Scholarly article on 4-chloro-2-(2-(3-chlorophenylamino)acetamido)benzoic acid 1262500-37-7 from Journal of Medicinal Chemistry p. 734

734 J. Med. Chem. 2011, 54, 734–750

DOI: 10.1021/jm101383f

Incorporation of Basic Side Chains into Cryptolepine Scaffold: Structure-Antimalarial Activity

Relationships and Mechanistic Studies

†

Joao Lavrado, Ghislain G. Cabal, Miguel Prudencio, Maria M. Mota, Jiri Gut, Philip J. Rosenthal, Cecılia Dıaz,

´ ´

‡

§

~

^

Rita C. Guedes,^†Daniel J. V. A. dos Santos,^†Elena Bichenkova,^{^}Kenneth T. Douglas,^{^}Rui Moreira,*^,†and Alexandra Paulo^†

^†Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon. Av. Prof. Gama Pinto,

1649-003 Lisbon, Portugal, ^‡Unidade de Malaꢀria, Instituto de Medicina Molecular, Universidade de Lisboa, 1649-028 Lisboa, Portugal,

^§Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, San Francisco, California 94143,

´

ꢀ

United States, Departamento de Bioquımica, Escuela de Medicina and Instituto Clodomiro Picado, Universidad de Costa Rica, 2060 San Jose,

Costa Rica, and ^{^}School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PT,

United Kingdom

Received June 7, 2010

The synthesis of cryptolepine derivatives containing basic side-chains at the C-11 position and their

evaluations for antiplasmodial and cytotoxicity properties are reported. Propyl, butyl, and cycloalkyl

diamine side chains significantly increased activity against chloroquine-resistant Plasmodium falcipa-

rum strains while reducing cytotoxicity when compared with the parent compound. Localization studies

inside parasite blood stages by fluorescence microscopy showed that these derivatives accumulate inside

the nucleus, indicating that the incorporation of a basic side chain is not sufficient enough to promote

selective accumulation in the acidic digestive vacuole of the parasite. Most of the compounds within this

series showed the ability to bind to a double-stranded DNA duplex as well to monomeric hematin,

suggesting that these are possible targets associated with the observed antimalarial activity. Overall,

these novel cryptolepine analogues with substantially improved antiplasmodial activity and selectivity

index provide a promising starting point for development of potent and highly selective agents against

drug-resistant malaria parasites.

Introduction

in part, to a chloroquine-like action, i.e. inhibition of hemo-

zoin (malaria pigment) formation.^17-20Degradation of hemo-

globin by malaria parasite proteases releases ferriproto-

porphyrin IX (FPIX), which is detoxified by crystallization to

hemozoin in the acidic digestive vacuole of the parasite.

Chloroquine (CQ^a), 2 (Figure 1), and related 4-aminoquino-

line antimalarials bind to FPIX via π-π stacking interaction

of the quinoline moiety with the porphyrin ring, thus inhibit-

ing detoxification.²¹It has been proposed that chloroquine

can also bind as a capping molecule to hemozoin crystals,

inhibiting crystal growth with consequent accumulation of

toxic free heme in the digestive vacuole of the parasite.²²

However, a cellular localization study by Arzel and co-

workers showed that cryptolepine accumulates into specific

structures of the parasite that could correspond to the parasite

nucleus.²³The data provided by Arzel and co-workers suggest

that the affinity of 1 for heme may not be sufficient to drive its

accumulation in the digestive vacuole in the absence of a pH-

dependent trapping mechanism, and indirectly point toward

additional (or alternative) mechanisms of action for cryp-

tolepine other than via hemozoin binding in the digestive

vacuole. Interaction of cryptolepine with parasite DNA might

be considered as an alternative mechanism contributing to the

antiprotozoal activity of cryptolepine analogues.²³

The emergence and spread of chloroquine-resistant Plas-

modium falciparum parasites has been a major global health

problem and contributes significantly to the continued high

prevalence of malaria.^1,2New, safe, and effective drugs active

against multidrug resistant P. falciparum strains are thus

urgently needed.^1,3Medicinal plants have long been used for

treating parasitic diseases, including malaria, and constitute

an important source of new molecules for lead optimization

programs, as exemplified by the success of artemisinin and its

derivatives.^4-8

Cryptolepine, 1 (Figure 1), is an indolo[3,2-b]quinoline

alkaloid first isolated in 1929⁹from the roots of Cryptolepis

sanguinolenta, a shrub used in traditional medicine for the

treatment of malaria in Central and West Africa. In addition,

cryptolepine also exerts antibacterial, antifungal, antihyper-

glycemic, anti-inflammatory, and antitumor activities.^10,11

Cryptolepine displays potent antiplasmodial activity against

both chloroquine-resistant and sensitive strains but unfortu-

nately also presents cross-resistance with chloroquine. More-

over, the cytotoxic properties of this alkaloid, which are likely to

result from intercalation of 1 into DNA GC-rich sequences and

inhibition of topoisomerase II,^12-14preclude its clinical use.^14-16

The antimalarial mechanism of action of cryptolepine is not

yet clarified, but it was shown that this activity is due, at least

^aAbbreviations: HUVEC, human umbilical vein endothelial cells;

PPA, polyphosphoric acid; CQ, chloroquine; SI, selectivity index; RI,

resistance index; GFP, green fluorescent protein; FP, falcipain.

*To whom correspondence should be addressed: Phone: þ351 217

946 477. Fax: þ351 217 946 470. E-mail: rmoreira@ff.ul.pt.

r

pubs.acs.org/jmc

Published on Web 01/05/2011

2011 American Chemical Society

Article

Recently, on the basis of the observation that a basic amino

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 735

nitrogen atoms as well as the substitution pattern and basicity

of the distal amino group. Antiplasmodial and cytotoxic activ-

ities of cryptolepine derivatives 3a-y were evaluated against

different P. falciparum strains and a mammalian cell line.

Taking advantage of the fluorescence properties of the in-

doloquinoline nucleus, fluorescence microscopy was used to

evaluate the subcellular localization of the compounds in

parasites. To identify possible biological targets, which could

potentially be linked to the antiplasmodial activity of cryp-

tolepine derivatives, additional experiments were performed

to determine the ability of cryptolepine analogues to interact

with hematin, with cysteine proteases involved in the degrada-

tion of host hemoglobin (falcipain-2 and -3) and with DNA

oligonucleotide duplex.

side chain is a major requirement for chloroquine accumula-

tion in the acid digestive vacuole of the parasite, we synthesized

a small series of cryptolepine analogues containing diamino-

alkane side chains (-NH-linker-NR¹R²) at C-11. These

compounds were very potent against the P. falciparum W2

chloroquine-resistant strain, with IC₅₀values ranging from

22 to 184 nM.²⁴From a preliminary cytotoxicity study using

human umbilical vein endothelial cells (HUVEC), several

compounds emerged with selectivity ratios that were improved

compared to 1. This promising result prompted us to inves-

tigate structure-activity relationships (SAR) to decipher the

structural requirements of side-chains at C-11 of the cryp-

tolepine scaffold, i.e. analogues3a-y (Figure 1), for improved

antiplasmodial activity and selectivity relative to the lead

compound. The diversity of the side chain was explored by

varying the nature and length of the linker between the two

Chemistry

Cryptolepine analogues 3a-y were synthesized according

to the route depicted in Scheme 1, via the 11-Cl intermediates

€

10, based on the procedure developed by Gorlitzer and

Weber^25,26and adapted by Bierer.^27,28Anthranilic acids, 4,

were treated with bromoacetyl bromide to afford the corre-

sponding bromoacetyl derivatives 5, which were then reacted

with the appropriate aniline to give compounds 6. Acid-

catalyzed cyclization of 6 with polyphosphoric acid (PPA)

gave the indolo[3,2-b]quinolin-11-ones (quindolones) 7, which,

by reaction with POCl₃, gave the corresponding substituted 11-

chloro-indolo[3,2-b]quinolines (11-chloroquindolines) 8. Hy-

drogenation of 8a with 10% Pd-C at 60 psi provided the

indolo[3,2-b]quinoline (quindoline) 9. N-5 Methylation of 8

and 9 was achieved by reaction with methyl triflate, and the

5-methyl-indolo[3,2-b]quinoline derivatives of 8 were then

treated with hydrochloric acid to afford the corresponding

cryptolepine chlorides, 10. Finally, cryptolepine derivatives

3a-n and 3r-y were obtained in 30-82% yield by aromatic

nucleophilic substitution on 10 with excess of appropriate

amine. Compounds 3o-q were synthesized by reaction of 3n

with isobutyraldehyde, benzylaldehyde, and salicylaldehyde

Figure 1. Structures of cryptolepine (1), chloroquine (2), and cryp-

tolepine analogues (3a-y).

Scheme 1. Synthesis of Cryptolepine (1) and Cryptolepine Analogues 3a-y^a

^aReagents and conditions: (i) bromoacetyl bromide, DMF/1,4-dioxane (1:1), rt; (ii) aniline, DMF, 100 ꢀC; (iii) PPA, 130 ꢀC; (iv) POCl₃, 130 ꢀC; (v) H₂

Pd-C 10% NaOAc, AcOH, 60 psi; (vi) (a) 8 or 9, methyl triflate, toluene, rt, (b) HCl in ether for 8; (vii) NH₂-linker-NR⁴R⁵, AcOEt, reflux.

736 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

and subsequent reduction with NaBH₃CN in 59, 66, and 30%

yields, respectively (Scheme 2).

Structures of all compounds 3a-y were established on the

basis of two-dimensional ¹H and ¹³C heterocorrelation NMR

experiments (HMQC and HMBC) and the position of the side

chain at C11 was confirmed by NOE difference experiments.

Figure 2 provides an example of such NOE connectivities

detected for 3h in the NMR experiments, which shows a

¹H-¹H connectivity between the side chain methylene group

adjacent to NH-C11 (δ_H4.21 ppm) with the proton of the

indole nitrogen (δ_H10.75 ppm), as well as the protons at

δ_H4.56 ppm from the N5-CH₃group with doublets at δ_H8.33

and 8.51 ppm, assigned to aromatic protons at C4 and C6,

respectively. Cryptolepine analogues 3 showed also a shield-

ing effect on ¹H chemical shifts of N5-CH₃of 0.1-0.45 ppm

when compared with 10. Additionally, the ¹³C chemical

shifts of C10a and C11a showed an upfield shift of the order of

10 ppm and of 3 ppm for N5-CH₃, when compared with 1 and

10, whereas the chemical shift of C11 showed a deshielding

effect of 10-15 ppm when compared with the parent com-

pounds 1 and 10.

For derivatives 3d, 3j, 3l, and 3t, it was necessary to

synthesize the corresponding diamines required for the sub-

stitution reaction with 10. Thus, N¹,N¹-dimethylpropane-1,2-

diamine, 12, used for the synthesis of 3d, was prepared by

reductive amination of N,N-dimethylaminopropanone, 11, with

ammonium acetate and NaBH₃CN (Scheme 3). 3-(Piperidin-

1-yl)propan-1-amine, 15, required for the synthesis of 3j,

was prepared via Gabriel synthesis, using phthalimide 13

as starting material (Scheme 3). N¹,N¹-Diethylbutane-1,4-

diamine, 18, required for the synthesis of 3l, was prepared

by reductive alkylationof16 with acetaldehyde and NaBH₃CN,

followed by removal of the Boc group with triflouroacetic acid

(Scheme 3). Compound 3t was synthesized by reaction of

10a with acetamide, 20, which was obtained by Mannich

reaction of phenol 19 with formaldehyde and diethylamine

(Scheme 3).

Scheme 2. Synthesis of Cryptolepine Analogues 3o-q^a

^aReagents and conditions: (i) BuⁱCHO, PhCH₂CHO or 2-OH-

C₆H₄CHO, anhydrous Na₂SO₄, NaBH₃CN, dry MeOH, rt, 24 h.

Figure 2. NOE correlations observed for cryptolepine derivative 3h

in the NMR experiments.

Scheme 3. Synthesis of Amines 12, 15, 18, and 20^a

^aReagents and conditions: (i) NH₄OAc, NaBH₃CN, anhydrous MgSO₄, dry MeOH, reflux; (ii) piperidine, triethylamine, CH₂Cl₂, reflux;

(iii) hydrazine; (iv) MeCHO, anhydrous MgSO₄, NaBH₃CN, dry MeOH, 0 ꢀC, 2h; (v) CH₂Cl₂:TFA (1:1), rt, 1 h; (vi) aq HCHO, diethylamine, EtOH,

reflux, 96 h.

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 737

Table 1. In Vitro Antiplasmodial Activity (IC₅₀) against P. falciparum W2, V1/S, 3D7, and D6 Strains, Cytotoxicity (IC₅₀) against Vero Cells,

Selectivity Index (SI), and Resistance Index (RI) of Chloroquine (2), Cryptolepine (1) and Derivatives (3a-y)

^aSelectivity index toward W2 strain, which is expressed by the ratio IC₅₀^Vero/IC₅₀

.

^bResistance index, which is expressed by the ratio IC₅₀

/

W2

3D7

IC₅₀

.

Results and Discussion

against both CQ-sensitive and CQ-resistant strains. The

exception seems to be derivatives 3 containing a terminal

primary amine, as these compounds displayed reduced ac-

tivity, particularly against 3D7 and D6 strains, when com-

pared with compounds with secondary (3e vs 3i) or tertiary

terminal amines (e.g., 3a vs 3c, 3e vs 3g and 3k vs 3l). The

reason for these differences in activity is not obvious, and an

extended evaluation of terminal amines will be necessary to

further understand the loss of potency in compounds 3 with a

terminal primary amine. Additional evidence for the impor-

tance of a basic distal amine for antiplasmodial activity comes

from the observation that compound 3y which lacks a distal

basic amine group is weakly active, with IC₅₀values of 1299-

2200 nM against CQ-sensitive and CQ- resistant strains.

Antiplasmodial Activity. Cryptolepine, 1, and derivatives

3a-y were evaluated for their antiplasmodial activity against

a panel of P. falciparum strains with different drug resistance

phenotypes: W2 (chloroquine-resistant), 3D7 (chloroquine-

sensitive), V1/S (chloroquine- and pyrimethamine-resistant),

and D6 (chloroquine-sensitive, mefloquine-resistant) (Table 1).

Cryptolepine was 2- to 3.5-fold more active against the CQ-

sensitive than the CQ-resistant strains, with resistance index

(RI), as expressed by the ratio IC₅₀^W2/IC₅₀^3D7, of about 3.

More importantly, cryptolepine derivatives containing a

secondary or tertiary terminal basic amine (e.g. 3b, 3f, 3g, 3i,

3j, and 3n) were shown to be potent antiplasmodials, with

IC₅₀values generally of 20-90 nM (Table 1). These results

indicate that the diaminoalkane side chain, when attached to

C-11 of the indolo[3,2-b]quinoline, leads to potent compounds

A comparison of the different linkers between the distal

basic center and the indolo[3,2-b]quinoline moiety reveals

738 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

Anti-Plasmodium Liver Stage Activity. Cryptolepine and

derivative 3n were also assessed against liver stages of the

rodent malaria parasite Plasmodium berghei at a concentra-

tion of 5 μM, using a recently described fluorescence acti-

vated cell sorting (FACS)-based method.³⁷This method is

based on the measurement of the fluorescence of Huh-7 cells,

a human hepatoma cell line, following infection with GFP-

expressing P. berghei sporozoites. Both compounds were

inactive in this assay, a result that points toward the selec-

tivity of 1 and derivatives 3 for erythrocytic stages of malaria

parasites.

