CNS and antimalarial activity of synthetic meridianin and psammopemmin analogs

Article abstract of DOI:10.1016/j.bmc.2011.08.033

The marine invertebrate-derived meridianin A, the originally proposed structure for psammopemmin A, and several related 3-pyrimidylindole analogs were synthesized and subsequently investigated for central nervous system, antimalarial, and cytotoxic activity. A Suzuki coupling of an indoleborate ester to the pyrimidine electrophile was utilized to form the natural product and derivatives thereof. The 3-pyrimidineindoles were found to prevent radioligand binding to several CNS receptors and transporters, most notably, serotonin receptors (<0.2 μM K_i for 5HT_2B). Two compounds also inhibited the human malaria parasite Plasmodium falciparum (IC₅₀ <50 μM). Only the natural product was cytotoxic toward A549 cells (IC ₅₀ = 15 μM).

Full text of DOI:10.1016/j.bmc.2011.08.033

Bioorganic & Medicinal Chemistry 19 (2011) 5756–5762
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
CNS and antimalarial activity of synthetic meridianin
and psammopemmin analogs
Matthew D. Lebar a, Kristopher N. Hahn a, Tina Mutka b, Patrick Maignan b, James B. McClintock c,
Charles D. Amsler c, Alberto van Olphen b, Dennis E. Kyle b, Bill J. Baker a,
⇑
a Department of Chemistry and Center for Molecular Diversity in Drug Design, Discovery and Delivery, South Florida, Tampa, FL 33620, USA
b Department of Global Health, University of South Florida, Tampa, FL 33620, USA
c Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The marine invertebrate-derived meridianin A, the originally proposed structure for psammopemmin A,
and several related 3-pyrimidylindole analogs were synthesized and subsequently investigated for cen-
tral nervous system, antimalarial, and cytotoxic activity. A Suzuki coupling of an indoleborate ester to the
pyrimidine electrophile was utilized to form the natural product and derivatives thereof. The 3-pyrimi-
dineindoles were found to prevent radioligand binding to several CNS receptors and transporters, most
Received 14 June 2011
Revised 12 August 2011
Accepted 15 August 2011
Available online 22 August 2011
notably, serotonin receptors (<0.2
parasite Plasmodium falciparum (IC50 <50
(IC50 = 15 M).
lM Ki for 5HT2B). Two compounds also inhibited the human malaria
Keywords:
l
M). Only the natural product was cytotoxic toward A549 cells
Central nervous system bioactivity
Antimalarial bioactivity
Cytotoxicity
l
Ó 2011 Elsevier Ltd. All rights reserved.
Tunicate natural products
Synthetic derivatives of natural products
1. Introduction
Our previous study utilized a Suzuki–Miyaura reaction to syn-
thesize the reported structure for psammopemmin A by coupling
4-amino-2-bromo-5-iodopyrimidine to a 4-hydroxyindole moiety.
Using this strategy, we now report the synthesis of meridianin A
(2), 4-methoxymeridian A (3), meridoquin (4), and 20-debromo-
20-chloro-1 (5). The remarkable biological activity previously
reported for natural occurring meridianins and synthetic analogs
thereof impelled us to examine the potential pharmacological
effects of our analogs.
We were prompted to investigate the binding affinity of several
of our 3-pyrimidylindoles to various serotonin receptors due to the
structural features (amine-containing indolol) common to our
compounds and serotonin (5-hydroxytryptamine, 5-HT). Serotonin
transmission is thought to play a role in central nervous system
(CNS) disorders. Compounds that bind to specific serotonin recep-
tor subtypes could lead to treatment of CNS diseases.15 Selective
antagonists of 5-HT2C helped to establish the receptor’s role in
behaviors such as feeding16 and anxiety.17 Neuropsychiatric disor-
ders such as major depression as well as anxiety,18 and migraine19
are currently being treated with 5-HT selective receptor ligands
while drugs that target the 5-HT2A receptor20 are under clinical
investigation for the treatment of schizophrenia.
We recently reported1 the 60N-HX salt of psammopemmin A (1),2
isolated from the Antarctic sponge Psammopemma sp., actually pos-
sesses the structure of meridianin A (2), a similar 3-pyrimidylindole
alkaloid. The meridianins, isolated from the Antarctic tunicates Apli-
dium meridianum,3–5 A. falklandicum,5 as well as Synoicum sp.,1 are
low micromolar inhibitors of various cyclin-dependent kinases,
glycogen synthase kinase-3, protein kinase A, and other protein ki-
nases.6,7 Meridianin A, which has been previously synthesized using
a Bredereck protocol8 and through a one pot Masuda borylation–Su-
zuki coupling,9 was found to inhibit CDK1 (IC50 = 2.5
(3.5 M), PKA (11.0 M), PKG (200 M), and GSK3-b (1.3
showing no toxic effects toward Hep2, HT29, and LMM3 cell lines
l
M), CDK5
l
l
l
lM) while
(IC50 >100
(IC50 = 3.10
toward SH-SY5Y cells (IC50 >30
l
l
M).6 Additionally, meridianin
M), CDK9 (2.40 M), CK1 (1.10
M).10 Naturally occurring meridia-
A
inhibited CDK2
lM) but was nontoxic
l
l
nins were found to deter predation against the common Antarctic
omnivorous predator Odontaster validus.5 A variety of synthetic
routes to form meridianins and related bioactive analogs have been
devised, and subsequently reviewed.11 Several more syntheses of
meridianin derivatives have been recently reported.7,12–14
Pyrimidine containing compounds (e.g., pyrimethamine) have
been used to combat malaria, a devastating disease affecting disad-
vantaged populations worldwide.21,22 Because many current treat-
ments for malaria are losing efficacy due to drug-resistant
⇑
Corresponding author. Tel.: +1 813 974 1967; fax: +1 813 974 1733.
