 75 MHz for 13C. Chemical shift (δ)
values are expressed in parts per million (ppm) and are refer-
enced to the residual solvent signals of CDCl3. The HRMS
spectra were measured using a Bruker MicroTOF with electro-
spray ionization. TLC analysis was carried out on silica gel G254
or aluminum oxide ALOX-100 UV254 μm. The purity of each
compound were tested to be g95% by LC/MS using an Agilent
HPLC connected to a Bruker MicroTOF with electrospray
ionization (Phenomenex Luna C18 4.6 mm ꢀ 150 mm 5 μm

70 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
J = 11.3), 3.87 (m), 1.79 (m), 1.42 (m), 1.18 (m). 13C NMR
(CDCl3) δ 142.7, 142.5, 139.3, 138.2, 136.7, 134.5, 130.1, 130.1,
129.3, 128.4, 128.2, 127.5, 123.4, 120.6, 120.4, 113.5, 113.4, 75.2,
70.3, 69.6, 63.8, 58.7, 57.1, 53.6, 52.4, 51.2, 44.6, 37.7, 37.5, 32.5,
31.3, 29.8, 29.8, 28.2, 27.6, 26.7, 24.6, 20.3, 19.1, 13.3. HRE-
SIMS m/z calcd for C40H53N4O [M þ H]þ 605.4219, found
605.4222.
1.57 (m), 1.77 (m), 1.37 (m), 1.62 (m), 1.48 (m), 1.51 (m), 2.38
(m), 2.73 (m), 2.01 (m), 2.99 (m), 2.30 (d, J = 11.3), 3.85 (t,
J=6.1), 1.68 (m), 1.57 (m), 2.31 (m), 3.92 (s). 13C NMR (CDCl3)
δ 174.8, 144.8, 144.2, 142.3, 138.6, 137.3, 134.1, 132.7, 129.9,
129.6, 128.9, 128.3(2), 122.0, 121.8, 120.0, 113.6, 112.0, 75.2,
69.2, 68.7, 63.6, 53.1, 52.7, 49.9, 49.3, 48.2, 46.0, 44.8, 42.0, 41.2,
35.8, 33.8, 29.7, 28.5, 26.9, 25.9, 25.8, 25.6, 25.4, 21.3. HRE-
SIMS m/z calcd for C42H55N4O3 [M þ H]þ 663.4274, found
663.4291.
9N-isobutylmanzamine A (6e): Yield 82%. 1H NMR (CDCl3) δ
8.47 (d, J=5.2), 7.74 (d, J=5.2), 7.57 (d, J=7.8), 7.35 (t, J=8.0),
7.57 (d, J=7.4), 7.54 (d, J=8.0), 5.75 (s), 1.56 (m), 2.24 (m), 2.46
(m), 2.74 (m), 5.55 (brs), 5.30 (brs), 1.56 (m), 1.85 (m), 1.33 (m) 1.55
(m), 1.47 (m), 1.54 (m), 2.36(m), 2.84 (m), 2.07 (m), 2.93 (m),
2.46 (m), 2.74 (m), 2.26 (m), 3.75 (s), 2.74 (m), 3.16 (m), 1.73 (m),
1.73 (m), 1.26 (m), 1.74 (m), 1.58 (m), 1.74 (m), 5.94 (brs), 5.64
(brs), 3.06 (m), 2.34 (m), 2.43 (m), 2.64 (d, J=11.3), 2.23 (d, J=
11.3), 3.82 (m), 1.76 (m), 1.23 (m), 1.23(m). 13C NMR (CDCl3) δ
142.4, 142.2, 139.2, 138.1, 136.8, 134.7, 130.5, 130.4, 129.1, 128.4,
128.2, 127.7, 123.2, 120.6, 120.6, 113.6, 113.3, 75.8, 70.5, 69.3, 63.6,
61.5, 58.4, 53.5, 52.2, 51.8, 44.2, 37.3, 37.2, 32.6, 31.6, 29.5, 28.3,
27.4. 27.2, 26.8, 24.8, 20.1, 18.6 (2C). HRESIMS m/z calcd for
C40H53N4O [M þ H]þ 605.4219, found 605.4225.
General Preparation of Compounds 3, 4, 5, and 12. 1 (54.8 mg,
0.1 mmol) was dissolved in 5 mL of ethanol, to which was added
5 mg of palladium charcoal, and stirred for 4 h under H2 gas. The
completion of the reaction was monitored by TLC, and the
solvent was removed in vacuo. The crude reaction mixture was
chromatographed on a silica gel column using hexane-ethyl
acetate gradient, yielding 3 as a major compound (68.3%) and 5
as a minor compound (6.5%). The similar reaction procedure
has been applied to 8-hydroxymanzamine A, yielding 4 as a
major compound (51.7%) and 12 as a minor compound (1.6%).
Tetrahydromanzamine A (3): Yield 68.3%. 1H NMR (CDCl3)
δ 8.64 (d, J=5.0), 7.83 (d, J=5.1), 8.09 (d, J=7.7), 7.26 (dd, J=
7.6, 7.5), 7.53 (dd, J=7.6, 7.5), 7.58 (d J=7.5), 6.61 (s), 2.02 (m),
1.83 (m), 2.30 (m), 2.21 (m), 1.53 (m), 1.68 (m), 2.57 (m), 1.57,
(m), 1.70 (m), 1.72 (m), 2.68 (m), 2.43 (m), 2.80 (m), 1.99 (m),
2.30 (m), 3.14 (m), 3.78 (s) 3.38 (m), 2.95 (m), 1.80 (m), 1.65 (m),
1.55 (m), 1.77 (m), 2.15 (m), 1.55 (m), 1.65 (m), 3.11 (m), 1.85
(m), 1.56 (m), 2.62 (brd, J=11.9), 2.30 (d, J=12.0). 13C NMR
(CDCl3) δ 142.3, 142.1, 139.4, 138.6, 136.9, 134.7, 131.4, 127.6,
123.4, 120.9, 120.7, 113.2, 112.9, 75.6, 70.4, 69.3, 63.3, 58.5, 53.3,
52.7, 51.6, 44.7, 37.6, 37.4, 34.1, 32.5, 32.3, 31.7, 31.7, 29.2, 28.0,
27.8, 27.2, 26.2, 24.9, 20.2. HRESIMS m/z calcd for C36H49N4O
[M þ H]þ 553.3906, found 553.3922.