Cytotoxicity. The in vitro cytotoxicity of compounds

3a-y and cryptolepine was evaluated using mammalian

Vero cells. Remarkably, most of the derivatives 3a-y were

less cytotoxic than the parent compound, with 3a, 3k, and 3n

being 60- to 85-fold less cytotoxic than cryptolepine (Table 1).

Moreover, a comparison of selectivity index (SI) results, as

expressed by the ratio IC₅₀^Vero/IC₅₀^W2, reveals that com-

pounds 3 with a terminal primary amine (i.e., 3a, 3e, and 3k,

and with the butyl and piperidine side-chains (3l and 3n,

respectively) were significantly more selective than the par-

ent compound. The presence of chlorine atoms in the indolo-

[3,2-b]quinoline led to an increase in cytotoxicity when

compared to the unsubstituted counterpart, 3n. Incorpora-

tion of isoquine’s R-aminocresol motif at C-11 (i.e., 3t),

resulted in a cytotoxicity level comparable to that of crypto-

lepine, despite the improvement in SI.

Distribution into P. falciparum-Infected Erythrocytes. Cryp-

tolepine, 1, was previously reported to accumulate in the nucleus of

schizont-infected erythrocytes, which led to the conclusion that

cryptolepine interferes with the parasite’s replication at schizont

stage due to its DNA intercalating ability.²³To investigate the

possible sites of action of newly synthesized cryptolepine ana-

logues 3, the intracellular distribution of 3n in blood stages of

P. falciparum was studied by fluorescence microscopy and

compared with that of 1. Figure 4 shows the contrast phase

and fluorescence images of P. falciparum-infected erythrocytes

after incubation of P. falciparum cultures with the compounds.

Our data shows that 1 and its derivative 3n accumulate inside

erythrocytic ring-stage parasites and the observed intensity of

fluorescence indicates that nucleus and food vacuole are parasite

organelles targeted by both compounds. It should be noted

that hematin very efficiently quenches the fluorescence of cryp-

tolepine derivative 3n, which results in around 60% quenching

of fluorescence intensity for a 1:1 ligand:hematin ratio, while

DNA only quenches 20% the fluorescence of 3n (Figures S3

and S4, Supporting Information). Quenching by hematin has

also been reported for the fluorescence of acridine conjugates.³⁸

These results suggest that quenching of 3n in the food vacuole

may occur as a result of the presence of hematin, thus under-

estimating the vacuolar signal when compared with the

nonvacuolar signal arising from accumulation in the nucleus.

Overall, this data indicates that incorporation of basic side

chains into the cryptolepine structure does not notably promote

selective accumulation of cryptolepine analogues toward acidic

digestive vacuole of the parasite.

Figure 3. Structures of amodiaquine and its isomer isoquine.

that compounds 3 containing a propyl or butyl linear side

chain are slightly more potent than their ethyl counterparts.

For example, the N,N-diethylaminopropyl and N,N-diethyl-

aminobutyl derivatives, 3g and 3l, are approximately 2.5

times more potent than their ethyl counterpart, 3c, against

the CQ-resistant W2 strain. These results are consistent with

reports that 4-aminoquinoline analogues with shortened side-

chains retain activity against CQ-resistant strains, although

no major difference in activity has been observed between the

ethyl and propyl linkers.^29,30

Branched linkers lead to a significant reduction in anti-

plasmodial activity when compared with their linear coun-

terparts (e.g., 3d vs 3b and 3h vs 3f-g,i-j). Furthermore, com-

pound 3m, containing a side chain identical to that of

chloroquine, displayed reduced activity against both CQ-

resistant and CQ-sensitive P. falciparum strains (IC₅₀rang-

ing from 120 to 275 nM), being 2- to 6-fold less potent than

its linear butyl counterpart 3l. These results are in line with

the observation that 4-aminoquinolines containing the chloro-

quine side chain give consistently lower potency than those

with a propyl side chain against both 3D7 and W2 strains.³¹

Compound 3n, with a piperidine side-chain, was highly

active against both CQ-resistant and CQ-sensitive strains,

suggesting that a conformational constraint between the distal

basic center and the indolo[3,2-b]quinoline scaffold may

improve potency. Appending a sterically demanding iso-

butyl or benzyl group to the nitrogen atom of the piperidine

moiety (3o and 3p, respectively) did not significantly alter

antiplasmodial activity. To our surprise, the derivative con-

taining a 2-hydroxybenzyl group, 3q, was 2- to 10-fold less

active than its benzyl counterpart, 3p. In contrast, compound

3t, which contains a 2-diethylaminomethylphenol moiety,

exhibited IC₅₀values of ca. 20 and 54 nM against the CQ-

sensitive and CQ-resistant strains, respectively. The 2-diethyl-

aminomethylphenol and related R-aminocresol motifs are

commonly found in potent antimalarials such as amodia-

quine and its isomer isoquine (Figure 3),^32-34and it has been

postulated that intramolecular hydrogen bonding between

the protonated amine (H-bond donor) and the hydroxyl

(H-bond acceptor) may be an important feature for activity

against CQ-resistant P. falciparum strains.³⁵

The 3-chloro and 3,8-dichloro derivatives, 3w and 3x,

respectively, were equipotent (IC₅₀≈ 45 nM) with their

unsubstituted counterpart, 3n, against the panel of CQ-

sensitive and CQ-resistant strains, suggesting that introduc-

tion of electron-withdrawing substituents in the indolo[3,2-b]-

quinoline moiety does not significantly affect antiplasmodial

activity when a diaminoalkane side-chain is present at C11.

Interestingly, it has been reported that 3-chlorocryptolepine

is equipotent with cryptolepine against the P. falciparum K1

strain, while its 8-chloro counterpart is inactive.³⁶In con-

trast, 2,8-dichlorocryptolepine was found to be 10-fold more

potent than cryptolepine in the same screen.^11,36

Binding to a Double-Stranded Oligonucletide. The observa-

tion that compounds 3 can accumulate in the nucleus prompted

us to explore alternative targets for their antiplasmodial

activity and study their binding properties toward DNA

using a 12-mer double-stranded d(GATCCTAGGATC)₂

oligonucleotide as a model system. This self-complementary

oligonucleotide was preferred over larger double-stranded

structures in order to avoid multiple binding modes that

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 739

Figure 4. Intracellular localization of 1 and 3n in P. falciparum-infected erythrocytes. Cells were incubated for 3 h at room temperature, in the

dark, with 5 μM of cryptolepine and its analogue 3n and immediately observed using a Zeiss Axiovert 200 M fluorescence microscope after

being washed with PBS. The yellow lines indicate the regions corresponding to the plot profiles on the right. Green, compounds 1 or 3n; blue,

nuclei; gray line, digestive vacuole (phase contrast image).

could preclude the determination of binding constants accu-

rately.

The equilibrium association constants of cryptolepine

derivatives 3a-y binding to d(GATCCTAGGATC)₂in phos-

phate buffer containing 0.1 M NaCl and at 25 ꢀC were

determined by spectrofluorometry. All emission spectra were

recorded at an excitation wavelength of 339 nm. Emission of

compounds 3 in aqueous solutions was characterized by broad

band at ca. 460 nm, and its fluorescence intensity was propor-

tional to their concentrations up to 20 μM, indicating that

there was no significant intermolecular stacking which would

give rise to a quenching effect. The fluorescence titration spectra

of compounds 3a-y (see Figure 5a for 3n as an example)

showed shifts of 5-10 nm and up to 60% quenching of

fluorescence intensity on addition of the oligonucleotide,

thus indicating the formation of a complex. The plots of

the change in the fluorescence intensity (see Supporting

Information), corrected for dilution, vs the concentration of

the oligonucleotide added were fitted by the equation for a

single-site binding (eq 1),

F ¼ F_max½DNAꢀ=ðK_dþ ½DNAꢀÞ

ð1Þ

where F is the observed value of emission intensity, F_maxits

limiting value at high values of [DNA], and K_dthe dissocia-

tion constant. The equilibrium association constants, K_ass

were determined as the inverse of K_d(eq 2),

,

K_ass¼ 1=K_d

ð2Þ

For compounds 3e, 3j, 3m-n, 3q-t, and 3x-y, there was

also evidence of an additional, much weaker binding site, as

revealed by a continuous, but very small, increase in the

fluorescence intensity at high concentration of oligonucleo-

tide (see Supporting Information). In these cases, the plots of

fluorescence intensity vs [DNA] were fitted by the simplified

equation for two independently acting binding sites (eq 3),

Figure 5. Fluorescence and UV-visible spectral changes at 25.0 ꢀC

upon titration of 3n and cryptolepine with the 12-mer DNA duplex

d(GATCCTAGGATC)₂in 0.01 M phosphate buffer at pH 7.40

containing 0.1 M NaCl. (a) Fluorescence emission spectra of 3n

(5 μM). The DNA:ligand ratio increased as follows: 0, 0.3, 0.5, 0.8,

1.1, 1.6, 2.1, 2.6, and 3.1 sequentially from the top spectrum.

(b) UV-visible spectra of 1 (5 μM). The DNA:ligand ratio increased

as follows: 0, 0.2, 0.5, 1.0, 1.6, 2.1, 2.6, 3.1, 4.0, 5.0, 6.4, 7.6, 8.8, 10.2,

11.3 and 12.3, sequentially from the top spectrum. The expanded

box shows details in the region of the 374 nm isosbestic point.

F ¼ ðfF_max½DNAꢀ=ðK_dþ ½DNAꢀÞg þ C½DNAꢀÞ

ð3Þ

where C = F⁰_max/K⁰_d, and K⁰_dis the dissociation constant for

the weaker process (see Experimental Section). The equilibrium

740 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

Table 2. Association Constants, K_ass, for Binding of Cryptolepine Derivatives 3 to d(GATCCTAGGATC)₂(ds-DNA) and Heme, along with IC₅₀

Values for the Inhibition of Falcipain-2 (FP-2) and -3 (FP-3)

ds-DNA

heme binding

FP-2 IC₅₀FP-3 IC₅₀

( μM)

ds-DNA K_ass

(ꢁ 10⁶M^-1

heme binding

FP-2 IC₅₀

( μM)

FP-3 IC₅₀

( μM)

compd

K

_ass(ꢁ 10⁶M^-1

)

K_ass(ꢁ 10⁶M^-1

)

( μM)

>50

compd

)

K_ass(ꢁ 10⁶M^-1

)

1

0.25 ( 0.01^a

0.85 ( 0.01^b

3.1 ( 0.7^b

1.14 ( 0.05^b

1.9 ( 0.1^b

0.25 ( 0.01^b

3.0 ( 0.2^b

9.1 ( 0.6^b

2.6 ( 0.4^b

5.0 ( 0.8^b

5.3 ( 0.3^b

1.4 ( 0.1^b

9 ( 1^b

0.045 ( 0.003

0.127 ( 0.007

0.120 ( 0.005

0.41 ( 0.03

0.148 ( 0.008

0.18 ( 0.02

0.066 ( 0.005

0.127 ( 0.007

0.069 ( 0.007

0.20 ( 0.01

0.161 ( 0.008

0.147 ( 0.006

0.15 ( 0.01

38 ( 9

3m

3n

3o

3p

3q

3r

4.3 ( 0.2^b

0.062 ( 0.07

0.154 ( 0.006

0.227 ( 0.008

0.125 ( 0.007

0.108 ( 0.005

0.155 ( 0.008

0.21 ( 0.02

0.075 ( 0.009

0.17 ( 0.01

0.11 ( 0.01

0.18 ( 0.01

11.6 ( 0.2

6.39 ( 0.04

10.5 ( 0.6

12.8 ( 0.9

14.8 ( 0.2

14 ( 2

8.2 ( 0.2

15.2 ( 0.9

9.32 ( 0.09

11 ( 1

7 ( 2

7.9 ( 0.5

16 ( 2

ND

3a

3b

3c

3d

3e

3f

3g

3h

3i

6.3 ( 0.2

>25

ND^c

ND

0.74 ( 0.03^b

0.74 ( 0.07^b

0.68 ( 0.09^b

0.6 ( 0.3^b

0.51 ( 0.06^b

3.4 ( 0.6^b

0.8 ( 0.3^b

1.0 ( 0.1^b

17 ( 1^b

>50

8.3 ( 0.5

11 ( 1

34 ( 5

14 ( 1

>50

ND

26 ( 7

ND

43 ( 6

ND

3s

ND

3t

ND

33.5 ( 0.8

>25

ND

3u

3v

3w

3x

3y

ND

3j

4.5 ( 0.4^b

1.6 ( 0.4^b

0.47 ( 0.06^b

5.6 ( 0.1

4.8 ( 0.3

18 ( 2

3k

3l

ND

0.12 ( 0.01

0.19 ( 0.01

ND

46.3 ( 0.8

^aUV-visble spectroscopy determination. ^bFluorescence spectroscopy determination. ^cND: not determined.

Figure 6. Job plots for (a) 1 and (b) 3v complexed with 12-mer d(GATCCTAGGATC)₂oligonucleotide at 0.01 M phosphate buffer (pH 7.4),

containing 0.1 M NaCl, at 25 ꢀC. The sum of the concentrations of ligand and oligonucleotide was kept constant at 12.5 μM for 1 and 2.5 μM for

3v. For 1, absorbance was measured at 368 nm, and for 3v, fluorescence was measured at 483 nm with excitation at 339 nm.

association constants, K_ass, for compounds 3a-y are presented

in Table 2.

ligand:oligonucleotide ratio of 2:1 for both complexes. The

2:1 stoichiometry for the cryptolepine:oligonucleotide com-

plex detemined in this research is consistent with the previously

reported crystal structure of cryptolepine:d(CCATGG)₂,

in which cryptolepine molecules were stacked between the

duplexes as well as at each extremity of the double-hexamer

duplex and intercalated in two CC sites.¹²

The spectrofluorometric assay failed with cryptolepine

triflate salt, 1, because no quenching was observed with

increasing concentrations of oligonucleotide, and thus bind-

ing of cryptolepine to the DNA duplex d(GATCCTAG-

GATC)₂was assessed by UV-visible spectroscopy. The

spectral changes of triflate salt of 1 showed bathochromic

and hypochromic shifts upon addition of oligonucleotide duplex

(Figure 5b). Sharp isosbestic points were present at 374 and

405 nm for oligonucleotide/ligand ratios from 0 to 2.6,

indicating a relatively strong binding of the triflate salt 1 to

d(GATCCTAGGATC)₂. Further increases in oligonucleo-

tide concentration caused a shift in the isosbestic point,

suggesting a second, weaker binding process at higher oli-

gonucleotide concentrations. This result is consistent with

two independent and noncooperative types of binding as

previously reported for cryptolepine.⁴⁰

To obtain a better insight into the nature of the interaction

with the double-stranded oligunucleotide, titrations were

also performed at a higher ionic strength. Titration of 1 with

d(GATCCTAGGATC)₂was performed in 0.01 M phos-

phate buffer (pH 7.4) containing 1 M NaCl. The increased

concentration of Na^þcounter cations resulted in a K_assvalue

of 1.0 ((0.1) ꢁ 10⁵M^-1, which is of the same order of

magnitude as that determined in phosphate buffer contain-

ing 0.1 M NaCl (Table 2). As expected, 1 maintains its

binding capabilities to the double-stranded oligonucleotide at

high ionic strength solutions, consistent with intercalation.^43,44

Incontrast,nointeractionwasobservedbetween3n and 3t with

the DNA duplex d(GATCCTAGGATC)₂in phosphate

buffer containing 1 M NaCl. This remarkable loss of binding

in the presence of high concentration of Na^þcounter cations

strongly suggests that binding properties of derivatives 3 to

the oligonucleotide duplex are largely dependent on electro-

static interactions between the two species, which can be

attributed to the interactions between the protonated pri-

mary, secondary, or tertiary amine groups 3a-y and nega-

tively charged sugar-phosphate backbones of the DNA

Job plot analysis^41,42was performed to measure the

stoichiometry of association of cryptolepine and its deriva-

tive showing the highest K_ass, 3v, with d(GATCCTA-

GGATC)₂in 0.01 M phosphate buffer at pH 7.40 containing

0.1 M NaCl. The maximum absorbance for 1 and 3v as a

function of the mole fraction of ligand was plotted to give

linear dependences at high and low molar fractions (Figure 6).

