Orally Effective Aminoalkyl 10H-Indolo[3,2-b]quinoline-11-carboxamide Kills the Malaria Parasite by Inhibiting Host Hemoglobin Uptake

DOI: 10.1002/cmdc.201800579

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Orally Effective Aminoalkyl 10H-Indolo[3,2-b]quinoline-11-

carboxamide Kills the Malaria Parasite by Inhibiting Host

Hemoglobin Uptake

Ramesh Mudududdla⁺,^[a]Dinesh Mohanakrishnan⁺,^[b]Sonali S. Bharate,^[a]

Ram A. Vishwakarma,^[a]Dinkar Sahal,*^[b]and Sandip B. Bharate*^[a]

A series of indolo[3,2-b]quinoline-C11-carboxamides were syn-

thesized by incorporation of aminoalkyl side chains into the

core of indolo[3,2-b]quinoline-C11-carboxylic acid. Their in vitro

antiplasmodial evaluation against Plasmodium falciparum led

to the identification of a 2-(piperidin-1-yl)ethanamine-linked

analogue {2-bromo-N-[2-(piperidin-1-yl)ethyl]-10H-indolo[3,2-

b]quinoline-11-carboxamide (3g)} (IC₅₀=1.3 mm) as the most

promising compound exhibiting good selectivity indices

against mammalian cell lines. The kill kinetics on erythrocytic-

stage parasites revealed that 3g caused complete killing of

only the trophozoite-stage parasites. Mechanistic studies

showed that 3g targets the food vacuole of the parasite and

inhibits hemoglobin uptake, b-hematin formation, and the

basic endocytic processes of the parasite. Analogue 3g was

found to be orally bioavailable, and its curative antimalarial

studies at 50 mg per kg p.o. against a Plasmodium berghei

(ANKA)-infected mouse model revealed that mice treated with

3g showed 27–35% suppression of parasitemia with an in-

crease in life span relative to untreated, control mice. Thus, the

present work demonstrated a proof of concept for the oral effi-

cacy of indolo[3,2-b]quinoline-C11-carboxamides.

Introduction

Malaria is a parasitic disease transmitted to humans by the

bites of infected female anopheles mosquito. Five species of

Plasmodium (P. falciparum, P. vivax, P. ovale, P. malariae, and

P. knowlesi) are known to cause malaria; P. falciparum and

P. vivax are the most common, but P. falciparum has a major

impact, as it is responsible for the largest number of malarial

deaths in humans. In 2016, approximately 216 million cases

and 445000 malaria associated deaths were reported around

the world, mostly among African children below 5 years of

age.^[1]Several medications for the treatment of malaria are

known, including 4-aminoquinolines [e.g., chloroquine (CQ)],

artemisinin derivatives (e.g., arteether), tetracyclines, and also

several combination therapies. Over the last several decades,

front-line antimalarial drugs, that is, chloroquine, antifolates,

and artemisinin, have been losing their efficacy owing to the

emergence of resistance.^[2]To overcome parasitic resistance,

there is a need to find more effective new antimalarial drugs.

Currently, several research groups are working toward the dis-

covery of more efficient antimalarial drugs, and several clinical

studies are in progress.^[3]Nature has been valuable in provid-

ing drugs against malaria (e.g., quinine and artemisinin are sec-

ondary metabolites of plants) and continues to be the best re-

pository for druggable lead compounds against several diseas-

es, including malaria.^[4]

Since ancient times, plants and plant-derived natural prod-

ucts have been used as remedies for various illnesses. Alkaloids

have contributed significantly to antimalarial drug discovery,

and currently, a large number of alkaloids are in clinic or in

clinical development.^[5]Indoloquinoline alkaloids are naturally

occurring fused cyclic compounds that can be isolated from

the West African climbing shrub Cryptolepis sanguinolenta.^[6]

The roots of this plant are used as a folk medicine in central

and West Africa as an antirheumatic and spasmolytic and for

the treatment of malaria, bacterial infection, hepatitis, and so

on.^[7]This plant was first studied in 1929,^[8]at which point the

major chemical constituent, cryptolepine (1), was isolated. In

1951, Gellꢀrt et al.^[9]assigned the exact chemical structure of

[a] Dr. R. Mudududdla,⁺Dr. S. S. Bharate, Dr. R. A. Vishwakarma,

Dr. S. B. Bharate

CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001

(India)

E-mail: sbharate@iiim.ac.in

Homepage: www.iiim.res.in

[b] Dr. D. Mohanakrishnan,⁺Dr. D. Sahal

Malaria Drug Discovery Laboratory, International Centre for Genetic Engi-

neering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067

(India)

E-mail: dinkar@icgeb.res.in

[⁺] These authors contributed equally to this work.

cryptolepine as

a linearly fused 5-methyl-10H-indolo[3,2-

b]quinoline.

Cryptolepine (1) is reported to possess antimalarial activities

against both CQ-sensitive and CQ-resistant strains^[10]and is

also endowed with anticancer,^[11]anti-hyperglycemic,^[12]anti-

bacterial/antifungal,^[13]and anti-infective activities.^[14]The cyto-

toxic properties of cryptolepine are due to DNA intercalation

by binding of the N5 quaternary methyl group at cytosine–cy-

Supporting Information (NMR spectra of all compounds) and the ORCID

identification number(s) for the author(s) of this article can be found

under:

https://doi.org/10.1002/cmdc.201800579.

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tosine sites, which causes inhibition of DNA synthesis.^[11,15]

However, the high cytotoxicity and low selectivity of this alka-

loid have prevented it from clinical development for the treat-

ment of malaria.^[16]Several studies^[17]have shown that the in-

corporation of basic amino functionalities at the C11 position

(e.g., structure 2, Figure 1) in the scaffold of cryptolepine en-

line under reflux conditions produced compound 10. Com-

pound 10 was cyclized by using polyphosphoric acid (PPA) at

1408C to give indolo[3,2-b]quinolin-11-one 11. The POCl₃-medi-

ated chlorination of 11 gave 11-chloro-10H-indolo[3,2-b]quino-

line (12). Hydrogenation of 12 was performed by using H₂/Pd

in AcOH and NaOAc at 60 psi to give 10H-indolo[3,2-b]quino-

line (13). The final step of N-methylation was done by treating

13 with methyl iodide in MeCN under reflux to yield cryptole-

pine iodide (1) as an orange-yellow solid.

For the synthesis of the C11 cryptolepine carboxamide series

of compounds, we proposed two major modifications to the

scaffold of cryptolepine (1). First, with the aim to enhance anti-

plasmodial potency, we planned to incorporate various carbox-

amide-linked aminoalkyl side chains at the C11 position of

quindoline. In a second modification aimed at reducing the

toxicity, we planned to remove the quaternary methyl group

of cryptolepine. Key C11 COOH intermediates 18a–d were pre-

pared starting from anthranilic acid (8) in four steps, as depict-

ed in Figure 2B. The reaction of anthranilic acid (8) with

chloroacetic acid in basic medium yielded 2-[(carboxymethyl)a-

mino]benzoic acid (14), which upon N-acetylation by using

Ac₂O produced 2-[N-(carboxymethyl)acetamido]benzoic acid

(15). Cyclization of 15 was done by acetic anhydride and trie-

thylamine to give 1-acetyl-1H-indol-3-yl acetate (16). Next,

base-mediated cyclization of acetate 16 with isatins 17a–d

produced substituted indolo[3,2-b]quinoline-C11-carboxylic

acids 18a–d. Treatment of C11 carboxylic acid derivatives

18a–d with aliphatic three-to-four carbon-chain amines 19 in

the presence of 3-[bis(dimethylamino)methyliumyl]-3H-benzo-

triazol-1-oxide hexafluorophosphate (HBTU)/diisopropylethyla-

mine (DIEA) as a peptide coupling agent produced compounds

3a–n. Suzuki coupling of 2-bromo compound 3a with arylbor-

onic acids 20a–c by using [1,1’-bis(diphenylphosphino)ferroce-

ne]dichloropalladium(II) [Pd(dppf)Cl₂] produced 2-aryl-C11-car-

boxamide derivatives 4a–c. Similarly, treatment of C11 carbox-

ylic acid intermediates 18a–d with secondary amines 21 pro-

duced carboxamides 5a–h. Furthermore, treatment of 18a–d

with benzylamines 22a–c and anilines 23a–c produced car-

boxamides 6a–c and 7a–c, respectively. All newly synthesized

derivatives were fully characterized by ¹H NMR and ¹³C NMR

spectroscopy in addition to MS, and their purity was checked

by HPLC and LC-HRMS (ESI) analysis.

Figure 1. Structures of cryptolepine (1), C11-substituted analogue 2, and C11

carboxamides 3–7.

hances the antimalarial potency. Cryptolepine shows antiplas-

modial activity against the P. falciparum W2 strain with an IC₅₀

value of 755 nm and cytotoxicity in the HUVEC cell line with a

CC₅₀value of 1180 nm. The incorporation of a 4-piperidinamino

group at C11 (in the cryptolepine structure) results in a 17-fold

improvement in antiplasmodial activity (compound 2, PfW2:

IC₅₀=44 nm) and reduced cytotoxicity in HUVEC cells (CC₅₀=

2042 nm). Cryptolepine (1) and compound 2 at 50 mg/kg/day

(p.o.) suppress parasitemia in mice by 43 and 55%.^[18]The

effect of C11 carboxamide-linked aminoalkyl chains has never

been investigated. Furthermore, the cytotoxicity of this class of

compounds is primarily associated with their planar structures

and quaternary nitrogen atoms; therefore, considering these

structural aspects, herein we designed a new series of indolo-

quinolines (i.e., compounds 3–7) comprising a saturated ami-

noalkyl linker at the C11 position to maintain (or to improve)

antimalarial activity and a non-quaternary quinoline nitrogen

atom. The synthesized compounds were tested for their anti-

plasmodial activity against CQ-sensitive and CQ-resistant

strains of P. falciparum.

The preclinical parameters (including aqueous solubility, cy-

tochrome P450 enzyme inhibition, caco-2 permeability) of the

best-identified compound were determined, which was fol-

lowed by oral pharmacokinetics and in vivo antimalarial effica-

cy. The detailed studies performed to understand the mecha-

nism of antimalarial action of fluorescent lead compound 3g

revealed that it targets the food vacuole, at which it inhibits

the uptake of hemoglobin and inhibits the detoxification of

heme to hemozoin.

In vitro antiplasmodial activity

Synthesized compounds 3a–n, 4a–c, 5a–h, 6a–c, and 7a–c

were all screened for their in vitro antiplasmodial activity

against CQ-sensitive (3D7) and CQ-resistant (Dd2 and Indo)

strains of P. falciparum. To assess the ability of these com-

pounds to kill Plasmodium parasites selectively, the cytotoxicity

of all compounds was checked in two mammalian cell lines:

HUH-7 and HEK-293T (Table 1). Compounds 3a–n from the first

series bearing a C2 halo group and a C11 aliphatic amino car-

boxamide moiety showed promising antiplasmodial activity

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

lethyl carboxamide analogue 3g displayed antiplasmodial ac-

tivity against P. falciparum 3D7, Dd2, and Indo strains with IC₅₀

Results and Discussion

Chemistry

Cryptolepine (1) was synthesized starting from commercially

available anthranilic acid (8), as depicted in Figure 2A.^[12,17a]Re-

action of anthranilic acid (8) with bromoacetyl bromide gave

N-bromoacetyl anthranilic acid (9), which on coupling with ani-

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Figure 2. A) Synthesis of cryptolepine (1): a) bromoacetyl bromide (1.2 equiv), 1,4-dioxane/DMF (1:1), 18 h, 82%; b) aniline (1.2 equiv), DMF, 1308C, 85%;

c) PPA (excess), 1308C, 2 h, 67%; d) POCl₃(excess), 2 h, 60%; e) H₂/10% Pd/C, AcOH, NaOAc, 60 psi, 2 h, 80%; f)CH₃I, DMF, 808C, 12 h, 70%. B)Synthesis of

indolo[3,2-b]quinoline-11-carboxamides 3–7: a)chloroacetic acid (1.1 equiv), Na₂CO₃(5.0 equiv), H₂O, 18 h, 82%; b) Ac₂O (1.2 equiv), Na₂CO₃(1.0 equiv), DMF,

80–85%; c) Ac₂O (10.0 equiv), Et₃N (3.0 equiv), 1008C, 2 h, 80%; d) KOH (2.0 equiv), H₂O, 6 h, reflux, 70–80%; e) HBTU (1.2 equiv), DIEA (3 equiv), DMF, 6 h, 40–

60%; f) Pd(dppf)Cl₂(0.2 equiv), 1,4-dioxane/H₂O, 808C, 12 h, 60–80%.

values of 1.3, 6.3, and 5 mm and with resistance indices of 4.8

(Dd2/3D7) and 3.8 (INDO/3D7). Also, derivative 3g showed

good selectivity indices of 47 and 33 against HUH-7 and HEK-

293T, respectively. The replacement of the C2 halo substituent

with an aryl moiety resulted in a significant improvement in

antiplasmodial activity (compound 3a vs. 4a–c) and resistance

index. It was observed that the piperidine as a terminal ring in

the carboxamide linker was favored over morpholine as a ter-

minal ring (3g vs. 3d). Furthermore, opening of the piperidine

ring to N-diethylamine (3g vs. 3i) resulted in a 13-fold de-

crease in antiplasmodial activity. A halogen substitution on the

quinoline ring led to an improvement in the antiplasmodial ac-

tivity. The third series of compounds bearing cyclic aminoalkyl

carboxamide groups, that is, 5a–h, were weakly active against

Plasmodium, and the best analogue showed an IC₅₀value of

28 mm. The fourth series of compounds bearing benzylamino

carboxamide groups, that is, 6a–c, were found to be inactive

with the lowest IC₅₀value of 87 mm. The final series bearing

anilino carboxamide groups, that is, 7a–c, showed promising

antiplasmodial activity against all three Plasmodium falciparum

strains. In this series, para-bromoanilino carboxamide 7b dis-

played antiplasmodial activity against the 3D7, Dd2, and Indo

strains with IC₅₀values of 2.1, 3.5, and 2 mm, respectively,

which were indicative of excellent resistance indices. Further-

more, 7b showed good selectivity indices of 41 (HUH-7) and

>48 (HEK-293T). On the basis of the in vitro antiplasmodial ac-

tivity and selectivity indices data presented in Table 1, com-

pound 3g was selected for further investigation.

