Synthesis and evaluation of β-carbolinium cations as new antimalarial agents based on π-delocalized lipophilic cation (DLC) hypothesis

Article abstract of DOI:10.1248/cpb.53.653

Several β-carbolines including naturally occurring substances and their corresponding cationic derivatives were synthesized and evaluated for antimalarial (antiplasmodial) activity in vitro and in vivo. A tetracyclic carbolinium salt was elucidated for antileishmanial and antitrypanosomal activities in vitro as well as antiplasmodial activity. Quarternary carbolinium cations showed much higher potencies in vitro than electronically neutral β-carbolines and a good correlation was observed between π-delocalized lipophilic cationic (DLC) structure and antimalarial efficacy. β-Carbolinium compounds exhibit medium suppressive activity in vivo against rodent malaria.

Full text of DOI:10.1248/cpb.53.653

June 2005
Chem. Pharm. Bull. 53(6) 653—661 (2005)
653
b
Synthesis and Evaluation of -Carbolinium Cations as New Antimalarial
p
Agents Based on -Delocalized Lipophilic Cation (DLC) Hypothesis
Kiyosei TAKASU, ,a Tsubasa SHIMOGAMA,a Chalerm SAIIN,a Hye-Sook KIM,b Yusuke WATAYA,b
*
c
,a
*
Reto BRUN, and Masataka IHARA
a Graduate School of Pharmaceutical Sciences, Tohoku University; Aobayama, Sendai 980–8578, Japan: b Faculty of
Pharmaceutical Sciences, Okayama University; 1–1–1 Tsushimanaka, Okayama 700–0082, Japan: and c Swiss Tropical
Institute; Socinstrasse 57, CH-4002 Basel, Switzerland.
Received February 15, 2005; accepted March 15, 2005; published online March 22, 2005
Several b-carbolines including naturally occurring substances and their corresponding cationic derivatives
were synthesized and evaluated for antimalarial (antiplasmodial) activity in vitro and in vivo. A tetracyclic car-
bolinium salt was elucidated for antileishmanial and antitrypanosomal activities in vitro as well as antiplas-
modial activity. Quarternary carbolinium cations showed much higher potencies in vitro than electronically neu-
tral b-carbolines and a good correlation was observed between p-delocalized lipophilic cationic (DLC) structure
and antimalarial efficacy. b-Carbolinium compounds exhibit medium suppressive activity in vivo against rodent
malaria.
Key words antimalarial agent; b-carbolinium salt; total synthesis; p-delocalized lipophilic cationic (DLC) hypothesis; tropical
disease; structure–activity relationship
Malaria, which is caused by plasmodium protozoa, is one for selective carcinoma cell killing. It has been reported by
of the most serious infectious disease as well as HIV and tu- Vaidya et al. that collapse of mitochondrial membrane poten-
berculosis. The disease exists in 100 countries but is mainly tial in a malarial parasites is observed by the treatment with
confined to tropical areas of Africa, Asia and South America. antimalarial drugs.12) Consequently, we envisaged that DLCs
More than one million people, most occurring in infants and would be a new candidate for the chemotherapeutics of
young children, die annually from malaria, and disease bur- malaria as well as another neglected diseases.
den is estimated at 42 million DALYs (disable adjusted life
b-Carboline alkaloids are widely found in a number of
years).1) Another tropical parasitic diseases, such as leishma- plants and mammalian species. Some of these alkaloids, such
niasis (Kara azar, oriental sore), African trypanosomasis as manzamine A, 10-hydroxycanthin-6-one and akagerine
(African sleeping sickness) and American trypanosomasis (Chart 1), exhibit a variety of biological and pharmaceutical
(Chagas disease), which are called as “neglected diseases”, potencies including antiplasmodial activity.13—15) Pavanand et
also prevent social and economical development in those al. isolated the simple b-carboline, 4-methoxy-1-vinyl-b-car-
countries.2) Despite of the tremendous toll, effective treat- boline; (MVC, 1a; Chart 1)16) and related compounds as an-
ments and prevention for the diseases are quite difficult; no tiplasmodial components from Picrasma javanica B1, a
vaccine is available in clinically use: limited chemotherapeu- medicinal plant used for the treatment of malaria.17) However
tics, even including some drawbacks, are available: parasites their antiplasmodial activities are not so strong as alterna-
develop resistance against clinically used drug: and so on.3) tives for a clinical use. We considered that b-carbolines
So that, development of new class of antiprotozoal including could be easily transformed into DLCs by means of quar-
antimalarial agents are required and the efforts are steadily ternarization of the pyridine nitrogen atom and the resulting
increasing in non-profit institutions.4)
b-carbolinium salts may have higher antiplasmodial proper-
Recently we have reported the rhodacyanines, having a p- ties than neutral carbolines. Herein, we report the synthesis
delocalized lipophilic cationic (DLC) structure, exhibit in of b-carboline compounds and their quarternary salts, and
vitro strong antiplasmodial and antileishmanial activities we evaluate their in vitro antiprotozoal activities and cell cy-
against Plasmodium falciparum5,6) and against Leishmania totoxicity. Moreover, antimalarial efficacy in vivo was evalu-
major,7) respectively, with low cytotoxicity against mam- ated using malaria-infected mice.18)
malian cell. The conceptual term, DLC, was originally pro-
posed by Chen in their anticancer research work.8) It has sub-
sequently been reported several DLC compounds exhibit se-
lective antitumor activity by their selective accumulation in
the mitochondria of carcinoma cells.9—11) Namely, lipophilic
cations possessing a delocalized positive charge (DLCs) pen-
etrate the hydrophobic barriers of the plasma and mitochon-
dria membrane in response to the negative inside transmem-
brane potentials. The higher mitochondrial membrane poten-
tials of carcinoma cells compared to normal cells induce the
selective transfer of DLCs into carcinoma mitochondria.
Since most DLCs are toxic to mitochondria at high concen-
trations, their selective accumulation in carcinoma mitochon-
dria and consequent mitochondrial toxicity provide a basis
Chart 1
∗ To whom correspondence should be addressed. e-mail: kay-t@mail.pharm.tohoku.ac.jp
© 2005 Pharmaceutical Society of Japan

654
Vol. 53, No. 6
Results and Discussion
(DIBAL-H) at ꢀ78 °C to produce aldehyde 1f (kumujancine)
b
b
Synthesis of -Carbolines and -Carbolinium Salts in 96% yield; Kumujancine is a natural b-carboline isolated
MVC (1a) was synthesized according as Cook’s procedure from the wood of Picrasma quassioide.24) Finally, Wittig ole-
for the synthesis of crenatine (1b)19) with some modifications fination of 1f afforded 1a in 51% yield. It is noteworthy that
(Chart 2).20,21) Namely, Pictet-Spengler reaction of trypta- we first achieved the total synthesis of the two natural prod-
mine hydrochloride (2) with ethyl glyoxylate in ethanol, fol- ucts, MVC (1a) and kumujancine (1f). Transformation of 1f
lowed by acylation with acetyl chloride, furnished tetrahy- into 1a was also achieved by stepwise synthesis via alcohol
dro-b-carboline 3a in 44% yield (2 steps). Treatment of 3a 1g by methylation, followed by dehydration.