9N-isopentylmanzamine A (6f): Yield 84%. 1H NMR (CDCl3)
δ 8.40 (d, J=5.2), 7.79 (d, J=5.2), 7.51 (d, J=7.8), 7.30 (t, J=
8.0), 7.56 (d, J=7.4), 7.59 (d, J=8.0), 5.78 (s), 1.53 (m), 2.21 (m),
2.47 (m) 2.79 (m), 5.54 (brs), 5.33 (brs), 1.55 (m), 1.85 (m), 1.33
(m), 1.59 (m), 1.44 (m), 1.53 (m), 2.36 (m), 2.82 (m), 2.07 (m),
2.94 (m), 2.46 (m), 2.75 (m), 2.28 (m), 3.72 (m), 2.75 (m), 3.16
(m), 1.78 (m), 1.77 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m),
5.92 (brs), 5.64 (brs), 3.05 (m), 2.33 (m), 2.43 (m), 2.62 (d, J=
11.3), 2.23 (d, J=11.3), 3.85 (m), 1.72 (m), 1.85 (m), 1.09 (m),
1.09 (m). 13C NMR (CDCl3) δ 142.8, 142.2, 139.6, 138.0, 136.7,
134.5, 130.3, 130.2, 129.7, 128.3, 128.2, 127.3, 123.6, 120.5,
120.4, 113.6, 113.6, 75.5, 70.6, 69.7, 63.8, 59.3, 58.2, 53.8, 52.1,
51.6, 44.1, 37.6, 37.3, 33.5, 32.4, 31.8, 29.7, 28.6, 27.4, 26.7, 26.6,
24.6, 21.8, 21.8, 20.4. HRESIMS m/z calcd for C41H55N4O [M þ
H]þ 619.4376, found 619.4381.
1
Dihydromanzamine A (5): Yield 6.5%. H NMR (CDCl3) δ
8.56 (d, J=5.0), 7.87 (d, J=5.1), 8.25 (d, J=7.7), 7.21 (dd, J=
7.6, 7.5), 7.49 (dd, J=7.6, 7.5), 7.46 (d, J=7.5), 6.59 (s), 2.10 (m),
1.87 (m), 2.36 (m), 2.22 (m), 1.52 (m), 1.63 (m), 2.52 (m), 1.53
(m), 1.79 (m), 1.79 (m), 2.61 (m), 2.47 (m), 2.87 (m), 1.93 (m),
2.33 (m), 3.18 (m), 3.72 (s), 3.31 (m), 2.97 (m), 1.84 (m), 1.67 (m),
1.53 (m), 1.73 (m), 5.89 (m), 5.61 (m), 3.19 (m) 1.81 (m), 1.53 (m),
2.61 (brd, J=11.9), 2.36 (d, J=12.0). 13C NMR (CDCl3) δ 142.9,
142.8, 139.8, 137.8, 136.2, 134.2, 130.6, 130.2, 129.7, 127.8,
123.7, 120.7, 120.2, 113.5, 113.2, 75.2, 70.8, 69.8, 63.8, 58.3,
53.9, 52.1, 51.3, 44.4, 37.8, 37.3, 32.9, 32.6, 31.8, 31.6, 28.6, 28.4,
27.6, 26.2, 24.4, 20.9. HRESIMS m/z calcd for C36H47N4O [M þ
H]þ 551.3750, found 551.3758.
9N-neopentylmanzamine A (6g): Yield 85%. 1H NMR
(CDCl3) δ 8.45 (d, J=5.2), 7.82 (d, J=5.2), 7.66 (d, J=7.8),
7.34 (t, J=8.0), 7.52 (d, J=7.4), 7.56 (d, J=8.0), 5.83 (s), 1.42
(m), 2.17 (m), 2.32 (m), 2.61 (m), 5.58 (brs), 5.34 (brs), 1.56 (m),
1.77 (m), 1.38 (m), 1.63 (m), 1.45 (m), 1.51 (m), 2.37 (m), 2.73
(m), 2.04 (m), 2.97 (m), 2.30 (m), 2.63 (m), 2.14 (m), 3.76 (s), 2.65
(m), 3.13 (m), 1.82 (m), 1.97 (m), 1.32 (m), 1.67 (m), 1.53 (m),
1.66 (m), 5.92 (brs), 5.63 (brs), 3.14 (m), 2.25 (m), 2.55 (m), 2.61
(d, J=11.3), 2.33 (d, J=11.3), 3.81 (m), 1.05 (s), 1.05 (s) 1.05 (s).
13C NMR (CDCl3) δ 142.9, 142.2, 139.9, 138.5, 136.4, 134.4,
130.7, 130.6, 129.3, 128.9, 128.3, 127.2, 123.4, 120.9, 120.7,
113.9, 113.3, 75.1, 70.2, 69.5, 68.3, 63.6, 58.5, 53.5, 52.7, 51.9,
44.6, 37.9, 37.0, 32.7, 31.4, 29.1, 28.9, 27.6, 27.2, 27.1(3C), 26.8,
24.3, 20.6. RESIMS m/z calcd for C41H55N4O [M þ H]þ
619.4376, found 618.4382.
Tetrahydro-8-hydroxymanzamine A (4): Yield 51.7%. 1H
NMR (CDCl3) δ 8.40 (d, J=5.2), 7.78 (d, J=5.2), 7.56 (d, J=
7.7), 7.03 (dd, J=7.7, 7.5), 6.96 (d, J=7.5), 6.78 (s), 2.07 (m), 1.60
(m), 1.42 (m), 1.73 (m), 1.58 (m), 1.55 (m), 1.64 (m), 1.43 (m) 2.45
(dd, J=12.0, 5.3), 2.32 (m), 2.97 (m), 2.34 (m), 2.05 (m), 1.74 (m),
3.36 (m), 3.76 (s), 3.44 (m), 2.97 (m), 1.70 (m), 2.05 (m), 1.75 (m),
1.58 (m), 1.75 (m), 1.72 (m), 2.99 (m), 2.40 (m), 2.62 (d, J=11.2),
2.18 (d, J=11.2). 13C NMR (CDCl3) δ 142.1, 139.1, 138.2, 136.6,
135.4, 132.7, 130.1, 129.7, 123.5, 120.5, 117.5, 113.5, 110.4, 75.3,
70.2, 69.8, 63.9, 58.2, 53.7, 52.4, 51.6, 44.9, 37.7, 37.7, 33.5, 32.7,
32.1, 31.9, 30.9, 29.3, 28.8, 28.6, 27.9, 26.2, 24.7, 20.3. HRE-
SIMS m/z calcd for C36H49N4O2 [M þ H]þ 569.3856, found
569.3961.
9N-dodecylmanzamine A (6h): Yield 81%. 1H NMR (CDCl3)
δ 8.40 (d, J=5.2), 7.78 (d, J=5.2), 7.51 (d, J=7.8), 7.36 (t, J=
8.0), 7.56 (d, J=7.4), 7.59 (d, J=8.0), 5.78 (s), 1.59 (m), 2.23 (m),
2.42 (m), 2.79 (m), 5.54 (br s), 5.30 (br s), 1.55 (m), 1.81 (m), 1.30
(m), 1.56 (m), 1.42 (m), 1.50 (m), 2.31 (m), 2.82 (m), 2.07 (m),
2.94 (m), 2.45 (m), 2.71 (m), 2.29 (m), 3.76 (s), 2.79 (m), 3.19 (m),
1.70 (m), 1.79 (m), 1.23 (m), 1.70 (m), 1.58 (m), 1.79 (m), 5.90
(brs), 5.66, (brs), 3.05 (m), 2.30 (m), 2.43 (m), 2.64 (d, J=11.3),
2.21 (d, J=11.3), 3.87 (s). 13C NMR (CDCl3) δ 142.7, 142.6,
139.2, 138.3, 136.8, 134.7, 130.5, 130.4, 129.8, 128.7, 128.5,
127.5, 123.5, 120.9, 120.8, 113.8, 113.7, 75.4, 70.5, 69.4, 63.5,
58.8, 57.8, 53.5, 52.2, 51.6, 44.4, 37.5, 37.4, 32.9, 31.8, 31.4 (4),
31.1, 30.3, 29.8, 29.3, 28.4, 27.8, 26.5, 26.3, 24.6, 24.4, 22.8, 20.8,
13.8. HRESIMS m/z calcd for C48H69N4O [M þ H]þ 717.5471,
found 717.5478.