Stage-specific kill kinetics of 3g against erythrocytic stages

of P. falciparum

To study the mechanistic properties of drug action, the in vitro

stage-specific kill kinetics of 3g were studied against the eryth-

rocytic stages of P. falciparum. The synchronized Pf3D7 culture

was treated with 3g (IC₉₀=3.5 mm) for different time points

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Table 1. In vitro antiplasmodial activity, cytotoxicity against mammalian cells, and b-hematin inhibition potencies of C2-halo-C11 aliphatic aminoalkyl car-

boxamides 3a–n, C2-aryl C11 aliphatic aminoalkyl carboxamides 4a–c, C11 cyclic aminoalkyl carboxamides 5a–h, C11 benzylamino carboxamides 6a–c,

and C11 anilino carboxamides 7a–c.^[a]

Entry

Compound

R₂

P. falciparum IC₅₀[mm]

PfDd2 PfIndo

Resistance index

Selectivity index

b-hematin

inhib.

IC₅₀[mm]

R₁

Pf3D7

PfDd2/

PfIndo/

Pf3D7

HUH7/

Pf3D7

HEK293T/

Pf3D7

1

–

0.61ꢀ0.07

9ꢀ1.1

0.22ꢀ0.03

0.26ꢀ0.04 0.36

0.43

1.1

68.9

5.8

52.5

>1000

3a

Br

22.8ꢀ3.0

9.8ꢀ1.3

2.5

>11.1

nd

3b

3c

3d

3e

3 f

F

>100

nd

2.1

nd

740ꢀ103

>100

nd

1000ꢀ110

660ꢀ86

530ꢀ58

395ꢀ55

840ꢀ76

Br

H

Cl

nd

4ꢀ0.5

9.4ꢀ1.1

8.2ꢀ1.1

2.4

>25

3g

3h

Br

1.3ꢀ0.2

36ꢀ4.7

6.3ꢀ0.8

68ꢀ9.5

5ꢀ0.7

4.8

1.9

3.8

0.7

46.9

33.0

25ꢀ3.5

>2.8

>1000

3i

Br

17.5ꢀ2.5

100ꢀ11.0

83ꢀ12.5

10.4ꢀ1.1

5.7

4.7

1.4

nd

3j

3k

3l

Cl

9ꢀ1.1

17ꢀ2.1

81ꢀ9.7

>25

16ꢀ2.1

nd

1.2

nd

3.3

2.4

8.6

1.9

1000ꢀ120

nd

<12.5

0.94

nd

960ꢀ140

3m

Br

F

43ꢀ5.6

nd

700ꢀ98

3n

4a

4b

42ꢀ5.9

2.7ꢀ0.3

4.7ꢀ0.6

30ꢀ3.6

10.8ꢀ1.1

8.2ꢀ1.2

28ꢀ3.4

0.7

2.9

0.7

1.4

0.3

>37

12.3

>1000

p-F

7.8ꢀ1.0

3.4ꢀ0.4

2.3ꢀ0.3

4

>37

930ꢀ74

580ꢀ41

p-

NHAc

1.7

18.3

8.3

4c

5a

p-Ac

6.5ꢀ0.7

33ꢀ4.6

4.7ꢀ0.8

17ꢀ2.3

0.7

0.4

nd

>15.4

>3

>1000

Cl

<12.5

0.51

>3

500ꢀ60

5b

5c

Cl

Br

30ꢀ3.9

36ꢀ4.3

36ꢀ4.7

34ꢀ4.1

30ꢀ4.2

36ꢀ4.3

1.2

1

>3.3

>2.8

>3.3

>2.8

690ꢀ97

540ꢀ43

0.94

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Table 1. (Continued)

Entry

Compound

P. falciparum IC₅₀[mm]

Resistance index

Selectivity index

b-hematin

inhib.

R₁

R₂

Pf3D7

PfDd2

PfIndo

PfDd2/

Pf3D7

PfIndo/

Pf3D7

HUH7/

Pf3D7

HEK293T/

Pf3D7

IC₅₀[mm]

5d

5e

F

100ꢀ14.0

nd

860ꢀ60

H

>100

nd

740ꢀ81

5 f

H

>100

nd

660ꢀ92

5g

5h

Cl

45ꢀ5.0

28ꢀ3.9

28ꢀ3.6

27ꢀ3.0

nd

0.6

0.8

0.6

>2.2

>3.5

670ꢀ40

930ꢀ74

28ꢀ3.4

>100

87ꢀ12.2

>100

23ꢀ2.8

nd

38ꢀ4.9

3.5ꢀ0.4

5ꢀ0.6

0.168ꢀ0.01

0.97

>3.5

6a

6b

6c

7a

7b

7c

Br

p-OCF₃

p-CF₃

m-CF₃

p-OCF₃

p-Br

nd

3.7

1.7

0.5

nd

3.7

1

nd

9.2

41

5

nd

>9.7

>48

2.5

820ꢀ33

745ꢀ60

815ꢀ49

nd

10.3ꢀ0.9

2.1ꢀ0.3

38ꢀ5.3

2ꢀ0.4

5ꢀ0.6

>1000

p-F

10ꢀ1.2

0.5

11

CQ–diphosphate

0.04ꢀ0.003

0.44ꢀ0.05 4.2

>200

1120ꢀ134

[a] nd: not determined; resistance index=IC₅₀(resistant strain)/IC₅₀(sensitive strain); selectivity index=CC₅₀(cytotoxicity)/IC₅₀(antimalarial activity); IC₅₀col-

umns show meanꢀstandard deviation of two independent experiments taking three replicates for each experiment.

(rings: 12, 24, 36, and 48 h; trophozoites: 3, 6, 18, and 24 h;

schizonts: 3, 6, and 12 h). After drug exposure, the drug pres-

sure was withdrawn by centrifugal removal of conditioned

medium followed by media wash. Further, the washed para-

sites were allowed to grow in drug-free media under normal

culture conditions for a total period of 96 (rings), 72 (tropho-

zoites), and 60 h (schizonts). After incubation, the growth of

the parasites was measured by microscopic evaluation of

Giemsa-stained smears (Figure 3). The results suggested that

cryptolepine analogue 3g actively inhibited the trophozoite-

stage parasites (Figure 3B). Further, removal of 3g by aspira-

tion of conditioned medium followed by maintenance in drug-

free medium failed to revive the parasites. This cytocidal action

of 3g was observed as early as 3 h. The action of 3g on ring-

stage parasite was relatively slow, and it failed to show cytoci-

dal drug action until an exposure time of 48 h (Figure 3A).

However, at earlier time points (12–36 h), rings treated with 3g

showed a significant (53–99%) decrease in parasitemia relative

to the control. Although rings treated with 3g matured into

trophozoites, these trophozoites failed to progress further in

the presence of the drug. However, the removal of 3g from

the ring-stage parasites treated with 3g for 12–36 h allowed

some of the parasites to revive from drug-caused stress, which

was evident from increased parasitemia (1.5 to 12%) of the

drug-treated culture compared with initial parasitemia (1%)

(Figure 3A). These results suggested possible cytostatic action

(but not cytocidal action) of short-term exposure of 3g on

ring-stage parasite. Treatment of schizonts with 3g for 3 h fol-

lowed by drug withdrawal resulted in a pronounced decrease

in parasitemia, which suggested cytocidal action of 3g against

schizonts. However, the killing was not complete, and approxi-

mately 3% parasitemia was observed at all time points of

treatment. The relative percent parasitemia due to rings, troph-

ozoites, and schizonts revealed an interesting phenomenon: a

time-dependent effect with a greater number of schizonts in

the 12 h treated parasites than in the 3 and 6 h treated para-

sites (Figure 3C).

Kinetics of 3g uptake by malaria parasite

The intrinsic fluorescence of 3g was used to study the uptake

kinetics of 3g by malaria parasite. As the kill kinetic studies in-

dicated the trophozoites to be the most sensitive to the action

of 3g, the trophozoite-stage-enriched Pf3D7 culture was incu-

bated with 3g (50 mm) at 378C for different time points. The

washed cultures were observed under fluorescence (DAPI

filter) and optical microscopy (Figure 4). At 15 min, diffuse,

low-intensity fluorescence was observed in the entire cytosolic

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Figure 3. Stage-specific kill kinetics of 3g against erythrocytic stages of Pf3D7. Synchronized ring (at 12, 24, 26, and 48 h), trophozoite (at 3, 6, 18, and 24 h),

and schizont (at 3, 6, and 12 h) stage parasites were treated (D⁺) with the IC₉₀value of 3g (3.5 mm). In each case, 3g was removed, and the culture was incu-

bated in drug-free (D^ꢁ) medium for a total period (D⁺+D^ꢁ) of 96 (ring), 72 (trophozoite), and 60 h (schizont). The effect of 3g treatment was evaluated by

Giemsa-stained blood smears under microscope. D⁺represents drug pressure period and D^ꢁrepresents drug-free period. The total parasitemia of each time

point was calculated by analyzing microscopic images and was plotted in a bar diagram. Data shown in the histograms (panels A–C) represent meanꢀstan-

dard deviation of three replicates.

region of infected red blood cells (RBCs). At 30 min, the fluo-

rescence was found to be significantly concentrated and asso-

ciated with the parasite. Continuous incubation of 3g with

parasite culture showed further reorganization of the stained

parasite at 1 h. However, at 2 h, a dramatic increase in fluores-

cence intensity emanating from what appeared to be a dense-

ly stained entity was observed. The size of the fluorescent spot

and its association with hemozoin suggested that 3g may

have targeted the food vacuole. Further, it was observed that

between 3 and 6 h, the fluorescence intensity from the highly

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Figure 4. Kinetics of 3g uptake by malaria parasite. Synchronized trophozoite culture (ꢂ4% parasitemia and 2% hematocrit, 200 mL) was incubated with 3g

(50 mm) at 378C for the different time points indicated (15 min to 6 h). After each incubation, the culture was washed with 1ꢂ PBS and was suspended again

in PBS before observing the wet mount of each sample under fluorescence and optical microscopy. Drug accumulation was observed in close proximity to

hemozoin crystals (seen as brown pigmented bodies in optical sections).

dense staining seen at 2 h began to spread out to a much

larger area. In this context, it is worth noting that 3g-mediated

complete inhibition of trophozoite was also observed at 3 h

(Figure 4). The temporal congruence of the staining character-

istics and killing of the parasite suggested that kinetics and

trafficking of 3g were crucial to our mechanistic understanding

of the action of 3g.

cleus but to the food vacuole, at which it seemed to interact

with hemozoin (Figure 5B).

It is well known that quinoline-based drugs actively inhibit

erythrocytic-stage parasites at the trophozoite stage, which is

the stage of maximum growth associated with extensive pro-

teolysis of hemoglobin and the resulting accumulation of a

high concentration of heme.^[21]In molecular terms, quinolines

are known to interact with free heme, preventing its detoxifi-

cation through the formation of hemozoin and resulting in

death of malaria parasite.^[21]Our microscopic studies also indi-

cated that 3g was more active against the trophozoite stage,

which indicated possible targets for 3g in the trophozoite

stage. Further, colocalization of 3g with LysoTracker proved its

accumulation in the parasite food vacuole. Compound 3g

could possibly target one or more of the following four bio-

chemical pathways operational in the food vacuole: 1) hemo-

globin uptake pathway; 2) hemoglobin digestion pathway;

3) heme detoxification pathway; 4) amino acid transport path-

way. To find the target of 3g, we checked the activity of 3g

against the b-hematin formation and hemoglobin uptake path-

ways.

Colocalization studies of 3g fluorescence with EtBr and

LysoTracker

Though cryptolepines are well known for their ability to inter-

calate with DNA;^[11]our observations suggested that 3g may

have targeted the food vacuole. To find the intracellular site of

accumulation of 3g in malaria parasite, we decided to study

the colocalization of 3g with both DNA and food vacuole

markers. We used EtBr, a well-known DNA-binding stain that

emits red fluorescence,^[19]and LysoTracker red, a fluorophore

that accumulates in acidic cell organelles such as food va-

cuoles^[20]for our colocalization studies. After 3 h incubation of

parasites with 3g (50 mm), the washed cells were incubated

with EtBr stain and LysoTracker red in separate experiments for

30 min at 378C, and the washed cells were observed under

fluorescence and optical microscopy (Figure 5A,B). The ring

and schizont stages failed to show colocalization of EtBr and

3g (Figure 5A). The fluorescence of 3g appeared as a blue

crown near red EtBr-stained genomic DNA eclipsing the nu-

cleus only partially. The colocalization of the dispersed hemo-

zoin with 3g suggested that it may have affinity for hemozoin.

Had it gained entry into the nucleus, it ought to have given

uniform stain with complete colocalization of the red and blue

stains. On the other hand, complete colocalization of the blue

fluorescence of 3g with the red fluorescence of LysoTracker

(Figure 5B) confirmed the accumulation of 3g in the food va-

cuole, in which the digestion of host hemoglobin takes place.

These results suggested that 3g was targeted not to the nu-

Inhibition of detergent-mediated b-hematin formation by

3g

The b-hematin formation method described by Sandlin et al.^[22]

was used with minor modifications. The b-hematin formation

inhibitory potency of all the compounds studied by us is

shown in Tables 1. In this assay, 3g with an IC₅₀value of

840 mm was found to show greater potency than CQ (IC₅₀=

1120 mm). This b-hematin inhibitory activity together with the

antiplasmodial activity of 3g suggested that, like CQ, 3g accu-

mulated in the food vacuole (Figures 4 and 5), in which it may

have complexed with free heme to prevent its detoxification

through the formation of hemozoin.^[22]

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Figure 5. Colocalization of 3g fluorescence with EtBr and LysoTracker (LT). The parasites were incubated with 3g (50 mm) for 3 h. After incubation, the

washed cultures were further incubated individually with EtBr and LysoTracker for 30 min. Then, the washed culture was observed under fluorescence micros-

copy as wet mount. A) EtBr and 3g failed to show colocalization. B) The colocalization of LysoTracker and 3g suggests the accumulation of 3g in the food va-

cuole.