E-mail addresses: bjbaker@usf.edu, bjbaker@cas.usf.edu (B.J. Baker).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.033

M. D. Lebar et al. / Bioorg. Med. Chem. 19 (2011) 5756–5762
5757
parasites,23–25 new drugs are required to overcome resistance.
Meridianin analogs substituted with both 20-piperidinyl and 60-aryl
moieties have been reported to display a minimum inhibitory con-
bromination resulting in the formation of 12 (85%),29 halogen–
lithium exchange followed by addition of borate 7 gave vinyl
borate 13 (not isolated). Coupling 13 to 2-amino-4-chloropyrimi-
dine with Pd0 (10 mol %) resulted in 3-pyrimidylindole 14 (two
steps, 57%). Upon treatment with TBAF, 4-methoxymeridianin A
was obtained (90%).
centration of 1–10
l
g/mL (MIC = ꢀ3–30
lM) versus the malaria
parasite Plasmodium falciparum NF-54.26 We were curious if our
3-pyrimidylindole compounds could also inhibit the malaria
parasite.
Inspired by meriolin,10,30 a synthetic hybrid of naturally occurring
meridianin and variolin, we envisioned a meridianin-chloroquine hy-
brid we have dubbed meridoquin (4, Fig. 1). By combining the bioac-
tive meridianin skeleton with characteristics from the widely used 7-
chloro-4-aminoquinoline antimalarial chloroquine, we hoped to
build a new bioactive scaffold as a starting point for future studies
as well as demonstrate the utility of our pyrimidine–indole coupling
protocol. It has been shown that the chloro- substituent in 4-amino-
quinoline antimalarials is essential for potent antiplasmodial activity
while alkylation of the terminal amine improves inhibition
moderately.31
The synthesis of meridoquin (4) was achieved thorough cou-
pling of a suitably substituted indole moiety to the desired pyrim-
idine (Scheme 2). A low yielding but rapid reaction of neat
diethylamine and 2,4-dichloropyrimidine (15) resulted in the for-
mation of 2-chloro-4-N,N-diethylaminopyrimidine (16, 59%) as
well as the desired 4-chloro-2-N,N-diethylaminopyrimidine (17,
13%). Construction of the indole-coupling partner began with the
one pot N1 silylation, 3-bromination of commercially available 6-
chloroindole (18) resulting in dihaloindole 19 (89%). Halogen–lith-
ium exchange of 19 with t-butyllithium then addition of 7 afforded
intermediate 20 which was then coupled to pyrimidine 17 using
the previously developed Suzuki reaction conditions. Due to the
electron withdrawing effects of the ring nitrogens, 4-halopyrimi-
dines are effectual electrophiles for use in Suzuki coupling reac-
tions,32 explaining the absence of any bisindoles in the reaction
mixture. The indole–pyrimidine coupling product (20) was ob-
served exclusively (two steps, 69%). Desilylation of 20 with TBAF
afforded meridoquin (4, 86%).
3-Pyrimidylindoles 1–5 were screened against a variety of CNS
receptors and transporters,27 the malaria parasite P. falciparum,
and A549 lung cancer cells affording valuable SAR insight as
small structural changes have a considerable effect on biological
activity.
R'
1'
N
6'
1'
N
NR'2
N3'
6'
R4
4
N3'
OH
3
4
3
NH2
2
6
R6
2
N
6
N
H
H
2: R4=OH, R6=H, R'=H, meridianin A
3: R4=OMe, R6=H, R'=H, 4-methoxymeridianin A
4: R4=H, R6=Cl, R'=Et, meridoquin
1: R'=Br, proposed psammopemmin A
5: R'=Cl, 2'-debromo-2'-chloro-1
2. Results and discussion
2.1. Chemistry
We formed meridianin A (2) using a synthetic route developed
earlier for the synthesis of proposed psammopemmin A (Scheme
1). Halogen–lithium exchange of previously reported disilyl-
protected-3-bromo-indole 61 then addition of borate 7 generated
heteroaryl borate 8 which was immediately coupled to 2-amino-
4-chloropyrimidine using a Suzuki coupling reaction (46%, two
steps).28 The resulting intermediate (9) bearing the 4-hydroxyme-
ridianin skeleton was deprotected with TBAF to afford meridianin
A in 87% yield. Physical data of synthetic meridianin A were identical
those of meridianin A reported from A. meridianum3 and isolated
from Synoicum sp.1
The 20-debromo-20-chloro analog of psammopemmin A (5) was
formed through a Suzuki coupling of 4-amino-2-chloro-5-iodopyr-
imidine33 and borate ester 8 (Scheme 1, two steps, 56%). As was
reported previously in the synthesis of proposed psammopemmin
A (20-bromo),1 deprotection of 21 with TBAF resulted in mainly
degradation with isolation of 5 in trace amounts while treatment
with HFꢁpyr gave higher yields to afford the desired 20-debromo-
20-chloro analog in 32% yield.