1
Dihydro-8-hydroxymanzamine A (12): Yield 16%. H NMR
(CDCl3) δ 8.31 (d, J=5.2), 7.81 (d, J=5.2), 7.58 (d, J=7.6), 7.12
(dd, J=7.7, 7.6), 7.05 (d, J=7.6), 6.18, (s), 1.55 (m), 1.45 (m),
1.46 (m), 1.55 (m), 1.57 (m), 1.66 (m), 1.70 (m), 2.58 (m), 2.25 (m)
3.11 (m), 2.03 (m), 2.34 (m), 1.67 (m), 1.85 (m), 4.93 (s), 4.65
(brd, J=4.9), 4.24 (m), 1.70, (m), 2.02 (m), 1.65 (m), 1.50 (m),
5.92 (m), 5.67 (m), 2.99 (m), 2.60 (m), 2.52 (d, J=11.3) 2.20 (m).
13C NMR (CDCl3) δ 142.6, 142.3, 139.4, 138.7, 136.9, 134.7,
133.9, 130.8, 130.6, 129.6, 127.8, 123.7, 120.3, 120.2, 113.2, 75.2,
70.9, 69.8, 63.5, 58.4, 53.4, 52.7, 51.7, 44.4, 37.9, 37.3, 32.3, 32.2,
31.8, 31.4, 29.4, 28.6, 27.2, 26.9, 24.3, 20.9. HRESIMS m/z calcd
for C36H47N4O2 [M þ H]þ 567.3699, found 567.3708.
9N-(4-methylcarboxybutyl)manzamine A (6i): Yield 76%. 1H
NMR (CDCl3) δ 8.42 (d, J = 5.2), 7.81 (d, J = 5.2), 7.69 (d,
J=7.8), 7.34 (t, J=8.0), 7.53 (d, J=7.4) 7.57 (d, J=8.0), 5.83 (s),
1.42 (m), 2.13 (m), 2.30 (m), 2.61 (m), 5.59 (brs), 5.34 (brs),

Article
General Preparation of Compounds 10 and 11. 1 (54.8 mg, 0.1
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 71
21,27-N-oxamanzamine A (13): Manzamine A (1) (56 mg) and
50 mg of 3-chloroperoxybenzoic acid were dissolved in 1 mL of
CH2Cl2. After 20 mg of sodium bicarbonate was added, the
solution was stirred overnight at room temperature. The reac-
tion mixture was washed with 5% sodium bicarbonate solution
and extracted with chloroform. The chloroform layer was
evaporated, and the residue was purified using silica gel pre-
parative TLC (CHCl3:MeOH 85:15) to get 13 (20 mg). 21,27-N-
oximanzamine A (13): yellow powder. 1H NMR (CDCl3-
CD3OD) δ 9.21 (s), 8.35 (d, J=5.2), 8.08 (d, J=7.6), 7.82 (d,
J=5.2), 7.49 (m), 7.26 (t, J=7.6), 6.21 (dt, J=10, 7.6), 6.12 (s),
5.71 (t, J=10), 5.60 (br), 5.44 (br), 4.49 (m), 3.91 (d, J=13.6),
3.84 (s), 3.35 (m), 3.11 (d, J=10.4), 2.97 (s), 2.72 (m), 2.51 (m),
2.29 (m), 2.10-1.39 (m). 13C NMR (CDCl3) δ 142.2, 140.7,
139.7, 137.6, 136.7, 133.4, 133.3, 133.2, 132.3, 130.7, 129.7,
128.8, 126.4, 121.5, 121.1, 120.1, 114.3, 112.0, 74.5, 73.7, 70.4,
61.9, 61.4, 49.6, 46.5, 43.1, 38.8, 37.5, 27.7, 25.7, 23.1, 23.1, 22.2,
19.4. HRESIMS m/z calcd for C36H45N4O3 581.3492 [M þ H]þ
found 581.3474 [M þ H]þ, 603.3301 [M þ Na]þ, 563.3359 [M -
mmol) was dissolved in 15 mL of dry acetone, with 16.4 mg (1.2
mmol) of anhydrous K2CO3 and 11 μL of anhydrous acetic
anhydride added. The reaction was refluxed for 9 h under
nitrogen. The completion of the reaction was monitored by
TLC, and the solvent was removed in vacuo. Then 100 mL of
ethyl acetate was added and washed with an equal amount of
brine solution. This was repeated twice and the organic layer
was dried on Na2SO4 (200 g), and the solvent was removed in
vacuo. The reaction mixture was chromatographed on a silica
gel column using hexane-ethyl acetate gradient yielded com-
pound 10 (49.8%). The similar reaction procedure has been
applied to 8-hydroxymanzamine A (2) to yield the compound 11
(46.4%).
12,13-Dehydromanzamine A (10): Yield 49.8%. 1H NMR
(CDCl3) δ 8.41 (d, J=5.3), 7.81 (d, J=5.3), 7.96 (d, J=7.5),
7.14 (dd, J=7.6, 7.5), 7.30 (d, J=7.6), 7.68 (d, J=7.6), 6.60 (s),
6.45 (dd, J=8.5, 7.0), 2.12 (m), 5.43 (m), 5.35 (m), 1.89 (m), 1.73
(m) 1.38 (m), 1.92 (m), 2.62 (m), 2.51 (m), 2.71 (m), 1.98 (m), 2.73
(m), 2.41 (m), 3.96 (m), 4.88 (s), 3.85 (dd, J=14.7, 4.7), 3.34 (dd,
J=14.7, 10.8), 1.38 (m), 1.88 (m), 1.60 (m), 2.15 (m), 5.87 (dd, J=
7.7, 7.6), 5.24 (m), 2.41 (m), 1.46 (d, J=12.9), 2.62 (d, J=10.9),
2.51 (d, J=10.9). 13C NMR (CDCl3) δ 142.9, 142.6, 142.4, 139.7,
138.5, 134.3, 130.8, 130.7, 129.7, 129.3, 127.8, 127.6, 123.6,
120.9, 120.8, 118.3, 115.8, 113.7, 113.7, 75.9, 69.6, 63.4, 58.7,
53.3, 52.9, 51.3, 44.8, 37.3, 32.3, 31.5, 29.3, 28.8, 27.8, 26.5, 24.8,
21.1. HRESIMS m/z calcd for C36H43N4 [M þ H]þ 531.3488,
found 531.3495.