Indeed, similar to quinoline antimalarial drugs, the indolo-

quinoline “cryptolepine” has also been reported to form a ferri-

protoporphyrin IX : cryptolepine complex {[FP:C]⁺m/z=848}

with FP.^[23]Therefore, we investigated whether non-quaternary

cryptolepine derivative 3g also formed a complex with heme.

LC–MS analysis of a mixture containing 40 mm heme and

100 mm cryptolepine/3g in MeOH/H₂O (1:1, v/v; pH 7) indicat-

ed the formation of the respective monomer complexes

{[FP:C]⁺m/z=848 and [FP:3g]⁺m/z=1066} (Section S2, Sup-

porting Information), which indicated the formation of the re-

spective complexes with heme.

low permeability of uninfected and ring-stage-infected RBCs

versus the high permeability of trophozoites and schizonts

allows selective lysis of the latter, which allows quantitative es-

timation of the released hemoglobin by recording the absorb-

ance at l=540 nm (A₅₄₀).^[25b]In the sorbitol-based assay, a mol-

ecule that inhibits hemoglobin uptake is expected to show a

higher A₅₄₀value in the treated sample than in the untreated

control. By comparing the A₅₄₀values by following the release

of hemoglobin from an equal number of trophozoite-stage-in-

fected RBCs at 0 and 15 h, we first calculated the A₅₄₀value

representing the amount of hemoglobin taken up by untreat-

ed parasite (Figure 6A). To measure the effect of 3g on hemo-

globin uptake, synchronized trophozoite stage (28 h post-inva-

sion) P. falciparum 3D7 cultures (4% parasitemia, 2% hematoc-

rit) were treated with the IC₉₀(3.5 mm) of 3g for 15 h. The rela-

tive hemoglobin contents of the treated and untreated parasi-

tized red blood cells (PRBCs) taken at identical numbers were

determined in terms of A₅₄₀by following sorbitol-mediated he-

molysis. As expected, sorbitol induced lysis from 15 h, and un-

treated culture showed less hemoglobin (A₅₄₀ꢂ0.28) than the

0 h untreated parasite culture (A₅₄₀ꢂ0.6) (Figure 6A). This de-

crease in hemoglobin amount to about half was due to vora-

cious consumption of hemoglobin in the trophozoite stage. In

contrast, we found a higher amount of hemoglobin (A₅₄₀

ꢂ0.45) in PRBCs treated with 3g for 15 h as compared with

untreated PRBCs (A₅₄₀ꢂ0.28). This higher amount of hemoglo-

bin release from 3g-treated PRBCs suggested that 3g inhibited

the uptake of hemoglobin by parasite. Microscopic observa-

tions of untreated and 3g-treated cultures at 0 and 15 h con-

firmed that the 3g-treated parasites were inhibited at the

trophozoite stage, whereas the untreated trophozoite stage

Although it is well known that cryptolepines may inhibit the

growth of malaria parasite by inhibiting the formation of he-

mozoin, it is also true that derivatives of cryptolepines may

have additional targets besides heme detoxification.^[10c]Hence,

we examined if 3g could also inhibit the endocytosis of hemo-

globin by the food vacuole.

Inhibition of hemoglobin uptake by 3g

Once we had confirmed the uptake and accumulation of 3g in

the food vacuole, we wanted to explore if 3g could also inhibit

the uptake of host hemoglobin and starve the parasite to

death. Hence, we resorted to sorbitol-induced selective hemol-

ysis^[24]to study the uptake of hemoglobin by the parasites and

the inhibition of the same by 3g. The principle of this method

was based on the fact that the malaria parasite induces new

permeation pathways (NPP) in the host red blood cell mem-

brane that allow the entry of electroneutral and cationic sol-

utes.^[25]These NPPs allow impermeable sorbitol into infected

RBCs, which causes osmotic cell swelling and hemolysis.^[26]The

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transited to the schizont stage at 15 h (Figure 5A). Our data

suggests that the retarded growth of 3g-treated parasite is

likely due to reduced endocytosis of hemoglobin.

FACS analysis of inhibition of FITC–dextran endocytosis by

malaria parasite

Invasion of merozoites into fluorescein isothiocyanate (FITC)–

dextran-loaded resealed RBCs enabled us to assess the inhibi-

tion of the endocytic machinery of 3g-treated malaria parasite

by using fluorescence-activated cell sorting (FACS). In such re-

sealed RBCs, the parasite was expected to endocytose both he-

moglobin and the fluorescent FITC–dextran and transfer them

both to the food vacuole. The high-intensity fluorescence

signal emitted by FITC–dextran can be detected by FACS and,

thus, provides a quantitative estimation of both the intensity

of the signal and the number of cells carrying the signal. The

18 h (late ring) and 24 h (early trophozoites) cultures were

treated with the IC₉₀value of 3g for 6 h, and then the treated

and untreated parasites were isolated from the host RBCs by

using saponin hemolysis. These two time points corresponded

to phases of the parasite life cycle at which the parasite dev-

oured less (at 18 h) and more (at 24 h) hemoglobin. The isolat-

ed parasites were further analyzed by FACS for their uptake of

FITC–dextran (Figure 6B–E). As shown in Figure 6B, the un-

treated controls of 18–30 h parasites showed a gradual in-

crease in the fluorescence intensity as a result of the continu-

ous uptake of FITC–dextran from resealed RBCs. In contrast, as

shown in Figure 6C, the sample treated with 3g from 18 to

24 h and analyzed by FACS at 24 h had 1.7-fold fewer fluores-

cent cells than the 24 h untreated sample, which suggested in-

hibition of endocytosis by the parasite at the late ring stage.

Figure 6D shows that in an analogous experiment done on a

24 h early trophozoite stage culture, the number of fluorescent

cells in the 30 h untreated control was twofold higher than

that in the 24 h untreated sample, which suggested continued

endocytosis during the 24–30 h time window. However, rela-

tive to the number of fluorescent cells for the 30 h control, the

number of fluorescent cells for the sample treated with 3g for

the 24–30 h duration was approximately twofold lower, which

suggested that inhibition of endocytosis by 3g continued until

30 h. Figure 6E displays the analysis of the FACS data at a fluo-

rescence intensity of 2ꢂ10³for 3g-treated samples and un-

treated controls and demonstrates the inhibitory effect of 3g

on the endocytosis process. The fact that angle b (24 h) is ap-

proximately twofold larger than angle a (18 h) indicates greater

inhibition of endocytosis at 24–30 h than at 18–24 h.

Figure 6. A) Inhibition of hemoglobin uptake by 3g. The effect of hemoglo-

bin uptake in the presence of 3g was studied by the sorbitol-induced he-

molysis method by using a spectrophotometer. Hemoglobin released was

measured by absorbance at l=540 nm. The significantly higher amount of

hemoglobin in the sample treated with 3g for 15 h as compared with the

corresponding untreated (UT) 15 h sample suggests inhibition of hemoglo-

bin uptake by the malaria parasite. Data shown in the histograms represent

meanꢀstandard deviation of three independently done experiments taking

two replicates for each experiment. B–E) FACS analysis of inhibition of FITC–

dextran endocytosis by malaria parasite. The 18 and 24 h trophozoite-stage

cultures, growing in FITC–dextran-resealed RBCs, were incubated without

and with 3g (at the IC₉₀value) for 6 h periods between a) 18–24 h and

b) 24–30 h, respectively, following which the parasites were isolated by sap-

onin hemolysis. Untreated control parasites at a) 18 h of growth and b) 24 h

of growth as well as test parasites treated with 3g for c) 18–24 h and d) 24–

30 h were analyzed for the FITC signal by FACS taking 2000 parasites in each

case. B) This panel shows a progressive increase in the intensity of FITC–dex-

tran in 18, 24, and 30 h parasite cultures. C) This panel shows data for 18–

24 h 3g-treated sample in comparison with the 18 and 24 h untreated con-

trols. D) This panel shows data for 24–30 h 3g-treated sample in comparison

with 24 and 30 h untreated controls. A decreased fluorescence signal in the

treated samples is observed at both of these time points. E) This panel rep-

resents the cell counts at 2ꢂ10³signal intensity for the three controls (18,

24, and 30 h) and the two 3g-treated (18–24 and 24–30 h) samples. Relative

to untreated control (blue line), there is decreased uptake of FITC–dextran

in the 3g-treated sample at time points 24 h (red line) and 30 h (green line).

The control shows a linear rise in uptake signal, whereas both the treated

samples show significantly diminished uptake signals. The fact that angle b

is approximately twofold larger than angle a indicates that inhibition of en-

docytosis is greater at 24–30 h than at 18–24 h.

Physicochemical properties, CYP P450 inhibition, Caco-2

permeability, and pharmacokinetics of 3g

The solubility of 3g was determined in water, phosphate-buf-

fered saline (PBS), simulated gastric fluid (SGF), and simulated

intestinal fluid (SIF). Compound 3g displayed low solubility (<

10 mgmL^ꢁ1) in water, PBS and SIF, whereas it showed high solu-

bility in SGF (400 mgmL^ꢁ1). Further, 3g was tested for inhibition

of five major CYP450 isoenzymes, namely, CYP3A4, CYP2D6,

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CYP2C9, CYP2C19, and CYP1A2, at 10 mm, and 47, 91, 26, 12,

and 56% inhibition, respectively, was observed. The results in-

dicated that compound 3g showed moderate or very low in-

hibition of the CYP3A4, CYP2C9, and CYP2C19 enzymes. How-

ever, it showed higher inhibition of CYP2D6. Further, the Caco-

2 permeability of 3g was also investigated. This assay was

done in a bidirectional (i.e., A2B and B2A) mode. The A2B ap-

parent permeability (P_app

) )

value of 3g (6.0ꢂ10^ꢁ6cms^ꢁ1

showed that it had high permeability through the membrane.

The B2A value (11.0ꢂ10^ꢁ6cms^ꢁ1) was approximately twofold

higher than the A2B value (efflux ratio=1.8). As per FDA ac-

ceptance criteria, if the efflux ratio is more than 2, it should be

considered as an efflux substrate. As the efflux ratio is 1.8, 3g

is not a substrate of efflux transporter pump. Hence, these re-

sults indicate that 3g has good solubility in gastric pH with no

CYP3A4 or efflux liability.

Figure 7. In vivo antimalarial efficacy of compound 3g (50 mgkg^ꢁ1body

weight, p.o.) in P. berghei infected BALB/c mice. The lines show mean per-

cent parasitemia and standard deviation for five mice used/group of untreat-

ed (blue) and treated mice (red). Histogram shows the percent parasitemia

suppression across days 5–19. Boxed data show the average survival time

for untreated and treated mice. The p value (0.0118) of survival time calcu-

lated by using the student t-test (GraphPad Prism Software) indicates that

there was a significant effect of 3g on the longevity of treated mice.

To check the plasma exposure of the compound in a mouse

model, the pharmacokinetics of compound 3g were studied in

healthy BALB/c mice following a single 10 mgkg^ꢁ1dose ad-

ministration orally (p.o.) and a 1.0 mgkg^ꢁ1dose administration

intravenously (i.v.). Plasma samples were collected at appropri-

ate time points between the range of 0 to 24 h and were ana-

lyzed by LC–MS/MS. Compound 3g showed good plasma ex-

posure (C_max=585 and 963 ngmL^ꢁ1) by both p.o. and i.v.

for 10 mgkg^ꢁ1oral dose was 585 ngmL^ꢁ1or 1.3 mm, which is

equal to the in vitro anti P. falcipraum IC₅₀value, we believe

that the 50 mgkg^ꢁ1oral dose given in the in vivo mouse ma-

laria experiment could have resulted in a substantially higher

plasma concentration of 3g. However, the inability of 3g to

show complete clearance of malaria parasite may be caused

by different susceptibilities of P. falciparum 3D7 (a laboratory

strain) and P. berghei ANKA (a mouse strain) and the possibility

that the pharmacokinetics of the drugs are different in healthy

and infected mice.

routes. Compound 3g displayed good availability by i.v. (C_max

=

963 ngmL^ꢁ1) as compared with p.o. (C_max=585 ngmL^ꢁ1) in

plasma. However, the increased half-life of orally administered

3g (t_1/2,b=3 h) as compared to i.v.-administered 3g (t_1/2,b

=

0.55 h) suggests that the oral route has its merits. Furthermore,

the AUC_0ꢁ1(area under the curve from t=0 extrapolated to

infinity) of 3g by the oral route was found to be

1243 nghmL^ꢁ1, with oral bioavailability (%F) of 23%. The maxi-

mum concentration of 3g in the plasma was observed at

585 ngmL^ꢁ1(1.29 mm) for 10 mgkg^ꢁ1of p.o. administration,

which is nearly equal to the in vitro antiplasmodial IC₅₀value

(1.3 mm).

Conclusions

In summary, a new series of indolo[3,2-b]quinoline-C11-carbox-

amides were identified as promising orally bioavailable antima-

larial agents. Aminoethylpiperidinylindolo[3,2-b]quinoline-C11-

carboxamide 3g displayed promising antiplasmodial activity

(IC₅₀=1.3 mm) against the CQ-sensitive Pf3D7 strain and also

showed good antiplasmodial activities against the CQ-resistant

Dd2 (IC₅₀=6.3 mm) and Indo (IC₅₀=5.0 mm) strains. Detailed

mechanistic studies revealed that 3g targeted the food va-

cuole of the parasite and inhibited the process of hemoglobin

uptake. Antimalarial compound 3g also showed acceptable

oral pharmacokinetic properties and discernible in vivo antima-

larial activity in P. berghei infected BALB/c mice. However, there

is scope for molecular tweaking to improve its pharmacokinet-

ics in infected animals. In summary, the present work demon-

strated a proof of concept for the oral efficacy of C11 carboxa-

mide non-quaternary cryptolepines in an animal model, open-

ing new directions for future development of this class of com-

pounds as novel antimalarial drugs. The fluorescent antimalari-

al compound 3g could be effectively used as a probe for

deeper understanding of its trafficking and mechanism of anti-

malarial action.