The 4-methoxy analog of meridianin A (3) was synthesized
using a route analogous to that producing meridianin A (Scheme
1). 4-Indolol (10) was refluxed with methyl iodide under basic con-
ditions to form 4-methoxyindole (11, 77%). After silylation and
N
N
N
N
NH2
NH2
c
OR
OR
Br
OR
OR
Bpin
a
b
N
N
N
H
N
TIPS
TIPS
TIPS
6: R=TBS (ref. 1)
8: R=TBS
2: R=H
9: R=TBS
12: R=Me
13: R=Me
3: R=Me
14: R=Me
f
O
O
B OiPr
Cl
N
e
N
7
g
OTBS
OMe
OH
5: 2'-chloropsammopemmin A
NH2
d
N
N
H
N
H
TIPS
11
10
21
Scheme 1. Reagents and conditions: (a) t-BuLi then 7, ꢂ78 °C; (b) 2-amino-4-chloropyrimidine, 10 mol % Pd(PPh3)4, 2 M Na2CO3, MeOH, benzene, reflux, 9: 46% from 6, 14:
57% from 12; (c) TBAF, THF, rt, 2: 87% from 9, 3: 90% from 14; (d). MeI, K2CO3, acetone, reflux, 77%; (e) n-BuLi, TIPSCl, THF, ꢂ79–ꢂ10 °C then NBS, ꢂ78 °C, 12: 85%; (f) 4-amino-
2-chloro-5-iodopyrimidine, Pd(PPh3)4, 2 M Na2CO3, MeOH, benzene, reflux, 56% from 6; (g) HFꢁpyr, pyridine, acetonitrile, 32%.

5758
M. D. Lebar et al. / Bioorg. Med. Chem. 19 (2011) 5756–5762
N
N
N
N
N
NH2
N
HN
+
N
H
Cl
N
H
Cl
N
4: meridoquin
(6-chloro-N,N-diethyl meridianin)
chloroquine
meridianin
skeleton
Figure 1.
N
N
N
a
+
Et2N
N
Cl
Br
Cl
N
17
NEt2
Cl
Cl
N
15
Cl
16
N
NEt2
N
b
c,d
e
4: meridoquin
N
H
N
N
Cl
Cl
TIPS
TIPS
18
19
20
Scheme 2. Reagents and conditions: (a) Et2NH, neat, 5 min, 59% 16; 13% 17; (b) n-BuLi, TIPSCl, THF, ꢂ79 to ꢂ10 °C then NBS, ꢂ78 °C, 89%; (c) t-BuLi then 7, ꢂ78 °C; (d) 17,
10 mol % Pd(PPh3)4, 2 M Na2CO3, MeOH, benzene, reflux, 62% from 19; (e) TBAF, THF, 86%.
2.2. Activity at CNS receptors and transporters
5-HT1D and 5-HT2C revealed 4-methoxymeridianin A did not inhibit
binding of the radiolabled compounds. Like meridianin A, the
4-methoxy analog significantly inhibited binding of radioligand
[3H]LSD with 5-HT2B (Ki = 88 nM). 4-Methoxymeridianin A also
inhibited, to a lesser extent, binding of [3H]8-OH-DPATto 5-HT1A
as well as [3H]LSD to 5-HT5A and 5-HT7. Secondaryscreening showed
proposed psammopemmin A (1) did not inhibit binding of [3H]LSD
to 5-HT1D. These initial findings indicate meridianin A is a slightly
more selective inhibitor of serotonin receptor subtypes than other
compounds tested while methylation of the 4-OH group leads to
more potent inhibition of radioligand binding to 5-HT2B as well as
broader but less potent activity versus other receptor subtypes.
In addition to 5-HT receptors, the binding effects of 3-pyrimidy-
lindoles on other CNS receptors and transporters were investigated.
The dopamine active transporter (DAT) was screened for radioligand
[3H]WIN35428 (a synthetic tropane, an analogue to cocaine) inhibi-
tion. Meridianin A (2) was the only compound to display activity in
The binding affinity of proposed psammopemmin A (1), merid-
ianin A (2), 4-methoxymeridianin A (3), and analog 5 toward
eleven 5-HT receptor subtypes was evaluated. Primary screening
was conducted in vitro by measuring the percent inhibition of radi-
oligand bound to the receptor in question (% inhibition = 100% ꢂ %
radioactively bound). Secondary screening was performed in vitro
on compounds showing >50% inhibition (Table 1).
Meridianin A inhibited binding of the radioligand to 5-HT1A
,
5-HT1D 5-HT2B and 5-HT2C in primary screening. Secondary
,
screening revealed meridianin A did not inhibit binding of 5-
HT1D and 5-HT2C with [3H]5-carboximidotryptamine and [3H]mes-
ulergine, respectively. Meridainin A did, however, significantly
inhibit the binding of radioligand [3H]lysergic acid diethylamide
([3H]LSD) with 5-HT2B (Ki = 150 nM) and also displayed weaker
binding inhibition of [3H]8-OH-DPAT (an aminotetralin analog) to
5-HT1A
Primary screening of 4-methoxymerididanin A (3) showed the
compound could inhibit radioligand binding to 5-HT1A, 5-HT2B
.
primary and secondary DAT screens (Ki = 2.35 lM). Analog 5 was
also found to inhibit (Ki = 7.04 l
M) radioligand binding ([3H]3-qui-
,
nuclidinyl benzilate, [3H]QNB) of the muscarinic acetylcholine
receptor (M5).