H2O]þ, 291.1766 [M þ 2H]2þ, 282.1719 [M þ 2H - H2O]2þ
.
General Preparation of Compounds 20a-d and 21a-d. Meth-
od A: 19, tryptamine derivatives, and molecular sieves were
stirred at room temperature in CH2Cl2 or EtOH (3 mL) for
2 h. TFA (50 μL) was then added to the reaction mixture and
further stirred for another 3 h at 50 ꢀC. The reaction mixture
was chromatographed over silica gel immediately to give the
Pictet-Spengler product.
12,13-Dehydro-8-O-acetylmanzamine A (11): Yield 46.4%.
1H NMR (CDCl3) δ 8.38 (d, J=5.6), 7.82 (d, J=5.6), 7.97 (d,
J=7.8), 7.23 (dd, J=7.8, 7.6), 7.29 (d, J=7.6), 6.50 (s) 6.43 (dd,
J=8.5, 7.0), 2.21 (m), 1.80 (m), 5.56 (m), 5.50 (m), 1.65 (m), 1.42
(m), 1.30 (m), 1.40 (m), 1.32 (m), 2.60 (m), 2.43 (m), 2.83 (m),
1.95 (m), 2.81 (m), 2.44 (m), 3.50 (s), 3.14 (dd, J=11.9, 8.7), 2.99
(dd, J=11.8, 4.7), 1.76 (m), 2.15 (m), 2.05 (m), 2.42 (m), 5.84 (dd,
J=7.7, 7.6), 5.21 (m), 3.99 (dd, J=8.3, 8.2), 2.42 (m), 1.49 (d, J=
12.9), 2.76 (d, J=11.5), 2.30 (m), 2.53 (s). 13C NMR (CDCl3) δ
167.8, 142.8, 142.8, 142.6, 139.2, 138.7, 136.9, 134.5, 134.4,
130.6, 130.6, 129.4, 128.3, 128.1, 127.8, 123.9, 120.2, 120.1,
117.4, 113.3, 75.8, 69.5, 63.5, 58.6, 53.9, 52.7, 51.2, 44.7, 37.2,
32.2, 31.4, 29.3, 28.2, 27.8, 26.4, 24.3, 20.2, 17.8.; HRESIMS m/z
calcd for C38H45N4O2 [M þ H]þ 589.3543, found 589.3548.
8-Acetoxymanzamine A (9): see ref 64.
Method B: To 19 and tryptamine derivatives in 1 mL anhy-
drous EtOH were added 0.2 mL of trifluoroacetic acid (TFA).
The mixture was then absorbed onto 1.5 g of silica gel. The silica
gel mixture was placed in a glass vessel and was irradiated in a
microwave oven at 390 W for 10 min. The crude mixture was
then eluted from the silica gel using acetone, concentrated, and
chromatographed over silica gel to furnish the Pictet-Spengler
product.
Manzamine D (20a): 19 (46.3 mg) and tryptamine (31 mg) in
CH2Cl2 were reacted as described in method A to give manza-
mine D (20a, 52.7 mg, 86%). Manzamine D (20a): white
1
amorphous. H NMR (CDCl3) δ 7.78 (br s), 7.51 (d, J=7.5),
7.35 (brd, J=7.5), 7.17 (t, J=7.5), 7.12(t, J=7.5), 5.95 (dd, J=
18.7), 5.77 (brs), 5.67 (q, J=9), 5.55 (td, J=10, 5), 5.30 (t, J=10),
4.60 (s), 4.17 (t, J = 7), 3.42 (s), 3.35 (m), 3.04 (m), 2.83(m),
2.75(m), 2.51 (m), and 1.20-2.40 (complex). 13C NMR (CDCl3)
δ 141.2, 136.8, 135.6, 134.5, 133.9, 132.2, 129.9, 128.8, 127.8,
121.4, 119.2, 118.0, 111.0, 109.4, 75.3, 69.6, 68.5, 59.7, 54.9, 53.4,
50.8, 49.7, 47.3, 44.6, 43.2, 40.7, 37.8, 33.0, 31.8, 28.2, 26.9, 26.0,
25.7, 22.4, 21.7. HRESIMS m/z calcd for C36H49N4O [M þ H]þ
553.3906, found 553.3898.
2N-Methylmanzamine D (20b): 19 (50.2 mg) and (2N)-methyl-
tryptamine (30.2 mg) in CH2Cl2 were reacted as described in
method A to give N(2)-methylmanzamine D (20b, 22.5 mg,
33%) and unreacted ircinal A (19, 27.9 mg). N(2)-methylman-
zamine D (20b): yellowish oil. 1H NMR (CDCl3) δ 7.47 (br s),
7.46 (d, J=7.6), 7.31 (brd, J=7.6), 7.12 (t, J=7.2), 7.07 (t, J=
7.2), 5.88 (q, J=8), 5.80 (s), 5.61 (q, J=9), 5.48 (td, J=10.5), 5.23
(t, J=9), 4.16 (brs), 3.82 (s), 3.40 (s), 3.11 (m), 2.99 (m), 2.88 (m),
2.40 (s), and 1.2-2.7 (complex). 13C NMR (CDCl3) δ 141.2,
136.5, 135.9, 134.8, 134.1, 132.3, 129.8, 128.9, 121.5, 119.5,
118.2, 111.3, 108.6, 75.5, 69.5, 68.5, 68.1, 55.3, 53.9, 53.6, 51.2,
49.9, 47.2, 44.6, 41.7, 37.1, 32.4, 32.2, 30.0, 29.5, 28.3, 27.1, 26.3,
25.9, 21.9, 21.6. HRESIMS m/z calcd for C37H51N4O [M þ H]þ
567.4063, found 567.4042.
6-Nitro-manzamine A (8) and 8-nitromanzamine A (7): Manza-
mine A (1) (55 mg) was dissolved in 3 mL of trifluoroacetic acid
and cooled to 0 ꢀC in ice bath. NaNO2 (11 mg) was added in one
portion and stirred at 0 ꢀC for 1.5 h. The mixture was poured
into water and the precipitate was collected by centrifuge. The
precipitate was separated using silica gel preparative TLC
(hexane:acetone:triethylamine 8:2:0.1) to yield 8 (6 mg) and 7
1
(6 mg). 6-Nitro-manzamine A (8): yellow powder. H NMR
(CDCl3) δ 9.04 (d, J=2.0), 8.49 (d, J=5.2), 8.41 (dd, J=9.2, 2.0),
7.89 (d, J=5.2), 7.77 (d, J=9.2), 6.50 (s), 6.19 (s), 5.58 (m), 5.38
(t, 10.8), 4.70 (br), 3.69 (s), 3.25 (t, 11.0), 2.93 (d, J = 9.0),
2.80-2.20 (m), 2.10-1.20 (m). 13C NMR (CDCl3) δ 144.5,
144.2, 142.7, 141.2, 141.1, 139.4, 135.7, 134.5, 133.2, 129.7,
127.0, 123.7, 123.5, 121.0, 118.4, 114.2, 112.9, 77.9, 71.2, 70.5,
57.6, 53.7 (2C), 49.4, 47.1, 44.9, 40.8, 39.5, 33.9, 28.7, 26.6, 26.6,
25.2, 24.8, 24.3, 20.9. HRESIMS m/z calcd for C36H44N5O3.