with 5 equivalent of 2,3-dichloro-5,6-dicyano-1,4-benzo-
Synthesis of unnatural 4-methoxy-1-methyl-b-carboline
quinone (DDQ) led to 4-oxocarboline 4a, which was difficult (1c) was started from tryptamine hydrochloride (2) and aque-
to be isolated owing to its instability, although the TLC pro- ous acetaldehyde. N-Benzoyl-tetrahydro-b-carboline 3c was
file of the reaction mixture indicated nearly single spot and synthesized by Pictet-Spengler reaction, followed by benzoy-
no remaining of the starting material 3a. It is known that lation. Oxidation of 3c with DDQ afforded desired 4-oxote-
treatment of tetrahydro-b-carbolines with DDQ promote C1- trahydrocarboline 4c and 2-acetylindole 5 in 43% and 20%
and C4- oxidation competitively.20) In this case, the introduc- yield, respectively. Compound 4c was isolable by silica gel
tion of electron-withdrawing group as a C-1 substituent chromatography and showed better stability than 4a. Byprod-
might inhibit C-1 oxidation owing to the electrochemical uct 5 would be produced by oxidation of 3c at C-1 position
reason (see below; for the synthesis of 1c). The and subsequent hydration. Oxidation of 1-methyltetrahydro-
crude product 4a was used in the following reaction carboline 3c would occur competitively at C-1 and C-4 posi-
without further purification. The reaction of 4a with tion because of the small electrochemical difference. Further
dimethoxypropane in the presence of p-TsOH under oxidation of 4c by p-chloranil, followed by methylation, gave
azeotropic conditions, followed by oxidative aromatization 1c. 4-Unsubstituted analogs 1e and 1h—j were prepared by
with p-chloranil,22) furnished 1-ethoxycarbonyl-4-methoxy- the similar procedure as the synthesis of 1a. Pictet-Spengler
b-carboline (1d) along with demethoxy compound (1e; ku- reaction of tryptamine hydrochloride (2) with ethyl glyoxy-
mujian A)23) in 30% and 7% yield (2 steps), respectively. late, followed by direct oxidative aromatization by Pd/C cata-
Ester 1d was reduced by diisobutylaluminum hydride lyst, yielded kumujian A (1e). Kumujian C (1h),23) alcohol
Chart 2

June 2005
655
Table 1. b-Carbolinium Salts 6
Run
Carbolinium
Carboline
R1
R2
R3
R4
R5
X
Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
6aa
6ab
6b
1a
1a
1b
1c
1c
1c
1c
1c
1c
1c
1c
1c
1c
1d
1e
1i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
CHꢁCH2
CHꢁCH2
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
CH3
C2H5
CH3
CH3
CH3
CH3
C2H5
C2H5
TsO
TsO
I
60
30
74
68
75
88
94
77
19
76
92
12
76
72
72
72
41
87
78
91
45
38
6ca
6cb
6cc
6cd
6ce
6cf
6cg
6ch
6ci
6cj
6d
6e
6i
6k
6l
6m
6o
6pa
6pb
I
TsO
Cl
TsO
Cl
Cl
Cl
Cl
Cl
Cl
TsO
TsO
TsO
TsO
TsO
TsO
TsO
TsO
Br
(CH2)2OH
(CH2)2OCH3
[(CH2)2O]2CH3
[(CH2)2O]3CH3
C2H5
CO2C2H5
CO2C2H5
CH(OH)CH3
–CHꢁCHC(ꢁO)–
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
1k
1l
1m
1o
1p
1p
H
H
OCH3
OCH3
OCH3
H
H
H
CH3
CH3
C6H5
CH3
CH3
CH3
(CH2)2OH
Chart 3
1i,25) and pavettine (1j),26) which all are naturally occurring
products, were prepared in due course. Canthin-6-one (1k)27)
was synthesized from aldehyde 1h in a single operation.28)
Thus, acetylation and consequent intramolecular aldol con-
densation occurred by the reaction of 1h with acetyl chloride
Chart 4
in the presence of triethylamine. Harmane (1l) and harmine mined. The biological results are summarized in Table 2.
(1o) were purchased, and crenatine (1b), 1m, 1n and 1p were Electronically neutral b-carbolines 1a—c, 1o and 1p dis-
synthesized according as known procedures.20,21,29)
played weak to medium inhibitory effects against P. falci-
b-Carbolinium salts 6, which correspond to DLCs, were parum (EC50ꢁ0.5—3.1ꢂ10ꢀ5 M), and their cytotoxicities
synthesized from the corresponding b-carbolines 1 by the were comparable to their antiplasmodial activity levels (runs
simple quarternarization with alkyl tosylate or alkylhalide 1—5). In contrast, N-alkyl carbolinium salts 6 displayed con-
(Chart 3). Preparation of chloride salts 6cc and 6ce—cj was siderably enhanced antiplasmodial activities (runs 6—23) ex-
achieved by ion exchange operation from the corresponding cept for 6d, 6m and 6n. Introduction of a methyl and ethyl
tosylate or iodide salts (Table 1).30) Dicationic salt 6n was group on the pyridine nitrogen atom of 1a resulted in a 5-fold
prepared from 1n by the treatment of excess amount of and a 39-fold increase in antiplasmodial activity, respectively
methyl tosylate, and tetracyclic compound 6q was synthe- (1a versus 6aa or 6ab). In addition, the cytotoxicity levels of
sized from harmine (1o) according as the reported procedure these substances were decreased 2—3-fold by quarternariza-
(Chart 4).31,32) Dihydro-b-carbolinium salt 8, which lacks of tion. A similar enhancement of antiplasmodial effectiveness
one conjugation in the aromatic system (thus, non-DLC), was observed by the transformation of carbolines 1 into the
was prepared from 7. N-Methyl deprotonated analogs 9l and corresponding carbolinium salts 6 (1b versus 6b, 1c versus
9o were quantitatively obtained by the treatment of the corre- 6c, 1o versus 6o, and 1p versus 6pa or 6pb). In particular,
sponding b-carbolinium salts 6l and 6o, respectively, with 6pa showed a 67-fold increase in an antiplasmodial activity
aqueous NaOH.33)
compared to the corresponding 1p. These results indicate
Evaluation of Antiplasmodial Activity in Vitro The that DLC compounds have increased antiplasmodial potency
antiplasmodial potencies of the b-carbolines 1, b-carbolin- within the class of b-carbolines by simple transformation. In
ium salts 6 and related compounds 7—9 were evaluated in contrast, quarternarization of dihydro-b-carboline had no ef-
vitro against P. falciparum (chloroquine sensitive FCR-3 fect in biological activity; both the electronically neutral mol-
strain) and their cytotoxicities were determined using mouse ecule 7 and cationic salt 8 displayed similarly low antiplas-
mammary tumor FM3A cells. Selective toxicities, defined by modial activities (runs 24, 25). Deprotonated analogs 9 hav-
the ratio EC50 (FM3A)/EC50 (P. falciparum), were deter- ing no charge were less active than the corresponding cations

656
Vol. 53, No. 6
6 (runs 26 versus 18, and 27 versus 21). On the other hand, these substances were decreased by these substitutions. In
dicationic salt 6n showed no inhibitory effect against P. falci- contrast, substitution of R3 with phenyl or ethoxycarbonyl
parum even in high concentration (ꢃ2ꢂ10ꢀ6 M) although its groups resulted in a remarkable decrease in antiplasmodial
cytotoxicity level was pretty high (run 20). Accordingly, activity (runs 14, 19), and introduction of an a-hydroxyethyl
these findings further indicate that a broad delocalized sys- substituent lead slightly loss in the activity (run 16). The in-
tem and the cationic charge on the delocalized system, that is troduction of a substituent at the indole-NH by a methyl
a DLC structure, are critical factors in the biological proper- group caused an increase in activity (6o versus 6pa). The
ties of this family of compounds.34—37)
protection of indole nitrogen atom might inhibit conversion
A structure–activity relationship (SAR) analysis concern- into an electronically neutral molecule under cellular condi-
ing R1—R5 substituents was also undertaken. The introduc- tions (Chart 5).38) Probably, according as the same reason,
tion of a methoxyl group as an R1 or R2 substituent caused a tetracyclic canthin-6-one analog 6k displayed good antiplas-
2-fold (6l versus 6o) or 3-fold (6l versus 6cb) enhancement modial property. Replacement of methyl group as an R5 sub-
in activity against P. falciparum, whereas the cytotoxicities of stituent by an ethyl group resulted in a 10-fold increase in an-
tiplasmodial potency for 6ab (versus 6aa), but a 2-fold de-
crease in activity was observed for 6ce (versus 6cc). b-Hy-
droxyethyl substituent for R5 group also lead decrease in ac-
tivity (6pa versus 6pb). It was observed that counter anion
(Xꢀ) affected antiplasmodial properties to some extent (runs
9, 11, 12). The difference might be caused by the solubility
of the substances in aqueous media. Among our tested com-
pounds, 2-ethyl-4-methoxy-1-vinyl-b-carbolinium p-toluene-
sulfonate (6ab) was found to display good antiplasmodial ef-
ficacy. Its selective toxicity was less than that of quinine,
which is an antimalarial medicine in current clinical use, but
its antiplasmodial activity against P. falciparum is compara-
ble. It is additionally noteworthy that 6ca is effective even
against chloroquine resistant parasites (P. falciparum K1
strain) with EC50 values of 3.6ꢂ10ꢀ7 M (run 10).