5-HT2C and 5-HT5A, and 5-HT7. Secondary screening toward
Table 1
Bioactivity of 3-pyrimidylindoles 1–5: CNS binding, antimalarial and cytotoxic activitya
D5b
DATb
M5b
P. falciparumc
A549c
b
b
b
b
b
b
5-HT1A
5-HT1D
5-HT2B
5-HT2C
5-HT5A
5-HT7
1
2
3
4
ꢃ
>10
>10
>10
—
ꢃ
ꢃ
ꢃ
ꢃ
>10
>10
>10
—
ꢃ
ꢃ
>33
12
40
200
>38
0.006j
>219
15
>420
>333
>256
<9.7k
0.684
1.354
—
0.150
0.088
—
>10
>10
—
ꢃ
ꢃ
2.350
ꢃ
>10
ꢃ
1.525
—
1.985
—
—
—
5
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
>10
ꢃ
7.036
control
0.049d
0.006e
0.003d
0.027f
0.014e
0.027f
0.124g
0.022h
0.0004i
a
b
c
d
e
f
Compound 4 was not assayed against CNS receptors or transporters.
Ki ( M; —, indicates not tested.
M); ꢃ, indicates primary screen IC50>10
IC50 M).
Methsergide.
Ergotamine.
Chlorpromazine.
SKF38393.
Vanoxerine.
Atropine.
Chloroquine.
Roridin E.
l
(
l
l
g
h
i
j
k

M. D. Lebar et al. / Bioorg. Med. Chem. 19 (2011) 5756–5762
5759
CNS therapeutic agents that target receptors in the brain (i.e.,
5-HT, M5) are required to pass the blood brain barrier (BBB).
Meridianin derivatives have the appropriate mass (Mr <400 Da) to
permeate the BBB.34 Psilocybin and 5-methoxy-N,N-dimethyltryp-
tamine,35 oxygenated indole-containing natural products similar
to meridianins but possessing a tertiary amine moiety, are able to
transverse the BBB and interact with CNS receptors. N-Alkylation
of meridianin derivatives should, presumptively, enhance BBB
permeability.36
purchased from Sigma–Aldrich and used as received. Low-temper-
ature baths of ꢂ78 °C were obtained with an immersion cooler
bath using acetone with dry ice. TLC was carried out using What-
man normal phase Silica gel 60 Å PartisilÒ. TLC plates were visual-
ized with UV (254 nm) or 5% phosphomolybdic acid (PMA) in EtOH
and heating. Products were chromatographed on a Teledyne Isco
Combiflash Companion MPLC instrument using normal phase silica
Gel cartridges purchased from Teledyne Isco. Melting points were
recorded on an Electrothermal Mel-Temp 3.0 instrument. HRMS
data were obtained on an Agilent LC/MSD TOF electrospray ioniza-
tion mass spectrometer. 1H and 13C NMR spectra were recorded on
a Varian 400 MHz or 500 MHz instrument using residual proton-
ated solvent as 1H internal standard or 13C absorption lines of sol-
vents for 13C internal standard in CDCl3 or DMSO-d6 (Cambridge
Isotope Labs).
2.3. Antimalarial and cytotoxic activity
The potential antimalarial activity and cytotoxicity of 3-pyri-
midylindoles 1-5 were investigated. Meridianin A (2), 4-methoxy-
meridianin A (3), and meridoquin (4) were active against the
malaria parasite P. falciparum in initial screening. Secondary screen-
ing was conducted to determine IC50 values toward P. falciparum.
4.2. General procedure for the synthesis of 2–4
Meridianin A was the most potent (IC50 = 12
l
M) but 4-methoxyme-
M) also
ridianin A (IC50 = 40 M) and meridoquin (IC50 = 200
l
l
To a stirring solution of mono- or disilyl- protected 3-pyrimidy-
lindole (0.15 mmol) in dry THF (2 mL) at rt was added dropwise tet-
ra-n-butylammonium fluoride (TBAF: 1.0 M; monosilyl: 0.15 mmol;
disilyl: 0.3 mmol). After 5 min aqueous sodium carbonate (Na2CO3,
2 M, 2 mL) was added. The mixture was then partitioned between
sat NH4Cl/10% MeOH in EtOAc. The aqueous layer was extracted
with another aliquot of 10% MeOH in EtOAc. The combined organic
layers were dried over anhyd MgSO4, concentrated, and purified
displayed modest antimalarial activity. Meridianin A was cytotoxic
toward A549 lung cancer cells at nearly equimolar concentrations
to its antimalarial activity (IC50 = 15
because cytotoxic effects of meridianin A toward Hep2, HT29,
LMM3 and SH-SY5Y cell lines (IC50 >100 M) were not observed,
lM). This was surprising
l
previously.6,10 Interestingly, Meijer et al. found all natural meridia-
nin analogs were cytotoxic with the exception of meridianin A.6 Both
4-methoxymeridianin A and meridoquin displayed no toxic effects
at the highest concentration examined. Proposed psammopemmin
A (1) and its 20-debromo-20-chloro analog (5) showed no activity
against P. falciparum or A549 at the highest concentration tested.