The stoichiometry of the formed complex was determined by

the intersections of these lines. These intersections at ligand

mole fractions of 0.67 (for 1) and 0.66 (for 3v) suggests a

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 741

duplex. This data provides an evidence that incorporation of

a basic side chain into cryptolepine structure alters to a

certain extent the mode of interaction of the ligand with

the DNA duplex by adding electrostatic component to that,

which seem to become highly essential, and might be linked

to the enhanced antiplasmodial activity seen for the cryp-

tolepine analogues.

From the data in Table 2, K_assvalues for cryptolepine

derivatives 3a-y span almost 2 orders of magnitude, ranging

from 0.25 to 17 ꢁ 10⁶M^-1. Overall, these derivatives interact

more strongly with the oligonucleotide than cryptolepine.

Interestingly, the K_assvalues also compare favorably with

that of chloroquine, which has been recently reported to bind

to DNA with a K_assvalue of ca. 10⁵M^{-1 39}

Inspection of

.

Table 2 shows that compounds 3 containing linear side

chains with a distal tertiary or secondary amine generally

display higher binding affinities toward the oligonucleotide

than those with a terminal primary amine (e.g., 3g vs 3e and

3l vs 3k), a trend similar to that observed with antiplasmodial

activity (Table 1). There appears to be no clear correlation

between the in vitro cytotoxicity and the binding of com-

pounds 3 to the oligonucleotide based on measured the corre-

sponding K_assvalues. In an attempt to understand the

observed structure-binding relationships, molecular proper-

ties of selected cryptolepine derivatives 3 were studied by density

functional theory (DFT; see Supporting Information). How-

ever, no clear-cut correlations emerged between molecular

parameters such as the atomic charges at N-10 and N-5 of 3

and the corresponding K_assvalues and thus this matter

requires further detailed investigations in a separate study.

Hematin Binding Studies. Taking in account that (i) the

antiplasmodial mechanism of cryptolepine is likely to be due,

at least in part, to inhibition of hemozoin formation¹⁹and (ii)

the fluorescence microscopy study could not rule out dis-

tribution of compounds 3 into the digestive vacuole of the

parasite, we decided to evaluate the binding of compounds 3

to hematin.

The equilibrium binding constants (K_ass) with hematin

monomer (FPIX-OH) were determined by UV-visible titra-

tion carried out in pH 5.5 buffer containing 40% DMSO

v/v to minimize porphyrin aggregation and μ-oxo dimer

formation.^34,45-48Spectral changes at the FPIX-OH Soret

band (λ = 402 nm)^49,50were recorded, and the resulting

absorbance data were first corrected for dilution and then

analyzed using GraphPad nonlinear curve fitting software.

Several binding processes have been considered for fitting

the data from quinoline and acridine antimalarials, the most

successful corresponding to (i) 1:1 association between drug

and FPIX-OH (model 1), (ii) 2:1 association of drug to FPIX-

OH in a stepwise fashion (model 2), and (iii) 1:2 stepwise

association of drug to FPIX-OH (model 3).^49,51,52The best fit

of absorbance data from compounds 3a-y and cryptolepine

was obtained with model 1 (Supporting Information), and the

resulting log K_assvalues are presented in Table 2. The 1:1 binding

stoichiometry as determined by titration was confirmed using

the continuous variation technique (Job’s plot)^41,42for cryp-

tolepine, chloroquine and 3c, changing the FPIX-OH molar ratio

and maintaining the sum of the concentrations of the ligand and

FPIX-OH constant at 10 μM. The Job’s plots in Figure 7a-c

show 1:1 complex formation for cryptolepine, chloroquine, and

3c, respectively. Titration with chloroquine was also performed

for comparison purposes, but in this case, both binding models 1

and 3 gave excellent fits to the absorbance data (r²of 0.997 and

0.999, respectively). The Job’s plot for chloroquine shows that

Figure 7. Job plots for (a) cryptolepine (1), (b) chloroquine (2), and

(c) derivative 3c with FPIX-OH in buffered 40% DMSO v/v, pH 5.5

at 25 ꢀC.

the intercept occurs at a molar ratio of 0.51, i.e. consistent with

1:1 binding stoichiometry (Figure 7b). This result is in line with

the observation of Egan and co-workers that the 1:1 complex

is the predominant species in pH 7.5 buffer containing 40%

DMSO, while the 1:2 complex is likely to occur only at lower

ratios of chloroquine to FPIX-OH.⁴⁹

Inspection of Table 2 shows that K_assvalues for 3a-y

range from 0.062 to 0.227 ꢁ 10⁶M^-1, while the K_assvalue for

cryptolepine is 0.045 ꢁ 10⁶M^-1, indicating that introduction

of a side-chain at C-11 of the cryptolepine aromatic nucleus

increases the interaction with FPIX-OH when compared

with the parent compound. Importantly, the K_assvalues for

3a-y compare favorably to that of chloroquine (0.085 ꢁ 10⁶

M^-1in our assay), indicating that cryptolepine analogues 3

share with chloroquine the ability to interact with hematin

monomer. Interestingly, the phenyl derivative 3r binds as

efficiently as most of the derivatives containing a terminal basic

group to hematin monomer, which suggests that a distal

basic amine is not an absolute requirement for binding.

742 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

It should be noted that chloroquine can also inhibit hemozoin

crystal growth by binding to its crystal faces.²²According to

this hemozoin crystal growth model, the flat and well

exposed face of the crystal shows repeats of propionic car-

boxylate groups separated by 8.0 A, which may be the target

of positively charged groups.²¹Remarkably, we found that

the distances between the positively charged N-5 nitrogen

atom and the distal amine groups of piperidine (3n), propyl

(3f ), and butyl (3l) derivatives are ≈8 A (Supporting In-

formation), a value consistent with binding to the hemozoin

crystal flat face.

Inhibition of Cysteine Proteases Falcipain-2 and -3. Cryp-

tolepine derivatives 3 were also screened for inhibition of

cysteine proteases from P. falciparum, falcipain-2 (FP-2) and

-3 (FP-3) (Table 2), which are of particular interest as

therapeutic targets due to their role in parasite development.⁵³

Both FP-2 and FP-3 are involved in the hydrolysis of host

hemoglobin in the parasite’s digestive vacuole, and falcipain

inhibitors have been reported to block the development of

cultured erythrocytic parasites⁵⁴and to cure mice infected

with lethal malaria infections.⁵⁵Inspection of the data in

Table 2 shows that compounds 3 are poor to moderate

inhibitors of FP-2 and FP-3. The IC₅₀values against FP-2

ranged from 4.8 μM for the dichloro derivative 3x to 34-50 μM

for derivatives incorporating linear or branched alkylamine

side chains. Furthermore, compounds 3 generally display

only moderate selectivity toward FP-2 when compared to

FP-3. The observation that falcipains are inhibited only in

the μM range, suggests that the indolo[3,2-b]quinoline is not

suitable for enzyme binding and that falcipains cannot be

considered as the main targets for cryptolepine derivatives 3.

accumulate inside the nucleus and, to some extent, in the

digestive vacuole of blood stages of parasite. These data

clearly indicated that incorporation of a diaminoalkane side

chain, as a strategy that has been extensively used as a pH-

trapping device in antimalarial drug design, was not sufficient

enough to provide cryptolepine derivatives with the ability to

accumulate selectively in the digestive vacuole. Instead, there

seem to be some other mechanisms which effectively promote

antimalarial activity of the studied analogues. Indeed, it was

demonstrated that compounds 3a-y generally displayed en-

hanced association constants toward the double-stranded

oligonucleotide duplex, presumably due to additional electro-

static interactions with the negatively charged sugar-phosphate

backbones, which might be linked to the improved antiplasmo-

dial activity via inhibition of parasite DNA replication.²²

The interactions between the cryptolepine analogues and

hematin could be an additional mechanism of antiplasmodial

activity seen for 3a-y derivatives, presumably via possible

inhibition of hemozoin crystal growth. However, the detailed

study of the mechanisms of action underpinning high potency

and improved selectivity of cryptolepine analogues will be a

subject of the separate investigations.

Overall, cryptolepine derivatives containing basic side

chains have been confirmed as promising lead compounds

for the development of new, highly potent, and selective

agents against the blood stages, but not the liver stage, of P.

falciparum malaria.

Experimental Section

Chemistry. Chemicals were purchased from Sigma-Aldrich

Chemical Co. Ltd., Spain and used without further purification

All compounds were characterized through NMR spectroscopy

and in some cases through mass spectrometry. NMR spectra

were acquired on a Bruker 400 Ultrashield spectrometer at

400 MHz (¹H NMR) and 100 MHz (¹³C NMR), using the

solvent as internal reference. Coupling constants (J) are given in

hertz. Mass spectra were recorded using a Micromass Autospec

spectrometer with electronic impact as ionization method and

high resolution mass spectra (accurate mass) were recorded on a

Bruker Microtof spectrometer with ESI-TOF as ionization

method (University of Santiago de Compostela, Spain). The

purity of compounds submitted for biological testing were in all

cases g95% as determined from elemental analysis (Supporting

Information). C, H, and N analyses were carried out by the Unit

Elemental Analysis, University of Santiago de Compostela,

Spain, on a LECO model CHNS-932 elemental analyzer. UV-

visible spectra for hematin binding studies were recorded on a

Shimadzu UV-1603 UV-visible spectrometer with a temperature-

controlled (25 ꢀC) cell holder. Purified 12-mer double-stranded

oligonucleotide d(GATCCTAGGATC)₂was purchased from

Atdbio, School of Chemistry, University of Southampton,

UK. UV-visible spectral scans and absorbance readings for

DNA binding studies were conducted using 1 cm path length

quartz cuvettes in a Cary 4000 (Varian) UV-visible spectro-

photometer equipped with a Peltier-thermostatted cuvette

holder. Fluorescence emission and excitation spectra were re-

corded in 1 cm path length quartz cuvettes using a Cary-Eclipse

(Varian) fluorescence spectrophotometer. Melting points were

determined using a Bock-Monoscop M apparatus and are

uncorrected. Reactions were monitored by thin-layer chroma-

tography (TLC) using coated silica gel plates (Merck, aluminum

sheets, silica gel 60 F₂₅₄).

Conclusion

On the basis of previous reports indicating that a basic

amino side-chain is a major requirement for chloroquine accu-

mulation in the acid digestive vacuole of the parasite, we

synthesized a series of cryptolepine analogues containing

diaminoalkaneside chains (-NH-linker-NR¹R²) atC-11with

the initial aim to facilitate localization of these ligands within

the plasmodial vacuole. These novel cryptolepine derivatives

were obtained by reaction of the key intermediate, 11-chloro-

cryptolepine, with the appropriate amine, to give moderate to

good yield.

A major outcome of this work is that several cryptolepine

analogues with a diaminoalkane side hain at C-11 position

showed significant increase of the antiplasmodial activity

against both CQ-resistant and CQ-sensitive P. falciparum strains

when compared with the parent compound and with the

currently used antimalarial drug chloroquine. Maximum

potency was achieved with propyl and butyl side chains, while

branched linkers seemed to be poorly tolerated. More im-

portantly, some of theseidentified agents (e.g., 3e, 3f, 3i, 3k, 3l,

and especially 3n) combine significant antiplasmodial activity

with a very low cytotoxicity. The cytotoxicity profile was

particularly improved for the derivative containing a confor-

mationally restricted piperidine side-chain, 3n, displaying a

selectivity index value of ca. 1400, i.e. a 1000-fold increase

compared with the parent compound.

The research was also focused on identification of possible

biological targets for this new class of antimalarial agents in

order to understand an origin of this substantial improvement

in potency and selectivity against P. falciparum strains. Fluo-

rescence microscopy revealed that these cryptolepine derivatives

General Procedure A. Preparation of 2-(2-Bromoacetamido)-

benzoic Acid (5a). A solution of 2-amino-benzoic acid (10.0 g,

72.9 mmol) in DMF (30 mL) and 1,4-dioxane (30 mL) was

cooled to 0 ꢀC. Bromoacetyl bromide (8.0 mL, 91.7 mmol) was

added dropwise over a 20 min period, keeping the internal

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 743

temperature between 0 and 1 ꢀC. After the addition was complete,

the ice bath was removed and stirring was continued overnight

at room temperature. The reaction mixture was added to water

(300 mL), and the light-yellow precipitate was filtered, washed

with water until neutral pH, and then dried to give 18.1 g (96%)

7.8 Hz, 1H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 167.77,

139.54, 139.05, 130.99, 129.44, 127.84, 125.57, 123.48, 123.24,

121.63, 120.87, 119.25, 118.28, 116.38, 112.98.

3-Chloro-indolo[3,2-b]quinolin-11-one (7b). A mixture of 6b

(2.0 g, 6.6 mmol) and PPA (60 g) was treated according to

general procedure C, giving 0.630 g (36%) of 7b as a light-green

1

of 5a as a white solid, mp 162-165 ꢀC. H NMR (400 MHz,

1

CD₃OD) δ_H(ppm) 8.59 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 8.0 Hz,

1H), 7.60 (dd, J = 8.5, 7.1 Hz, 1H), 7.21 (dd, J = 8.0, 7.1, Hz,

1H), 4.13 (s, 2H). ¹³C NMR (100 MHz, CD₃OD) δ_C(ppm)

170.53, 166.71, 140.96, 134.61, 131.97, 124.02, 120.72, 117.33,

29.56.

solid, mp >300 ꢀC. H NMR (400 MHz, DMSO) δ_H(ppm)

12.56 (NH, 1H), 11.81 (NH, 1H), 8.35 (d, J = 8.7 Hz, 1H), 8.15

(d, J = 8.1 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H), 7.5 (d, J = 6.2 Hz,

1H), 7.49 (dd, J = 7.3, 6.2 Hz 1H), 7.30 (dd, J = 8.7, 1.8 Hz, 1H),

7.23 (dd, J = 8.1, 7.3 Hz, 1H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 167.30, 140.02, 139.08, 135.65, 129.40, 128.10, 127.94,

123.64, 121.96, 121.31, 121.15, 119.62, 117.13, 116.14, 113.17.