Antiprotozoal Activity of Tetracyclic Cation 6q in Vitro
For tetracyclic compound 6q, various antiprotozoal proper-
ties including antimalarial activity were examined in vitro
(Table 3). Antileishmanial (Kara azar), anti-African-try-
panosomal (African sleeping sickness), anti-American-try-
panosomal (Chagas disease) and antigiargial (diarrhoeal gia-
rdiasis) activities were determined as EC50 values against the
following parasites; L. donovani, T. brucei rhodesiense, T.
cruzi and Giardia lamblia, respectively.33) G. lamblia is cate-
gorized as a class of diplonadida parasites, which have no
mitochondrial organelle in its cell. The other tested parasites
possess parasitic mitochondria in their cells. Cell cytotoxicity
was tested using L-6 (a rat skeletal myoblast cell line). Inter-
estingly, compound 6q showed good inhibitory effect within
a range of 2.6—10ꢂ10ꢀ7 M against all parasites except for G.
lamblia. The efficacies of 6q against the parasites were com-
Table 2. In Vitro Antimalarial Activity and Cytotoxicity for b-Carbolines,
b-Carbolinium Salts, and Their Related Compounds
EC50 (M)
Selective
Run Compound
toxicityc)
P. falciparuma)
FM3Ab)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1a
1b
1c
1o
1p
6aa
6ab
6b
6ca
6ca
6cb
6cc
6ce
6d
6e
6i
6k
6l
6m
6n
6o
6pa
6pb
7
5.0ꢂ10ꢀ6
1.6ꢂ10ꢀ5
2.2ꢂ10ꢀ5
2.2ꢂ10ꢀ5
3.1ꢂ10ꢀ5
1.1ꢂ10ꢀ6
1.3ꢂ10ꢀ7
9.5ꢂ10ꢀ7
3.7ꢂ10ꢀ7
3.6ꢂ10ꢀ7 d)
7.1ꢂ10ꢀ7
1.1ꢂ10ꢀ6
2.1ꢂ10ꢀ6
1.9ꢂ10ꢀ5
8.8ꢂ10ꢀ6
8.8ꢂ10ꢀ6
8.2ꢂ10ꢀ7
2.0ꢂ10ꢀ6
1.3ꢂ10ꢀ5
NAe)
3.8ꢂ10ꢀ6
1.8ꢂ10ꢀ5
0.76
1.1
0.82
0.82
1.0
1.8ꢂ10ꢀ5
1.8ꢂ10ꢀ5
3.2ꢂ10ꢀ5
8.4ꢂ10ꢀ6
7.6
77
ꢃ32
81
83
ꢃ41
37
ꢃ2.0
1.2
1.7
ꢃ3.1
1.0ꢂ10ꢀ5
ꢃ3.0ꢂ10ꢀ5 f)
3.0ꢂ10ꢀ5
3.0ꢂ10ꢀ5
ꢃ2.9ꢂ10ꢀ5 g)
4.1ꢂ10ꢀ5
ꢃ4.3ꢂ10ꢀ6 h)
2.2ꢂ10ꢀ5
1.5ꢂ10ꢀ5
ꢃ2.7ꢂ10ꢀ5 i)
ꢃ2.6ꢂ10ꢀ5 j)
3.5ꢂ10ꢀ5
ꢃ32
18
ꢃ1.8
—
17
48
ꢃ2.3ꢂ10ꢀ5 k)
5.2ꢂ10ꢀ7
1.1ꢂ10ꢀ6
4.6ꢂ10ꢀ7
1.1ꢂ10ꢀ6
1.8ꢂ10ꢀ5
1.6ꢂ10ꢀ5
4.8ꢂ10ꢀ6
2.6ꢂ10ꢀ6
1.1ꢂ10ꢀ7
1.9ꢂ10ꢀ5
2.2ꢂ10ꢀ5
ꢃ7.1ꢂ10ꢀ5 l)
4.5ꢂ10ꢀ5
ꢃ65
2.1
ꢃ2.4
6.0
7.3
910
8
9l
ꢃ3.8ꢂ10ꢀ5 m)
2.9ꢂ10ꢀ5
9o
Quinine
1.9ꢂ10ꢀ5
1.0ꢂ10ꢀ4
a) Chloroquine sensitive strain (FCR-3) except for run 10. b) Mouse mammary
tumor FM3A cells representing a model of host. c) Selective toxicityꢁEC50 value for
FM3A/EC50 for P. falciparum. d) Chloroquine resistant P. falciparum K-1 was used.
e) NA means no activity (99% growth of P. falciparum was observed in 2.2ꢂ10ꢀ6
M
treatment). f) EC37 value (63% growth of FM3A was observed). g) EC13 value
(87% growth). h) EC36 value (64% growth). i) EC15 value (85% growth). j) EC16
value (84% growth). k) EC10 value (90% growth). l) EC14 value (86% growth).
m) EC16 value (84% growth).
Chart 5
Table 3. In Vitro Antiprotozoal Properties of 6q
Run
Parasites
EC50 of 6q (M)
EC50 of std. (M)
1
2
3
4
5
6
7
Plasmodium falciparum K-1
Leishmania donovani axenic
Leishmania donovani inf, macrophages
Trypanosoma brucei rhodesiense
Trypanosoma cruzi
3.5ꢂ10ꢀ7
2.6ꢂ10ꢀ7
1.0ꢂ10ꢀ6
6.3ꢂ10ꢀ7
4.5ꢂ10ꢀ7
Inactive
1.5ꢂ10ꢀ6 (chloroquine)
2.9ꢂ10ꢀ7 (miltefosine)
1.2ꢂ10ꢀ6 (miltefosine)
6.0ꢂ10ꢀ9 (meralsporol)
8.7ꢂ10ꢀ7 (benznidazole)
—
Giargia lamblia
Cytotoxicity L-6
4.9ꢂ10ꢀ6
2.4ꢂ10ꢀ8 (podophyllotoxin)

June 2005
657
Table 4. In Vivo Antimalarial Properties of b-Carbolinium Salts
malarial medicinal chemistry. Tetracyclic b-carbolinium ana-
log 6q also displays good antileishmanial and antitrypanoso-
mal properties as well as antimalarial activity.
Dose schedule
Suppression of
Run
Compound
(mg kgꢀ1 for 4 d)
parasitemia (%)
Experimental
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6ab
6ab
6ab
6ab
6ce
6ce
6ce
6cf
6cg
6ch
6ci
10 (ip)
20 (ip)
2.5 (iv)
5.0 (iv)
10 (ip)
3.0 (iv)
10 (iv)
10 (ip)
10 (ip)
10 (ip)
10 (ip)
10 (ip)
10 (ip)
10 (ip)
25.9
28.2
22.7
23.9
21.3
12.4
—
12.7
2.1
8.6
0.6
13.1
33.5
38.0
Melting points (mp) were measured with a Yanaco micro melting point
apparatus and are uncorrected. UV and fluorescence spectra were recorded
with a BECKMAN DU-640 and a Hitachi F 2000 spectrometers, respec-
tively. IR spectra were recorded with a SHIMADZU FTIR-8300. 1H- and
13C-NMR spectra (400, 100 MHz, respectively) were recorded on a JEOL
AL400 spectrometer. Chemical shifts were relative to tetramethylsilane
(TMS). Fast atom bombardment (FAB) mass spectra were determined with a
JEOL JMS DX-303 mass spectrometer. Electron ionization (EI) mass spec-
tra were recorded on a JEOL JMS AX-500 instrument. Elemental analyses
were performed on Yanagimoto MT-3, and the results (C, H, N) were within
ꢄ0.4% of theoretical values. All reactions were carried out under an argon
atmosphere. Unless otherwise described, the materials and the solvent were
obtained by reaction of commercial suppliers and used without further pu-
rification.
a)
6cj
6g
6q
Ethyl 2-Acetyl-1,2,3,4-tetrahydro-b-carboline-1-carboxylate (3a) To
a suspension of tryptamine hydrochloride (2; 17.1 g, 86.9 mmol) in ethanol
(200 ml) was added a solution of glyoxylic acid ethyl ester (21.4 g,
105 mmol) in toluene (50% v/v) at 0 °C. After the reaction mixture was
stirred over night at ambient temperature, solvent was removed under re-
duced pressure. The resulting residue was treated with sat. NaHCO3, and
was extracted with ethyl acetate. The combined organic phases were washed
with brine, dried over Na2SO4 and concentrated in vacuo. The crude product
was used in the next step without further purification. To a solution of the
above residue and DMAP (0.131 g, 0.925 mmol) in CH2Cl2 (200 ml) at 0 °C
were added Et3N (15.5 ml, 111 mmol) and AcCl (7.89 ml, 111 mmol). The
mixture was stirred over night at ambient temperature. After concentration
under reduced pressure, the residue was treated with sat. NaHCO3, and was
extracted with ethyl acetate. The combined organic phases were washed with
brine, dried over Na2SO4 and concentrated in vacuo. The residue was chro-
matographed on silica gel with AcOEt–hexane (1 : 1) to give compound 3a
(11.0 g, 44% for 2 steps) as colorless solids, mp 193—195 °C. IR (KBr)
a) All mice died in 24 h due to an acute toxicity.
parable to the corresponding chemotherapeutics in clinically
use, and the cytotoxicity level of 6q was about 10-fold less
than its antiprotozoal activity. Antileishmanial activity of 6q
was observed against not only axenic leishmania parasites
but also ones infected to macrophages (runs 2, 3).