4-Hydroxy methylation of meridianin A appears to significantly de-
crease the cytotoxicity of the compound, while retaining antipara-
sitic activity. It is important to note that BBB permeability of
meridianin derivatives, while desirable for CNS drugs, would be det-
rimental to their use as antimalarials. Although their size is appro-
priate for BBB permeability, meridianin derivatives may not be
lipophilic enough to cross phospholipid bilayers. For instance, sero-
tonin itself is unable to traverse from blood to the brain.37
via MPLC (silica gel,
EtOAc:hexanes).
2 and 3: 10% MeOH/DCM; 4: 35%
4.2.1. Meridianin A (2)
Yellow needles from MeOH/H2O; 87% yield; mp = 168 °C; 1H
NMR (400 MHz, DMSO-d6) d (multiplicity, J (Hz), integration):
6.36 (d, 7.6, 1H), 6.73 (br s, 2H), 6.79 (d, 7.9, 1H), 6.96 (dd, 7.6,
7.9, 1H), 7.10 (d, 5.4, 1H), 8.10 (d, 5.4, 1H), 8.21 (d, 2.6, 1H),
11.73 (br s, 1H), 13.58 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
102.4, 104.4, 105.5, 113.8, 114.4, 124.4, 128.5, 139.3, 152.0,
158.5, 160.5, 161.8; ESI HRMS [M+H]+ calcd for C12H11N4O:
227.0927, found: 227.0929.
4.2.2. 4-Methoxymeridianin A (3)
3. Conclusion
Yellow solid; 90% yield; mp = 213–215 °C; 1H NMR (400 MHz,
DMSO-d6) d (multiplicity, J (Hz), integration): 3.87 (s, 3H), 6.27
(br s, 2H), 6.64 (d, 7.2, 1H), 7.08 (m, 1H), 7.09 (m, 1H), 7.25 (d,
5.3, 1H), 7.84 (d, 2.6, 1H), 8.15 (d, 5.3, 1H), 11.64 (br s, 1H); 13C
NMR (100 MHz, DMSO-d6) d: 55.0, 101.2, 105.6, 109.6, 114.5,
115.4, 122.7, 127.5, 138.8, 153.3, 157.0, 161.8, 163.1; ESI HRMS
[M+H]+ calcd for C13H13N4O: 241.1084, found: 241.1084.
In summary, several meridianin and psammopemmin analogs
were synthesized and examined for biological activity. Meridianin
A (2) inhibited binding of [3H]LSD to 5-HT2B, inhibited radioligand
binding to DAT, and inhibited growth of P. falciparum but was,
unfortunately, found to be cytotoxic versus A549. 4-Methoxyme-
ridianin A (3) inhibited binding of [3H]LSD with 5-HT2B, 5-HT5A
,
and 5-HT7, inhibited growth of P. falciparum, and displayed no
cytotoxic effects at the highest concentrations tested (>300 M).
4.2.3. Meridoquin (4)
l
White solid; 86% yield; mp = 166 °C; 1H NMR (500 MHz, CDCl3)
d (multiplicity, J (Hz), integration): 1.29 (t, 6.9, 6H), 3.75 (q, 6.9,
4H), 6.78 (dd, 5.3, 2.1, 1H), 7.24 (d, 8.6, 1H), 7.43 (dd, 2.1, 2.1,
1H), 7.83 (s, 1H), 8.27 (dd, 5.3, 2.1, 1H), 8.45 (d, 8.6, 1H), 8.60 (br
s, 1H). 13C NMR (125 MHz, CDCl3) d: 13.2 (2C), 42.1 (2C), 104.3,
111.2, 116.5, 121.9, 123.1, 124.2, 126.5, 128.7, 137.2, 156.9,
161.1, 161.7; ESI HRMS [M+H]+ calcd for C16H18ClN4: 301.1215,
found: 310.1220.
Meridoquin (4) had moderate antimalarial activity and was found
to be nontoxic. Proposed psammopemmin A (1) was devoid of
activity but its 20debromo-20-chloro analog (5) inhibited radioli-
gand binding of M5. Further study on 3-pyrimidylindole analogs
is ongoing and will reveal if the preliminary promising bioactivity
and selectivity findings reported herein can be improved.