[M þ H]þ 594.3444, found 594.3435. 8-Nitromanzamine A (7):
1
yellow powder. H NMR (CDCl3) δ 10.4 (s), 8.57 (d, J=5.2),
8.48 (d, J=8.0), 8.45 (d, J=8.0), 7.87 (d, J = 5.2), 7.40 (t, 8.0),
6.45 (s), 5.96 (m), 5.69 (m), 5.55(m), 5.32 (t, J = 10), 4.27
(br), 3.58 (s), 3.11 (m), 2.61 (m), 2.50-1.6 (m), 1.4 (m). 13C
NMR (CDCl3) δ 144.6, 1443.6, 142.7, 141.4, 141.2, 139.4, 135.3,
134.6, 133.2, 130.0, 127.1, 123.8, 123.7, 121.1, 118.5, 114.4,
113.0, 77.8, 71.2, 70.6, 57.7, 53.7, 53.72, 53.68, 49.5, 47.1, 44.8,
40.5, 39.8, 34.1, 28.8, 26.7, 26.6, 25.3, 24.9, 24.3, 21.0. HRE-
SIMS m/z calcd for C36H44N5O3. [M þ H]þ 594.3444, found
594.3426.
6-Methoxymanzamine D (20c): 19 (38.8 mg) and 5-methoxy-
tryptamine (6, 56.1 mg) in CH2Cl2 were reacted as described in
method A to give 6-methoxymanzamine D (20c, 48.6 mg, 89%).
Ircinal A (19, 29.5 mg) and 5-methoxytryptamine (26.4 mg) in
anhydrous EtOH were reacted as described in method B to give
6-methoxymanzamine D (20c, 35.1 mg, 83%). 6-Methoxyman-
1
zamine D (20c): white amorphous. H NMR (CDCl3) δ 7.52

72 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
(brs), 7.21 (d, J=8.8), 6.93 (d, J=1.6), 6.79 (dd, J=8.8, 1.6), 5.90
(q, J=8.3), 5.73 (brs), 5.62 (q, J=8.5), 5.50 (td, J=10.4, 4.4),
5.22 (t, J= 9.2), 4.14 (brs), 3.85 (s), 3.33 (m), and 1.00-3.20
(complex). 13C NMR (CDCl3) δ 153.4, 141.1, 136.8, 134.8,
134.5, 132.2, 130.6, 129.9, 128.7, 128.2, 111.6, 111.1, 109.2,
100.4, 75.3, 69.5, 68.5, 59.8, 55.9, 54.8, 53.4, 50.7, 49.6, 47.3,
44.5, 43.2, 40.6, 37.7, 33.0, 31.8, 28.5, 26.8, 25.9, 25.7, 22.5, 21.7.
HRESIMS m/z calcd for C37H51N4O2 [M þ H]þ 583.4012,
found 583.4033.
Methyl Manzamine D-3-carboxylate (20d): 19 (83.2 mg) and
D-tryptophan methyl ester HCl (67.1 mg) in EtOH were reacted
as described in method A to give 20d (87.6 mg, 70%). Ircinal A
(19, 105.6 mg) and D-tryptophan methyl ester HCl (67.7 mg) in
anhydrous EtOH were reacted as described in method B to give
20d (95.4 mg, 61%). 20d: white amorphous. 1H NMR (CDCl3) δ
7.60 (brs), 7.48 (d, J=7), 7.35 (d, J=7), 7.16 (td, J=7.1), 7.10 (t,
J=7), 5.90 (q, J=10), 5.86 (brs), 5.64 (q, J=8), 5.52 (td, J=10.4),
5.22 (t, J=9), 4.75 (brs), 4.17 (t, J=6), 3.82 (s), 3.37 (br s), 3.12
(brd, J=15), 3.02 (m), 2.88 (brt, J=12), 2.65 (d, J=11), and
1.20-2.55 (complex). 13C NMR (CDCl3) δ 173.4, 171.1, 142.8,
136.2, 132.7, 132.6, 127.5, 127.1, 121.7, 119.3, 117.8, 111.4,
108.7, 70.5, 70.1, 60.4, 59.7, 56.8, 56.2, 53.4, 52.5, 52.1, 49.3,
47.1, 44.2, 40.0, 37.7, 32.9, 28.3, 26.5, 25.7, 25.1, 24.8, 24.6, 21.0,
21.0, 14.2. HRESIMS m/z calcd for C38H51N4O3 [M þ H]þ
611.3961, found 611.3947.
General Preparation of Compounds (21a) and (21b). To a
solution of manzamine D derivatives in CH2Cl2 (2 mL) at room
temperature was added DDQ (2,3-dichloro-5,6-dicyanobenzo-
quinone) in benzene (2 mL) and the mixture was stirred for 1 h.
The mixture was then washed with saturated NaHCO3 (5 mL),
extracted with CH2Cl2 (2 ꢀ 5 mL), dried (MgSO4), and evapo-
rated to give brownish oil. Chromatography of the reaction
products over silica gel gave the products.
6-Methoxymanzamine A (21a): 20c (141.52 mg) and DDQ
(69.5 mg) were reacted as described above to give 6-methoxy-
manzamine A (21a, 46.4 mg, 33%). 6-Methoxymanzamine A
(21a): yellowish oil. 1H NMR (CDCl3) δ 8.63 (brs), 8.40 (d, J=
5), 7.78 (d, J=5), 7.53 (d, J=2), 7.40 (dd, J=8, 2), 6.40 (s), 5.91
(brs), 5.62 (q, J=8.5), 5.52 (td, J=10.5), 5.26 (t, J=9), 4.31 (brs),
3.92 (s), 3.50 (d, J=4), 3.08 (m), 2.78 (m), 2.56 (m), and 1.2-2.5
(complex). 13C NMR (CDCl3) δ 154.2, 143.5, 139.9, 138.2,
137.4, 135.1, 134.5, 134.2, 132.3, 129.9, 129.2, 128.5, 122.2,
118.3, 117.2, 113.3, 112.5, 103.5, 75.1, 70.0, 68.7, 56.0, 55.0,
53.5, 50.9, 49.6, 47.1, 44.7, 40.9, 40.3, 32.7, 31.7, 28.1, 26.8, 26.0,
25.7, 21.7. HRESIMS m/z cacld for C37H47N4O2. [M þ H]þ
579.3699, found 579.3704.