Evaluation of Antimalarial Activity in Vivo Several b-
carbolinium salts 6 were subjected to in vivo assay for their
effects on the suppression of parasitemia in malaria (P.
berghei)-infected mice. Preliminary toxic study has revealed
that compounds 6ab and 6q show acute toxicity to normal
mice by intraperitoneal (i.p.) injection at a dose of more than
50 and 200 mg kgꢀ1 dꢀ1, respectively. In vivo antimalarial
assay was carried out using Peters’ 4-d suppressive test.39,40)
Compound 6ab displayed an antimalarial activity at a dose of
10 mg kgꢀ1 dꢀ1 of body weight by i.p. injection (26% sup-
pression of parasitemia; Table 4, run 1). However, no signifi-
cant increase in efficacy was observed even at a dose of
20 mg kgꢀ1 dꢀ1 (run 2). The antimalarial properties were dis-
played by intravenous (i.v.) injection even at smaller doses
(2.5—5.0 mg kgꢀ1 dꢀ1) (runs 3, 4). However, treatment by i.v.
injection at a higher dose produced side effects (acute toxic-
ity).
1
cmꢀ1: 3277, 1744, 1643. H-NMR (CDCl3) d: 8.39 (1H, br s), 7.49 (1H, d,
Jꢁ7.8 Hz), 7.37 (1H, d, Jꢁ8.1 Hz), 7.20 (1H, t, Jꢁ8.1 Hz), 7.11 (1H, t,
Jꢁ7.8 Hz), 6.16 (1H, s), 4.24 (2H, q, Jꢁ7.1 Hz) , 4.30—4.14 (1H, m),
3.67—3.60 (1H, m), 2.88—2.85 (2H, m), 2.29 (3H, s), 1.30 (3H, t,
Jꢁ7.1 Hz). 13C-NMR (CDCl3) d: 170.4, 168.7, 136.2, 126.7, 126.1, 122.4,
119.6, 118.2, 111.1, 109.4, 62.0, 52.3, 43.1, 21.7, 14.3. LR-MS (EI) m/z:
286 (Mꢅ). Anal. Calcd for C16H18N2O3: C, 67.12; H, 6.44; N, 9.78. Found:
C, 67.06; H, 6.44; N, 9.84.
Ethyl 4-Methoxy-b-carboline-1-carboxylate (1d) Compound 3a
(3.00 g, 10.5 mmol) and DDQ (11.9 g, 52.4 mmol) were mixed together until
a uniform color was observed. The mixture of powders was cooled on a dry
ice–acetone bath. The aqueous THF (THF–H2Oꢁ9 : 1 v/v; 70 ml) was added
slowly to the mixture of powders. The reaction mixture was stirred at
As the results, all compounds were tested at doses of
10 mg kgꢀ1 of body weight by i.p. injection. The results are ꢀ78 °C for 1 h, and then allowed to warm to ambient temperature. After the
reaction mixture was stirred over night at ambient temperature, solvent was
removed under reduced pressure. The residue was treated with sat. NaHCO3,
and extracted with CHCl3. The combined organic phases were washed with
brine, dried over Na2SO4 and concentrated in vacuo. The crude product of
summarized in Table 4. The similar toxic effect was observed
for compound 6ce; it showed 21% suppression by i.p. injec-
tion, whereas deaths of all mice by acute toxicity were ob-
served by i.v injection at the same dose (run 5 versus run 7).
Some of the tested compounds 6g and 6q were found to dis-
play a greater in vivo activity than compound 6ab (runs 13,
14). These in vivo results indicate that further studies for bio-
logical effects in host animals, such as bioavailability and
clearance, are indispensable.
In summary, we have synthesized b-carbolines and their
corresponding salts, including several naturally occurring
compounds, and evaluated them for their antimalarial po-
tency in vitro. The b-carbolinium salts, which have a DLC
(p-delocalized lipophilic cationic) structure, show a higher
antiplasmodial potency and better selective toxicity than non-
DLC compounds. Some of the b-carbolinium salts have in
vivo antimalarial potency to a certain extent. Thus, this study
indicates that transformation into a DLC structure may prove
4a was used in the next step without further purification. A solution of p-
toluenesulfonic acid monohydrate (2.40 g, 12.6 mmol) in benzene (70 ml)
was heated under azeotropic conditions for 2 h, and then cooled to ambient
temperature. To the mixture were added 2,2-dimethoxypropane (3.87 ml,
31.5 mmol), followed by the above the crude product of 4a, and the resulting
mixture was stirred at ambient temperature for 30 min. Then, to the mixture
was added p-chloranil (5.16 g, 21.0 mmol), and was further stirred over night
at the same temperature. After concentration of the solution in vacuo, the
residue was treated with CHCl3 and sat. NaHCO3. The aqueous phase was
extracted with CHCl3 and the combined organic phases were washed with
brine, dried over Na2SO4 and concentrated. The residue was chro-
matographed on silica gel with AcOEt–hexane (1 : 1) to give 1d (884 mg,
30%) as colorless solids and 1e (195 mg, 7%) as colorless solids. For 1d, re-
crystallization from CH2Cl2–diisopropylether gave colorless needles, mp
168—170 °C. IR (KBr) cmꢀ1: 3404, 3018, 2980, 2947, 2905, 1663. 1H-
NMR (CDCl3) d: 9.88 (1H, br s), 8.30 (1H, d, Jꢁ8.1 Hz), 8.22 (1H, 1H),
7.57—7.52 (2H, m), 7.31 (1H, t, Jꢁ8.1 Hz), 4.57 (2H, q, Jꢁ7.2 Hz), 4.23
(3H, s), 1.52 (3H, t, Jꢁ7.2 Hz). 13C-NMR (CDCl3) d: 166.4, 154.1, 139.4,
to be a highly effective modification methodology in the anti- 138.5, 128.0, 124.0, 123.9, 122.4, 120.7, 120.3, 118.6, 111.2, 61.6, 56.4,

658
Vol. 53, No. 6
14.6. LR-MS (EI) m/z: 270 (Mꢅ). Anal. Calcd for C15H14N2O3: C, 66.66; H, ent temperature, solvent was removed under reduced pressure. The resulting
5.22; N, 10.36. Found: C, 66.43; H, 5.30; N, 10.34. For 1e, recrystallization
residue was treated with sat. NaHCO3, and was extracted with ethyl acetate.
from acetone gave colorless solids, whose spectral data were identical with The combined organic phases were washed with brine, dried over Na2SO4
reported ones.23)
and concentrated in vacuo. The crude product was used in the next step
without further purification. To a solution of the resulting crude residue in p-
xylene (100 ml) at 0 °C was added 10% Pd/C (2.17 g), and the mixture was
stirred over night at 140 °C under an air atmosphere. The resulting solids
were removed through Celite® and washed with CHCl3. After concentration
of the filtrate, the resulting residue was chromatographed on silica gel with
toluene–acetone (7 : 1) to give compound 1e (4.01 g, 41%). Recrystallization
4-Methoxy-b-carboline-1-carbaldehyde (Kumujancine; 1f) To
a
stirred solution of 1d (468 mg, 1.73 mmol) in CH2Cl2 (30 ml) was added
DIBAL-H solution (1.0 M in hexane; 12.1 ml, 12.1 mmol) at ꢀ50 °C. The
mixture was stirred at ꢀ50 °C for 10 min and quenched by sequential addi-
tion of MeOH (14.0 ml) and 10% NaOH (9.53 ml) at ꢀ50 °C. Then the mix-
ture was stirred at ambient temperature for an additional 1 h. The precipi-
tates were removed through a Celite® and washed with CHCl3–MeOH from acetone gave colorless solids, whose spectral data were identical with
(10 : 1). The combined filtrate was washed with brine, dried over Na2SO4 reported ones.23)
and concentrated under reduced pressure. Recrystallization from
b-Carboline-1-carbaldehyde (Kumujian C; 1h) Compound 1h was
CH2Cl2–hexane gave compound 1f (376 mg, 96%) as yellow solids. The obtained in a similar manner to the synthesis of 1f (89% yield). The spectral
spectral data were identical with reported ones.24)
data were identical with reported ones.23)
1-(1-Hydroxyethyl)-4-methoxy-b-carboline (1g) To a stirred solution
of 1f (100 mg, 0.442 mmol) in THF (2.0 ml) was added MeLi (1.2 M in Et2O;
1.33 ml, 1.60 mmol) at ꢀ78 °C. The mixture was stirred at the sam tempera-
ture for 10 min, quenched by addition of MeOH, and concentrated in vacuo.