3,8-Dichloro-indolo[3,2-b]quinolin-11-one (7c). Compound 6c

(2.0 g, 5.90 mmol) reacted with PPA (60 g) according to general

procedure C. After the extraction, the solvent was removed at

reduced pressure and the residue purified by flash chromatog-

raphy (silica gel 230-400 mesh, 40-63 μm, AcOEt:hexane, 6:4)

to give 0.271 g (15%) of 7c as light-green solid, mp >300 ꢀC. ¹H

NMR (400 MHz, DMSO) δ_H(ppm) 12.27 (s, NH), 11.33 (s, NH),

8.36 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 8.8 Hz, 1H), 7.49 (d, J =

0.9 Hz, 1H), 7.46 (d, J = 7.1 Hz, 1H), 7.33 (dd, J = 8.8, 2.0 Hz,

1H), 7.24 (dd, J = 7.1, 0.9 Hz, 1H). ¹³C NMR (100 MHz,

DMSO) δ_c(ppm) 167.49, 140.50, 139.88, 135.94, 128.69, 127.60,

127.45, 126.41, 124.33, 121.87, 121.73, 119.76, 118.64, 113.84,

112.24.

2-(2-Bromoacetamido)-4-chlorobenzoic Acid (5b). Reaction of

2-amino-4-chloro benzoic acid (10 g, 58.2 mmol) and bromo-

acetyl bromide (6.3 mL, 72.8 mmol) according to general proce-

dure A, gave 17.1 g (99%) of 5b as a light-yellow solid, mp

166-168 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 11.73

(s, NH), 8.54 (d, J = 2.1 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.24

(dd, J = 8.6, 2.1 Hz, 1H), 4.28 (s, 2H). ¹³C NMR (100 MHz,

DMSO) δ_C(ppm) 169.03, 165.92, 141.54, 138.95, 133.26,

123.73, 119.61, 115.91, 30.98.

General Procedure B. Preparation of 2-(2-(Phenylamino)-

acetamido)benzoic Acid (6a). A solution of 5a (15.0 g, 58.1 mmol)

and aniline (19.0 mL, 208.3 mmol) in DMF (30 mL) was heated

at 120 ꢀC for 18 h. After cooling, the reaction mixture was poured

into ice-water (500 mL), the pH adjusted to 10-11 with 5% KOH,

and then extracted with dichloromethane (3 ꢁ 300 mL). The

combined dichloromethane extracts were set aside, and the aqueous

layer was acidified to pH 3 with a solution of 5% HBr. The pre-

cipitate was collected, washed with water, and then dried, yielding

General Procedure D. Preparation of 11-Chloro-10H-indolo-

[3,2-b]quinoline (8a). A solution of 7a (1.5 g, 6.58 mmol) in

POCl₃(20 mL) was refluxed for 2 h. The reaction mixture was

cooled, poured into ice, and neutralized with a cold KOH

solution. The aqueous solution was then extracted with AcOEt

(3 ꢁ 200 mL), and then the combined organic phases were

washed with water and brine, dried with anhydrous Na₂SO₄,

and then concentrated to give 0.95 g (60%) of impure 8a, as a

light-brown solid, mp 180-185 ꢀC. ¹H NMR (400 MHz, DMSO)

1

11.0 g (70%) of 6a as a white solid, mp 194-197 ꢀC. H NMR

(400 MHz, CD₃OD) δ_H(ppm) 8.72 (d, J = 8.0 Hz, 1H), 8.04 (d,

J = 7.6 Hz, 1H), 7.57 (dd, J = 8.0, 7.5 Hz, 1H), 7.15 (dd, J = 7.5,

7.6 Hz, 1H), 7.14 (dd, J= 7.5, 8.0 Hz, 2H), 6.69 (d, J= 7.5 Hz, 1H),

6.65 (d, J = 8.0 Hz, 2H), 3.91 (s, 2H). ¹³C NMR (100 MHz,

CD₃OD) δ_C(ppm): 171.95, 170.81, 148.72, 141.15, 134.30, 132.03,

129.45, 123.56, 120.79, 118.47, 116.89, 113.37, 49.95.

δ

_H(ppm) 11.82 (s, NH), 8.28 (d, J = 7.7 Hz, 1H), 8.21 (m, 2H),

7.69 (m, 2H), 7.61 (m, 2H), 7.27 (m, 1H). ¹³C NMR (100 MHz,

DMSO) δ_C(ppm) 145.22, 143.32, 142.99, 129.55, 129.28,

128.43, 126.01, 125.54, 122.79, 121.30, 120.87, 120.34, 119.40,

117.14, 111.23.

4-Chloro-2-(2-(phenylamino)acetamido)benzoic Acid (6b). Re-

action of 5b (15 g, 51.3 mmol) with aniline (16.3 mL, 179.6 mmol)

according to general procedure B gave 14.9 g of 6b (96%) as a

white solid, mp 217-220 ꢀC. ¹H NMR (400 MHz, DMSO)

3,11-Dichloro-10H-indolo[3,2-b]quinoline (8b). According with

general procedure D, 7b (1.5 g, 4.42 mmol) was reacted with POCl₃

(20 mL) to give 1.0 g of 8b (66%) as a light-orange solid, mp

171-176 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 11.98

(s, NH), 8.33 (d, J = 7.8 Hz, 1H), 8.29 (d, J = 2.1 Hz, 1H), 8.28

(d, J = 9.2 Hz, 1H), 7.73 (m, 1H), 7.68 (dd, J = 7.0, 1.1 Hz, 1H),

7.63 (d, J = 8.1 Hz, 1H), 7.35 (dd, J = 7.8, 7.0 Hz, 1H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 147.43, 144.85, 144.34, 131.76,

131.27, 130.69, 128.07, 127.20, 124.66, 122.74, 122.35, 121.31,

120.89, 118.68, 112.64.

δ

_H(ppm) 12.13 (s, NH), 8.83 (d, J = 2.2 Hz, 1H), 7.94 (d, J =

8.6 Hz, 1H), 7.21 (dd, J = 8.6, 2.2 Hz, 1H), 7.10 (dd, J = 8.0,

7.4 Hz, 2H), 6.63 (d, J = 7.4 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H),

3.86 (s, 2H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 171.96,

168.80, 148.46, 141.99, 139.03, 133.33, 129.45, 123.08, 119.16,

117.63, 115.16, 112.90, 49.36.

4-Chloro-2-(2-(3-chlorophenylamino)acetamido)benzoic Acid

(6c). Reaction of 5b (3.0 g, 10.2 mmol) and 3-chloroaniline

(3.34 mL, 31.7 mmol) in DMF (10 mL) was heated at 90 ꢀC for

72 h and treated according to general procedure B to give 3.0 g of

6c (84%) as a white solid, mp 223-226 ꢀC. ¹H NMR (400 MHz,

DMSO) δ_H(ppm) 12.10 (s, NH), 8.80 (d, J = 2.2 Hz, 1H), 7.95

(d, J = 8.6 Hz, 1H), 7.20 (dd, J = 8.6, 2.2 Hz, 1H), 7.11 (dd, J =

8.0 Hz, 1H), 6.84 (s, NH), 6.55 (m, 2H), 6.54 (dd, J = 8.2, 1.4 Hz,

1H), 3.92 (d, J = 5.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 171.29, 168.95, 149.99, 141.93, 139.03, 134.00, 133.34,

131.02, 123.13, 119.16, 117.04, 115.24, 112.41, 111.32, 48.80.

General Procedure C. Preparation of Indolo[3,2-b]quinolin-

11-one (7a). A mixture of 6a (6.0 g, 21.5 mmol) and polyphos-

phoric acid (PPA, 200 g) was heated with mechanical stirring

at 130 ꢀC for 2 h. The reaction mixture was poured into ice-

water (500 mL), neutralized with saturated KOH solution, and

then extracted with AcOEt (2 ꢁ 250 mL). The extract was

washed with water and brine, dried with anhydrous Na₂SO₄,

and then concentrated to give 3.47 g (67%) of impure 7a as a

light-brown solid, mp >300 ꢀC. ¹H NMR (400 MHz, DMSO)

3,8,11-Trichloro-10H-indolo[3,2-b]quinoline (8c). Reaction of

7c (0.25 g, 1.6 mmol) with POCl₃(10 mL) according with general

procedure D gave 0.13 g of 8c (49%) as a strong orange solid, mp

167-170 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 12.14

(s, NH), 8.34 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.30

(d, J = 4.9 Hz, 1H), 7.77 (dd, J = 9.1, 2.0 Hz, 1H), 7.62 (s, 1H),

7.37 (dd, J = 8.3, 1.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 146.48, 145.34, 144.56, 135.68, 132.23, 130.99, 128.04,

127.57, 124.82, 123.84, 122.82, 121.28, 120.19, 119.36, 112.29.

10H-Indolo[3,2-b]quinoline (9). To a portion of 8a (0.27 g,

1.07 mmol), sodium acetate (1.1 g, 13.4 mmol), and 10% Pd/C

in acetic acid (25 mL) was hydrogenated at 60 psi for 2 h. The

reaction mixture was filtered to remove the catalyst, the

solvent evaporated to a small volume and basified with ice-

cold saturated solution of NaHCO₃, and then extracted with

AcOEt (3 ꢁ 25 mL). The combined organic extracts were

washed with water and brine, dried (anhydrous Na₂SO₄), and

the solvent removed under reduced pressure to give 0.199 g (85%) of

δ

_H(ppm) 12.41 (s, NH), 11.64 (s, NH), 8.29 (d, J = 7.9 Hz, 1H),

8.13 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.69 (dd, J =

8.3, 7.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 8.4,

7.7 Hz, 1H), 7.29 (dd, J = 8.3, 7.9 Hz, 1H), 7.20 (dd, J = 7.7,

1

pure 9, as a yellow solid, mp 200-203 ꢀC. H NMR (400 MHz,

DMSO) δ_H(ppm) 11.43 (s, NH), 8.36 (d, J = 7.7 Hz, 1H), 8.29

744 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

(s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.61

(m, 4H), 7.29 (dd, J = 7.8, 7.0, 1H). ¹³C NMR (100 MHz,

DMSO) δ_C(ppm) 145.04, 143.34, 142.71, 131.75, 128.98, 128.01,

126.80, 126.03, 125.32, 124.15, 120.67, 120.29, 118.63, 112.31,

110.81.

(26.3 mg) of pure 3a, as a light-yellow solid, mp 293-295 ꢀC.

¹H NMR (400 MHz, DMSO) δ_H(ppm) 12.29 (s, NH) 9.24

(s, NH), 8.84 (d, J = 8.2 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.44

(s, NH₂), 8.31 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.97

(dd, J = 8.8, 7.4 Hz, 1H), 7.68 (dd, J = 8.3, 7.6 Hz, 1H), 7.64

(dd, J = 8.2, 7.4 Hz, 1H), 7.32 (d, J = 8.4, 7.6 Hz, 1H), 4.58

(s, 3H), 4.44 (m, 2H), 3.32 (m, 2H). ¹³C NMR (100 MHz,

DMSO) δ_C(ppm) 143.46, 142.90, 137.02, 132.27, 130.52,

126.72, 124.63, 124.29, 123.88, 120.75, 117.24, 115.95, 115.31,

114.32, 113.72, 42.41, 38,85, 38.08.

5-Methyl-10H-indolo[3,2-b]quinolin-5-ium Trifluoro Methanesul-

fonate (1). A suspension of 9 (0.085 mg, 0.38 mmol) in anhydrous

toluene (2 mL) was treated with methyl triflate (70 μL,

0.638 mmol), and the mixture was stirred at room temperature

for 24 h. Diethyl ether was added (5 mL), and the resulting

orange precipitate was filtered, washed with diethyl ether, and

dried to give 0.092 g (62%) of pure 1, mp >300 ꢀC. ¹H NMR

(400 MHz, DMSO) δ_H(ppm) 12.75 (s, NH), 9.15 (s, 1H), 8.68

(d, J = 7.7 Hz, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.45 (d, J = 7.4 Hz,

1H), 8.06 (dd, J = 8.5, 6.4 Hz, 1H), 7.84 (m, 2H), 7.72 (d, J =

8.0 Hz, 1H), 7.42 (dd, J = 8.2, 7.7 Hz, 1H), 4.92 (s, 3H). ¹³C

NMR (100 MHz, DMSO) δ_C(ppm) 144.73, 137.06, 134.34, 132.97,

132.27, 131.44, 128.85, 126.09, 125.29, 125.24, 123.85, 120.41,

116.84, 112.86, 112.20, 39.21.

General Procedure E. Preparation of 5-Methyl-11-chloro-

10H-indolo[3,2-b]quinolin-5-ium Chloride (10a). To a suspension

of 8a (2.0 g, 7.91 mmol) in anhydrous toluene (50 mL) was added

methyl triflate (2.55 mL, 23.7 mmol), and the mixture stirred at

room temperature for 48 h. The solid was filtered, suspended in

sodium carbonate 5% (400 mL), and extracted with chloroform

(3 ꢁ 400 mL). The chloroform extracts were washed with water

and brine, dried (anhydrous Na₂SO₄), and concentrated to a

small volume. The solution was acidified with HCl in diethyl

ether to give, after filtration and drying, 2.15 g (90%) of pure

10a, as an orange solid, mp 269-272 ꢀC. ¹H NMR (400 MHz,

DMSO) δ_H(ppm) 13.39 (s, NH), 8.88 (d, J = 9.4 Hz, 1H), 8.85

(d, J = 8.9 Hz, 1H), 8.69 (d, J = 8.3 Hz, 1H), 8.30 (dd, J = 9.4,

7.7 Hz, 1H), 8.12 (dd, J = 8.3, 7.7 Hz, 1H), 8.01 (dd, J = 8.4, 7.7

Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 8.9, 7.7 Hz, 1H),

5.04 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 146.33,

139.03, 136.15, 134.90, 133.20, 132.11, 129.30, 128.79, 126.98,

124.97, 123.73, 122.45, 119.21, 114.78, 113.94, 40.97.

5-Methyl-3,11-dichloro-10H-indolo[3,2-b]quinolin-5-ium Chloride

(10b). A suspension of 8b (0.15 g, 0.52 mmol) was treated with

methyl triflate (114 μL, 1.04 mmol) according to general proce-

dure E, giving 0.139 g of 10b (79%) as an orange solid, mp

234-237 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 13.63 (NH,

1H), 9.00 (d, J = 1.7 Hz, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.60 (d,

J = 9.1 Hz, 1H), 8.09 (dd, J = 9.1, 1.7 Hz, 1H), 7.96 (dd, J =

8.5, 7.4 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.53 (dd, J = 8.5,

7.4 Hz, 1H), 4.98 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm)

146.60, 139.54, 138.36, 136.52, 135.29, 132.30, 129.40, 129.34,

127.10, 126.98, 122.67, 122.52, 118.68, 114.73, 114.06, 41.27.