7.6), 7.00 (t, J=7.6), 6.92 (s) 6.89 (d, J=7.6), 6.39 (s), 5.02 (m),
5.40 (ddd, J=10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt,
J=4.5), 2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16
(m), 1.99 (m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m).
HRESIMS m/z calcd for C44H51N4O4 [M þ H]þ 699.3910,
found 699.3888. [M þ 2H]þ2 350.2013.
31-(4-Methoxyphenyl)manzamine F (17b): Yield 50%. 1H
NMR (CDCl3) δ 8.22 (d, J=5.2), 7.71 (d, J=5.2), 7.50 (d, J=
7.6), 7.00 (t, J=7.6), 6.92 (s) 6.89 (d, J=7.6), 6.39 (s), 5.01 (m),
5.40 (ddd, J=10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt,
J=4.5), 2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16
(m), 1.99 (m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). 13C
NMR (CDCl3) δ 197.3, 159.3, 143.5, 143.3, 143.3, 140.2, 138.6,
138.2, 133.6, 132.3, 132.0, 131.8, 130.9, 129.7, 128.6, 123.4,
121.7, 121.2, 114.1, 113.6, 81.3, 76.2, 68.7, 63.6, 55.2, 53.1,
52.8, 49.7, 47.2, 46.6, 41.8, 41.5, 39.0, 33.3, 33.1, 26.7, 25.4,
24.9, 24.1, 21.8. HRESIMS m/z calcd for C44H50N4O4 [M þ H]þ
699.3910, found 699.3887. [M þ 2H]þ2 350.1977.
1
31-(4-Nitrophenyl)manzamine F (17c): Yield 45%. H NMR
(CDCl3) δ 8.21 (d, J=5.2), 7.7 (d, J=5.2), 7.50 (d, J=7.6), 7.00
(t, J=7.6), 6.92 (s) 6.89 (d, J=7.6), 6.39 (s), 5.01 (m), 5.40 (ddd,
J=10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, J=4.5), 2.84 (brs),
2.64 (brd, J = 10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99 (m),
1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). HRESIMS m/z
calcd for C43H48N5O5 [M þ H]þ 714.3655, found 714.3687.
[M þ 2H]þ2 357.6822.
31-(2,6-Dichlorophenyl)manzamine F (17d): Yield 40%. 1H
NMR (CDCl3) δ 8.20 (d, J=5.2), 7.71 (d, J=5.2), 7.52 (d, J=
7.6), 7.00 (t, J=7.6), 6.92 (s) 6.89 (d, J=7.6), 6.40 (s), 5.01 (m),
5.41 (ddd, J=10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, 4.5),
2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99
(m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). HRESIMS m/z
calcd for C43H47Cl2N4O3 [M þ H]þ 737.3025, found 737.3028.
[M þ 2H]þ2 369.1557.
31-(3,4-Dimethoxyphenyl)manzamine F (17e): Yield 47%. H
NMR (CDCl3) δ 8.20 (d, J=5.2), 7.71 (d, J=5.2), 7.50 (d, J=
7.6), 7.00 (t, J=7.6), 6.91 (s) 6.89 (d, J=7.6), 6.39 (s), 5.01 (m),
5.41 (ddd, J=10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt,
J=4.5), 2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16
(m), 1.99 (m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m).
HRESIMS m/z calcd for C45H53N4O5 [M þ H]þ 729.4016,
found 729.4018. [M þ 2H]þ2 365.2025.
31-(4-Bromophenyl)manzamine F (17f): Yield 40%. 1H NMR
(CDCl3) δ 8.23 (d, J=5.2), 7.72 (d, J=5.2), 7.50 (d, J=7.6), 7.00
(t, J=7.6), 6.94 (s), 6.89 (d, J=7.6), 6.39 (s), 5.01 (m), 5.40 (ddd,
J=10.8, 10.8, 4), 3.91 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J=4.5),
2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99
(m), 1.77-1.50(m), 1.45 (m), 1.31 (m), 1.06 (m). HRESIMS m/z
calcd for C43H48BrN4O3 [M þ H]þ 747.2910, found 747.2930.
[M þ 2H]þ2 374.1516.
Methyl Manzamine A-3-carboxylate (21b): 20d (26.9 mg) and
DDQ (17.3 mg) were reacted as described above to gave 21b
1
(18.1 mg, 68%). 21b: yellowish oil. H NMR (CDCl3) δ 9.25
(brs), 8.92 (s), 8.32 (d, J=8), 7.77 (d, J=8), 7.74 (t, J=8), 7.50 (t,
J=8), 6.59 (s), 6.11 (br s), 5.79 (q, J=8), 5.70 (td, J=10.5), 5.44
(t, J=9), 4.20 (s), 3.72 (brs), and 1.4-3.4 (complex). 13C NMR
(CDCl3) δ 166.8, 143.4, 140.4, 139.5, 138.2, 137.2, 134.8, 132.3,
129.7, 129.3, 128.7, 128.4, 122.1, 121.6, 120.9, 116.5, 112.1, 74.9,
69.9, 68.6, 55.1, 53.6, 52.4, 50.9, 49.6, 47.2, 46.9, 44.8, 40.8, 40.5,
34.7, 33.1, 31.6, 28.2, 26.8, 25.9, 25.7, 21.6. HRESIMS m/z cacld
for C38H47N4O3. [M þ H]þ 607.3648, found 607.3655.
General Preparation of Compounds 17a-g. 14 (54.8 mg, 0.1
mmol) was dissolved in 15 mL of chloroform and a catalytic
amount of piperidine was added to the aromatic aldehyde (2.33
mg, 0.12 mmol) and stirred for an additional 5 min and then
refluxed for 8 h. The completion of the reaction was monitored
by TLC, and the chloroform in reaction mixture was evaporated
and the residue was washed with water and extracted with
chloroform. The crude product was chromatographed on a
silica gel column using a hexane/acetone (7:3). The analogues
of 14 (17a-g) were synthesized using the same reaction condi-
tions.
31-(4-Fluorophenyl)manzamine F (17g): Yield 45%. 1H NMR
(CDCl3) δ 8.21 (d, J=5.2), 7.71 (d, J=5.2), 7.50 (d, J=7.6), 7.00
(t, J=7.6), 6.92 (s) 6.89 (d, J=7.6), 6.39 (s), 5.01 (m), 5.40 (ddd,
J=10.8, 10.8, 4), 3.90 (brs), 3.80 (s), 3.64 (s), 3.23 (brt, J=4.5),
2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99
(m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). HRESIMS m/z
calcd for C43H48FN4O3 [M þ H]þ 687.3710, found 687.3713.
[M þ 2H]þ2 344.1875.