1-(1-Hydroxyethyl)-b-carboline (1i) Compound 1i was obtained in a
similar manner to the synthesis of 1h (79% yield). The spectral data were
identical with reported ones.25)
1-Vinyl-b-carboline (Pavettine; 1j) Compound 1j was obtained from
The residue was treated with sat. NaHCO3, and extracted with ethyl acetate. 1i in a similar manner to the synthesis of 1a from 1g (18% yield). The spec-
The combined organic phases were washed with brine, dried over Na2SO4 tral data were identical with reported ones.26)
and concentrated in vacuo. Recrystallization from CH2Cl2–hexane gave
Canthin-6-one (1k) To a stirred suspension of sodium hydride (5.0 mg,
0.125 mmol) in THF (0.5 ml) were added 1h (20 mg, 0.102 mmol) and, then,
AcCl (9.0 ml, 0.127 mmol) at 0 °C. The mixture was stirred for 5 h at ambi-
ent temperature. After removal of the solvent under reduced pressure, the re-
compound 1g (102 mg, 95%) as colorless solids, mp 190 °C. IR (KBr) cmꢀ1
:
3263. 1H-NMR (CDCl3) d: 8.98 (1H, br s), 8.32 (1H, d, Jꢁ8.3 Hz), 7.92
(1H, s), 7.50 (2H, m), 7.28 (1H, m), 5.34 (1H, q, Jꢁ 6.5 Hz), 4.12 (3H, s),
1.69 (3H, d, Jꢁ6.5 Hz). 13C-NMR (CD3OD) d: 152.7, 142.6, 141.5, 134.9, sulting residue was treated with sat. NaHCO3 and extracted with ethyl ac-
128.3, 124.7, 121.4, 120.6, 120.0, 119.3, 112.4, 71.0, 56.5, 23.3. LR-MS
etate. The combined organic phases were washed with brine, dried over
(EI) m/z: 242 (Mꢅ). HR-MS Calcd for C14H14N2O2, 242.1055; found Na2SO4 and concentrated. The residue was chromatographed on silica gel
242.1042.
with AcOEt–hexane (1 : 1) to give compound 1k (6.9 mg, 31%) as yellow
solids. The spectral data were identical with reported ones.27)
General Procedure for Quarternarization of b-Carbolines (Synthesis
of b-Carbolinium Salts) To a stirred solution of 1 in CH3CN was added
4-Methoxy-1-vinyl-b-carboline (MVC; 1a) From 1f: To a stirred solu-
tion of methyltriphenylphosphonium iodide (413 mg, 1.02 mmol) in THF
(5 ml) was added n-BuLi (1.59 M in hexane; 0.611 ml, 0.971 mmol) at 0 °C.
The mixture was then stirred at ambient temperature for 2 h. A solution of 1f alkylating agent (2 eq). The mixture was then stirred over night at 80 °C
(55 mg, 0.243 mmol) in THF (5 ml) was added to the above mixture at 0 °C,
and then the whole were heated at 70 °C for 2 h. The reaction mixture was
quenched by addition of NH4Cl at 0 °C, followed by NaHCO3, and extracted
with CHCl3. The combined organic phases were washed with brine, dried
over Na2SO4, and concentrated. The residue was chromatographed on silica
under an open atmosphere. After concentration in vacuo, the crude residue
was purified by recrystallization from appropriate solvent.
General Procedure for Ion Exchange (Preparation of Chloride Salts)
Compound 6 was dissolved in 10% KOH aq. (ca. 10 ml), and the resulting
mixture was extracted with AcOEt. The combined organic phases were
gel with AcOEt–hexane (1 : 1) to 1a (27.5 mg, 51%) as yellow solids. The washed with brine, dried over Na2SO4, and concentrated in vacuo. To a solu-
spectral data were identical with reported ones.16)
tion of the resulting residue in CHCl3 was added anhydrous HCl (1.0 M Et2O
solution). The resulting precipitates were corrected by filtration.
4-Methoxy-2-methyl-1-vinyl-b-carbolin-2-ium p-Toluenesulfonate
(6aa) Compound 6aa was obtained from 1a in 60% yield by recrystalliza-
From 1g: To a solution of 1g (50 mg, 0.206 mmol) in CH2Cl2 (0.50 ml) at
0 °C were added Et3N (144 ml, 1.03 mmol) and MsCl (24.0 ml, 0.310 mmol).
The reaction mixture was stirred at ambient temperature for 8 h, and then
quenched by addition of H2O at 0 °C. After extraction with CHCl3, the com- tion from CH3CN–Et2O as yellow solids, mp 208—220 °C. IR (KBr) cmꢀ1
:
bined organic phases were washed with brine, dried over Na2SO4 and con- 3422, 3038, 1622, 1541. UV-vis lmax (MeOH) nm (log e): 380 (3.80), 317
centrated. The residue was chromatographed on silica gel with toluene–ace-
(4.04). 1H-NMR (CD3OD) d: 8.35 (1H, d, Jꢁ8.3 Hz), 8.24 (1H, s), 7.73
tone (7 : 1) to give compound 1a (8.6 mg, 19%) as yellow solids, whose (2H, d, Jꢁ3.7 Hz), 7.65 (2H, d, Jꢁ7.9 Hz), 7.45—7.41 (1H, m), 7.18 (1H,
spectral data were identical with reported ones.16)
dd, Jꢁ17.8, 11.7 Hz), 7.15 (2H, d, Jꢁ7.9 Hz), 6.34 (1H, d, Jꢁ11.7 Hz), 6.28
2-Benzoyl-1-methyl-1,2,3,4-tetrahydro-b-carboline (3c) Compound
(1H, d, Jꢁ17.8 Hz), 4.35 (3H, s), 4.25 (3H, s), 2.30 (3H, s). 13C-NMR
3c was obtained in a similar manner to the synthesis of 3a. Yield 81% for 2 (CD3OD) d: 187.5, 153.2, 144.4, 143.5, 141.5, 135.0, 131.9, 130.2, 129.6,
1
steps, white amorphous. IR (KBr) cmꢀ1: 3200, 1609. H-NMR (CDCl3) d: 126.8, 126.1, 125.3, 123.3, 123.1, 120.6, 119.0, 113.5, 58.1, 46.4, 21.3. LR-
8.22 (1H, br s), 7.45—7.08 (9H, m), 5.87 (1H, m), 3.91 (1H, m), 3.45 (1H, MS (FAB) m/z: 239 (Mꢅ). HR-MS Calcd for C15H15N2Oꢅ, 239.1179; found
m), 2.88 (1H, m), 2.74 (1H, m), 1.62 (3H, m). LR-MS (EI) m/z: 290 (Mꢅ). 239.1176.
HR-MS Calcd for C19H18N2O, 290.1419; found 290.1408.