4. Experimental
4.3. Synthesis of 20-debromo-20-chloro analog (5)
4.1. General experimental procedures
The synthesis of compound 5 was carried out according to the
reported procedure.1
Unless otherwise stated, all experiments were performed under
inert atmosphere (N2 or Ar) in oven- or flame-dried glassware
equipped with a magnetic stir bar and a rubber septum. All sol-
vents used were reagent grade. Anhydrous THF was obtained by
distillation from sodium/benzophenone. All other chemicals were
Tan powder; 32% yield; 1H NMR (400 MHz, DMSO-d6) d (multi-
plicity, J (Hz), integration): 6.39 (d, 7.3, 1H), 6.89 (d, 8.3, 1H), 6.90
(vbr s, 2H), 6.92, (dd, 7.3, 7.8, 1H), 7.27 (s, 1H), 7.87 (s, 1H), 9.30 (s,
1H), 11.28 (br s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 103.1,

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M. D. Lebar et al. / Bioorg. Med. Chem. 19 (2011) 5756–5762
103.8, 105.6, 112.6, 115.5, 122.6, 123.7, 138.6, 151.2, 156.4, 157.0,
164.0; ESI HRMS [M+H]+ calcd for C12H10ClN4O: 261.0538,
found:261.0536.
position): 0.10 (s, 6H), 0.83 (s, 9H), 1.16 (d, 7.3, 18H), 1.71 (sept,
7.3, 3H), 5.40 (br s, 2H), 6.55 (d, 7.8, 1H), 7.05 (dd, 7.8, 8.2, 1H),
7.09 (s, 1H), 7.16, (d, 8.2, 1H), 8.02 (s, 1H); 13C NMR (100 MHz,
CDCl3) d: ꢂ4.3 (2C), 12.8 (3C), 18.1 (6C), 18.2, 25.6 (3C), 107.7,
108.9, 109.1, 113.5, 121.6, 123.0, 130.2, 143.7, 149.4, 156.1,
158.6, 164.1; ESI HRMS [M+H]+ calcd for C27H43ClN4OSi2Na:
553.2556, found: 553.2540.
4.4. General procedure for the synthesis of 9, 14, 20, and 21
To a stirring solution of 61 or 19 (1.0 mmol) in dry THF (5 mL) at
ꢂ78 °C under N2 was added dropwise tert-butyllithium (1.6 M in
pentane) until the solution remained yellow then and additional
aliquot (1.1 mmol) was added dropwise. The solution stirred for
15 min. Neat 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane (7) was added dropwise until the solution became colorless
(ꢀ1.5 mmol). The mixture stirred for 1 h at ꢂ78 °C and was
quenched with sat. NH4Cl. The mixture warmed to rt and was parti-
tioned between Et2O/satd NH4Cl. The aqueous layer was extracted 2
times with EtO2. The combined organic layers were dried (anhyd
MgSO4), concentrated to afford crude borate ester which was used
immediately in the next reaction without further purification.
A stirring mixture of crude borate ester, tetrakis(triphenylphos-
phine)palladium (0.1 mmol), the suitably substituted pyrimidine
(2-amino-4-chloropyrimidine, 17, or 4-amino-2-chloro-5-iodopyr-
imidine, 0.9 mmol), benzene (25 mL, degassed by sparging with
N2), methanol (5 mL, degassed), and aqueous sodium carbonate
(1.25 mL, 2 M, degassed) was refluxed under N2 for 24 h. The mix-
ture was allowed to cool to rt, diluted with EtOAc and dried with
anhyd MgSO4. The filtrate was concentrated onto silica and puri-
fied via MPLC (silica gel, 9 and 14: 40% EtOAc/hexanes; 20 and
21: 18% EtOAc/hexanes).
4.5. The synthesis of 4-methoxy-1H-indole (11)
A stirring mixture of K2CO3 (3.9 g, 28.5 mmol), 101 (380 mg,
2.85 mmol), and iodomethane (4.05 g, 28.5 mmol, 1.8 mL) in dry
acetone (10 mL, Sigma–Aldrich) under N2 was refluxed for 6 h. The
mixture was then cooled to rt, and partitioned between EtOAc/
H2O. The aqueous layer was extracted 2ꢃ with aliquots of EtOAc.
Thecombined organic layers were dried over anhyd. MgSO4, concen-
trated, and purified via MPLC (silica, eluting at 22% EtOAc:Hex) to af-
ford 11 as white crystals (326 mg, 2.22 mmol, 78% yield).
mp = 66 °C; 1H NMR (400 MHz, CDCl3) d (multiplicity, J (Hz), integra-
tion): 3.98 (s, 3H), 6.55 (d, 7.7, 1H), 6.68 (m, 1H), 7.05 (d, 8.2, 1H),
7.12 (m, 2H), 8.16 (br s, 1H); 13C NMR (100 MHz, CDCl3) d: 55.3,
99.5, 99.8, 104.4, 118.5, 122.61, 122.72, 137.18, 153.33; ESI HRMS
[M+H]+ calcd for C9H10NO: 148.0757, found: 148.0757.
4.6. General procedure for the synthesis of 12 and 19
The syntheses of compounds 12 and 19 were carried out
according to the reported procedures.1,29
4.6.1. 3-Bromo-4-methoxy-1-(triisopropylsilyl)-1H-indole (12)
White crystalline solid; 85% yield; mp = 65 °C; 1H NMR
(400 MHz, DMSO-d6) d (multiplicity, J (Hz), integration): 1.06 (d,
7.4, 18H), 1.71 (sept., 7.6, 3H), 3.84 (s, 3H), 6.61 (d, 7.6, 1H), 7.10
(d, 8.2, 1H), 7.13 (dd, 8.24, 7.6, 1H), 7.26 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 11.9 (3C), 17.76 (6C), 55.24, 89.5, 101.1,
107.3, 118.0, 123.4, 129.4, 141.4, 153.0; ESI HRMS [M+H]+ calcd
for C18H28BrNOSiNa: 404.1016, found: 404.1025.