In vivo antimalarial activity of 3g in a P. berghei infected

mouse model of malaria

The curative antimalarial potency of cryptolepine derivative 3g

was evaluated in a P. berghei (ANKA) infected mouse model of

malaria. In view of the good pharmacokinetic properties with

oral administration, compound 3g (50 mgkg^ꢁ1BW) with 2%

(hydroxypropyl)methyl cellulose in a solution of 2% Tween 80

in saline as a vehicle was administered orally to infected mice

for four consecutive days. Microscopic evaluation of the results

indicated that mice treated with 3g showed 27–35% suppres-

sion of parasitemia from day 7 to day 13. The suppression of

parasitemia also resulted in increased life span of the treated

mice (average survival time: 27.8ꢀ5 days) as compared to un-

treated control (average survival time: 20.2ꢀ2.3 days)

(Figure 7). Given that cryptolepines are known for their toxic

properties, increased life span of treated mice suggests that

cryptolepine derivative 3g possesses good antimalarial activity

along with low cytotoxicity. As the pharmacokinetic analysis

showed that the maximum availability (C_max) of 3g in plasma

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7.9, 12 Hz, 1H), 7.20 ppm (dd, J=7.7, 12 Hz, 1H); MS (ESI): m/z=

234.0 [M+H]⁺.

Experimental Section

General

11-Chloro-10H-indolo[3,2-b]quinoline (12, RMS-104): A solution

of 11 (1.5 g, 6.58 mmol) in POCl₃(20 mL) was heated at reflux for

2 h. The mixture was cooled to room temperature and poured into

crushed ice and neutralized with cold KOH solution. The product

was extracted with EtOAc (3ꢂ150 mL), and combined organic layer

was dried with anhydrous Na₂SO₄and concentrated under vacuum

to give 12 (partially pure) as a light-brown solid (0.95 g, 60%): mp:

180–1858C; ¹H NMR (400 MHz, [D₆]DMSO): d=11.82 (s, 1H), 8.28

(d, J=7.7 Hz, 1H), 8.21 (m, 2H), 7.69 (m, 2H), 7.61 (m, 2H),

7.27 ppm (m, 1H); MS (ESI): m/z=253.0 [M+H]⁺.

All chemicals were obtained from Sigma–Aldrich Company and

were used as received. H, ¹³C, and DEPT NMR spectra were record-

ed with Bruker Avance DPX FT-NMR 500 and 400 MHz instruments.

Chemical data for H NMR spectra are reported in parts per million

1

(ppm) downfield from tetramethylsilane and are referenced to the

residual proton in the NMR solvent (CD₃OD). ¹³C NMR spectra were

recorded at 125 or 100 MHz: chemical data for carbon atoms are

reported in parts per million (ppm) downfield from tetramethylsi-

lane and are referenced to the carbon resonance of the solvent

(CD₃OD: d=49.00 ppm). ESI-MS and HRMS spectra were recorded

with Agilent 1100 LC-Q-TOF and HRMS-6540-UHD machines. IR

spectra were recorded with a PerkinElmer IR spectrophotometer.

Melting points were recorded with a digital melting point appara-

tus. All compounds that were tested in biological assays were

>95% pure. All animal experiments performed in the manuscript

were conducted in compliance with institutional guidelines.

10H-Indolo[3,2-b]quinoline (13, RMS-105):

A solution of 12

(200 mg, 1 equiv), sodium acetate (1.0 g, 10 equiv), and 10% Pd/C

in acetic acid (25 mL) was hydrogenated at 60 psi for 2 h by using

a hydrogenation chamber. The mixture was filtered and washed

with a small amount of AcOH, and acetic acid was evaporated and

neutralized with ice-cold saturated NaHCO₃solution. The product

was extracted with EtOAc (3ꢂ25 mL), and the combined organic

layer was dried with anhydrous Na₂SO₄. The solvent was evaporat-

ed under vacuum to give 13 as a yellow solid (180 mg, 80%): mp:

200–2038C; ¹H NMR (400 MHz, [D₆]DMSO): d=11.43 (s, 1H), 8.36

(d, J=7.7 Hz, 1H), 8.29 (s, 1H), 8.20 (d, J=8.5 Hz, 1H), 8.11 (d, J=

8.1 Hz, 1H), 7.61 (m, 4H), 7.29 ppm (dd, J=7.0, 12 Hz, 1H); MS

(ESI): m/z 219.0=[M+H]⁺.^[10c]

Synthesis

2-(2-Bromoacetamido)benzoic acid (9, RMS-101): A solution of

anthranilic acid (8; 5.0 g, 36.4 mmol) in DMF and 1,4-dioxane (1:1,

30 mL) was cooled to 08C, and bromoacetyl bromide (4.0 mL,

45.8 mmol) was added dropwise over a 20 min period. The ice

bath was then removed, and stirring was continued at room tem-

perature for overnight. The mixture was added to water (150 mL),

which resulted in the formation of a light-yellow precipitate. The

precipitate was filtered and washed with an excess amount of

water until neutral pH, and the solid residue was dried under

vacuum to give compound 9 as a white solid (9.1 g, 96%): mp:

Cryptolepine (1): Methyl iodide (3 equiv) was added to a stirred

solution of indolo[3,2-b]quinoline 13 (500 mg, 1 equiv) in DMF

(5 mL), and the mixture was heated at reflux at 1008C for 6–8 h.

The mixture was then cooled to room temperature, and EtOAc

(10 mL) was added, which resulted in the formation of an orange-

red solid. The solid was filtered, washed with an excess amount of

EtOAc, and dried under vacuum to give cryptolepine (1, 70%):

¹H NMR (400 MHz, [D₆]DMSO): d=12.89 (s, 1H), 9.29 (s, 1H), 8.79

(dd, J=8.4, 13.6 Hz, 2H), 8.59 (d, J=8.0 Hz, 1H), 8.16 (t, J=7.6 Hz,

1H), 7.94 (dd, J=7.6, 12.0 Hz, 2H), 7.84 (d, J=8.4 Hz, 1H), 7.51 (t,

J=7.6 Hz, 1H), 5.03 ppm (s, 3H); ¹³C NMR (100 MHz, [D₆]DMSO):

d=145.6, 138.0, 135.2, 133.9, 133.2, 132.4, 129.8, 127.0, 126.2,

1

162–1658C; H NMR (400 MHz, CD₃OD): d=8.46 (d, J=8.0 Hz, 1H),

8.00 (d, J=8.0 Hz, 1H), 7.46 (t, J=8.4 Hz, 1H), 7.07 (t, J=8.4 Hz,

1H), 3.99 ppm (s, 2H); MS (ESI): m/z=256.9 [M+H]⁺.

2-[2-(Phenylamino)acetamido]benzoic acid (10, RMS-102): A solu-

tion of benzoic acid derivative 9 (5.0 g, 19.3 mmol) and aniline

(6.3 mL, 69.4 mmol) in DMF (10 mL) was heated at 1208C for 18 h.

The mixture was then cooled to room temperature and poured

into ice water (250 mL), and the pH was adjusted to 10–11 by

using 5% KOH. Then, the mixture was extracted with dichlorome-

thane (3ꢂ100 mL), and combined dichloromethane layer was kept

aside. The aqueous layer was acidified to pH 3 with a solution of

5% HBr, which resulted in the formation of a white precipitate. The

precipitate was collected, washed with water, and dried to yield 10

126.1, 124.7, 121.3, 117.8, 113.8, 113.1, 54.4 ppm; IR (CHCl₃): n˜_max

=

3742, 3435, 2920, 2850, 1637, 1609, 1577, 1503, 1452, 1384, 1252,

1034 cm^ꢁ1MS (ESI): m/z=233.0 [M]⁺; HRMS: m/z calcd for

;

C₁₆H₁₃N₂⁺: 233.1073; found: 233.1071.^[17b]

2-[(Carboxymethyl)amino]benzoic acid (14, RMS-120): Sodium

carbonate (200 g, 18.8 mmol) was added to a stirred solution of

chloroacetic acid (347 g, 3.67 mol) in water (500 mL) at room tem-

perature. The solution was then heated at 40–508C and was quick-

ly added to a mixture of anthranilic acid (8; 500 g, 3.65 mmol) in

water (340 mL) and 35% aq NaOH solution (320 mL). The resulting

mixture was then heated at 40–458C for 4 days, and the solid mix-

ture was treated with a solution of NaOH (150 g, 3.75 mmol) in

water (4 L). The mixture was heated at 608C and filtered while hot.

The solid residue was washed with 20% aq NaOH until the residue

was dissolved. The combined filtrate was acidified with 37% aq

HCl to pH 3, and the resulting precipitate was then filtered off and

dried at 1008C to yield 14 as a cream-colored solid (567 g, 80%):

1

as a white solid (3.66 g, 70%): mp: 194–1978C; H NMR (400 MHz,

CD₃OD): d=8.60 (d, J=8.0 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.43 (t,

J=8.8 Hz, 1H), 7.01 (t, J=7.6 Hz, 3H), 6.54 (m, 3H), 3.78 ppm (s,

2H); MS (ESI): m/z=270.0 [M+H]⁺.

Indolo[3,2-b]quinolin-11-one (11, RMS-103): A mixture of 10

(3.0 g, 21.5 mmol) and polyphosphoric acid (PPA, 200 g) was

heated at 1308C and stirred by using a mechanical stirrer for 2 h.

The mixture was cooled to room temperature and poured into

crushed ice (250 mL), neutralized with saturated KOH solution, and

then extracted with EtOAc (2ꢂ250 mL). The organic layer was

washed with water and brine. The combined organic layer was

dried with anhydrous Na₂SO₄, and the solvent was evaporated

under reduced pressure to give partially pure 11 as a light-brown

solid (1.75 g, 67%): mp: >3008C; ¹H NMR (400 MHz, [D₆]DMSO):

d=12.41 (s, 1H), 11.64 (s, 1H), 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=7.4, 12 Hz, 1H),

7.51 (d, J=8.4 Hz, 1H), 7.49 (dd, J=7.7, 12.4 Hz, 1H), 7.29 (dd, J=

1

mp: 2208C; H NMR (400 MHz, CD₃OD): d=8.30 (d, J=8.4 Hz, 1H),

7.70 (s, 1H), 7.42 (d, J=7.6 Hz, 1H), 7.28–7.17 (m, 2H), 3.20 ppm (s,

2H); MS (ESI): m/z=195.0 [M+H]⁺.

2-[N-(Carboxymethyl)acetamido]benzoic acid (15, RMS-123):

(Carboxymethylamino)benzoic acid (14; 10.0 g, 0.84 mmol) was

added in small portions to a stirred solution of Na₂CO₃(8.9 g,

0.84 mmol) in water (83.0 mL) at room temperature. Acetic anhy-

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dride (8.56 0.84 mmol) was then added at room temperature with

stirring. The mixture was stirred for 30 min and 37% aq HCl

(14.0 mL) was added dropwise, which resulted in the formation of

a precipitate. The solid product was filtered after 12 h, washed

with water (3ꢂ15 mL), and air dried to give 15 as a light-brown

solid (11.0 g): mp: 208–2108C; H NMR (400 MHz, CD₃OD): d=7.97

(d, J=8.4 Hz, 1H), 7.56 (m, 2H), 7.44 (m, 1H), 3.60 (d, J=8.4,

17.6 Hz, 2H), 1.69 ppm (s, 3H); MS (ESI): m/z=260.0 [M+Na]⁺.

was evaporated under reduced pressure, and the obtained crude

product was purified by silica gel (#100–200) column chromatogra-

phy (5–10% MeOH/CH₂Cl₂) to give C11-carboxamide 3a–n, 5a–h,

6a–c, or 7a–c (40–60%).

2-Bromo-N-(3-morpholinopropyl)-10H-indolo[3,2-b]quinoline-11-

carboxamide (3a, RMS-126): Yellow amorphous solid; mp: 247–

1

2498C; H NMR (400 MHz, CD₃OD): d=8.36 (d, J=7.6 Hz, 1H), 8.24

(d, J=2.0 Hz, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.70 (dd, J=9.2, 2.0 Hz,

1H), 7.57 (t, J=7.6 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.25 (t, J=

7.2 Hz, 1H), 3.60 (m, 4H), 3.20 (m, 4H), 2.64 (m, 4H), 1.93 ppm (m,

2H); ¹³C NMR (100 MHz, [D₆]DMSO): d=164.4, 146.8, 144.6, 141.5,

131.3, 130.5, 129.0, 126.1, 123.8, 121.4, 120.4, 120.0, 119.6, 118.7,

111.9, 66.1, 56.0, 53.3, 37.8, 25.6 ppm; IR (CHCl₃): n˜_max=3436, 2065,

1632, 1465, 1019 cm^ꢁ1; MS (ESI): m/z=468.9 [M+H]⁺; HRMS: m/z

calcd for C₂₃H₂₂BrN₄O₂+H⁺: 467.1077; found: 467.1066; HPLC: t_R=

16.02 min, purity: 95.8%.

1-Acetyl-1H-indol-3-yl acetate (16, RMS-124): 2-[N-(Carboxyme-

thyl)acetamido]benzoic acid (15; 1.36 g, 0.48 mmol) was added to

a stirred solution of Ac₂O (4.6 g, 4.5 mmol) and triethylamine (1.3 g,

1.4 mmol). The mixture was heated at reflux for 20 min and con-

centrated under vacuum to give an oily residue (35 mL), which was

refrigerated overnight. The solid product was filtered off and dried

under vacuum to give product 16 as a light-green solid (80%): mp:

72–748C; ¹H NMR (400 MHz, CDCl₃): d=8.36 (d, J=7.9 Hz, 1H),

7.90 (s, 1H), 7.41 (m, 3H), 2.62 (s, 3H), 2.39 ppm (s, 3H); MS (ESI):

m/z=217.0 [M+H]⁺.