5-Methyl-3,8,11-trichloro-10H-indolo[3,2-b]quinolin-5-ium Chlo-

ride (10c). A suspension of 8c (50 mg, 0.16 mmol) was treated

with methyl triflate (35 μL, 0.32 mmol) according to general

procedure E, giving 26.1 mg of 10c (46%) as an orange solid, mp

229-233 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 13.47

(s, NH), 9.00 (s, 1H), 8.82 (d, J = 9.0 Hz, 1H), 8.62 (d, J = 9.1

Hz, 1H), 8.12 (d, J = 9.1 Hz, 1H), 7.81 (d, J = 1.5 Hz, 1H), 7.57

(dd, J = 9.0, 1.5 Hz, 1H), 4.95 (s, 3H). ¹³C NMR (100 MHz,

DMSO) δ_C(ppm) 146.69, 140.11, 139.05, 138.83, 136.77, 132.64,

129.97, 129.63, 128.70, 127.06, 123.19, 122.77, 118.73, 113.73,

113.41, 41.26.

General Procedure F. Preparation of 5-Methyl-11-(2-amino-

ethylamino)-10H-indolo[3,2-b]quinolin-5-ium Chloride (3a). To a

suspension of 10a (40 mg, 0,132 mmol) in AcOEt (5 mL) was

added ethane-1,2-diamine (17.7 μL, 0.263 mmol) The reaction

mixture was refluxed for 24 h, and the resulting precipitate

collected, washed with dry diethyl ether, and evaporated. The

solid was suspended in 5% sodium carbonate (20 mL) and

extracted with chloroform (3 ꢁ 20 mL). The combined organic

extracts were washed with water, dried with brine (20 mL) and

anhydrous Na₂SO₄, and dried under reduced pressure. Recrys-

tallization from chloroform with HCl in diethyl ether gave 61%

5-Methyl-11-[2-(Dimethylamino)ethylamino]-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3b). To a suspension of 10a (40.0 mg,

0.132 mmol) in AcOEt (5 mL) was added commercial N¹,N¹-

dimethylethane-1,2-diamine (29 μL, 0.264 mmol). The reaction

mixture was refluxed for 24 h, and the resulting precipitate was

collected, washed with diethyl ether, and dried. The product was

recrystallized from methanol-AcOEt to give 62% (29.2 mg)

1

yield of pure 3b, as a dark-yellow solid, mp 260-262 ꢀC. H

NMR (400 MHz, CD₃OD) δ_H(ppm) 8.71 (d, J = 8.4 Hz, 1H),

8.48 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 8.03 (dd, J =

8.1, 7.6 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.73 (dd, J = 8.4,

7.6 Hz, 1H), 7.69 (dd, J= 8.2, 7.5 Hz, 1H), 7.40 (dd, J=8.2,7.5Hz,

1H), 4.64 (s, 3H), 4.61 (t, J = 6.2 Hz, 2H), 3.72 (t, J = 6.2 Hz,

2H), 3.05 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ_C(ppm)

143.73, 143.16, 137.37, 136.93, 132.41, 131.06, 124.51, 123.78,

123.65, 121.19, 116.85, 116.74, 115.78, 114.67, 113.35, 56.55,

42.68, 40.51, 37.51.

5-Methyl-11-[2-(diethylamino)ethylamino]-10H-indolo[3,2-b]-

quinolin-5-ium Chloride (3c). After recrystallization from metha-

nol with ethyl acetate, the compound 3c was obtained in 35%

yield (18.4 mg) as a dark-yellow solid, mp >330 ꢀC, from 10a

(40.0 mg, 0.132 mmol) and commercially available N¹,N¹-

diethylethane-1,2-diamine (37.1 μL, 0.264 mmol) according to

general procedure F. ¹H NMR (400 MHz, CD₃OD) δ_H(ppm)

8.82 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 8.1 Hz, 1H), 8.30 (d, J =

8.2 Hz, 1H), 7.98 (dd, J = 8.2, 7.7 Hz, 1H), 7.77 (d, J = 8.6 Hz,

1H), 7.70 (dd, J = 8.6, 7.2 Hz, 1H), 7.66 (dd, J = 8.2, 7.7 Hz,

1H), 7.34 (dd, J = 8.1, 7.2 Hz, 1H), 4.57 (s, 3H), 4.14 (s, 2H),

2.98 (s, 2H), 2.71 (q, J = 7.0 Hz, 4H), 0.95 (t, J = 7.0 Hz, 6H).

¹³C NMR (100 MHz, CD₃OD) δ_C(ppm) 144.50, 143.39, 137.53,

135.48, 132.52, 130.71, 124.74, 124.51, 124.19, 120.83, 118.07,

117.68, 115.60, 115.55, 114.01, 53.41, 47.66, 44.62, 38.24, 11.27.

5-Methyl-11-[1-(dimethylamino)propan-2-ylamino]-10H-indolo-

[3,2-b]quinolin-5-ium chloride (3d). This compound was obtained

in 28% yield (13.9 mg) as an orange solid, mp 272-276 ꢀC, by

reaction of 10a (40.0 mg. 0.132 mmol) and N¹,N¹-dimethylpro-

pane-1,2-diamine 12 (26.6 mg, 0.264 mmol), according to gen-

eral procedure F, and recrystallization from CHCl₃:MeOH (1:1)

with AcOEt:Et₂O (3:2). ¹H NMR (400 MHz, DMSOþD₂O)

δ

_H(ppm) 8.69 (d, J = 8.5 Hz, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.30

(d, J = 8.9 Hz, 1H), 8.04 (dd, J = 7.1, 8.9 1H), 7.86 (d, J =

8.5 Hz, 1H), 7.77 (dd, J = 7.4, 8.5 Hz, 1H), 7.73 (d, J = 7.1, 8.5 Hz,

1H), 7.42 (dd, J = 7.4, 8.4 Hz 1H), 5.11 (m, 1H), 4.62 (s, 3H),

3.81 (t, J = 11.7 Hz 1H), 3.45 (d, J = 11.9 Hz 1H), 2.88 (s broad,

6H), 1.44 (d, J = 6.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO þ

D₂O) δ_C(ppm) 143.73, 143.11, 137.49, 135.73, 133.30, 132.04,

130.69, 125.17, 125.04, 124.82, 122.02, 117.76, 116.30, 115.01,

114.07, 60.90, 47.64, 44.03, 38.47, 20.56.

5-Methyl-11-(3-Aminopropylamino)-10H-indolo[3,2-b]quinolin-

5-ium Chloride (3e). To a suspension of 10a (40.0 mg, 0.132 mmol)

was added commercial propane-1,3-diamine (22 μL, 0.264 mmol)

according to general procedure F. The product was recrystallized

from CH₂Cl₂:MeOH (8:2) with Et₂O to give 24.3 mg (54%) of 3e as

yellow solid, mp 243-245 ꢀC. ¹H NMR (400 MHz, DMSO)

δ_H(ppm) 12.28 (s, NH), 9.27 (s, NH), 8.90 (d, J = 8.6 Hz, 1H),

8.56 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 8.9 Hz, 1H), 8.20 (s, NH₂),

8.03 (d, J = 8.0 Hz, 1H), 8.01 (dd, J = 8.9, 8.1 Hz, 1H), 7.73 (dd,

J = 8.0, 7.6 Hz, 1H), 7.68 (dd, J = 8.6, 8.0 Hz, 1H), 7.37 (dd, J =

8.5, 7.6 Hz, 1H), 4.61 (s, 3H), 4.32 (d, J = 6.0 Hz, 2H), 3.01 (d, J =

5.7 Hz, 2H), 2.17 (dd, J = 6.0, 5.7 Hz, 2H). ¹³C NMR (100 MHz,

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 745

DMSO) δ_C(ppm) 144.01, 143.21, 137.65, 135.83, 132.76, 130.88,

124.89, 124.74, 124.39, 121.17, 117.83, 116.59, 115.68, 114.75,

114.18, 42.86, 38.51, 36.75, 28.05.

(ppm) 12.31 (s, NH), 9.21 (s, NH), 8.87 (d, J = 8.1 Hz, 1H), 8.55

(d, J = 8.1 Hz, 1H), 8.34 (d, J = 8.9 Hz, 1H), 8.01 (m, 2H), 7.71

(dd, J = 7.5, 6.5 Hz, 1H), 7.68 (dd, J = 8.1, 4.9 Hz, 1H), 7.36

(dd, J = 8.1, 6.5 Hz, 1H), 4.60 (s, 3H), 4.31 (m, J = 5.5 Hz, 2H),

3.43 (m, 2H), 3.23 (m, 2H), 2.86 (m, 2H), 2.30 (m, 2H), 1.78 (m,

4H), 1.69 (m, 1H), 1.38 (m, 1H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 143.41, 142.61, 137.02, 135.16, 132.12, 130.20, 124.44,

124.09, 123.70, 120.52, 117.15, 116.06, 115.02, 114.17, 113.58,

53.24, 51.81, 42.93, 37.89, 23.86, 22.12, 21.28.

5-Methyl-11-[3-(dimethylamino)propylamino]-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3f).After recrystallization from methanol-

ethyl acetate, 3f was obtained in 34% yield (16.3 mg) as a dark-

yellow solid, mp 277-280 ꢀC, from 10a (40.0 mg, 0.132 mmol)

and commercial N¹,N¹-dimethylpropane-1,3-diamine (33.2 μL,

0.264 mmol), according to general procedure F. ¹H NMR

(400 MHz, CD₃OD) δ_H(ppm) 8.65 (d, J = 8.4 Hz, 1H), 8.53 (d,

J = 8.1 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 8.05 (dd, J = 8.1, 7.0 Hz,

1H), 7.92 (d, J= 8.1 Hz, 1H), 7.77 (d, J=8.1,7.1Hz,1H),7.73(dd,

J = 8.4, 7.0 Hz, 1H), 7.44 (dd, J = 8.1, 7.1 Hz, 1H), 4.67 (s, 3H),

4.32 (t, J = 6.9 Hz, 2H), 3.37 (t, J = 7.9 Hz, 2H), 2.39 (quintet, J =

7.5 Hz, 2H), 2.92 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ_C(ppm)

143.63, 143.34, 137.46, 136.45, 134.47, 132.22, 130.71, 124.22,

123.62, 123.23, 120.93, 116.63, 115.51, 114.63, 113.13, 54.79,

42.50, 42.08, 37.22, 25.09.

5-Methyl-11-[3-(diethylamino)propylamino]-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3g). The title compound was obtained

in 82% yield (43.1 mg) as a dark-yellow solid, mp 274-277 ꢀC,

by reaction of 10a (40.0 mg, 0.132 mmol) and commercially

available N¹,N¹-diethylpropane-1,3-diamine (41.6 μL, 0.264 mmol),

according to general procedure F. ¹H NMR (400 MHz,

CD₃OD) δ_H(ppm) 8.62 (d, J = 8.5 Hz, 1H), 8.47 (d, J =

8.1 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.04 (dd, J = 8.5, 7.4 Hz,

1H), 7.91 (d, J = 8.4 Hz, 1H), 7.74-7.70 (m, 2H), 7.41 (dd, J =

8.1, 7.7 Hz, 1H), 4.59 (s, 3H), 4.29 (t, J = 7.0 Hz, 2H), 3.38 (t,

J = 7.8 Hz, 2H), 3.26 (q, J = 7.2 Hz, 4H), 2.36 (quintet, J =

7.5 Hz, 2H), 1.34 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz,

CD₃OD) δ_C(ppm) 143.57, 143.36, 137.46, 136.35, 132.37,

130.81, 124.35, 123.68, 123.39, 121.08, 116.70, 116.53, 115.52,

114.63, 113.27, 49.08, 47.27, 42.84, 37.33, 24.65, 7.87.

5-Methyl-11-[3-(dimethylamino)-2,2-dimethylpropyl amino]-

10H-indolo[3,2-b]quinolin-5-ium Chloride (3h). Reaction of 10a

(51.2 mg, 0.164 mmol) and commercial N¹,N¹-2,2-tetramethyl-

propane-1,3-diamine (0.26 mL, 1.64 mmol), according to gen-

eral procedure F, gave 3h in 78% yield (52.6 mg), as an orange

solid, mp 171-173 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm)

10.75 (s, NH), 8.51 (d, J = 8.1 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H),

8.20 (d, J = 7.9 Hz, 1H), 8.00 (dd, J = 8.5, 7.5 Hz, 1H), 7.93 (d,

J = 8.1 Hz, 1H), 7.75 (dd, J = 7.9, 7.5 Hz, 1H), 7.67 (dd, J =

8.1, 7.6 Hz, 1H), 7.32 (dd, J = 8.1, 7.6 Hz, 1H), 4.56 (s, 3H), 4.21

(s, 2H), 2.59 (s, 2H), 2.44 (s, 6H), 1.13 (s, 6H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 144.59, 143.08, 137.67, 135.60,

132.62, 130.61, 124.83, 124.56, 123.11, 120.99, 118.02, 116.63,

115.58, 114.88, 114.18, 70.03, 57.33, 48.18, 38.31, 35.51, 25.38.

5-Methyl-11-[3-(isopropylamino)propylamino]-10H-indolo[3,2-b]-

quinolin-5-ium Chloride (3i). After recrystallization from metha-

nol-ethyl acetate, 3i was obtained in 56% yield (28.7 mg) as

a yellow solid, mp >330 ꢀC, from reaction of 10a (40.0 mg,

0.132 mmol) with commercial N¹-isopropylpropane-1,3-di-

amine (36.9 μL, 0.264 mmol) according to general procedure

F. ¹H NMR (400 MHz, CD₃OD) δ_H(ppm) 8.67 (d, J = 8.3 Hz,

1H), 8.54 (d, J = 8.2 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.05 (dd,

J = 8.4, 7.3 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.75 (dd, J = 8.1,

7.2 Hz, 1H), 7.73 (dd, J= 8.3, 7.3 Hz, 1H), 7.44 (dd, J= 8.2, 7.2 Hz,

1H), 4.68 (s, 3H), 4.34 (t, J = 7.0 Hz, 2H), 3.42 (quintet, J =

6.5 Hz, 1H), 3.27 (t, J = 7.6 Hz, 2H), 2.43 (quintet, J = 7.3 Hz,

2H), 1.37 (d, J = 6.5 Hz, 6H). ¹³C NMR (100 MHz, CD₃OD) δ_C

(ppm) 143.86, 143.76, 137.68, 135.73, 132.43, 130.92, 124.43,

123.82, 123.43, 121.16, 116.85, 115.72, 114.79, 113.30, 50.86,

42.65, 42.22, 37.43, 26.93, 17.91.

5-Methyl-11-[4-aminobutylamino]-10H-indolo[3,2-b]quinolin-

5-ium Chloride (3k). To a suspension of 10a (40.0 mg, 0.132 mmol)

was added commercial butane-1,4-diamine (26.6 μL, 0.264 mmol)

according to general procedure F. The product was recrystallized

from CH₂Cl₂:MeOH (7:3) with Et₂O to give 21.2 mg (45%) of3k as

1

a yellow solid, mp 263-265 ꢀC. H NMR (400 MHz, DMSO)

δ_H(ppm) 8.89(d, J= 8.5 Hz, 1H), 8.55 (d, J= 8.2 Hz, 1H), 8.34 (d,

J = 8.5 Hz, 1H), 8.00 (dd, J = 8.3, 7.3 Hz, 1H), 7.95 (d, J = 8.3 Hz,

1H), 7.71 (dd, J = 7.8, 7.3 Hz, 1H), 7.67 (dd, J = 8.3, 7.3 Hz, 1H),

7.36 (dd, J = 8.2, 7.3 Hz, 1H), 4.60 (s, 3H), 4.22 (t, J = 6.6 Hz, 2H),

2.85 (t, J = 7.4 Hz, 2H), 1.89 (m, 2H), 1.75 (m, 2H). ¹³C NMR

(100MHz,DMSO) δ_C(ppm) 144.20, 143.13, 143.08, 137.77, 137.73,

132.70, 130.77, 125.01, 124.69, 124.26, 121.12, 117.78, 115.67,

114.93, 114.18, 45.18, 38.78, 38.45, 27.12, 24.75.