4.4.1. 4-(4-(tert-Butyldimethylsilyloxy)-1-(triisopropylsilyl)-1H-
indol-3-yl)pyrimidin-2-amine (9)
White solid; 46% yield; mp = 83–85 °C; 1H NMR (500 MHz,
CDCl3) d (multiplicity, J (Hz), integration): 0.10 (s, 6H), 0.90 (s,
9H), 1.17 (d, 7.6, 18H), 1.73 (sept., 7.6, 3H), 5.01 (br s, 1H), 5.04
(br s, 1H), 6.63 (d, 7.8, 1H), 7.05 (dd, 7.8, 8.1, 1H), 7.15 (d, 8.1,
1H), 7.22 (d, 5.3, 1H), 7.69 (s, 1H), 8.19 (d, 5.3, 1H); 13C NMR
(125 MHz, CDCl3) d: ꢂ0.4 (2C), 12.8 (3C), 18.2 (6C), 18.6, 26.0
(3C), 107.9, 110.9, 113.4, 118.4, 120.7, 122.5, 133.7, 144.2, 149.5,
156.6, 162.5, 163.1; ESI HRMS [M+H]+ calcd for C27H45N4OSi2:
497.3126, found: 497.3116.
4.6.2. 3-Bromo-6-chloro-1-(triisopropylsilyl)-1H-indole (19)
White crystalline solid; 89% yield; mp = 52 °C; 1H NMR
(400 MHz, CDCl3) d (multiplicity, J (Hz), integration): 1.15 (d, 7.6,
18H), 1.67 (q, 7.6, 3H), 7.18 (dd, 8.3, 1.4, 1H), 7.22 (s, 1H), 7.46 (d,
1.4, 1H), 7.48 (d, 8.3, 1H). 13C NMR (100 MHz, CDCl3) d: 12.7 (3C),
18.0 (6C), 93.6, 113.8, 119.9, 121.3, 128.6, 128.7 130.4, 140.4; ESI
HRMS [M+H]+ calcd for C17H26BrClNSi:386.0701, found: 386.0698.
4.4.2. 4-(4-Methoxy-1-(triisopropylsilyl)-1H-indol-3-
yl)pyrimidin-2-amine (14)
White solid; 57% yield; mp = 106 °C; 1H (400 MHz, DMSO-d6) d
(multiplicity, J (Hz), integration): 1.11 (d, 7.5, 18H), 1.74 (sept., 7.5,
3H), 3.86 (s, 3H), 6.33 (br s, 2H), 6.71 (d, 7.7, 1H), 7.16 (m, 3H), 7.77
(s, 1H), 8.16 (d, 5.2, 1H); 13C NMR (100 MHz, DMSO-d6) d: 12.0 (3C),
17.9 (6C), 55.0, 102.0, 107.5, 110.2, 117.4, 118.5, 123.1, 133.0,
143.1, 153.3, 157.0, 161.14, 163.2; ESI HRMS [M+H]+ calcd for
C22H33N4OSi: 397.2418, found: 397.2418.
4.7. The synthesis of aminochloropyrimidines 16 and 17
At rt, neat diethylamine (2 mL) was slowly added to 2,4-dichlo-
ropyrimidine (15, 500 mg, 3.38 mmol). The mixture stirred for
1 min. The resulting white precipitate was dissolved in EtOAc
and concentrated onto silica. Purification via MPLC (silica, gradient
from 0 to 35% EtOAc:hexane) afforded 4-chloro-2-N,N-diethylami-
nopyrimidine (17, eluting at 12% EtOAc:Hex, 54 mg, 0.44 mmol,
13% yield) and 2-chloro-4-N,N-diethylaminopyrimidine (16, elut-
ing at 26% EtOAc:hexane, 372 mg, 2.0 mmol, 59% yield).
4.4.3. 4-(6-Chloro-1-(triisopropylsilyl)-1H-indol-3-yl)-N,N-
diethylpyrimidin-2-amine (20)
White solid; 62%; mp = 126 °C; 1H NMR (400 MHz, CDCl3) d
(multiplicity, J (Hz), integration): 1.18 (d, 7.4, 18H), 1.29 (t, 7,
6H), 1.72 (sept., 7.3, 3H), 3.76 (q, 7.3, 4H), 6.80 (d, 5.5, 1H), 7.22
(dd, 8.7, 1.8, 1H), 7.49 (d, 1.8, 1H), 7.88 (s, 1H), 8.26 (dd, 5.4, 1H),
8.46 (d, 8.7, 1H); 13C NMR (100 MHz, CDCl3) d: 12.7 (3C), 13.3
(2C), 18.06 (6C), 42.04 (2C), 104.3, 113.8, 118.0, 121.7, 122.8,
127.4, 128.1, 133.8, 142.4, 157.1, 161.2, 161.6; ESI HRMS [M+H]+
calcd for C25H38ClN4Si: 457.2549, found: 457.2569.