31-(2-Chlorophenyl)manzamine F (17h): Yield 45%. 1H NMR
(CDCl3) δ 8.22 (d, J=5.2), 7.73 (d, J=5.2), 7.51 (d, J=7.6), 7.02
(t, J=7.6), 6.93 (s) 6.89 (d, J=7.6), 6.39 (s), 5.03 (m), 5.42 (ddd,
J=10.8, 10.8, 4), 3.92 (brs), 3.82 (s), 3.64 (s), 3.23 (brt, J=4.5),
2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16 (m), 1.99
(m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). HRESIMS m/z
calcd for C43H48ClN4O3 [M þ H]þ 703.3415, found 703.3445.
[M þ 2H]þ2 352.1750.
Manzamine F-31-hydrazone (15): 14 (58 mg) 2 mL of anhy-
drous ethanol. Hydrazine hydrate (200 μL) was added and
refluxed for 2 h. The temperature was allowed to cool down to
room temperature and 15 crystals formed. The mother liquid
31-(3-Methoxyphenyl)manzamine F (17a): Yield 55%. 1H
NMR (CDCl3) δ 8.21 (d, J=5.2), 7.71 (d, J=5.2), 7.50 (d, J=

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 73
was purified using silica gel preparative TLC (CHCl3:MeOH
95:5), which gave additional amount of 15. 15: pale yellow
crystals. 1H NMR δ 9.13 (s), 8.43 (d, J=4.4), 8.09 (d, J=7.6),
7.82 (d, J=4.4), 7.53 (br), 7.25 (s), 6.42 (s), 5.90 (m), 5.62 (m),
5.52 (m), 5.26 (t, J=9.6), 4.35 (br), 3.52 (s), 3.05 (m), 2.78 (m),
2.58 (m), 2.43-1.86 (m), 1.80-1.3 (m). HRESIMS m/z calcd for
C36H47N6O2 [M þ H]þ 595.3761, found 595.3782.
Mycobacterium intracellulare ATCC 23068. Susceptibility test-
ing was performed using a modified version of the CLSI
(formerly NCCLS) methods.68-70 M. intracellulare is tested
using a modified method of Franzblau, et al.71 Samples were
serially diluted in 20% DMSO/saline and transferred in dupli-
cate to 96-well flat bottom microplates. Microbial inocula were
prepared by correcting the OD630 of microbe suspensions in
incubation broth to afford final target inocula. Drug controls
(ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and am-
photericin B (ICN Biomedicals, Ohio) for fungi) were included
in each assay. All organisms were read at either 630 nm using the
EL-340 Biokinetics Reader (Bio-Tek Instruments, Vermont) or
544ex/590em, (M. intracellulare) using the Polarstar galaxy
plate reader (BMG LabTechnologies, Germany) prior to and
after incubation.
31β-Hydroxymanzamine F (16): First, 58 mg of 14 was
dissolved in 1 mL of methanol and cooled to 0 ꢀC. Then 20
mg of NaBH4 was added and stirred for 10 min. Then 10 mL of
water was added and extracted with ether 5 mL ꢀ 3. Ether was
evaporated and the product was purified by silica gel prepara-
tive TLC (CHCl3:MeOH 9:1) to yield 16 (24 mg). 16: yellow
powder. 1H NMR (CDCl3) δ 8.21 (d, J=5.2), 7.71 (d, J=5.2),
7.50 (d, J=7.6), 7.00 (t, J=7.6), 6.89 (d, J=7.6), 6.39 (s), 5.01
(m), 5.40 (ddd, J=10.8, 10.8, 4), 3.90 (brs), 3.64 (s), 3.23 (brt, J=
4.5), 2.84 (brs), 2.64 (brd, J=10.8), 2.44 (m), 2.16 (m), 2.16 (m),
1.99 (m), 1.77-1.50 (m), 1.45 (m), 1.31 (m), 1.06 (m). 13C NMR
(CDCl3) δ 137.8, 136.7, 132.6, 127.6, 120.9, 113.8, 113.2, 112.5,
81.8, 70.3, 68.5, 63.2, 53.3, 52.6, 49.4, 48.0, 41.7, 40.4, 36.5, 35.5,
33.0, 30.8, 26.5, 25.5, 25.1, 24.9, 21.5. HRESIMS m/z calcd for
C36H47N4O3 [M þ H]þ 583.3648, found 583.3664.
Antineuroinflammatory Assay. Rat neonatal microglia (2 ꢀ
105 cells) were seeded into each well of 24-well flat-bottom
culture clusters and stimulated with Escherichia coli lipopoly-
saccharide (LPS) (0.3 ng/mL) in Dulbecco’s modified Eagle
medium þ 10% fetal bovine serum þ penicillin þ streptomycin
for 17 h in a humidified 5% CO2 incubator at 35.9 ꢀC as
described.13,34 Media were then removed, microglia washed
with warm (37 ꢀC) Hanks’ balanced salt solution (HBSS), and
then incubated with compounds (0.01-10 μM) or vehicle
(DMSO) for 15 min prior to stimulation with phorbol 12-
myristate 13-acetate (PMA) (1 μM). All experimental treat-
ments were run in duplicate and in a final volume of 1 mL.
Then 70 min after PMA stimulation, HBSS was aspirated from
each well and superoxide anion (O2-), thromboxane B2 (TXB2),
and lactated dehydrogenase (LDH) release were determined as
described.13,34 Table 3 shows data from 1-3 experiments and is
expressed as the compound’s inhibitory concentration 50%
31-Ethylmanzamine F (18): 14 (58 mg) was dissolved in 2 mL
of tetrahydrofuran and cooled in dry ice acetone bath, then 10
equiv of 3 M EtMgBr in ether was added. The solution was
warmed up to room temperature and stirred for 24 h. The
mixture was poured into 5% NH4Cl under stirring and ex-
tracted using chloroform. The chloroform extract was purified
using silica gel preparative TLC (CHCl3:MeOH 85:15) twice to
yield 18 (18 mg). 18: yellow powder. 1H NMR (CDCl3) δ 11.15
(s), 10.1 (s), 8.80 (s), 8.35 (d, J=4.8), 7.82 (d, J=4.2), 7.59 (d, J=
7.6), 7.13 (t, J=7.6), 7.05 (d, J=7.6), 6.88 (s), 4.19 (s), 3.31 (t, J=
12.8), 2.98 (d, J=12.8), 2.83 (m), 2.55 (m), 2.26-2.40 (m), 2.16
(d, J=11.6), 2.00 (t, J=10.8), 1.80 (m), 1.53-1.22 (m), 0.88 (t, J=
7.2). 13C NMR (DEPT 135) δ 137.9, 134.1, 132.8, 127.6, 121.1,
114.3, 114.2, 112.0, 82.4, 67.7, 64.4, 54.2, 53.2, 49.8, 45.2, 43.4,
40.5, 38.8, 37.1, 34.3, 33.5, 26.9, 25.5, 24.9, 24.6, 23.7, 21.3, 7.87.
HRESIMS m/z calcd for C38H51N4O3 [M þ H]þ 611.3961,
found 611.3953.