2-Fluoro-N-(3-morpholinopropyl)-10H-indolo[3,2-b]quinoline-11-

carboxamide (3b, RMS-172): Yellow amorphous solid; mp: 274–

1

General procedure for synthesis of 10H-indolo[3,2-b]quinoline-

11-carboxylic acids 18a–d: A solution of 1-acetyl-1H-indol-3-yl

acetate (16; 6.1 g, 0.23 mmol) in water (50 mL) was stirred for

30 min. A solution of isatin 17 (0.23 mmol) and KOH (26 g,

0.46 mmol) in water (50 mL) was added slowly. The mixture was

heated at reflux for 4 h and was then cooled to 708C. Air was bub-

bled through the mixture for 20 min. The mixture was filtered, and

the resulting filtrate was acidified to pH 1 with concd HCl. The pre-

cipitate was collected and washed with water and dried under

vacuum to give carboxylic acid 18, which was used in the next

step without further purification.

2758C; H NMR (400 MHz, [D₆]DMSO): d=11.39 (s, 1H), 8.91 (t, J=

5.2 Hz, 1H), 8.26 (m, 2H), 7.71 (d, J=10.8 Hz, 1H), 7.57 (m, 3H),

7.25 (t, J=6.8 Hz, 1H), 3.30 (m, 6H), 3.23 (m, 2H), 2.37 (m, 4H),

1.76 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO): d=164.5, 160.3

(d, ¹J_CF=242.0 Hz), 146.1, 144.3, 140.2, 131.8, 130.2, 129.1, 123.3,

123.2, 121.2, 120.5, 119.9, 116.3 (d, ²J_CF=23.8 Hz), 111.9, 107.4 (d,

²J_CF=23.5 Hz), 66.1, 56.0, 53.3, 37.8, 25.6 ppm; IR (CHCl₃): n˜_max

=

3406, 2918, 2849, 2343, 1649, 1454, 1022 cm^ꢁ1; MS (ESI): m/z=

407.2 [M+H]⁺, HRMS: m/z calcd for C₂₃H₂₃FN₄O₂+H⁺: 407.1878;

found: 407.1880; HPLC: t_R=35.33 min, purity: 99.5%.

2-Fluoro-N-(2-morpholinoethyl)-10H-indolo[3,2-b]quinoline-11-

carboxamide (3c, RMS-171): Yellow amorphous solid; mp: 219–

10H-Indolo[3,2-b]quinoline-11-carboxylic acid (18a, RMS-125b):

1

Light-green solid; mp: >3008C; H NMR (400 MHz, [D₆]DMSO): d=

2218C; H NMR (400 MHz, [D₆]DMSO): d=11.50 (s, 1H), 8.96 (s, 1H),

11.52 (s, 1H), 9.38 (s, 1H), 8.37 (dd, J=8.0, 17.4 Hz, 2H), 8.23 (d, J=

8.8 Hz, 1H), 7.84–7.78 (m, 2H), 7.69 (t, J=7.6 Hz, 1H), 7.36 ppm (m,

2H); MS (ESI): m/z=262.0 [M+H]⁺.

8.39 (m, 2H), 8.23 (d, J=9.6 Hz, 1H), 7.70 (m, 2H), 7.38 (t, J=

8.0 Hz, 1H), 4.12 (s, 4H), 3.75 (s, 2H), 2.69 (m, 2H), 2.61 ppm (m,

4H); ¹³C NMR (100 MHz, [D₆]DMSO): d=164.6, 160.7 (d, J=

241.8 Hz), 146.0, 144.2, 140.3, 131.8, 130.1, 129.3, 123.4, 121.1,

120.5, 119.9, 116.4 (d, J=26.2 Hz), 111.9, 108.0 (d, J=23.4 Hz), 66.0,

57.0, 53.1, 35.9 ppm; IR (CHCl₃): n˜_max3401, 2918, 2850, 1741, 1632,

1576, 1539, 1512, 1492, 1463, 1400, 1333, 1214, 1194, 1116, 1148,

1079, 1021 cm^ꢁ1; MS (ESI): m/z=393.1 [M+H]⁺; HRMS: m/z calcd

for C₂₂H₂₁FN₄O₂+H⁺: 393.1721; found: 393.1748; HPLC: t_R=

29.94 min, purity: 95.3%.

2-Bromo-10H-indolo[3,2-b]quinoline-11-carboxylic acid (18b,

RMS-125a): Brown-red solid; mp: >3008C; ¹H NMR (400 MHz,

[D₆]DMSO): d=11.54 (s, 1H), 9.40 (s, 2H), 8.39 (d, J=7.6 Hz, 1H),

8.25 (d, J=8.9 Hz, 1H), 7.86–7.69 (m, 3H), 7.40 ppm (t, J=7.8 Hz,

1H); IR (CHCl₃): n˜_max=3670, 3645, 3584, 3418, 2921, 2348, 2054,

1612, 1460, 1317, 1122, 1048 cm^ꢁ1; MS (ESI): m/z=341.2 [M+H]⁺.

2-Fluoro-10H-indolo[3,2-b]quinoline-11-carboxylic acid (18c,

RMS-170): Brown-red solid; mp: >3008C; ¹H NMR (400 MHz,

[D₆]DMSO): d=11.50 (s, 1H), 8.90 (d, J=10 Hz, 1H), 8.34 (m, 2H),

7.80 (d, J=8.8 Hz, 1H), 7.69–7.61 (m, 2H), 7.35 ppm (t, J=7.6 Hz,

1H); MS (ESI): m/z=280.0 [M+H]⁺.

2-Bromo-N-(2-morpholinoethyl)-10H-indolo[3,2-b]quinoline-11-

carboxamide (3d, RMS-138): Yellow amorphous solid; mp: 250–

2528C; ¹H NMR (400 MHz, CD₃OD): d=8.53 (s, 1H), 8.51 (d, J=

7.6 Hz, 1H), 8.20 (d, J=9.2 Hz, 1H), 7.93 (s, 1H), 7.85 (d, J=9.2 Hz,

1H), 7.71 (t, J=8.0 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.39 (t, J=

7.2 Hz, 1H), 3.85 (m, 6H), 2.81 ppm (m, 6H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=164.4, 146.7, 144.5, 141.6, 131.2, 130.5, 129.18,

129.12, 126.4, 123.9, 121.4, 120.4, 120.0, 119.7, 118.7, 111.9, 66.2,

57.3, 53.2, 36.0 ppm; IR (CHCl₃): n˜_max=3437, 2922, 1617, 1380,

1033, 1019 cm^ꢁ1; MS (ESI): m/z=452.8 [M+H]⁺; HRMS: m/z calcd

for C₂₂H₂₁BrN₄O₂+H⁺: 453.0921; found: 453.0907; HPLC: t_R=

8.4 min, purity: 97.7%.

2-Chloro-10H-indolo[3,2-b]quinoline-11-carboxylic acid (18d,

RMS-187): Dark-green solid; mp: 310–3128C; ¹H NMR (400 MHz,

[D₆]DMSO): d=11.52 (s, 1H), 9.22 (s, 1H), 8.37 (d, J=7.6 Hz, 1H),

8.30 (d, J=9.2 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 7.74–7.66 (m, 2H),

7.36 ppm (t, J=7.2 Hz, 1H); MS (ESI): m/z=296.0 [M+H]⁺.

General procedure for synthesis of 10H-indolo[3,2-b]quinoline-

C11-carboxamides 3a–n, 5a–h, 6a–c, and 7a–c: HBTU (1.2 equiv)

and diisopropylethylamine (3 equiv) were added to a stirred solu-

tion of 10H-indolo[3,2-b]quinoline-C11-carboxylic acid 18 (300 mg,

1 equiv) in anhydrous DMF (10 mL) under a N₂atmosphere at

room temperature. After stirring for 10 min, alkylamine 19, 21, or

22 (1.1 equiv) was added to the stirring solution. The mixture was

stirred for another 24 h, and then ice-cold water was added. The

product was extracted with ethyl acetate (3ꢂ50 mL), and the ob-

tained organic layer was dried with anhydrous Na₂SO₄. The solvent

N-(2-Morpholinoethyl)-10H-indolo[3,2-b]quinoline-11-carboxa-

mide (3e, RMS-173): Yellow amorphous solid; mp: 240–2418C;

¹H NMR (400 MHz, [D₆]DMSO): d=11.27 (s, 1H), 8.80 (t, J=5.6 Hz,

1H), 8.29 (m, 2H), 8.18 (d, J=8.4 Hz, 1H), 7.65–7.52 (m, 4H), 7.24

(t, J=8.0 Hz, 1H), 3.61 (m, 2H), 3.24 (m, 4H), 2.58 (m, 2H),

2.47 ppm (m, 4H); ¹³C NMR (100 MHz, [D₆]DMSO): d=164.9, 146.1,

144.2, 143.1, 130.0, 129.0, 128.7, 126.1, 125.5, 124.8, 122.7, 121.3,

120.8, 120.6, 119.7, 111.7, 66.2, 57.0, 53.1, 36.1 ppm; IR (CHCl₃):

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n˜_max=3401, 2920, 2851, 1741, 1643, 1461, 1491, 1461, 1336, 1115,

1021 cm^ꢁ1; MS (ESI): m/z=375.0 [M+H]⁺; HRMS: m/z calcd for

C₂₂H₂₂N₄O₂+H⁺: 375.1816; found: 375.1820; HPLC: t_R=4.62 min,

purity: 100%.

n˜_max=3401, 2920, 2851, 1627, 1487, 1462, 1336, 1212, 1193,

1022 cm^ꢁ1; MS (ESI): m/z=367.3 [M+H]⁺; HRMS: m/z calcd for

C₂₀H₁₉ClN₄O+H⁺: 367.1320; found: 367.1343; HPLC: t_R=4.87 min,

purity: 100%.

2-Chloro-N-[2-(pyrrolidin-1-yl)ethyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3 f, RMS-200): Yellow amorphous solid; mp:

250–2518C; H NMR (400 MHz, [D₆]DMSO): d=11.59 (s, 1H), 9.08 (s,

2-Chloro-N-[3-(dimethylamino)propyl]-10H-indolo[3,2-b]quino-

line-11-carboxamide (3k, RMS-202): Yellow amorphous solid; mp:

216–2188C; H NMR (400 MHz, [D₆]DMSO): d=11.56 (s, 1H), 9.11 (s,

1

1H), 8.38 (d, J=7.6 Hz, 1H), 8.28 (d, J=8.8 Hz, 2H), 7.72 (m, 2H),

7.63 (d, J=8.0 Hz, 1H), 7.34 (t, J=7.2 Hz, 1H), 3.72 (m, 2H), 3.43

(m, 6H, merged with [D₆]DMSO signal), 1.91 ppm (m, 4H); ¹³C NMR

(125 MHz, [D₆]DMSO): d=164.7, 146.7, 144.4, 141.4, 131.1, 130.6,

130.2, 129.4, 126.6, 123.2, 121.5, 120.4, 120.1, 111.8, 53.4, 22.9 ppm;

IR (CHCl₃): n˜_max=3417, 2921, 2852, 1743, 1621, 1540, 1463, 1384,

1214, 1021 cm^ꢁ1; MS (ESI): m/z=393.3 [M+H]⁺; HRMS: m/z calcd

for C₂₂H₂₁ClN₄O+H⁺: 393.1477; found: 393.1494; HPLC: t_R=

8.78 min, purity: 98.6%.

1H), 8.38 (d, J=8.0 Hz, 1H), 8.29 (d, J=9.2 Hz, 1H), 8.13 (s, 1H),

7.73–7.63 (m, 3H), 7.35 (t, J=7.2 Hz, 1H), 3.20 (m, 2H), 2.83 (s, 6H),

2.55 (m, 2H), 2.05 ppm (m, 2H); ¹³C NMR (125 MHz, [D₆]DMSO): d=

164.7, 146.8, 144.5, 141.3, 131.2, 130.6, 130.2, 129.1, 126.6, 123.2,

122.8, 121.5, 120.5, 120.2, 119.1, 111.9, 54.9, 42.4, 36.6, 24.1 ppm; IR

(CHCl₃): n˜_max=3401, 2920, 2850, 1741, 1619, 1541, 1463, 1384,

1021 cm^ꢁ1; MS (ESI): m/z=381.3 [M+H]⁺; HRMS: m/z calcd for

C₂₁H₂₁ClN₄O+H⁺: 381.1477; found: 381.1500; HPLC: t_R=4.87 min,

purity: 100%.

2-Bromo-N-[2-(piperidin-1-yl)ethyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3g, RMS-148): Yellow amorphous solid; mp:

259–2618C; ¹H NMR (400 MHz, CD₃OD): d=10.14 (s, 1H), 8.36 (d,

J=8.0 Hz, 1H), 8.32 (s, 1H), 8.07 (d, J=8.8 Hz, 1H), 7.65 (d, J=

8.8 Hz, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.32 (t, J=7.2 Hz, 2H), 7.27 (m,

1H), 3.91 (m, 2H), 2.82 (m, 2H), 2.68 (m, 4H), 1.68 (m, 4H),

1.52 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO): d=164.3, 146.7,

144.4, 141.6, 131.2, 130.5, 129.2, 129.1, 126.4, 123.9, 121.4, 120.5,

120.0, 119.6, 118.7, 111.8, 57.43, 54.8, 53.9, 36.4, 25.4, 23.9 ppm; IR

(CHCl₃): n˜_max3439, 2959, 2925, 2852, 2096, 1930, 1640, 1596, 1575,

1491, 1454, 1436, 1407, 1389, 1367, 1294, 1192, 1175, 1158, 1129,

1083, 1046, 1023 cm^ꢁ1; MS (ESI): m/z=452.9 [M+H]⁺; HRMS: m/z

calcd for C₂₃H₂₃BrN₄O+H⁺: 451.1128; found: 451.1124; HPLC: t_R=

24.53 min, purity: 97.9%.