2-Benzoyl-1-methyl-4-oxo-1,2,3,4-tetrahydro-b-carboline (4c) Com- Compound 6ab was obtained from 1a in 30% yield, yellow solids from
pound 4c was obtained in a similar manner to the synthesis of 4a. Recrystal-
CH3CN–Et2O, mp 108 °C. IR (KBr) cmꢀ1: 3431, 3059, 1624, 1537. 1H-
2-Ethyl-4-methoxy-1-vinyl-b-carbolin-2-ium p-Toluenesulfonate (6ab)
lization from AcOEt gave compound 4c (4.32 g, 43%) as yellow solids, mp NMR (CD3OD) d: 8.40 (1H, d, Jꢁ8.1 Hz), 8.33 (1H, s), 7.75—7.74 (2H,
1
240 °C. IR (KBr) cmꢀ1: 3061, 1666, 1609. H-NMR (CDCl3) d: 11.0 (1H, m), 7.66 (2H, d, Jꢁ8.1 Hz), 7.46—7.42 (1H, m), 7.26 (1H, dd, Jꢁ17.8,
br s), 8.12 (1H, m), 7.61—7.07 (8H, m), 6.50 (1H, m), 4.41 (1H, d, 11.7 Hz), 7.17 (2H, d, Jꢁ8.1 Hz), 6.37 (1H, d, Jꢁ11.7 Hz), 6.32 (1H, d,
Jꢁ17.4 Hz), 4.20 (1H, d, Jꢁ17.4 Hz), 1.73 (3H, d, Jꢁ6.4 Hz). LR-MS (EI)
Jꢁ17.8 Hz), 4.74 (2H, q, Jꢁ7.3 Hz), 4.28 (3H, s), 2.32 (3H, s), 1.63 (3H, t,
m/z: 304 (Mꢅ). Anal. Calcd for C19H16N2O2: C, 74.98; H, 5.30; N, 9.20. Jꢁ7.3 Hz). 13C-NMR (CD3OD) d: 153.8, 144.6, 143.4, 141.4, 132.0, 130.7,
Found: C, 75.14; H, 5.41; N, 9.19.
129.7, 126.8, 125.8, 125.7, 123.4, 123.2, 120.7, 117.5, 113.5, 58.1, 54.9,
21.3, 16.4. LR-MS (FAB) m/z: 253 (Mꢅ). HR-MS Calcd for C16H17N2Oꢅ,
253.1335; found 253.1328.
4-Methoxy-1-methyl-b-carboline (1c) Compound 1c was obtained in a
similar manner to the synthesis of 1d. Yield 43%, as colorless solids, mp
1
179 °C. IR (KBr) cmꢀ1: 3072, 1624, 1589. H-NMR (CDCl3) d: 9.64 (1H,
4-Methoxy-1,2-dimethyl-b-carbolin-2-ium p-Toluenesulfonate (6cb)
br s), 8.33 (1H, d, Jꢁ8.0 Hz), 7.98 (1H, 2), 7.48—7.46 (2H, m), 7.29—7.24 Compound 6cb was obtained from 1c in 75% yield, white solids from
(1H, m), 4.10 (3H, s), 2.75 (3H, s). 13C-NMR (CDCl3) d: 150.7, 139.5, CH3CN, mp 249—250 °C. IR (KBr) cmꢀ1: 3437, 3068, 1632, 1549. 1H-
135.5, 135.0, 127.1, 124.1, 121.4, 120.3, 120.1, 117.8, 111.0, 56.1, 19.7. NMR (CD3OD) d: 8.32 (1H, d, Jꢁ8.0 Hz), 8.17 (1H, s), 7.71 (2H, m), 7.65
LR-MS (EI) m/z: 212 (Mꢅ). Anal. Calcd for C13H12NO2: C, 73.56; H, 5.70; (2H, d, Jꢁ8.0 Hz), 7.43—7.39 (1H, m), 7.14 (2H, d, Jꢁ8.0 Hz), 4.35 (3H,
N, 13.20. Found: C, 73.19; H, 5.72; N, 12.90.
Ethyl b-Carboline-1-carboxylate (Kumujian A; 1e) A suspension of
tryptamine hydrochloride (2; 8.00 g, 40.7 mmol) in ethanol (150 ml) was
added a solution of glyoxylic acid ethyl ester (12.5 g, 61.2 mmol) in toluene 6cc was prepared from 6cb in 88% yield, yellow solids from CHCl3, mp
(50% v/v) at 0 °C. After the reaction mixture was stirred over night at ambi-
s), 4.21 (3H, s), 2.97 (3H, s), 2.30 (3H, s). LR-MS (FAB) m/z: 227 (Mꢅ).
HR-MS Calcd for C14H15N2Oꢅ, 227.1179; found 227.1190.
4-Methoxy-1,2-dimethyl-b-carbolin-2-ium Chloride (6cc) Compound
1
276—278 °C. IR (KBr) cmꢀ1: 3385, 3053, 1630, 1545. H-NMR (CD3OD)

June 2005
659
d: 8.33 (1H, d, Jꢁ8.4 Hz), 8.21 (1H, s), 7.72 (2H, m), 7.42 (1H, m), 4.38
1.66 (3H, t, Jꢁ7.2 Hz). 13C-NMR (CD3OD) d: 152.5, 145.1, 136.8, 136.0,
(3H, s), 4.23 (3H, s), 3.00 (3H, s). LR-MS (FAB) m/z: 227 (Mꢅ). HR-MS 131.7, 125.5, 122.9, 122.4, 119.0, 118.3, 111.3, 58.2, 55.2, 34.0, 16.4, 16.1.
Calcd for C14H15N2Oꢅ, 227.1179; found 227.1175.
LR-MS (FAB) m/z: 255 (Mꢅ). HR-MS Calcd for C16H19N2Oꢅ, 255.1492;
2-Ethyl-4-methoxy-1-methyl-b-carbolin-2-ium p-Toluenesulfonate found 255.1499.
(6cd) Compound 6cd was obtained from 1c in 94% yield, white solids
1-Ethoxycarbonyl-4-methoxy-2-methyl-b-carbolin-2-ium p-Toluene-
from CH3CN, mp 171—172 °C. IR (KBr) cmꢀ1: 3445, 3061, 1628, 1545.
sulfonate (6d) Compound 6d was obtained from 1d in 72% yield as yel-
1H-NMR (CD3OD) d: 8.30 (1H, d, Jꢁ8.0 Hz), 8.23 (1H, s), 7.77—7.69 (2H, low solids (from CH3CN–Et2O), mp 210—212 °C. IR (KBr) cmꢀ1: 3425,
m), 7.60 (2H, d, Jꢁ8.2 Hz), 7.40 (1H, dd, Jꢁ8.0, 8.0 Hz), 7.09 (2H, d, 2924, 2855, 1728, 1624, 1601. H-NMR (CDCl3) d: 12.11 (1H, br s), 8.59
1
Jꢁ8.2 Hz), 4.74 (2H, q, Jꢁ7.2 Hz), 4.21 (3H, s), 3.19 (3H, s), 2.27 (3H, s),
1.63 (3H, t, Jꢁ7.2 Hz). 13C-NMR (CD3OD) d: 152.8, 145.6, 143.5, 141.4,
137.3, 136.3, 131.9, 129.6, 126.7, 125.9, 123.0, 119.5, 118.2, 111.5, 58.0,
(1H, s), 8.15 (1H, d, Jꢁ8.2 Hz), 7.91 (2H, d, Jꢁ7.9 Hz), 7.70 (1H, d,
Jꢁ7.6 Hz), 7.62 (1H, dd, Jꢁ8.2, 8.2 Hz), 7.29—7.26 (1H, m), 7.21 (2H, d,
Jꢁ7.9 Hz), 4.77 (2H, q, Jꢁ7.0 Hz), 4.66 (3H, s), 4.08 (3H, s), 2.38 (3H, s),
55.2, 34.0, 21.3, 16.3, 16.1. LR-MS (FAB) m/z: 241 (Mꢅ). HR-MS Calcd for 1.41 (3H, t, Jꢁ7.0 Hz). 13C-NMR (CDCl3) d: 192.8, 159.6, 152.3, 143.4,
C15H17N2Oꢅ, 241.1335; found 241.1340.
2-Ethyl-4-methoxy-1-methyl-b-carbolin-2-ium Chloride (6ce) Com- 114.1, 65.0, 58.3, 48.7, 21.4, 14.2. LR-MS (FAB) m/z: 285 (Mꢅ). Anal.
pound 6ce was prepared from 6cd in 77% yield, white yellow from CHCl3, Calcd for C23H24O6N2S: C, 60.51; H, 5.30; N, 6.14. Found: C, 60.16; H,
mp 262—263 °C. IR (KBr) cmꢀ1: 3396, 3061, 1628, 1541. 1H-NMR 5.53; N, 5.98.