4.7.1. 4-(N,N-Diethyl)-2-chloroaminopyrimidine (16)
Viscous colorless liquid, solidifies in freezer; 1H NMR (400 MHz,
CDCl3) d (multiplicity, J (Hz), integration): 1.18 (t, 7.0, 6H), 3.48 (br
s, 4H), 6.26 (d, 5.9, 1H), 7.96 (d, 5.9, 1H); 13C NMR (125 MHz,
CDCl3) d: 12.5, 42.4, 100.9, 156.5, 160.7, 161.7; ESI HRMS [M+H]+
calcd for C8H13ClN3: 186.0793, found: 186.0794.
4.4.4. 5-(5-(4-(tert-Butyldimethylsilyloxy)-1-(triisopropylsilyl)-
1H-indol-3-yl)-2-chloropyrimidin-4-amine (21)
4.7.2. 2-(N,N-Diethyl)amino-4-chloropyrimidine (17)
Viscous colorless liquid; 1H NMR (400 MHz, CDCl3) d (multiplic-
ity, J (Hz), integration): 1.19 (t, 7.1, 6H), 3.61 (q, 7.1, 4H), 6.44 (d,
Compound 21 (123 mg, 0.23 mmol, 56% yield); mp = 83–85 °C;
1H NMR (400 MHz, CDCl3) d (multiplicity, J (Hz), integration,

M. D. Lebar et al. / Bioorg. Med. Chem. 19 (2011) 5756–5762
5761
4.7, 1H), 8.14 (d, 4.7, 1H); 13C NMR (100 MHz, CDCl3) d: 12.9, 42.0,
108.0, 158.7, 160.9, 161.0;ESI HRMS [M+H]+ calcd for C8H13ClN3:
186.0793, found: 186.0791.
and prepare and dispense test compounds to the 96 well plates.
Positive and negative controls were included on each assay plate.
After the incubation period, cell proliferation was assessed using
Promega’s CellTiter 96 Aqueous One Solution Cell Proliferation As-
4.8. In vitro P. falciparum assay
say reagent. Into each well 20 lL reagent was added followed by
incubation for 1 h at 37 °C and 5% CO2. A Biotek plate reader was
used to read absorbance at 490 nM and IC50 values were deter-
mined for each test compound.
The transgenic P. falciparum clone 3D7 expressing luciferase
was grown in continuous culture using the method outlined by
Trager and Jenson.38 Briefly, parasites were grown in RPMI 1640
media without phenol-red and contained 10% heat-inactivated
type A+ human plasma, sodium bicarbonate (2.4 g/L), HEPES
(5.94 g/L) and 4% washed human type A+ erythrocytes. Cultures
were gassed with a 90% N2, 5% O2 and 5% CO2 mixture followed
by incubation at 37 °C. P. falciparum 3D7 cultures at P4% parasite-
mia and having >70% of parasites in the ring-stage were used in the
assay. Parasites were diluted to 0.5% parasitemia and 1.5% hemat-
ocrit using the RPMI 1640 media described above. Test compounds
Acknowledgments
Inspiration for this work derived from research supported by
the Office of Polar Programs of the National Science Foundation
(ANT-0838776 to BJB; ANT-0838773 to C.D.A., J.B.M.) and facili-
tated by the staff of Raytheon Polar Services Company. Ki determi-
nations and receptor binding profiles were generously provided by
the National Institute of Mental Health’s Psychoactive Drug
Screening Program, Contract # HHSN-271-2008-00025-C (NIMH
PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at
the University of North Carolina at Chapel Hill and Project Officer
Jamie Driscol at NIMH, Bethesda MD, USA. Screening against Plas-
modium falciparum and A549 was generously supported by Medi-
cines for Malaria Venture (MMV 008/0105 to B.J.B., A.V.O. and
D.E.K.). We thank Lindsay Morton for technical assistance with ma-
laria screening and divers of group S-022, including Margaret Ams-
ler, Alan Maschek, Craig Aumack and Philip Buculo, who assisted
with the original collections of tunicates that yielded meridianins.
were diluted to a final concentration of 100 lg/mL and then seri-
ally diluted in duplicate over 11 concentrations using a Beckman
Coulter Biomek 3000. The final test compound concentrations ran-
ged from 100 to 0.098
of 90 l/well of 0.5% parasitized erythrocytes at 1.5% hematocrit
was added on top of 10 L/well of the test compound. A separate
lg/mL. In 96-well microtiter plates a volume
l
l
96 well plate containing chloroquine, dihydroartemisinin and ato-
vaquone was added to each set of assay plates as control drugs. The
Beckman Coulter Biomek 3000 was used to dispense test com-
pounds, control drugs and parasitized erythrocytes into the micro-
titer plates. Positive and negative controls were included in each
microtiter plate. Positive controls consisted of drug-free parasit-
ized erythrocytes and negative controls consisted of parasitized
erythrocytes dosed with a high concentration of chloroquine or
dihydroartemisinin that ensured 100% parasite death. Assay plates
were placed into a plastic gassing chamber and equilibrated with a
90% N2, 5% O2 and 5% CO2 mixture then incubated at 37 °C for 72 h.
After 72 h of incubation excess culture media was aspirated off of
each well using the Biomek 3000. Plates were then frozen at
ꢂ20 °C until later processed for parasite growth determinations.
For processing, assay plates were removed from ꢂ20 °C and al-
lowed to thaw at room temperature. Assay plates were processed
using Luciferase Assay System reagents purchased from Promega.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.033.
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