2-Chloro-N-[3-(diethylamino)propyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3l, RMS-203): Yellow amorphous solid; mp: 218–

2228C; H NMR (400 MHz, [D₆]DMSO): d=11.58 (s, 1H), 9.12 (s, 1H),

1

8.28 (d, J=8.0 Hz, 1H), 8.29 (d, J=8.8 Hz, 1H), 8.13 (s, 1H), 7.74–

7.62 (m, 3H), 7.35 (t, J=7.6 Hz, 1H), 3.39 (m, 2H, merged with

[D₆]DMSO), 3.17 (m, 6H), 2.02 (m, 2H), 1.21 ppm (t, J=7.2 Hz, 6H);

¹³C NMR (125 MHz, [D₆]DMSO): d=164.7, 146.9, 144.5, 141.4, 131.2,

130.6, 130.2, 129.1, 126.6, 123.2, 122.7, 121.5, 120.4, 120.2, 119.2,

111.9, 48.8, 46.6, 36.7, 23.4, 8.6 ppm; IR (CHCl₃): n˜_max=3411, 2920,

2851, 1741, 1618, 1577, 1455, 1384, 1021 cm^ꢁ1; MS (ESI): m/z=

409.4 [M+H]⁺; HRMS: m/z calcd for C₂₃H₂₅ClN₄O+H⁺: 409.1790;

found: 409.1812; HPLC: t_R=3.83 min, purity: 97.2%.

2-Bromo-N-[2-(dimethylamino)ethyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3m, RMS-204): Yellow amorphous solid; mp:

1

2-Bromo-N-(3-hydroxypropyl)-10H-indolo[3,2-b]quinoline-11-car-

boxamide (3h, RMS-149): Yellow amorphous solid; mp: 277–

237–2388C; H NMR (400 MHz, [D₆]DMSO): d=8.98 (s, 1H), 8.45 (s,

1H), 8.37 (d, J=8.0 Hz, 1H), 8.19 (d, J=8.8 Hz, 1H), 7.80 (d, J=

9.2 Hz, 1H), 7.68 (d, J=7.2 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.34 (t,

J=7.6 Hz, 1H), 3.16 (m, 2H), 2.72 (m, 2H), 2.45 ppm (s, 6H);

¹³C NMR (125 MHz, [D₆]DMSO): d=164.6, 146.7, 144.4, 141.1, 131.1,

130.6, 129.6, 129.1, 126.4, 123.8, 121.5, 120.4, 120.1, 119.3, 118.8,

111.8, 57.1, 54.8, 44.4, 36.6 ppm; IR (CHCl₃): n˜_max=3432, 2921, 2851,

1737, 1651, 1577, 1538, 1462, 1213, 1021 cm^ꢁ1; MS (ESI): m/z=

413.3 [M+H]⁺; HRMS: m/z calcd for C₂₀H₁₉BrN₄O+H⁺: 411.0815;

found: 411.0831; HPLC: t_R=5.43 min, purity: 100%.

1

2788C; H NMR (400 MHz, CD₃OD): d=11.12 (brs, 1H), 8.30 (d, J=

8.0 Hz, 1H), 8.20 (s, 1H), 7.98 (d, J=9.2 Hz, 1H), 7.79 (s, 1H), 7.63

(d, J=8.8 Hz, 1H), 7.53 (t, J=8.0 Hz, 1H), 7.44 (d, J=8.0 Hz, 1H),

7.21 (t, J=7.6 Hz, 1H), 3.72 (t, J=6.0 Hz, 2H), 3.64 (t, J=6.8 Hz,

2H), 1.93 ppm (m, 2H); ¹³C NMR (100 MHz, CD₃OD): d=167.6,

148.5, 146.3, 142.9, 132.0, 131.23, 131.18, 130.9, 127.5, 125.4, 122.9,

121.65, 121.6, 120.9, 120.7, 112.8, 61.1, 38.8, 32.9 ppm; IR (CHCl₃):

n˜_max=3493, 2919, 2850, 1730, 1649, 1465, 1286, 1072 cm^ꢁ1; MS

(ESI): m/z=398.0 [M+H]⁺; HRMS: m/z calcd for C₁₉H₁₆BrN₃O₂+H⁺:

398.0499; found: 398.0512; HPLC: t_R=27.45 min, purity: 94.9%.

N-[2-(Dimethylamino)ethyl]-2-fluoro-10H-indolo[3,2-b]quinoline-

11-carboxamide (3n, RMS-212): Yellow amorphous solid; mp:

1

2-Bromo-N-[2-(diethylamino)ethyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3i, RMS-127): Yellow amorphous solid; mp: 203–

187–1898C; H NMR (400 MHz, [D₆]DMSO): d=8.93 (s, 1H), 8.37 (d,

J=7.6 Hz, 1H), 8.31 (dd, J=5.6, 9.2 Hz, 1H), 7.99 (d, J=10.8 Hz,

1H), 7.69–7.59 (m, 3H), 7.35 (t, J=7.6 Hz, 1H), 3.38 (m, 2H, merged

with [D₆]DMSO), 2.67 (m, 2H), 2.42 ppm (s, 6H); ¹³C NMR (100 MHz,

1

2058C; H NMR (400 MHz, CD₃OD): d=8.45 (d, J=8.0 Hz, 1H), 8.42

(s, 1H), 8.14 (d, J=8.8 Hz, 1H), 7.79 (d, J=9.2 Hz, 1H), 7.67 (t, J=

8.0 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.34 (t, J=7.2 Hz, 1H), 3.84 (t,

J=6.4 Hz, 2H), 3.15 (s, 2H), 3.06 (m, 4H), 1.36–1.26 ppm (m, 6H);

¹³C NMR (100 MHz, CDCl₃+CD₃OD): d=168.1, 148.5, 146.0, 143.1,

132.3, 132.0, 131.3, 130.1, 127.7, 125.5, 123.4, 121.9, 121.5, 119.6,

112.8, 52.4, 48.3, 38.1, 11.2 ppm; IR (CHCl₃): n˜_max=3436, 2070, 1633,

1464, 1157, 1019 cm^ꢁ1; MS (ESI): m/z=439.0 [M+H]⁺; HRMS: m/z

calcd for C₂₂H₂₃BrN₄O+H⁺: 439.1128; found: 439.1116; HPLC: t_R=

45.10 min, purity: 95.4%.

1

[D₆]DMSO): d=164.7, 160.7 (d, J_CF=237.9 Hz), 146.1, 144.1, 140.3,

2

131.6, 130.3, 129.9, 123.2, 121.3, 120.6, 120.0, 119.6, 116.4 (d, J_CF

=

25.4 Hz), 111.8, 107.7 (d, ²J_CF=24.4 Hz), 57.3, 44.5, 36.8 ppm; IR

(CHCl₃): n˜_max=3440, 2920, 2850, 2090, 1633, 1460, 1338, 1215,

1021 cm^ꢁ1; MS (ESI): m/z=351.2 [M+H]⁺; HRMS: m/z calcd for

C₂₀H₁₉FN₄O+H⁺: 351.1616; found: 351.1625; HPLC: t_R=4.66 min,

purity: 95.2%.

(2-Bromo-10H-indolo[3,2-b]quinolin-11-yl)(4-methylpiperazin-1-

yl)methanone (5a, RMS-210): Yellow amorphous solid; mp: 261–

2-Chloro-N-[2-(dimethylamino)ethyl]-10H-indolo[3,2-b]quinoline-

11-carboxamide (3j, RMS-201): Yellow amorphous solid; mp: 231–

2338C; ¹H NMR (400 MHz, [D₆]DMSO): d=8.99 (s, 1H), 8.38–8.25

(m, 3H), 7.71–7.61 (m, 3H), 7.34 (m, 1H), 3.45 (m, 2H), 2.75 (m,

2H), 2.47 ppm (s, 6H); ¹³C NMR (125 MHz, [D₆]DMSO): d=164.6,

146.7, 144.4, 141.4, 131.0, 130.5, 130.2, 129.7, 126.6, 123.3, 123.2,

121.5, 120.5, 120.1, 119.3, 111.8, 57.1, 44.3, 36.5 ppm; IR (CHCl₃):

1

2628C; H NMR (400 MHz, [D₆]DMSO): d=11.77 (s, 1H), 8.43 (d, J=

7.2 Hz, 1H), 8.35 (d, J=5.6 Hz, 1H), 7.19 (s, 1H), 7.78–7.66 (m, 3H),

7.40 (t, J=6.8 Hz, 1H), 4.20 (m, 1H), 3.78 (m, 1H), 3.22 (m, 2H),

2.78 (m, 1H), 2.45–2.36 (m, 2H), 2.26 (s, 3H), 2.05 ppm (m, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d=163.6, 146.5, 144.5, 141.3, 131.3,

130.5, 130.4, 128.3, 126.8, 123.1, 122.3, 121.5, 120.5, 120.1, 118.3,

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Full Papers

111.8, 54.9, 54.1, 46.1, 45.4, 41.2 ppm; IR (CHCl₃): n˜_max=3404, 2920,

2850, 2806, 1616, 1490, 1470, 1446, 1397, 1338, 1295, 1252, 1230,

1195, 1170, 1145, 1109, 1062, 1032 cm^ꢁ1; MS (ESI): m/z=379.1 [M+

H]⁺; HRMS: m/z calcd for C₂₁H₂₀ClN₄O+H⁺: 379.1320; found:

379.1319; HPLC: t_R=20.5 min, purity: 100%.

125.9, 124.6, 123.8, 121.8, 121.3, 120.7, 119.7, 111.7, 61.2, 58.8, 47.9,

27.2, 23.7 ppm; IR (CHCl₃): n˜_max=3433, 2921, 2851, 2809, 1618,

1494, 1463, 1396, 1337, 1219, 1139, 1021 cm^ꢁ1; MS (ESI): m/z=

346.0 [M+H]⁺; HRMS: m/z calcd for C₂₁H₁₉N₃O₂+H⁺: 346.1550;

found: 346.1557; HPLC: t_R=20.15 min, purity: 95.7%.

4-(2-Bromo-10H-indolo[3,2-b]quinoline-11-carbonyl)piperazine-

1-carbaldehyde (5b, RMS-211): Yellow amorphous solid; mp: 279–

2808C; H NMR (400 MHz, [D₆]DMSO): d=11.76 (s, 1H), 8.38 (d, J=

(S)-(2-Chloro-10H-indolo[3,2-b]quinolin-11-yl)[2-(hydroxymethyl)-

pyrrolidin-1-yl]methanone (5g, RMS-196): Yellow amorphous

solid; mp: 277–2788C; H NMR (400 MHz, [D₆]DMSO): d=11.76 (s,

1

8.0 Hz, 1H), 8.29 (d, J=8.4 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.92 (s,

1H), 7.72–7.60 (m, 3), 7.37 (t, J=7.6 Hz, 1H), 4.03–3.88 (m, 2H),

3.78 (m, 1H), 3.34 (m, 2H), 3.25–3.17 ppm (m, 3H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=164.0, 161.2, 146.5, 144.5, 141.4, 131.2,

130.6, 130.5, 128.4, 126.9, 123.1, 122.4, 121.5, 120.5, 120.2, 118.0,

111.8, 46.9, 45.3, 42.0, 39.8 ppm; IR (CHCl₃): n˜_max=3419, 2919, 1660,

1616, 1489, 1469, 1437, 1397, 1296, 1280, 1251, 1213, 1195, 1148,

1024, 1002 cm^ꢁ1; MS (ESI): m/z=393.1 [M+H]⁺; HRMS: m/z calcd

for C₂₁H₁₇ClN₄O₂+H⁺: 393.1113; found: 393.1123; HPLC: t_R=

27.1 min, purity: 98.9%.

1H), 11.34 (brs, 1H), 8.37 (s, 1H), 8.28 (d, J=8.8 Hz, 1H), 7.93 (d,

J=25.6 Hz, 1H), 7.71–7.59 (m, 3H), 7.34 (d, J=7.2 Hz, 1H), 5.10 (s,

1H), 4.45 (m, 1H), 3.86 (m, 1H), 3.11–3.00 (m, 2H), 2.05 (m, 2H),

1.88–1.72 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO): d=163.7,

146.7, 144.5, 141.4, 140.5, 131.2, 130.4, 127.8, 126.7, 123.2, 122.6,

122.4, 121.4, 120.7, 120.2, 111.8, 60.6, 58.9, 48.0, 27.0, 23.6 ppm; IR

(CHCl₃): n˜_max=3429, 2923, 2082, 1633, 1465, 1405, 1215, 1022 cm^ꢁ1

;

MS (ESI): m/z=379.1 [M]⁺, 377.1 [Mꢁ1]⁺; HRMS: m/z calcd for

C₂₁H₁₈ClN₃O₂+H⁺: 380.1160; found: 380.1166; HPLC: t_R=

23.14 min, purity: 96.2%.

(2-Bromo-10H-indolo[3,2-b]quinolin-11-yl)(morpholino)metha-

none (5c, RMS-190): Yellow amorphous solid; mp: 260–2628C;

¹H NMR (400 MHz, [D₆]DMSO): d=11.78 (s, 1H), 8.41 (d, J=7.6 Hz,

1H), 8.25 (d, J=9.2 Hz, 1H), 8.09 (s, 1H), 7.86 (d, J=8.8 Hz, 1H),

7.73 (t, J=7.2 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.39 (t, J=7.2 Hz,

1H), 4.05 (m, 4H), 3.88 (m, 2H), 3.22 ppm (m, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=163.8, 146.6, 146.6, 144.6, 141.5, 131.4,

130.6, 129.3, 128.3, 125.6, 123.7, 121.5, 120.5, 120.2, 119.0, 117.8,

111.8, 66.4, 65.9, 46.6, 41.8 ppm; IR (CHCl₃): n˜_max=3416, 2921, 2852,

1615, 1488, 1468, 1440, 1397, 1337, 1304, 1271, 1234, 1213, 1192,

1147, 1112, 1059, 1028 cm^ꢁ1; MS (ESI): m/z=409.9 [M+H]⁺; HRMS:

m/z calcd for C₂₀H₁₆BrN₃O₂+H⁺: 410.0499; found: 410.0495; HPLC:

t_R=32.3 min, purity: 96.9%.