139.6, 134.9, 131.6, 128.7, 125.8, 124.4, 122.5, 122.4, 122.1, 120.3, 117.7,
(CD3OD) d: 12.4 (1H, br s), 8.34 (1H, d, Jꢁ8.0 Hz), 8.26 (1H, s), 7.73—
7.72 (2H, m), 7.43—7.39 (1H, m), 4.74 (2H, q, Jꢁ7.2 Hz), 4.24 (3H, s),
1-(1-Hydroxyethyl)-2-methyl-b-carbolin-2-ium
p-Toluenesulfonate
(6i) Compound 6i was obtained from 1i in 72% yield as colorless solids
3.05 (3H, s), 1.65 (3H, t, Jꢁ7.2 Hz). 13C-NMR (CD3OD) d: 153.0, 144.0, (from CHCl3), mp 183—185 °C. IR (KBr) cmꢀ1: 3207, 1634, 1522. 1H-
136.8, 135.4, 131.6, 125.6, 122.9, 122.0, 120.5, 117.4, 113.3, 58.0, 54.4, NMR (CD3OD) d: 12.0 (1H, br s), 8.47 (1H, d, Jꢁ6.6 Hz), 8.38—8.36 (2H,
16.3, 14.6. LR-MS (FAB) m/z: 241 (Mꢅ). HR-MS Calcd for C15H17N2Oꢅ,
241.1335; found 241.1341.
m), 7.83—7.74 (2H, m), 7.63 (2H, d, Jꢁ7.8 Hz), 7.43 (1H, t, Jꢁ6.6 Hz),
7.14 (2H, d, Jꢁ7.8 Hz), 5.81 (1H, q, Jꢁ6.6 Hz), 4.41 (3H, s), 2.31 (3H, s),
2-(2-Hydroxyethyl)-4-methoxy-1-methyl-b-carbolin-2-ium Chloride 1.74 (3H, d, Jꢁ6.6 Hz). 13C-NMR (CD3OD) d: 146.9, 145.3, 143.3, 141.5,
(6cf) Compound 6cf was synthesized from 1c and 2-iodoethenol, followed 135.4, 135.0, 134.4, 133.0, 129.6, 126.7, 123.7, 122.8, 120.4, 117.0, 114.1,
by ion exchange, 19% overall yield, yellow solids from CHCl3, mp 259— 66.8, 45.7, 21.4. LR-MS (FAB) m/z: 227 (Mꢅ). HR-MS Calcd for
261 °C. IR (KBr) cmꢀ1: 3250, 3061, 1632, 1543. 1H-NMR (CD3OD) d: 12.4 C14H15N2Oꢅ, 227.1149; found 227.1175.
(1H, br s), 8.36 (1H, d, Jꢁ8.0 Hz), 8.22 (1H, s), 7.73—7.72 (2H, m), 7.44—
3-Methylcanthin-6-on-3-ium p-Toluenesulfonate (6k) Compound 6k
was obtained from 1k in 41% yield, colorless needles from CH3CN, mp
231—233 °C. IR (KBr) cmꢀ1: 3435, 1693, 1661. 1H-NMR (CD3OD) d: 8.98
(1H, d, Jꢁ6.4 Hz), 8.70 (1H, d, Jꢁ6.4 Hz), 8.65 (1H, d, Jꢁ8.3 Hz), 8.47
7.40 (1H, m), 4.83 (2H, t, Jꢁ5.2 Hz), 4.24 (3H, s), 4.11 (2H, t, Jꢁ5.2 Hz),
ꢅ
3.10 (3H, s). LR-MS (FAB) m/z: 257 (Mꢅ). HR-MS Calcd for C15H17N2O2
,
257.1285; found 257.1296.
4-Methoxy-2-(2-methoxyethyl)-1-methyl-b-carbolin-2-ium Chloride (1H, d, Jꢁ8.3 Hz), 8.45 (1H, d, Jꢁ10.2 Hz), 7.96 (1H, t, Jꢁ8.3 Hz), 7.72
(6cg) Compound 6cg was synthesized from 1c and 2-methoxyethyl p- (1H, t, Jꢁ8.3 Hz), 7.62 (2H, d, Jꢁ8.1 Hz), 7.30 (1H, d, Jꢁ10.2 Hz), 7.16
toluenesulfonate, followed by ion exchange, 76% overall yield, yellow solids (1H, d, Jꢁ8.1 Hz), 4.66 (3H, s), 2.32 (3H, s). LR-MS (FAB) m/z: 235 (Mꢅ).
from CHCl3, mp 238—239 °C. IR (KBr) cmꢀ1: 3385, 3040, 1630, 1543. 1H-
HR-MS Calcd for C15H11N2Oꢅ, 235.0866; found 235.0874.
NMR (CD3OD) d: 12.1 (1H, br s), 8.01 (1H, s), 7.89 (1H, d, Jꢁ8.0 Hz), 7.57
7-Methoxy-1,2-dimethyl-b-carbolin-2-ium p-Toluenesulfonate (6o)
(1H, dd, Jꢁ8.0, 8.0 Hz), 7.49 (1H, d, Jꢁ8.0 Hz), 7.21 (1H, dd, Jꢁ8.0, Compound 6o was obtained from 1o in 91% yield, colorless needles from
8.0 Hz), 4.77 (2H, t, Jꢁ4.8 Hz), 4.13 (3H, s), 3.93 (2H, t, Jꢁ4.8 Hz), 3.35
MeOH–diisopropylether, mp 191—192 °C. IR (KBr) cmꢀ1: 3427, 3074,
(3H, s), 2.90 (3H, s). 13C-NMR (CD3OD) d: 155.2, 143.5, 136.4, 136.0, 1628. UV-vis lmax (MeOH) nm (log e): 329 (4.29), 201 (5.30). 1H-NMR
131.4, 125.2, 122.6, 121.6, 120.0, 118.0, 113.0, 72.4, 59.4, 58.1, 58.0, 15.5. (CD3OD) d: 8.28 (1H, m), 8.15 (1H, dd, Jꢁ6.2, 6.2 Hz), 8.10—8.06 (1H,
LR-MS (FAB) m/z: 271 (Mꢅ). HR-MS Calcd for C16H19N2O2ꢅ, 271.1441; m), 7.66 (2H, d, Jꢁ8.3 Hz), 7.14 (2H, d, Jꢁ8.3 Hz), 7.07 (1H, m), 6.98 (1H,
found 271.1453.
m), 4.28 (3H, s), 3.93 (3H, s), 2.97 (3H, s), 2.29 (3H, s). 13C-NMR (CD3OD)
4-Methoxy-2-[2-(2-methoxyethoxy)ethyl]-1-methyl-b-carbolin-2-ium
d: 164.9, 147.4, 143.4, 141.5, 140.1, 136.2, 135.4, 133.2, 129.6, 126.8,
Chloride (6ch) Compound 6ch was synthesized from 1c and 2-(2- 124.9, 115.0, 114.3, 95.1, 56.3, 45.0, 21.3, 15.4. LR-MS (FAB) m/z: 227
methoxyethoxy)ethyl iodide, followed by ion exchange, 92% overall yield, (Mꢅ). HR-MS Calcd for C14H15N2Oꢅ, 227.1179; found 227.1192.
yellow solids from CHCl3, mp 131—132 °C. IR (KBr) cmꢀ1: 3348, 2874,
7-Methoxy-1,2,9-trimethyl-b-carbolin-2-ium p-Toluenesulfonate (6pa)
1
1628, 1541. H-NMR (CD3OD) d: 8.08 (2H, m), 7.64—7.60 (2H, m), 7.29 Compound 6pa was obtained from 1p in 45% yield, colorless needles from
(1H, m), 4.80 (2H, m), 4.18 (3H, s), 4.02 (2H, t, Jꢁ4.8 Hz), 3.59—3.57 (2H, MeOH–diisopropylether, mp 244—245 °C. IR (KBr) cmꢀ1: 3462, 1628,
m), 3.43—3.41 (2H, m), 3.17 (3H, s), 2.96 (3H, s). 13C-NMR (CD3OD) d: 1521. UV-vis lmax (MeOH) nm (log e): 332 (4.22), 201 (5.60). 1H-NMR
152.3, 143.7, 136.6, 131.6, 125.4, 122.8, 121.8, 120.2, 118.3, 113.2, 72.8,
71.5, 70.9, 59.0, 58.2, 58.0, 15.5. LR-MS (FAB) m/z: 315 (Mꢅ). HR-MS Jꢁ8.8 Hz), 7.60 (2H, d, Jꢁ8.3 Hz), 7.10 (1H, s), 7.09 (2H, d, Jꢁ8.3 Hz),
Calcd for C18H23N2O3ꢅ, 315.1703; found 315.1713.