5-Methyl-11-[4-(diethylamino)butylamino]-10H-indolo [3,2-b]-

quinolin-5-ium chloride (3l). The title compound was obtained in

70% yield (44.9 mg) as an orange solid, mp 268-272 ꢀC, by

reaction of 10a (47.0 mg, 0.155 mmol) with N¹,N¹-diethylbu-

tane-1,4-diamine 18 (44.8 mg, 0.31 mmol), according to general

procedure F, and recrystallization from MeOH with AcOEt:

1

Et₂O (2:3). H NMR (400 MHz, DMSO) δ_H(ppm) 12.36 (s,

NH), 9.24 (d, J = 1.3 Hz, NH), 8.89 (d, J = 8.1 Hz, 1H), 8.55 (d,

J = 8.1 Hz, 1H), 8.33 (d, J = 8.6 Hz, 1H), 8.01 (dd, J = 8.6,

7.6 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.72 (dd, J = 8.1, 7.6 Hz,

1H), 7.68 (dd, J = 8.1, 7.6 Hz, 1H), 7.38 (dd, J = 8.1, 7.6 Hz,

1H), 4.60 (s, 3H), 4.23 (q broad, 2H), 3.10 (m, 6H), 1.89 (m, 4H),

1.21 (t, J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm)

143.75, 142.58, 137.38, 135.13, 132.78, 132.23, 130.28, 124.58,

124.18, 123.76, 120.63, 117.26, 115.16, 114.40, 113.67, 50.27,

45.97, 44.76, 37.95, 26.82, 20.51, 8.34.

5-Methyl-11-[5-(diethylamino)pentan-2-ylamino]-10H-indolo-

[3,2-b]quinolin-5-ium Chloride (3m). Reaction of 10a (40.0 mg,

0.132 mmol) and N¹,N¹-diethylpentane-1,4-diamine (51 μL,

0.264 mmol), according to general procedure F, and recrystalli-

zation from MeOH with AcOEt:Et₂O (1:1), gave 3m in 30%

yield (17.2 mg), as an orange solid, mp 240-243 ꢀC. ¹H NMR

(400 MHz, DMSO) δ_H(ppm) 8.85 (d, J = 8.6 Hz, 1H), 8.58

(d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.04 (dd, J = 8.5,

7.8 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.76 (dd, J = 8.5, 7.7 Hz,

1H), 7.71 (dd, J = 8.6, 7.8 Hz, 1H), 7.39 (dd, J = 8.1, 7.7 Hz,

1H), 4.92 (m, 1H), 4.64 (s, 3H), 3.13-2.93 (m, 6H), 2.14-1.98

(m, 2H), 1.91-1.81 (m, 2H), 1.47 (d, J = 6.2 Hz, 3H), 1.14 (t,

J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 144.24,

143.36, 140.38, 138.04, 136.08, 132.93, 131.09, 125.73, 124.98,

124.46, 121.39, 118.04, 116.04, 115.25, 114.41, 51.01, 46.88,

46.69, 38.87, 34.15, 22.55, 20.75, 9.01.

5-Methyl-11-(piperidin-4-ylamino)-10H-indolo[3,2-b]quinolin-

5-ium Chloride (3n). Reaction of 10a (50.0 mg, 0.165 mmol) and

piperidin-4-amine (31.1 μL, 0.296 mmol), according to general

procedure F, gave 3n in 66% yield (40.3 mg), as an orange solid,

mp 325-327 ꢀC. ¹H NMR (400 MHz, DMSO) δ_H(ppm) 8.66 (d,

J = 8.3 Hz, 1H), 8.56 (d, J = 8.6 Hz, 1H), 8.29 (d, J = 6.2 Hz,

1H), 8.09 (dd, J = 8.6, 5.6 Hz, 1H), 7.97 (d, J = 6.3 Hz, 1H), 7.84

(dd, J = 6.2, 5.6 Hz, 1H), 7.81 (dd, J = 6.3, 5.6 Hz, 1H) 7.44 (dd,

J = 8.3, 5.6 Hz, 1H), 4.80 (s, 3H), 4.14 (d, J = 12.8 Hz, 2H), 3.85 (t,

J= 11.6 Hz, 2H), 3.50 (m, 1H), 2.28 (t, J= 11.6 Hz, 2H), 2.10-1.97

(m, 2H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 147.18, 144.35,

138.14, 137.77, 132.73, 132.51, 126.54, 125.71, 125.57, 125.00,

121.64, 120.67, 118.66, 114.74, 114.12, 51.16, 47.64, 39.63, 30.70.

5-Methyl-11-(1-isobutylpiperidin-4-ylamino)-10H-indolo[3,2-b]-

quinolin-5-ium Chloride (3o). To a solution of 3n (40.0 mg,

5-Methyl-11-[3-(piperidin-1-yl)propylamino]-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3j). The title compound was obtained in

71% yield (38.3 mg) as a light-orange solid, mp 289-293 ꢀC,

after reaction of 10a (40.0 mg, 0.132 mmol) with 3-piperidine-

1yl-propan-1-amine 15 (37.5 mg, 0.264 mmol), according to

general procedure F, and recrystallization from CHCl₃:MeOH

(5:1) with AcOEt:Et₂O (8:1). ¹H NMR (400 MHz, DMSO) δ_H

746 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

0.108 mmol), anhydrous Na₂SO₄(2 g) and isobutyraldehyde

(31.2 μL, 0.432 mmol) in dry MeOH (5 mL) was added

NaBH₃CN (33.9 mg, 0.54 mmol) and the reaction allowed to

proceed at room temperature for 24 h. The anhydrous Na₂SO₄

was filtered off and solvent removed under reduced pressure.

The resulting residue was dissolved in water (5 mL) and basified

with aqueous KOH 2 M to pH 12. The aqueous solution was

then extracted with CH₂Cl₂(3 ꢁ 20 mL) and the combined

organic extracts washed with water and brine, dried (anhydrous

Na₂SO₄), and concentrated to a small volume under reduced

pressure. HCl in Et₂O was added, and the resultant precipitate

was collected, washed with Et₂O, and dried, yielding 27.3 mg

(59%) of 3o as a orange solid, mp 270-274 ꢀC. ¹H NMR

(400 MHz, CD₃OD) δ_H(ppm) 8.64 (d, J = 8.2 Hz, 1H), 8.51

(d, J = 8.3 Hz, 1H), 8.46 (d, J = 8.2 Hz, 1H), 8.13 (dd, J = 8.3,

7.6 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.88 (dd, J = 8.2, 7.6 Hz,

1H), 7.84 (dd, J = 8.4, 7.3 Hz, 1H), 7.50 (dd, J = 8.2, 7.3 Hz, 1H),

4.90 (s, 3H), 4.27 (d, J = 12.6 Hz, 2H), 3.92 (t, J = 12.2 Hz, 2H),

3.70 (m, 1H), 3.09 (d, J = 7.2 Hz, 2H), 2.52 (d, J = 11.2 Hz, 2H),

2.26 (qd, J = 12.1, 3.6 Hz, 2H), 2.16 (m, 1H), 1.16 (d, J = 6.7 Hz,

6H). ¹³C NMR (100 MHz, CD₃OD) δ_C(ppm) 148.15, 146.06,

139.80, 139.53, 133.83, 133.81, 127.47, 126.81, 126.74, 126.07,

122.92, 122.55, 118.88, 115.94, 114.71, 56.97, 53.37, 52.21, 39.89,

30.22, 27.66, 20.52.

(21.7 μL, 0.24 mmol) to give 3r (75%), mp 269-271 ꢀC, after

recrystallization with MeOH:AcOEt (1:2) and Et₂O. ¹H NMR

(400 MHz, DMSO) δ_H(ppm) 8.49 (d, J = 8.6 Hz, 1H), 8.44 (d,

J = 8.5 Hz, 1H), 8.34 (d, J = 8.7 Hz, 1H), 8.03 (dd, J = 8.7, 7.6

Hz, 1H), 7.69 (dd, J = 8.6, 7.6 Hz, 1H), 7.65 (dd, J = 8.2, 7.6 Hz,

1H), 7.53 (d, J = 8.2 Hz, 1H), 7.41 (dd, J = 7.8, 7.5 Hz, 2H), 7.35

(dd, J = 8.5, 7.6 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.18 (d, J =

7.8 Hz, 2H), 4.65 (s, 3H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 143.26, 139.75, 138.78, 137.94, 137.48, 133.65,

132.87, 130.98, 130.51, 126.69, 126.09, 125.34, 125.04, 122.76,

122.32, 118.39, 118.18, 115.27, 114.22, 38.83.

5-Methyl-11-[4-(piperidin-1-yl)phenylamino]-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3s). Reaction of 4-(piperidin-1-yl)-

benzenamine (41.6 mg, 0.236 mmol) with 10a (40 mg, 0.132 mmol),

according with general procedure F, gave 3s as a yellow solid,

mp 311-312 ꢀC in 66% yield, after recrystallization using

1

MeOH:AcOEt (1:3) and Et₂O. H NMR (400 MHz, DMSO)

δ

_H(ppm) 8.65 (d, J = 8.1 Hz, 1H), 8.57 (d, J = 8.1 Hz, 1H),

8.42 (d, J = 8.1 Hz, 1H), 8.10 (dd, J = 8.1, 7.7 Hz, 1H), 7.76

(dd, J = 8.1, 7.7 Hz, 1H), 7.71 (dd, J = 8.0, 7.6 Hz, 1H), 7.60 (d,

J = 8.0 Hz, 1H), 7.43 (dd, J = 8.1, 7.6 Hz, 1H), 7.31 (d, J = 8.1

Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 4.81 (s, 3H), 3.37 (s broad,

4H), 1.80 (s broad, 4H), 1.69 (d, J = 4.6 Hz, 2H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 152.10, 143.89, 141.13, 139.22,

137.94, 133.79, 132.58, 132.35, 129.48, 125.95, 125.87, 125.31,

125.21, 122.48, 118.32, 118.25, 116.21, 114.45, 113.30, 51.89,

39.01, 26.85, 25.23.

5-Methyl-11-(1-benzylpiperidin-4-ylamino)-10H-indolo [3,2-b]-

quinolin-5-ium Chloride (3p). To a solution of 3n (40.0 mg,

0.108 mmol) and anhydrous Na₂SO₄(2 g) in dry MeOH (5 mL)

was added benzaldehyde (43.6 μL, 0.432 mmol) and the mixture

stirred for 1 h. NaBH₃CN (33.9 mg, 0.54 mmol) was added and

the mixture left to react for a further 20 h. The anhydrous Na₂SO₄

was filtered off and the solvent removed under reduced pressure.

The residue was dissolved in H₂O (5 mL) and the pH adjusted to

12 with 2 M KOH. The aqueous solution was extracted with

CH₂Cl₂(3 ꢁ 20 mL) and the combined extracts washed with

water and brine and dried with anhydrous Na₂SO₄. After con-

centration under reduced pressure, HCl in Et₂O was added to

precipitate the product as a salt, which was filtered and recrys-

tallized with MeOH:AcOEt (2:5) and Et₂O to give 3p as a deep-

yellow solid, mp 275-277 ꢀC, in 66% yield. ¹H NMR (400 MHz,

DMSO) δ_H(ppm) 8.63 (d, J = 8.2 Hz, 1H), 8.50 (d, J = 8.5 Hz,

1H), 8.47 (d, J = 8.2 Hz, 1H), 8.12 (dd, J = 8.5, 7.6 Hz, 1H), 7.93

(d, J = 8.1 Hz, 1H), 7.87 (dd, J = 8.2, 7.6 Hz, 1H), 7.83 (dd, J =

8.1, 7.6 Hz, 1H), 7.66 (d, J = 5.9 Hz, 2H), 7.56 (d, J = 6.7 Hz,

1H), 7.52 (dd, J = 6.7, 5.9 Hz, 2H), 7.48 (dd, J = 8.2, 7.6 Hz,

1H), 4.88 (s, 3H), 4.44 (s, 2H), 4.27 (d, J = 12.5 Hz, 2H), 3.92 (t,

J = 12.5 Hz, 2H), 3.77 (t, J = 10.3 Hz, 1H), 2.58 (d, J = 11.6 Hz,

2H), 2.28 (q, J = 11.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO)

δ_C(ppm) 146.67, 144.61, 138.35, 138.06, 135.98, 132.38, 132.35,

131.28, 129.71, 129.37, 129.01, 126.03, 125.32, 124.61, 121.46,

121.12, 117.42, 114.47, 113.27, 54.92, 50.73, 48.32, 38.46, 28.93.

5-Methyl-11-[1-(2-hydroxybenzyl)piperidin-4-ylamino]-10H-

indolo[3,2-b]quinolin-5-ium Chloride (3q). Reaction of 3n (40.0 mg,

0.108 mmol) with salicylaldehyde (45 μL, 0.432 mmol) according to

the procedure described for 3o, gave 3q, in 30% yield (15.8 mg), as an

orange solid, mp 277-280 ꢀC. ¹H NMR (400 MHz, DMSO)

5-Methyl-11-{4-[(diethylamino)methyl]-3-hydroxyphenyl amino}-

10H-indolo[3,2-b]quinolin-5-ium Chloride (3t). A solution of

N-(4-((diethylamino)-methyl)-3-hydroxyphenyl)-acetamido

20 (47.2 mg, 0.199 mmol) and 10a (40 mg, 0.132 mmol) in HCl

6 M (10 mL) was refluxed for 20 h. After this period, the

solution was concentrated under reduced pressure and the residue

dissolved in Na₂CO₃5% (25 mL). The aqueous solution was

extracted with CH₂Cl₂(3 ꢁ 20 mL) and the combined frac-

tions washed with water and then dried with NaCl (satd) and

with anhydrous Na₂SO₄. After concentration under reduced

pressure, HCl in Et₂O was added and the precipitate formed,

filtered off, and dried to give 3t as an orange solid, mp

228-231 ꢀC, in 31% yield. ¹H NMR (400 MHz, DMSO)

δ_H(ppm) 11.84 (s, 1H), 10.98 (s, 1H), 10.63 (s, 1H), 8.67 (d,

J = 8.9 Hz, 1H), 8.60 (d, J = 8.5 Hz, 1H), 8.58 (d, J = 8.5 Hz,

1H), 8.09 (dd, J = 8.5, 7.0 Hz, 1H), 7.78 (dd, J = 8.2, 6.2 Hz,

1H), 7.76 (d, J = 8.2 Hz, 1H), 7.74 (dd, J = 8.5, 7.0 Hz, 1H),

7.46 (d, J = 8.3 Hz, 1H), 7.43 (dd, J = 8.9, 6.2 Hz, 1H), 6.95

(d, J = 2.0 Hz, 1H), 6.67 (dd, J = 8.3, 2.0 Hz, 1H), 4.83 (s,

3H), 4.20 (d, J = 4.8 Hz, 2H), 3.13 (m, 4H), 1.29 (t, J = 7.1 Hz,

6H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 157.28, 144.25,

142.71, 142.22, 137.68, 136.58, 136.19, 133.43, 132.91, 132.32,

125.61, 125.22, 124.93, 120.99, 118.48, 117.94, 114.94, 113.60,

113.36, 111.83, 107.90, 49.49, 46.05, 39.32, 8.50.