(2-Chloro-10H-indolo[3,2-b]quinolin-11-yl)(morpholino)metha-

none (5h, RMS-197): Yellow amorphous solid; mp: 240–2418C;

¹H NMR (400 MHz, [D₆]DMSO): d=11.74 (s, 1H), 8.38 (d, J=7.6 Hz,

1H), 8.29 (d, J=8.8 Hz, 1H), 7.90 (s, 1H), 7.71 (dd, J=8.4, 15.6 Hz,

2H), 7.62 (d, J=8.0 Hz, 1H), 7.35 (t, J=7.6 Hz, 1H), 4.06–3.92 (m,

2H), 3.87–3.79 (m, 2H), 3.50 (m, 2H), 3.19 ppm (m, 2H); ¹³C NMR

(125 MHz, [D₆]DMSO): d=163.7, 146.5, 144.5, 141.4, 131.3, 130.6,

130.4, 128.3, 126.8, 123.1, 122.4, 121.5, 120.5, 120.2, 118.0, 111.8,

66.4, 65.8, 46.6, 41.8 ppm; IR (CHCl₃): n˜_max=3435, 2921, 2852, 2089,

1618, 1490, 1470, 1440, 1397, 1338, 1305, 1271, 1213, 1194, 1147,

1112, 1089, 1064, 1030 cm^ꢁ1; MS (ESI): m/z=366.1 [M+H]⁺; HRMS:

m/z calcd for C₂₀H₁₆ClN₃O₂+H⁺: 366.1004; found: 366.1014; HPLC:

t_R=23.28 min, purity: 97.7%.

(2-Fluoro-10H-indolo[3,2-b]quinolin-11-yl)(morpholino)metha-

none (5d, RMS-192): Yellow amorphous solid; mp: 274–2758C;

¹H NMR (400 MHz, [D₆]DMSO): d=11.70 (s, 1H), 8.34 (m, 2H), 7.65

2-Bromo-N-[4-(trifluoromethoxy)benzyl]-10H-indolo[3,2-b]quino-

line-11-carboxamide (6a, RMS-158): Yellow amorphous solid; mp:

246–2478C; H NMR (400 MHz, [D₆]DMSO): d=11.63 (s, 1H), 9.58 (t,

1

(m, 4H), 7.34 (m, 1H), 4.03–3.79 (m, 4H), 3.48 (m, 2H), 3.18 ppm

J=5.6 Hz, 1H), 8.37 (d, J=7.6 Hz, 1H), 8.20 (d, J=8.8 Hz, 1H), 8.15

(s, 1H), 7.80 (d, J=9.2 Hz, 1H), 7.71–7.62 (m, 4H), 7.42 (d, J=

8.0 Hz, 2H), 7.34 (t, J=7.2 Hz, 1H), 4.74 ppm (d, J=5.6 Hz, 2H);

¹³C NMR (100 MHz, [D₆]DMSO): d=164.7, 147.3, 146.8, 144.5, 141.4,

138.4, 133.1, 131.2, 130.6, 129.6, 129.0, 125.9, 123.8, 121.5, 121.0,

1

(m, 2H); ¹³C NMR (125 MHz, [D₆]DMSO): d=163.9,160.0 (d, J_CF

=

223.6 Hz), 145.9, 144.3, 140.2, 132.0, 130.3, 128.4, 123.1, 121.3,

1

120.6, 120.1, 118.3, 116.6 (d, ²J_CF=26.0 Hz), 111.8, 107.0 (d, J_CF

=

23.0 Hz), 66.4, 65.8, 46.6, 41.7 ppm; IR (CHCl₃): n˜_max=3429, 2920,

2851, 2350, 2089, 1626, 1516, 1494, 1478, 1442, 1398, 1338, 1306,

1272, 1215, 1197, 1149, 1111, 1068, 1031 cm^ꢁ1; MS (ESI): m/z=350.1

[M+H]⁺; HRMS: m/z calcd C₂₀H₁₆FN₃O₂+H⁺: 350.1299; found:

350.1317; HPLC: t_R=20.14 min, purity: 99.6%.

120.3, 120.2 (m), 119.0, 118.7, 111.9, 42.4 ppm; IR (CHCl₃): n˜_max

=

3436, 2923, 2323, 1644, 1624, 1510, 1486, 1466, 1396, 1335, 1263,

1215, 1163, 1110, 1019 cm^ꢁ1; MS (ESI): m/z=514.0 [M+H]⁺; HRMS:

m/z calcd for C₂₄H₁₅BrF₃N₃O₂+H⁺: 514.0406; found: 514.0425;

HPLC: t_R=37.68 min, purity: 96.2%.

(10H-Indolo[3,2-b]quinolin-11-yl)(morpholino)methanone

(5e,

RMS-193): Yellow amorphous solid; mp: 234–2358C; ¹H NMR

(400 MHz, [D₆]DMSO): d=11.62 (s, 1H), 8.38 (d, J=7.6 Hz, 1H), 8.27

(d, J=7.6 Hz, 1H), 7.93 (d, J=7.6 Hz, 1H), 7.67 (m, 4H), 7.33 (m,

1H), 4.03–3.79 (m, 4), 3.47 (m, 2H), 3.15 ppm (m, 2H); ¹³C NMR

(125 MHz, [D₆]DMSO): d=164.3, 146.0, 144.4, 143.0, 130.2, 129.2,

128.0, 126.3, 126.0, 123.8, 122.3, 121.4, 120.7, 119.8, 118.9, 111.7,

66.4, 65.9, 46.7, 41.7 ppm; IR (CHCl₃): n˜_max=3433, 2921, 2851, 1615,

1494, 1463 1396, 1219, 1138, 1021 cm^ꢁ1; MS (ESI): m/z=332.1 [M+

H]⁺; HRMS: m/z calcd C₂₀H₁₇N₃O₂+H⁺: 332.1394; found: 332.1380;

HPLC: t_R=22.15 min, purity: 95.1%.

2-Bromo-N-[4-(trifluoromethyl)benzyl]-10H-indolo[3,2-b]quino-

line-11-carboxamide (6b, RMS-160): Yellow amorphous solid; mp:

291–2928C; ¹H NMR (400 MHz, [D₆]DMSO): d=8.57 (d, J=8.8 Hz,

1H), 8.13 (s, 1H), 8.07 (d, J=12.0 Hz, 1H), 7.80 (s, 1H), 7.70 (d, J=

12.0 Hz, 1H), 7.63 (s, 3H), 7.58 (d, J=8.0 Hz, 1H), 7.52 (d, J=8.8 Hz,

1H), 7.26 (t, J=8.0 Hz, 1H), 4.5 ppm (s, 2H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=164.8, 148.0, 146.9, 144.6, 143.7, 141.5, 131.3, 130.5,

129.1, 129.0, 128.4, 127.9, 125.9, 125.3, 122.9, 121.4, 120.4, 120.1,

118.8, 118.7, 112.0, 42.7 ppm; IR (CHCl₃): n˜_max=3436, 2923, 2072,

1633, 1462, 1483, 1325, 1159, 1122, 1019 cm^ꢁ1; MS (ESI): m/z=

497.9 [M+H]⁺; HRMS: m/z calcd for C₂₄H₁₅BrF₃N₃O+H⁺: 500.0403;

found: 500.0396; HPLC: t_R=37.14 min, purity: 100%.

(S)-[2-(Hydroxymethyl)pyrrolidin-1-yl](10H-indolo[3,2-b]quinolin-

11-yl)methanone (5 f, RMS-194): Yellow amorphous solid; mp:

1

180–1868C; H NMR (400 MHz, [D₆]DMSO): d=11.60 (s, 1H), 11.23

2-Bromo-N-[3-(trifluoromethyl)benzyl]-10H-indolo[3,2-b]quino-

line-11-carboxamide (6c, RMS-162): Yellow amorphous solid; mp:

(s, 1H), 8.37 (m, 1H), 8.26 (m, 1H), 7.97.83 (m, 1H), 7.78–7.58 (m,

3H), 7.31 (m, 1H), 5.07 (s, 1H), 4.40–3.88 (m, 2H), 3.00 (m, 2H),

2.07 (m, 2H), 1.87–1.67 ppm (m, 2H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=164.4, 146.2, 144.3, 143.1, 130.1, 129.2, 127.4, 126.3,

1

270–2718C; H NMR (400 MHz, CD₃OD): d=8.35 (d, J=8.0 Hz, 1H),

8.1 (s, 1H), 8.04 (d, J=9.2 Hz, 1H), 7.75 (s, 1H), 7.68 (m, 2H), 7.59–

7.48 (m, 4H), 7.24 (t, J=7.6 Hz, 1H), 4.78 ppm (s, 2H); ¹³C NMR

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ÝÝ These are not the final page numbers!

Full Papers

1

(125 MHz, [D₆]DMSO): d=164.8, 146.9, 144.6, 141.4, 140.4, 131.8,

131.3, 130.6, 129.5, 129.3, 129.1, 125.8, 124.0, 123.8, 121.5, 120.3,

120.1, 118.9, 118.8, 112.0, 42.6 ppm; IR (CHCl₃): n˜_max=3437, 2066,

1650, 1640, 1633, 1463, 1327, 1166, 1128, 1019 cm^ꢁ1; MS (ESI): m/

z=497.8 [M+H]⁺; HRMS: m/z calcd for C₂₄H₁₆BrF₃N₃O+H⁺:

500.0403; found: 500.0405; HPLC: t_R=36.93 min, purity: 97.9%.

[D₆]DMSO): d=166.4, 163.6 (d, J_CF=246.1 Hz), 147.3, 143.8, 143.5,

137.6, 131.1, 130.6, 130.4, 128.9, 128.8, 125.7, 122.5, 122.0, 121.5,

121.2, 120.7, 117.8, 116.0 (d, ²J_CF=21.3 Hz), 111.3, 66.2, 58.3, 53.4,

29.7, 24.1 ppm; IR (CHCl₃): n˜_max=3436, 2066, 1650, 1633, 1464,

1019 cm^ꢁ1; MS (ESI): m/z=483.1 [M+H]⁺; HRMS: m/z calcd for

C₂₉H₂₇FN₄O₂+H⁺: 483.2191; found: 483.2197; HPLC: t_R=5.3 min,

purity: 97%.

2-Bromo-N-[4-(trifluoromethoxy)phenyl]-10H-indolo[3,2-b]quino-

line-11-carboxamide (7a, RMS-163): Yellow amorphous solid; mp:

2-(3-Acetamidophenyl)-N-(3-morpholinopropyl)-10H-indolo[3,2-

b]quinoline-11-carboxamide (4b, RMS-129): Yellow amorphous

solid; mp: 254–2568C; H NMR (400 MHz, CDCl₃): d=8.30 (m, 1H),

8.24 (s, 1H), 8.19 (m, 1H), 7.92 (s, 1H), 7.86 (m, 1H), 7.53 (m, 1H),

7.48 (m, 2H), 7.36 (m, 2H), 7.23 (m, 1H), 3.62 (t, J=6.8 Hz, 2H),

3.47 (t, J=4.4 Hz, 4H), 2.52 (t, J=7.2 Hz, 2H), 2.39 (s, 4H), 2.09 (s,

3H), 1.90 ppm (m, 2H); ¹³C NMR (125 MHz, CD₃OD): d=171.7,

168.1, 147.8, 146.1, 143.7, 142.5, 140.6, 139.5, 131.7, 130.1, 130.5,

129.7, 127.3, 124.2, 124.1, 122.9, 122.8, 121.9, 121.7, 121.3, 120.3,

1

245–2478C; H NMR (400 MHz, [D₆]DMSO): d=11.79 (s, 1H), 11.23

1

(s, 1H), 8.4 (d, J=7.6 Hz, 1H), 8.30 (s, 1H), 8.25 (d, J=9.2 Hz, 1H),

8.01 (d, J=8.8 Hz, 2H), 7.85 (d, J=9.2 Hz, 1H), 7.69 (t, J=8.0 Hz,

1H), 7.62 (d, J=8.0 Hz, 1H), 7.48 (d, J=8.8 Hz, 2H), 7.36 ppm (t, J=

7.2 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d=163.4, 147.0, 144.6,

144.3, 141.3, 137.9, 131.2, 130.8, 129.3, 129.0, 125.7, 123.6, 121.7,

121.5, 121.4, 120.3, 120.2, 119.2, 118.8, 118.7, 111.9 ppm; IR (CHCl₃):

n˜_max=3416, 2921, 2851, 1788, 1729, 1654, 1626, 11534, 1505, 1485,

1462, 1411, 1317, 1297, 1251, 1205, 1158, 1072, 1021 cm^ꢁ1; MS

(ESI): m/z=501.8 [M+H]⁺; HRMS: m/z calcd for C₂₃H₁₃BrF₃N₃O₂+

H⁺: 500.0216; found: 500.0234; HPLC: t_R=39.38 min, purity: 100%.

120.0, 112.7, 67.3, 57.8, 54.5, 39.5, 26.7, 24.0 ppm; IR (CHCl₃): n˜_max

=

3436, 2921, 2851, 2066, 1639, 1464, 1019 cm^ꢁ1; MS (ESI): m/z=

522.1 [M+H]⁺; HRMS: m/z calcd for C₃₁H₃₁N₅O₃+H⁺: 522.2500;

found: 522.2507; HPLC: t_R=8.37 min, purity: 98.6%.