In vitro Antimalarial Assay. Antimalarial activity was deter-
mined in vitro against chloroquine sensitive (D6, Sierra Leone)
and resistant (W2, Indo China) strains of Plasmodium falciparum by
measuring plasmodial LDH activity as described earlier.43,65 Test
compounds were dissolved in DMSO (2 mg/mL). A 200 μL
suspension of P. falciparum culture (2% parasitemia and 2%
hematocrit in RPMI 1640 medium supplemented with 10% human
serum and 60 μg/mL amikacin) was added to the wells of a 96-well
plate containing 10 μL of serially diluted samples. The plate was
flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2 and
incubated at 37 ꢀC for 72 h in a modular incubation chamber.
Plasmodial LDH activity was determined by using Malstat reagent
(Flow Inc., Portland, OR).66 Briefly, 20 μL of the incubation
mixture was mixed with 100 μL of the Malstat reagent and
incubated for 30 min. Then 20 μL of a 1:1 mixture of NBT/PES
(Sigma, St. Louis, MO) was added and the plate is further
incubated for 1 h in dark. The reaction was stopped by adding
100 μL of a 5% acetic acid solution. The plate was read at 650 nm.
Chloroquine and artemisinin were used as the positive controls.
To determine the selectivity index of antimalarial activity of
compounds, their in vitro cytotoxicity to mammalian cells (Vero
cells) was also determined. Cells were seeded at a density of 25000
cells/well and incubated for 24 h. Serially diluted samples were
added and again incubated for 48 h. The number of viable cells was
determined by Neutral Red assay.67 Doxorubicin was used as a
positive control. IC50 values were obtained from dose response
curves.
-
(IC50) for either O2 or TXB2. LDH release from microglia
was determined spectrophotometrically as described.35 Micro-
glia LDH release was expressed as the compound concentration
(LDH50) that yielded 50% percent of LDH release observed
after treatment of control microglia with Triton X-100 (0.1%).
Assay for Transport across BBB. MDR-MDCK cell line was
grown in Dulbecco’s Modified Eagles Medium as described
earlier.72 For the transport experiment, cells with passage
number 12-40 were seeded at a density of 75000 cells/cm2 and
grown for eight days in 12-well Transwell plates to form a
monolayer. The bidirectional transport assay was conducted
in HBSS, pH 7.4, as the transport medium. Caffeine (5 μM) was
used as a standard drug for BBB transport. The transport of
manzamine A free base and its salt (50 μM) was monitored in
absorptive and secretory directions across MDR-MDCK
monolayer for two hours. The concentration of manzamine A
in the donor chamber as well as in the samples collected at
different time intervals from the receiver chamber was deter-
mined by LC-MS method. The concentration of caffeine was
determined by an HPLC method as reported previously.72 The
cumulative amount of the transported drug was plotted against
time to obtain the rate of transport (as shown in Figure 1), and
the apparent permeability coefficient (as shown in Figure 2) was
calculated from the equation:
Papp ¼ ðdq=dtÞꢀ1=Co ꢀ 1=A
with dq/dt=rate of transport, Co=initial concentration in the
donor chamber, A=surface area of the filter.
LC/MS Analytical. A stock solution of manzamine A was
prepared by dissolving in DMSO. The standard solution was
diluted with 4% DMSO and 2% cyclodextrin in HBSS at a level
of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, and 5.0 μg/mL. Agilent HP1100
with Bruker micro-TOF was used. The analysis was performed
on a C8 column (4.6 mm ꢀ 150 mm, 5 μm, Phenomenex Luna)
using a gradient of MeOH - 2 mM NH4Ac (0.05% formic acid)
from 40:60 to 80:20 over 8 min at a flow rate of 0.8 mL/min. An
after column splitter at ratio of 1:5 was utilized. The mass
spectrometer was operated in positive ESI mode. The nebulizer
In Vitro Antimicrobial Assay. All organisms were obtained
from the American Type Culture Collection (Manassas,
VA) and include the fungi Candida albicans ATCC 90028
and Cryptococcus neoformans ATCC 90113 and the bacterium

74 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Peng et al.
pressure was 2 bar, source temperature was 200 ꢀC, and drying
gas was 4 L/min. Extracted ion at m/z 549.4 was integrated and
used for quantification. The standard curve was linear over the
range of 0.05-5 μg/mL (R2=0.9975). The precision at level of
0.05, 0.5, 2.0 μg/mL were 3.85%, 3.21%, and 2.85%, respec-
tively. The recovery at level of 0.05, 0.5, and 2.0 μg/mL were
109.0%, 94.71%, and 101.2%, respectively. Samples collected
from donor or receiver side of the monolayer were directly
injected into LC/MS.
In Vivo Antimalarial Assay. The in vivo antimalarial activity
of 1 was determined in mice infected with Plasmodium berghei
(NK-65 strain), originally obtained from the Walter Read Army
Institute of Research, Silver Spring, MD. For multiple dose
studies, the four days Peter’s suppressive test was modified to a
three days treatment schedule as recommended by Medicines for
Malaria Venture (www.mmv.org) for evaluation of suppressive
and curative activities. Male mice (Swiss Webster strain) weigh-
ing 18-20 g were intraperitoneally inoculated with 2 ꢀ 107
parasitized red blood cells obtained from a highly infected donor
mouse. Mice were divided into different groups with at least five
mice in each group. 1 stock was prepared in SSV or 0.1 M HCl,
as specified in respective studies. Further dilutions of 1 were
prepared in SSV or 0.01 M HCl. The mice infected with P.
berghei were orally administered 100 μL of the compound. The
animals were closely observed for at least 2 h after every dose for
any apparent signs of toxicity. Blood smears were prepared on
different days starting from 5 days post infection (through 28
days) by clipping the tail end, stained with Giemsa, and the slides
were observed under microscope for determination of parasite-
mia. Mice without parasitemia through day 28 postinfection
were considered cured.
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Acknowledgment. We gratefully acknowledge the Na-
tional Institutes of Health (1R01A136596 and 5K02A-
101502) and the Medicines for Malaria Venture (MMV) for
financial support. We thank Dr. Abbas G. Shilabin, Marsha
Wright and John Trott for scale up preparation, biological
testing, which was supported by the NIH, NIAID, Division of
AIDS, grant no. AI 27094 and the USDA Agricultural
Research Service Specific Cooperative Agreement no. 58-
6408-2-0009. Additional support to AMSM from Midwestern
University and technical assistance by Mary Hall are grate-
fully acknowledged. R.J.D. acknowledges funding from
National Science Foundation (EPS-0556308), Centers for
Disease Control and Prevention National Center for Zoo-
notic, Vector-Borne, and Enteric Diseases (NCZVED),
American Association of Colleges of Pharmacy New Investi-
gator Program, NIH National Center for Research Re-
sources (NCRR) grants P20 RR021929 and C06 RR-14503-
01, and University of Mississippi’s Office of Research and
Sponsored Programs, Partners and Provost’s Associates
Grants. S.P. is a CORE-NPN Natural Products Neuroscience
fellow.
Supporting Information Available: Complete references 20
and 58, full list of Tables 1-3, and cytotoxicity studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.

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