5-Methyl-11-(pyridin-3-ylamino)-10H-indolo[3,2-b]quinolin-

5-ium Chloride (3u). To a solution of 10a (40.0 mg, 0.132 mmol)

in DMF (5 mL) was added pyridin-3-amine (37.2 mg, 0.396 mmol)

and the mixture heated at 100 ꢀC for 48 h. Solvent was removed

under reduced pressure. The remaining crude mixture was

dissolved in Na₂CO₃5% (20 mL) and extracted with CH₂Cl₂

(3 ꢁ 30 mL). The combined organic extracts were washed with

water and brine and dried with anhydrous Na₂SO₄. After

concentration under reduced pressure, HCl in Et₂O was added

and the formed precipitate was filtered, washed with Et₂O, and

dried in a vacuum line, to give 3u in 48% yield as a brown solid

(22.6 mg), mp 157-159 ꢀC. ¹H NMR (400 MHz, DMSO)

δ_H(ppm) 11.64 (s, NH), 8.79 (d, J = 8.4 Hz, 1H), 8.69 (d,

J = 8.4 Hz, 1H), 8.64 (s, 1H), 8.63 (d, J = 7.2 Hz, 1H), 8.46 (d,

J = 8.2 Hz, 1H), 8.13 (dd, J = 8.2, 7.2 Hz, 1H), 7.82 (dd, J =

8.4, 7.2 Hz, 1H), 7.79 (dd, J = 7.8, 7.5 Hz, 1H), 7.67 (d, J =

7.8 Hz, 1H), 7.63 (d, J = 5.7 Hz, 1H), 7.51 (dd, J = 8.2, 5.7 Hz,

1H), 7.44 (dd, J = 8.4, 7.2 Hz, 1H), 4.86 (s, 3H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 143.65, 143.62, 141.60, 137.89,

δ_H(ppm) 12.18 (s, NH), 9.47 (d, J = 1.2 Hz, NH), 8.67 (d, J =

8.1 Hz, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.10

(dd,J= 8.8, 7.1 Hz, 1H), 7.99 (d, J= 8.6 Hz, 1H), 7.86 (dd, J=8.3,

7.1 Hz, 1H), 7.82 (dd, J= 8.6, 7.3 Hz, 1H), 7.56 (d, J=7.2Hz, 1H),

7.46 (dd, J = 8.1, 7.3 Hz, 1H), 7.28 (dd, J = 7.8, 6.9 Hz, 1H), 7.04

(d, J= 7.8 Hz, 1H), 6.89 (dd, J= 7.2, 6.9 Hz, 1H), 4.82 (s, 3H), 4.23

(s, 2H), 4.17(d, J=12.2Hz,2H),3.85(t, J= 11.5 Hz, 2H), 3.53 (m,

1H), 2.46 (d broad, 2H), 2.23 (q, J = 5.8 Hz, 2H). ¹³C NMR

(100MHz,DMSO) δ_C(ppm) 155.99, 146.54, 143.94, 137.56, 137.28,

132.21, 132.19, 131.95, 131.63, 130.34, 125.97, 125.04, 124.60,

121.15, 121.07, 119.04, 118.24, 118.13, 115.44, 114.19, 113.67,

53.78, 50.74, 42.04, 39.27, 28.39.

5-Methyl-11-(phenylamino)-10H-indolo[3,2-b]quinolin-5-ium

Chloride (3r). Following general procedure F, a suspension of

10a (40.0 mg, 0.132 mmol) was allowed to react with aniline

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 747

137.44, 137.42, 132.83, 132.58, 128.32, 125.62, 125.53, 125.37,

124.77, 124.55, 121.95, 121.60, 118.69, 118.49, 115.16, 113.88, 49.05.

3-Chloro-5-methyl-11-[3-(diethylamino)propylamino]-10H-

indolo[3,2-b]quinolin-5-ium Chloride (3v). To a suspension of

10b (40.0 mg, 0.118 mmol) in AcOEt (5 mL) was added

commercial N¹,N¹-diethylpropane-1,3-diamine (21.4 μL,

0.212 mmol), according to general procedure F, giving 25.1

mg (53%) of 3v, as a dark-yellow solid, mp 231-234 ꢀC.

¹H NMR (400 MHz, DMSO): δ_H(ppm) 12.49 (s, NH), 9.41 (s,

NH), 8.91 (d, J = 9.1 Hz, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.46

(s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.72

(dd, J = 9.1, 7.4 Hz, 1H), 7.37 (dd, J = 8.4, 7.4 Hz, 1H), 4.59

(s, 3H), 4.32 (s broad, 2H), 3.30 (s broad, 3H), 3.11 (q broad,

2H), 2.26 (s broad, 2H), 1.22 (t, J = 6.7 Hz, 6H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 144.12, 143.29, 138.45, 137.87,

135.95, 131.01, 127.17, 124.71, 124.56, 121.35, 117.52, 117.24,

116.94, 114.79, 114.41, 48.56, 46.49, 43.60, 38.83, 24.39, 8.87.

3-Chloro-5-methyl-11-(piperidin-4-ylamino)-10H-indolo[3,2-b]-

quinolin-5-ium Chloride (3w). To a suspension of 10b (30.0 mg,

0.088 mmol) in AcOEt (5 mL) was added piperidin-4-amine

(18.6 μL, 0.177 mmol), according to general procedure F, giving

18.8 mg (53%) of 3w, as a dark-yellow solid, mp 320-322 ꢀC. ¹H

NMR (400 MHz, DMSO) δ_H(ppm) 12.37 (s, NH), 8.71 (s, 1H),

8.66 (d, J = 8.2 Hz, 1H), 8.63 (s, NH), 8.28 (d, J = 9.1 Hz, 1H),

8.03 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H), 7.82 (dd, J =

8.0, 7.7 Hz, 1H), 7.44 (dd, J = 8.2, 7.7 Hz, 1H), 4.79 (s, 3H), 4.18

(d, J = 11.6 Hz, 2H), 3.92 (t, J = 10.7 Hz, 2H), 3.54 (m, 1H),

2.33 (d, J = 11.6 Hz, 2H), 2.07 (q, J = 10.7 Hz, 2H). ¹³C NMR

(100 MHz, DMSO) δ_C(ppm) 147.35, 144.48, 138.78, 137.90,

137.69, 132.52, 128.75, 125.82, 125.59, 124.90, 121.76, 119.25,

118.00, 114.63, 114.33, 51.38, 47.54, 39.49, 30.72.

concentrated HCl to pH 2. The solvent was removed at reduced

pressure and the crude product dissolved in water (50 mL). The

aqueous solution was washed with Et₂O (3 ꢁ 20 mL), treated

with NaOH 10% to pH 10, and extracted with CH₂Cl₂(3 ꢁ

20 mL). The organic extracts were washed with water and brine,

dried (anhydrous Na₂SO₄), and the solvent removed under

reduced pressure to give 12, 24% (204.2 mg), as a light-brown

oil. ¹H NMR (400 MHz, CDCl₃) δ_H(ppm) 3.00 (m, 1H), 2.18 (s,

6H), 2.12 (d, J = 10.1 Hz, 1H), 2.04 (dd, J = 4.2, 4.1 Hz, 1H),

1.01 (d, J = 6.89 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)

δ

_C(ppm) 68.20, 45.82, 44.14, 21.07.

2-(3-(Piperidin-1-yl)-propyl)isoindoline-1,3-dione (14). To a solu-

tion of N-(3-bromopropyl)phthalimide 13 (2.71 g, 10.1 mmol) and

TEA (1.67 mL, 24.2 mmol) in CH₂Cl₂(40 mL) was added

piperidine (1 mL, 10.1 mmol). The reaction mixture was refluxed

30 h, and after this period, the solvent was removed under reduced

pressure. The crude residue was purified by column chromatogra-

phy (CH₂Cl₂and CH₂Cl₂:MeOH (9:1)) to afford 14, 1.45 g (51%)

as a white solid, mp 266-267 ꢀC. ¹H NMR (400 MHz, CDCl₃) δ_H

(ppm) 7.83(dd, J= 5.2, 3.1 Hz, 2H), 7.73 (dd, J= 5.2, 3.1 Hz, 2H),

3.78 (t, J = 6.57 Hz, 2H), 3.06-2.66 (m, 6H), 2.23 (quintet, J = 7.2

Hz, 2H), 1.90 (m, 4H), 1.57 (m, 2H). ¹³C NMR (100 MHz, CDCl₃)

δ_C(ppm) 168.18, 134.08, 131.81, 123.28, 55.63, 53.64, 35.66, 23.93,

23.38, 22.58.

3-Piperidin-1-yl-propan-1-amine (15). To a solution of 14 (0.5 g,

1.83 mmol) in EtOH (30 mL) was added hydrazine (0.552 mL,

4.38 mmol). The reaction mixture was refluxed 3 h, and the resulting

precipitate was collected and the solvent removed under reduced

pressure. The crude product was dissolved in CH₂Cl₂, the precipi-

tate was again collected and the solvent evaporated under reduced

1

pressure to give 15, 71% yield (178 mg) as a light-brown oil. H

3,8-Dichloro-5-methyl-11-(piperidin-4-ylamino)-10H-indolo-

[3,2-b]quinolin-5-ium Chloride (3x). To a suspension of 10c

(21.7 mg, 0.058 mmol) in AcOEt (5 mL) was added piperidin-

4-amine (11.3 μL, 0.116 mmol), according to general proce-

dure F, giving 7.7 mg (30%) of 3x as an orange solid, mp

315-317 ꢀC. ¹H NMR (400 MHz, CD₃OD) δ_H(ppm) 8.55 (d,

J = 8.9 Hz, 1H), 8.53 (s, 1H), 8.35 (d, J = 9.1 Hz, 1H), 7.90 (s,

1H), 7.80 (d, J = 9.1 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 4.75 (s,

3H), 4.23 (d, J = 12.1 Hz, 2H), 3.91 (t, J = 12.1 Hz, 2H), 3.64

(s broad, 1H), 2.38 (d, J = 11.4 Hz, 2H), 2.15 (d, J = 11.4 Hz,

2H). ¹³C NMR (100 MHz, DMSO) δ_C(ppm) 148.61, 145.94,

140.28, 140.16, 139.76, 139.18, 129.34, 127.27, 126.67, 124.91,

123.55, 120.84, 118.38, 114.47, 114.23, 52.29, 49.22, 39.93, 31.62.

5-Methyl-11-(diethylamino)-10H-indolo[3,2-b]quinolin-5-ium

chloride (3y). To a solution of 10a (40 mg, 0.132 mmol) in DMF

(10 mL) was added diethylamine (54.8 μL, 0.528 mmol) and the

mixture allowed to react overnight at room temperature. The

solvent was removed at reduced pressure, and the solid was

suspended in 5% sodium carbonate (20 mL) and extracted with

chloroform (3 ꢁ 20 mL). The combined organic extracts were

washed with water and brine (20 mL) and dried with anhydrous

Na₂SO₄, and the solvent was removed under reduced pressure.

Recrystallization from chloroform with HCl in diethyl ether

gave 65% (29.3 mg) of pure 3y, as a light-yellow solid, mp

251-254 ꢀC. ¹H NMR (400 MHz, CD₃OD) δ_H(ppm) 8.54 (d,

J = 8.5 Hz, 1H), 8.45 (d, J = 8.5 Hz, 1H), 8.41 (d, J = 9.0 Hz,

1H), 8.02 (dd, J = 9.0, 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.75

(dd, J = 8.5, 8.4 Hz, 1H), 7.71 (dd, J = 8.4, 7.7 Hz, 1H), 7.39

(dd, J = 8.5, 7.7 Hz, 1H), 4.80 (s, 3H), 4.00 (q, J = 7.1 Hz, 4H),

1.30 (t, J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, CD₃OD) δ_C

(ppm) 145.73, 142.53, 136.62, 135.73, 130.64, 130.41, 125.44,

125.35, 123.26, 122.90, 120.89, 119.68, 115.68, 113.10, 111.49,

45.78, 36.73, 11.27.

NMR (400 MHz, CDCl₃) δ_H(ppm) 2.98 (s, 1H), 2.75 (t, J = 6.3

Hz, 2H), 2.39 (m, 6H), 1.63 (quintet, J = 6.9 Hz, 2H), 1.54 (m, 4H),

1.38 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ_C(ppm) 57.19, 54.34,

40.69, 28.86, 25.62, 24.07.

tert-Butyl 4-(diethylamino)butylcarbamate (17). A mixture of

tert-butyl 4-aminobutylcarbamate 16 (0.1 mL, 0.52 mmol),

anhydrous MgSO₄(2 g), and NaBH₃CN (70.4 mg, 1.25 mmol)

in dry MeOH (5 mL) was cooled to 0 ꢀC. Acetaldehyde (1 mL)

was added and reaction allowed to continue for 2 h in an ice

bath. After this period, the anhydrous MgSO₄was collected by

filtration and the solvent removed under reduced pressure. The

crude product was dissolved in water (5 mL), basified to pH

10 with aqueous KOH 5%, and extracted with CH₂Cl₂(3 ꢁ

20 mL). The combined organic extracts were washed with water

and brine, dried (anhydrous Na₂SO₄), and evaporated under

reduced pressure to give 17, in 94% yield (119.9 mg) as yellow

oil. ¹H NMR (400 MHz, CDCl₃) δ_H(ppm) 5.42 (s, NH), 2.95 (d,

J = 5.3 Hz, 2H), 2.43 (q, J = 7.1 Hz, 4H), 2.32 (t, J = 6.5 Hz,

2H), 1.36 (s broad, 4H), 1.27 (s, 9H), 0.90 (t, J = 7.1 Hz, 6H). ¹³

C

NMR (100 MHz, CDCl₃) δ_C(ppm) 155.77, 78.21, 51.96, 46.09,

39.89, 27.99, 27.65, 23.55, 10.55.

N¹,N¹-Diethylbutane-1,4-diamine (18). A solution of 17

(119.9 mg, 0.49 mmol) in CH₂Cl₂:TFA (1:1, 2 mL) was stirred

at room temperature for 1 h. Solvent was removed under

reduced pressure, and the crude residue neutralized with aqueous

KOH (2 M). The aqueous solution was then extracted with

CH₂Cl₂(3 ꢁ 10 mL). The combined organic extracts were

washed with water and brine, dried (anhydrous Na₂SO₄),

and evaporated under reduced pressure to give 18, 63% yield

(44.8 mg), as a yellow oil. ¹H NMR (400 MHz, CDCl₃)

δ_H(ppm) 4.02 (s, NH₂), 2.70 (t, J = 6.2 Hz, 2H), 2.50 (q,

J = 7.1 Hz, 4H), 2.39 (t, J = 6.7 Hz, 2H), 1.50 (m, 4H), 0.97

(t, J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ_C(ppm)

52.14, 45.98, 40.92, 30.01, 24.12, 10.60.