7.01 (1H, d, Jꢁ8.8 Hz), 4.31 (3H, s), 4.15 (3H, s), 3.97 (3H, s), 3.17 (3H, s),
(CD3OD) d: 8.32 (1H, d, Jꢁ6.6 Hz), 8.18 (1H, d, Jꢁ6.6 Hz), 8.11 (1H, d,
4-Methoxy-2-[2-{(2-methoxyethoxy)ethoxy}ethyl]-1-methyl-b-car- 2.27 (3H, s). LR-MS (FAB) m/z: 240 (Mꢅ). HR-MS Calcd for C15H17N2Oꢅ,
bolin-2-ium Chloride (6ci) Compound 6ci was synthesized from 1c and
2-[(2-methoxyethoxy)ethoxy]ethyl p-toluenesulfonate, followed by ion ex-
241.1335; found 241.1350.
2-(2-Hydroxyethyl)-7-methoxy-1,9-dimethyl-b-carboli-2-ium Bromide
change, 12% overall yield, yellow solids from CHCl3, mp 200—201 °C. IR (6pb) Compound 6pb was obtained from 1p in 38% yield, colorless nee-
(KBr) cmꢀ1: 2868, 1630, 1543. 1H-NMR (CD3OD) d: 8.25 (1H, d, dles from MeOH–Et2O, mp 234—235 °C. IR (KBr) cmꢀ1: 3319, 1626. UV-
1
Jꢁ8.0 Hz), 8.20 (1H, s), 7.72—7.70 (2H, m), 7.41—7.38 (1H, m), 4.88 (2H, vis lmax (MeOH) nm (log e): 366 (3.88), (4.32). H-NMR (CD3OD) d: 8.40
t, Jꢁ4.8 Hz), 4.23 (3H, s), 4.05 (2H, t, Jꢁ4.8 Hz), 3.60—3.58 (2H, m),
(1H, d, Jꢁ6.4 Hz), 8.29 (1H, d, Jꢁ8.7 Hz), 8.17 (1H, d, Jꢁ8.7 Hz), 7.19
3.53—3.51 (2H, m), 3.42—3.40 (2H, m), 3.33—3.28 (2H, m), 3.15 (3H, s), (1H, s), 7.04 (1H, d, Jꢁ8.7 Hz), 4.83 (2H, m), 4.24 (3H, s), 4.07 (2H, m),
3.05 (3H, s). 13C-NMR (CD3OD) d: 152.5, 144.0, 136.9, 136.5, 131.8, 4.01 (3H, s), 3.33 (3H, s). 13C-NMR (CD3OD) d: 165.6, 149.6, 141.0, 136.1,
125.7, 123.0, 122.2, 120.5, 118.4, 113.3, 72.7, 71.7, 71.4, 71.2, 70.9, 58.9,
134.1, 125.0, 114.7, 114.5, 114.1, 94.0, 61.9, 60.0, 56.6, 34.3, 17.0. LR-MS
58.3, 58.0, 15.5. LR-MS (FAB) m/z: 359 (Mꢅ). HR-MS Calcd for (FAB) m/z: 271 (Mꢅ). HR-MS Calcd for C15H17N2Oꢅ, 271.1441; found
C20H27N2O4ꢅ, 359.1965; found 359.1967.
2-Ethyl-4-methoxy-1,9-dimethyl-b-carbolin-2-ium Chloride (6cj)
271.1459.
2-Methyl-1-(1-methyl-4-pyridinio)-b-carbolin-2-ium Di-(p-toluenesul-
Compound 6cd (100 mg, 0.242 mmol) was treated with 10% KOH aq. fonate) (6n) Compound 6n was obtained from 1n in 38% yield, yellow
(10 ml), and extracted with AcOEt. The combined organic phases were solids from CH3CN, mp 239—241 °C. IR (KBr) cmꢀ1: 3462, 3061, 1636.
washed with brine, dried over Na2SO4, and concentrated. Mixture of the re-
sulting residue and methyl p-toluenesulfonate (90.2 mg, 0.484 mmol) was
stirred for 18 h at 80 °C. After cooling to ambient temperature, the resulting
1H-NMR (CD3OD) d: 9.30 (2H, d, Jꢁ6.4 Hz), 8.77—8.71 (2H, m), 8.55
(2H, d, Jꢁ6.4 Hz), 8.45 (1H, d, Jꢁ8.0 Hz), 7.79 (1H, t, Jꢁ8.0 Hz), 7.61 (5H,
m), 7.51 (1H, t, Jꢁ8.0 Hz), 7.16 (4H, d, Jꢁ7.6 Hz), 4.61 (3H, s), 4.29 (3H,
precipitate was collected by filtration, which was subjected to ion exchange s), 2.32 (6H, s). LR-MS (FAB) m/z: 275 (Mꢅ). HR-MS Calcd for
polymer (Amberlyte IRA-400) to give 6cj (53.5 mg, 76%) as yellow solids, C18H17N3ꢅ, 275.1411; found 275.1405.
mp 214—215 °C. IR (KBr) cmꢀ1: 3404, 1624, 1603, 1531. 1H-NMR
Biological Experiment. Malaria Parasites Chloroquine-sensitive
(CD3OD) d: 8.23 (1H, s), 8.08 (1H, m), 7.66 (1H, m), 7.59 (1H, m), 7.29 Plasmodium falciparum (ATCC 30932, FCR-3 strain) and chloroquine-re-
(1H, m), 4.75 (2H, q, Jꢁ7.2 Hz), 4.21 (3H, s), 4.08 (3H, s), 3.13 (3H, s), sistant P. falciparum (K1; a clone originating from Thailand) were used in

660
Vol. 53, No. 6
our study. P. falciparum was cultivated by a modification of the method of
parasitemia for the tested compounds was calculated by the formula: [(aver-
Trager and Jensen41) using a 5% hematocrit of type Aꢅ human red blood age % parasitemia in controls (sham-treated)ꢀaverage % parasitemia, in
cells suspended in RPMI 1640 medium (Gibco, NY, U.S.A.) supplemented
treated mice)/average % parasitemia in controls (sham-treated)]ꢂ100. Five
infected, 0.9% NaCl solution (or olive oil)-dosed mice were used as a con-
with heat-inactivated 10% type Aꢅ human serum. The plates were placed in
a CO2–O2–N2 incubator (5% CO2, 5% O2, and 90% N2 atmosphere) at trol. The care and treatment of mice were in accordance with the guidelines
37 °C, and the medium was changed daily until 5% parasitemia (which (No. 141, 1987) issued by the Science and International Affairs Bureau of
means that 5 parasite-infected erythrocytes in every 100 erythrocytes were
existing).
the Japanese Ministry of Education, Culture, Science and Technology.
Mammalian Cells Mouse mammary tumor FM3A cells (wild-type,
subclone F28-7)42) were supplied by the Japanese Cancer Research Re-
sources Bank (JCRB). FM3A cells were maintained in suspension culture at
37 °C in a 5% CO2 atmosphere in plastic bottles containing ES medium
(Nissui Pharmaceuticals, Tokyo, Japan) supplemented with 2% heat-inacti-
vated fetal bovine serum (Gibco, NY, U.S.A.).
Acknowledgements This work was financially supported by a Grant-in-
Aid from the Takeda Science Foundation, a Grant-in-Aid from the Tokyo
Biochemical Research Foundation, a Grant-in-Aid from Japan Science and
Technology Agency (JST).
References and Notes
Evaluation of in Vitro Antiplasmodial Activity The following proce-
dures were used for assay of antimalarial activity (except for run 24 in Table
2)43,44) Asynchronously cultivated P. falciparum were used. Various concen-
trations of compounds in dimethylsulfoxide (DMSO) were prepared. 5 ml of
each solution was added to individual wells of a multidish, 24 wells. Ery-
throcytes with 0.3% parasitemia were added to each well containing 995 ml
of culture medium to give a final hematocrit level of 3%. The plates were in-
cubated at 37 °C for 72 h in a CO2–O2–N2 incubator (5% CO2, 5% O2, and
90% N2 atmosphere). To evaluate the antimalarial activity of test compound,
we prepared thin blood films from each culture and stained them with
Giemsa (E. Merck, Germany). Total 1ꢂ104 erythrocytes/1 thin blood film
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duplicate at each concentration. Drug-free control cultures were run simulta-
neously. All data points represent the mean of three experiments. Para-
sitemia in control reached between 4% and 5% at 72 h. The EC50 value
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Evaluation of Cytotoxicity against Mammalian Cell Line FM3A
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wells. The plates were incubated at 27 °C in a 5% CO2 atmosphere for 48 h.
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