N¹,N¹-Dimethylpropane-1,2-diamine (12). To a solution of

NH₄OAc (5.26 g, 68.2 mmol), NaBH₃CN (0.634 g, 10 mmol),

and anhydrous MgSO₄(8 g) in dry MeOH was added 1-

(dimethylamino)propan-2-one 11 (0.863 g, 8.5 mmol, 1 mL).

The reaction mixture was refluxed for 20 h. After this period, the

MgSO₄was removed and the reaction mixture acidified with

N-{4-[(Diethylamino)methyl]-3-hydroxyphenyl}acetamide (20).

To a solution of N-(3-hydroxyphenyl)acetamide 19 (2 g,

13.2 mmol) and formaldehyde (0.73 mL, 26.4 mmol) in EtOH

(20 mL) was added diethylamine (2.74 mL, 26.4 mmol). The

mixture was refluxed for 96 h, and after this period, the solvent

748 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3

Lavrado et al.

was removed under reduced pressure. The remaining residue was

dissolved in CH₂Cl₂(20 mL) and extracted with HCl 0.1 M (4 ꢁ

50 mL). The combined aqueous extracts were basified to pH 10

with KOH 5% and extracted with CH₂Cl₂(3 ꢁ 20 mL). The

organic extracts were washed with H₂O and dried with NaCl

(satd) and anhydrous Na₂SO₄. Evaporation of the solvent gave

20 as yellow oil, in 55% yield. ¹H NMR (400 MHz, CDCl₃) δ_H

(ppm) 8.08 (s, OH), 7.05 (d, J = 7.9 Hz, 1H), 6.90 (s, 1H), 6.88 (d,

J = 7.9 Hz, 1H), 3.71 (s, 2H), 2.60 (q, J = 6.7 Hz, 4H), 2.11 (s,

3H), 1.08 (t, J = 6.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ_C

ppm 168.46, 158.08, 138.12, 128.25, 117.41, 110.34, 107.19, 55.86,

45.70, 24.01, 10.63.

solution (0.027 or 0.27 mM) of 12-mer oligonucleotide duplex to

a solution (0.5, 1, or 5 μM) of the ligand in 0.01 M phosphate

buffer (pH 7.4) containing 0.1 M NaCl. All the spectrophoto-

fluorimetric titration spectra were recorded at an excitation

wavelength of 339 nm and an emission wavelength between 450

and 500 nm. Dissociation constants of cryptolepine analogues

in complex with the 12-mer DNA oligonucleotide d(GAT-

CCTAGGATC)₂were determined by fitting the experimental

data to the single binding site model (eq 1) using least-squares

nonlinear regression analysis. For some compounds, the re-

quired equation was that for two binding sites sequestering

ligand independently of each other and with relatively weak

binding in the second binding equilibrium detectable at higher

values of [DNA]. The general equation for the two-site binding

model is

In Vitro Cytotoxicity. Vero cells (monkey kidney epithelium)

were kindly donated by the Departamento de Virologı

tad de Microbiologıa, Universidad de Costa Rica. Cells were

´

a, Facul-

´

maintained in Dulbecco essential medium supplemented with

10% fetal bovine serum, 2 mmol/L of glutamine, 100 IU/mL of

penicillin, and amphotericin B in a 37 ꢀC humidified incubator

under an atmosphere of 7% CO₂on air. For the experiments,

cells were cultured in 96-well plates (15000 cells/well) and allowed to

adhere overnight. Sample stocks were prepared in DMSO at a

concentration of 30 mg/mL. Log dilutions made on culture

medium were tested on cells for 48 h. Doxorubicin hydrochlo-

ride (Sigma-Aldrich) was used as a standard for the experiments.

After cells were treated with the compounds, MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent

(final concentration of 0.5 mg/mL) was added to the culture

medium and, after incubation for 2 h at 37 ꢀC, it was carefully

removed and 95% ethanol was added to the wells to dissolve

formazan crystals.⁵⁶Absorbances were read at 570 nm, and

results were calculated as viability percentages, using samples

incubated with DMSO dissolved in culture medium as 100%

viability values. IC₅₀values were obtained from a concentration

vs viability curve using SlideWrite Plus 6.1 (Advanced Graphics

Software, Inc., Carlsbad, CA).

In Vitro Antiplasmodial Activity in Human Red Blood Cells.

Human red blood cells infected with 1% ring stage P. falciparum

strains synchronized with 5% sorbitol were incubated with

tested compounds in 96-well plates at 37 ꢀC for 48 h in RPMI-

1640 medium, supplemented with 25 mM HEPES pH 7.4, 10%

heat inactivated human serum (or 0.5% Albumax, 2% human

serum), and 100 μM hypoxanthine under an atmosphere of 3%

O₂, 5% CO₂, 91% N₂. After 48 h, the cells were fixed in 2%

formaldehyde in PBS, transferred into PBS with 100 mM

NH₄Cl, 0.1% Triton X-100, 1 nM YOYO-1, and then analyzed

in a flow cytometer (FACSort, Beckton Dickinson; EX 488 nm,

EM 520 nm.) Values of IC₅₀were calculated using GraphPad

PRISM software.

In Vitro Antiplasmodial Activity in Human Hepatoma Cells. In

vitro infection of Huh7 cells, a human hepatoma cell line,

was determined by analyzing cells infected with GFP-expressing

P. berghei parasites by fluorescence-activated cell sorting

(FACS), as described previously.³⁷Cell samples for FACS

analysis were washed with 1 mL of phosphate buffered saline

(PBS), incubated with 100 μL of trypsin for 5 min at 37 ꢀC, and

collected in 400 μL of 10% v/v FCS in PBS at the selected time

points post sporozoite addition. Cells were then centrifuged at

0.1g for 5 min at 4 ꢀC and resuspended in 150 μL of 2% v/v FCS

in PBS. Cells were analyzed on a Becton Dickinson FACScali-

bur with the appropriate settings for the fluorophore used. Data

acquisition and analysis were carried out using the CELLQuest

(version 3.2.1fl1, Becton Dickinson) and FlowJo (version 6.3.4,

FlowJo) software packages, respectively. Invasion was asses-

sed by determining the percentage of GFP-positive cells 2 h

after sporozoite addition; intracellular parasite development

was assessed by measuring the fluorescence intensity of GFP-

positive cells 48 h after addition of the parasites to the cells.

Binding to d(GATCCTAGGATC)₂. Titrations were carried

out using spectrophotofluorimetry or UV-visible spectropho-

tometry in 1 cm quartz cuvettes by adding aliquots of a stock

F ¼ fF_max½DNAꢀ=ðK_dþ ½DNAꢀÞg

þ fF⁰_max½DNAꢀ=ðK⁰_dþ ½DNAꢀÞg

ð4Þ

where F ⁰_maxand K⁰_dare the binding parameters for the second

equilibrium. If K⁰_d. [DNA], this reduces to eq 3. Data were fit

to the equations supplied with GraphPad PRISM computer

program (GraphPad software, version 5.00, San Diego, CA) or

to appropriate variants written into that program. Best models

were decided by χ-squared parameters and the distribution of

the residuals. Association constants were calculated from dis-

sociation constants.

Heme Binding Studies. Titrations of FPIX-OH in buffered

40% DMSO (v/v) at pH 5.5, with chloroquine, cryptolepine,

and analogues, were made according to the following proce-

dure. Stock solutions of hematin and ligands were obtained by

dissolving compounds in UV-spectroscopy grade DMSO to a

concentration of 1 mM, with storage in the dark. Aqueous buffered

DMSO (40% v/v, 1 mL) solutions of hematin, chloroquine,

cryptolepine, and analogues were prepared daily from stock

solution (100 μM). Hematin solutions (10 μM) were prepared

with buffered 40% DMSO (v/v) solution and transferred to

cuvettes. Solutions of chloroquine, cryptolepine, and analogues

(100 μM) were initially added to the cuvette in amounts as small

as 2 μL (gradually increasing the volume in subsequent additions)

until final concentrations higher than the hematin concentration

were achieved. After each addition, the cuvette was stirred for 1

min before the absorbance was read. UV-visible titrations were

performed between 230 and 500 nm to incorporate the Soret

band of porphyrin. The UV-visible spectrum obtained after

each titrated addition was analyzed and stacked against the

corresponding absorbances. Dissociation constants of chloro-

quine, cryptolepine, and analogues complexed with FPIX-OH

were determined by fitting the experimental data to the appro-

priate equation models^49,52using least-squares nonlinear regres-

sion analysis with GraphPad PRISM software. Models were

analyzed by χ-squared parameters. Association constants, K_ass

were calculated from dissociation constants.

,

Binding Stoichiometries. Binding stoichiometries for cryp-

tolepine analogues:12-mer ds-DNA complexes were monitored

by spectrophotofluorimetry or UV-visible spectrophotometry

using the Job method of continuous variation.^41,57The total

concentration of DNA and cryptolepine ligand in the solutions

was kept constant, and the changes in fluorescence or absor-

bance intensity monitored as a function of the mole fraction of

cryptolepine ligand. The intercept of the two best-fit lines

obtained by least-squares linear regression analysis indicated

the binding stoichiometry of the complex.

Binding stoichiometries for chloroquine, cryptolepine, and

analogues with FPIX-OH were monitored by UV-visible spec-

trophotometry using the Job method of continuous variation^41,57in

buffered 40% v/v DMSO (pH 5.5). Buffered 40% DMSO solution

was prepared in a 250 mL volume using DMSO (100 mL),

aqueous HEPES (1M, 5 mL), and deionized water. The sum of

the concentrations of the ligand and FPIX-OH was kept constant

Article

Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 749

([ligand] þ [FPIX-OH] = 10 μM). The absorbance changes of

the Soret band was measured at 402 nm. In Figure 7, y is the

corrected absorbance (y = -{A - (ε_FPIX-OH[FPIX-OH] þ

(11) Wright, C. W. Recent developments in naturally derived antima-

larials: cryptolepine analogues. J. Pharm. Pharmacol. 2007, 59,

899–904.

(12) Lisgarten, J. N.; Coll, M.; Portugal, J.; Wright, C. W.; Aymami, J.

The antimalarial and cytotoxic drug cryptolepine intercalates into

DNA at cytosine-cytosine sites. Nature Struct. Biol. 2002, 9, 57–

60.

(13) Dassonneville, L.; Bonjean, K.; De Pauw-Gillet, M. C.; Colson, P.;

Houssier, C.; Quetin-Leclercq, J.; Angenot, L.; Bailly, C. Stimula-

tion of topoisomerase II-mediated DNA cleavage by three DNA-

intercalating plant alkaloids: cryptolepine, matadine, and serpen-

tine. Biochemistry 1999, 38, 7719–7726.

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West African anti-malarial herbal Cryptolepis sanguinolenta and its

major alkaloid cryptolepine. Toxicology 2005, 208, 141–147.

(17) Bray, P. G.; Mungthin, M.; Ridley, R. G.; Ward, S. A. Access to

hematin: the basis of chloroquine resistance. Mol. Pharmacol. 1998,

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P. M.; Ward, S. A. Relationship between antimalarial drug activity,

accumulation, and inhibition of heme polymerization in Plasmo-

dium falciparum in vitro. Antimicrob. Agents Chemother. 1998, 42,

682–686.

ε

_compound[compound])b}), where A is the measured absorbance,

_FPIX-OHand ε_compoundare the molar absorptivities of hematin and

ε

ligands, respectively, and bthe optical path length.⁵⁸The intercept of

the two best-fit lines obtained by least-squares linear regression

analysis indicated the binding stoichiometry of the complex.

Compound Localization in P. falciparum-Infected Erythrocytes.

P. falciparum blood stages were cultured in human erythrocytes

as previously described.⁵⁹Intracellular localization of com-

pounds 1 and 3n was performed by fluorescence microscopy

on a Zeiss Axiovert 200 M microscope equipped with an oil-

immersion 100ꢁ Plan-apochromat 1.4NA objective. A human

red blood cell culture with 1.9% parasitemia was incubated with

5 μM of each of the compounds or with an equivalent amount of

the DMSO solvent (control) for 3 h, at room temperature, in the

dark, and observed on poly-Lys-coated coverslips after gentle

washing with PBS to remove nonadherent cells. Fluorescence

intensity quantification and plot profiles were made with Im-

ageJ software.

~

Acknowledgment. This work was supported by Fundac-ao

^

para a Ciencia e Tecnologia (project PTDC/SAU-FAR/114864/

2009), British Council (B-4/07), FEES-Vicerrectorıa de In-

´

ꢀ

vestigacion, Universidad de Costa Rica (project 809-A8-518),

and CYTED network RIBIOFAR (RT-0282). J.L. thanks

FCT for a Ph.D. grant (SFRH/BD/29202/2006). M.P. was

(20) Wright, C. W.; Addae-Kyereme, J.; Breen, A. G.; Brown, J. E.;

Cox, M. F.; Croft, S. L.; Gokcek, Y.; Kendrick, H.; Phillips, R. M.;

Pollet, P. L. Synthesis and evaluation of cryptolepine analogues for

their potential as new antimalarial agents. J. Med. Chem. 2001, 44,

3187–3194.

(21) Weissbuch, I.; Leiserowitz, L. Interplay Between Malaria, Crystal-

line Hemozoin Formation, and Antimalarial Drug Action and

Design. Chem. Rev. 2008, 108, 4899–4914.

^

hired under the Ciencia program of the Portuguese Ministry

of Science and Technology. We acknowledge Rosangela Frita

^

for the P. falciparum-infected human blood culture.

Supporting Information Available: NMR spectra of cryp-

tolepine derivatives 3a-y, elemental analysis (C, H, N), high

resolution mass spectrometry, data from equilibrium binding

studies with FPIX-OH (determination of stoichiometry, asso-

ciation constants of complexes, and fluorescence quenching)

and DNA (determination of stoichiometry and association con-

stants of complexes) of cryptolepine and analogues, as well as

geometry optimization images of some cryptolepine analogues,

at an ab initio quantum mechanics level by using density functional

theory (DFT). This material is available free of charge via the

Internet at http://pubs.acs.org.

(22) Buller, R.; Peterson, M. L.; Almarsson, O.; Leiserowitz, L. Quino-

line binding site on malaria pigment crystal: a rational pathway for

antimalarial drug design. Cryst. Growth Des. 2002, 2, 553–562.

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F.; Gaspard, C.; Marsais, F.; Godard, A.; Queguiner, G. New

synthesis of benzo-delta-carbolines, cryptolepines, and their salts: in

vitro cytotoxic, antiplasmodial, and antitrypanosomal activities of

delta-caribolines, benzo-delta-carbolines, and cryptolepines. J. Med.

Chem. 2001, 44, 949–960.

(24) Lavrado, J.; Paulo, A.; Gut, J.; Rosenthal, P. J.; Moreira, R.

Cryptolepine analogues containing basic aminoalkyl side-chains

at C-11: synthesis, antiplasmodial activity, and cytotoxicity.

Bioorg. Med. Chem. Lett. 2008, 18, 1378–81.

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Indolo[3,2-b]Quinoline 5-Oxide (Dioxyquindoline). Arch. Pharm.

(Weinheim, Ger.) 1981, 314, 850–852.

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Article Doi

Incorporation of basic side chains into cryptolepine scaffold: Structure-antimalarial activity relationships and mechanistic studies

DOI: 10.1021/jm101383f

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