2-Bromo-N-(4-bromophenyl)-10H-indolo[3,2-b]quinoline-11-car-

boxamide (7b, RMS-164): Yellow amorphous solid; mp: 242–

2448C; ¹H NMR (400 MHz, [D₆]DMSO): d=11.80 (s, 1H), 11.17 (s,

1H), 8.40 (d, J=7.6 Hz, 1H), 8.27 (s, 1H), 8.25 (d, J=9.2 Hz,

1H),7.86 (m, 3H), 7.65 (m, 4H), 7.35 ppm (t, J=7.2 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d=163.3, 147.0, 144.6, 141.3, 138.0,

131.6, 131.2, 130.8, 129.3, 128.9, 125.5, 123.6, 122.2, 121.5, 120.3,

120.2, 119.1, 118.8, 116.0, 111.9 ppm; IR (CHCl₃): n˜_max=3427, 2962,

2874, 2311, 1724, 1649, 1599, 1581, 1528, 1488, 1469, 1447, 1394,

1377, 1341, 1303, 1286, 1123, 1073, 1039 cm^ꢁ1; MS (ESI): m/z=

495.8 [M+H]⁺; HRMS: m/z calcd for C₂₂H₁₃Br₂N₃O+H⁺: 495.9478;

found: 495.9465; HPLC: t_R=37.7 min, purity: 95.3%.

2-(4-Acetylphenyl)-N-(3-morpholinopropyl)-10H-indolo[3,2-

b]quinoline-11-carboxamide (4c, RMS-130): Yellow amorphous

solid; mp: 226–2288C; ¹H NMR (400 MHz, CDCl₃): d=9.27 (s, 1H),

8.58 (m, 2H), 8.49 (d, J=7.6 Hz, 1H), 8.42 (d, J=8.8 Hz, 1H), 8.10

(d, J=8.0 Hz, 2H), 7.94 (d, J=8.8 Hz, 1H), 7.83 (d, J=8.0 Hz, 2H),

7.60 (t, J=7.6 Hz, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.35 (t, J=7.2 Hz,

1H), 3.82 (m, 2H), 3.0 (s, 4H), 2.67 (s, 3H), 2.51 (t, J=6.0 Hz, 2H),

2.19 (m, 4H), 1.86 ppm (m, 2H); ¹³C NMR (100 MHz, CD₃OD): d=

166.9, 164.7, 146.7, 144.5, 142.8, 135.8, 131.6, 131.5, 130.3, 130.0,

129.1, 129.0, 128.6, 127.2, 125.2, 122.7, 122.5, 121.3, 120.9, 120.6,

119.8, 111.9, 66.1, 56.1, 53.3, 37.8, 27.1, 18.8 ppm; IR (CHCl₃): n˜_max

=

3436, 2924, 2856, 1647, 1620, 1401, 1269, 1019 cm^ꢁ1; MS (ESI): m/

z=507.1 [M+H]⁺; HRMS: m/z calcd for C₃₁H₃₀N₄O₃+H⁺: 507.2391;

found: 507.2391; HPLC: t_R=5.34 min, purity: 95.6%.

2-Bromo-N-(4-fluorophenyl)-10H-indolo[3,2-b]quinoline-11-car-

boxamide (7c, RMS-165): Yellow amorphous solid; mp: 307–

3088C; ¹H NMR (400 MHz, [D₆]DMSO): d=11.79 (s, 1H), 11.10 (s,

1H), 8.40 (d, J=7.6 Hz, 1H), 8.31 (s, 1H), 8.25 (d, J=8.8 Hz, 1H),

7.91 (m, 2H), 7.85 (d, J=8.8 Hz, 1H), 7.69 (d, J=7.2 Hz, 1H), 7.63

(d, J=8.0 Hz, 1H), 7.32 ppm (m, 3H); ¹³C NMR (100 MHz,

[D₆]DMSO): d=163.0, 159.5, 157.6, 147.0, 144.7, 141.5, 135.2, 131.4,

130.6, 129.2, 128.9, 125.8, 123.7, 122.0, 121.5, 120.4, 120.2, 119.0,

115.5, 115.3, 111.9 ppm; IR (CHCl₃): n˜_max=3436, 2921, 2851, 1737,

1651, 1617, 1508, 1463, 1384, 1217, 1021 cm^ꢁ1; MS (ESI): m/z=

435.8 [M+H]⁺; HRMS: m/z calcd for C₂₂H₁₃BrFN₃O+H⁺: 434.0299;

found: 434.0304; HPLC: t_R=36.79 min, purity: 96.4%.

Biological methods

Measurement of inhibition of P. falciparum growth in culture:^[27]

In this study, CQ-sensitive 3D7 and CQ-resistant Dd2 and Indo

strains of P. falciparum were cultivated in vitro by the method of

Trager and Jensen^[28]with minor modifications. Cultures were main-

tained in fresh O⁺human erythrocytes at 4% hematocrit in com-

plete medium (RPMI 1640 with 0.2% sodium bicarbonate, 0.5% Al-

bumax, 45 mgL^ꢁ1hypoxanthine, and 50 mgL^ꢁ1gentamicin) at

378C under reduced O₂(gas mixture 5% O₂, 5% CO₂, and 90% N₂).

Stock solutions of chloroquine were prepared in water (Milli-Q

grade). Test compounds were dissolved in DMSO. All stocks were

then diluted with culture medium to achieve the required concen-

trations (in all cases, the final concentration of DMSO was 0.4%,

which was found to be nontoxic to the parasite). Compounds were

then placed in 96-well flat-bottomed tissue culture grade plates to

yield triplicate wells with drug concentrations ranging from 0 to

100 mm in a final well volume of 100 mL for primary screening. The

antimalarial activity of the compounds was measured by the fluo-

rescence-based SYBR green I assay against CQ-sensitive and CQ-re-

sistant strains.^[29]Parasite culture was synchronized at the ring

stage with 5% sorbitol. Synchronized culture was aliquoted to a

drug containing 96-well plates at 2% hematocrit and 1% parasite-

mia. After 48 h of incubation under standard culture conditions,

plates were harvested and read by the SYBR green I fluorescence-

based method by using a 96-well fluorescence plate reader (Victor,

PerkinElmer), with excitation and emission wavelengths at 485 and

2-Aryl-10H-indolo[3,2-b]quinoline-11-carboxamides

4a–c:

Pd(dppf)Cl₂(0.2 equiv) and arylboronic acid 20 (1.2 equiv) were

added to a solution of 10H-indolo[3,2-b]quinoline-C11-carboxamide

3a (1 equiv) in 1,4-dioxane/water 50:50). The mixture was heated

at reflux at 808C for 12 h. The solvent was evaporated, and the

product was extracted with EtOAc (3ꢂ50 mL). The combined or-

ganic layer was dried with anhydrous sodium sulfate and was con-

centrated under reduced pressure. The crude product was purified

by silica gel column chromatography (EtOAc/hexane) to give 2-aryl

10H-indolo[3,2-b]quinoline 4 (60–80%).

2-(4-Fluorophenyl)-N-(3-morpholinopropyl)-10H-indolo[3,2-

b]quinoline-11-carboxamide (4a, RMS-128): Yellow amorphous

solid; mp: 220–2218C; ¹H NMR (400 MHz, CDCl₃): d=9.31 (s, 1H),

8.56 (s, 1H), 8.45 (d, J=8.0 Hz, 1H), 8.40 (s, 1H), 8.35 (d, J=8.5 Hz,

1H), 7.84 (d, J=7.5 Hz, 1H), 7.67 (dd, J=5.0, 8.0 Hz, 2H), 7.57 (t,

J=7.5 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.32 (t, J=7.5 Hz, 1H), 7.19

(t, J=8.5 Hz, 2H), 3.81 (m, 2H), 3.07 (m, 3H), 2.89 (m, 1H), 2.55 (m,

2H), 2.25 (m, 4H), 1.88 ppm (m, 2H); ¹³C NMR (125 MHz,

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530 nm, respectively. The fluorescence readings were plotted

against drug concentration, and the IC₅₀values were obtained by

visual matching of the drug concentration giving 50% inhibition of

growth. ICEstimator 1.2 online software was also used to calculate

the IC₅₀and IC₉₀values.

Kinetics of 3g uptake by malaria parasite: Trophozoite-stage-en-

riched Pf3D7 culture (ꢂ4% parasitemia and 2% hematocrit,

200 mL) was incubated with 3g (50 mm) at 378C for different time

points (15 min to 6 h). After incubation for each time point, the

culture was washed twice with 1ꢂ PBS (500 mL) by using centrifu-

gation (1500 RPM, 2 min) and was reconstituted in 100 mL 1ꢂ PBS.

Wet mount of each sample was observed under fluorescence

(DAPI filter) and optical microscopy.

In vitro cytotoxicity in mammalian cell lines: Animal cell lines

(HUH-7 and HEK-293T) were used to determine drug toxicity by

using the MTT assay.^[30]HUH-7 and HEK-293T cells were cultured in

complete RPMI containing 10% fetal bovine serum, 0.2% sodium

bicarbonate, and 50 mgmL^ꢁ1gentamicin. Briefly, cells (10⁴cells per

200 mL per well) were seeded into 96-well flat-bottomed tissue-cul-

ture plates in complete culture medium. Compound solutions (in

all cases, the final concentration of DMSO was 0.4%) were added

after overnight seeding and were incubated for 24 h in a humidi-

fied atmosphere at 378C and 5% CO₂. DMSO (final concentration:

10%) was added as a positive control. An aliquot of a stock solu-

tion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) (5 mgmL^ꢁ1in 1ꢂ phosphate-buffered saline) was added at

20 mL per well, and the samples were incubated for another 3 h.

After spinning the plate at 1500 rpm for 5 min, supernatant was re-

moved and 100 mL of the stop agent DMSO was added to each

well. Formation of formazan, an index of growth, was read at l=

570 nm by a 96-well plate reader (Versa Max), and the IC₅₀values

were determined by analysis of dose–response curves. Selectivity

Colocalization of fluorescence from 3g (blue) with EtBr/Lyso-

Tracker (red): The parasite cultures were incubated with test com-

pounds (50 mm) for 3 h. After incubation, the cells were washed

with 1ꢂ PBS by centrifugation (1500 rpm, 2 min). Further, the

washed cells were incubated with EtBr stain (10 mgmL^ꢁ1, Sigma), a

DNA binding stain that emits red fluorescence, and LysoTracker

red (50 nm), a fluorophore that accumulates in acidic cell organ-

elles such as food vacuoles in separate experiments for 30 min at

378C. The cells were washed twice with 1ꢂ PBS (500 mL) by centri-

fugation (1500 rpm, 2 min) and were reconstituted in 1ꢂ PBS

(100 mL). After washing, wet mount of each sample was prepared

and observed under fluorescence and optical microscopy.

Inhibition of detergent-mediated b-hematin formation assay:

The detergent-mediated b-hematin formation method was adopt-

ed as previously described by Sandlin et al.^[22a]with minor modifi-

cations. A hemin solution (25 mm) was prepared by dissolving

hemin (Fluka, Switzerland) in DMSO, and the solution was filtered

through a 0.22 mm membrane syringe filter. This stock solution was

diluted by vortexing to prepare a 228 mm hemin suspension in 2m

acetate buffer (pH 4.9). A 348 mm solution of NPꢁ40 (Fluka, Swit-

zerland) was prepared in Sterile and filtered Milli-Q water. Stock

solutions of chloroquine (CQ, 50 mm) and test compounds

(25 mm) were prepared in water and DMSO, respectively. The

stocks of CQ and test compounds were diluted in their respective

solvents to achieve the required concentrations. In all cases, the

final concentration of DMSO was 4% (v/v), which showed no effect

on b-hematin formation. Test compounds (4 mL) were then placed

in triplicate wells of 96-well flat-bottomed plates followed by water

(31.25 mL), NPꢁ40 solution (8.75 mL), acetone (12.25 mL), and heme

suspension (43.75 mL) in a final well volume of 100 mL. The final

concentrations of the test compounds/CQ were 0, 125, 250, 500,

750, and 1000 mm, whereas additional concentrations up to 2 mm

were tested for CQ. Further, the plates were incubated (6 h) in a

shaking incubator (378C, 50 rpm), following which the samples in

the wells were processed by using the pyridine–ferrochrome

method by adding acetone (26.25 mL) and pyridine solution [(50%

pyridine, 20% acetone, and 200 mm HEPES, pH 7.4 in water)]

(14 mL). Then, the plates were kept under shaking for 30 min to dis-

solve the free heme. After incubation, the plates were read at l=

405 nm by using a SpectraMax multiwell plate reader. Inhibition of

b-hematin formation by test compounds was assessed by compar-

ing the positive (CQ) and negative controls (4% DMSO). Finally,

dose–responses curves were generated to calculate the IC₅₀values

of the test compounds.

index was calculated as IC_{50(mammalian cell)}/IC_50(Pf3D7)

.

Aqueous solubility, hrCYP P450 isoenzyme inhibition, and Caco-

2 permeability: The thermodynamic aqueous solubility in PBS

(pH 7.4), SGF (pH 1.2), and SIF (pH 6.8) was determined by using a

protocol described earlier.^[31]The hrCYP P450 inhibition of five

major isoenzymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and

CYP1A2, was determined at 10 mm as described earlier.^[32]Similarly,

the caco-2 permeability study was conducted as described earli-

er.^[32]

Microscopic evaluation of the stage-specific kill kinetics of 3g

against P. falciparum stages: To find if there was stage specificity

in the action of 3g, highly synchronized Pf3D7 cultures at ring (R),

trophozoite (T), and schizont (S) stages were treated with 3g at

the IC₉₀value (3.5 mm) by the drug pressure followed by drug with-

drawal method as described below. The ring-stage P. falciaprum

culture was tightly synchronized for two continuous life cycles by

using 5% sorbitol to maintain the synchronicity of the Plasmodium

stages, and the synchrony of each stage was confirmed by micros-

copy. One percent of synchronized early rings (0–6 h), early tropho-

zoites (ꢂ24 h post-synchronization), and early schizonts (ꢂ40 h

post-synchronization) was used for the stage-specific drug-action

studies. Ring-stage cultures were treated with the test sample at

the IC₉₀value for 12, 24, 36, and 48 h. Similarly, trophozoite-stage

(3, 6, 18, and 24 h) and schizont-stage (3, 6, and 12 h) cultures

were treated with the test sample at the IC₉₀value. Following drug

treatment, as indicated above, the drugs were withdrawn by cen-

trifugal removal of conditioned medium followed by media wash.

The washed cells were maintained under standard culture condi-

tions in drug-free cRPMI medium for a total of 96, 72, and 60 h for

ring, trophozoites, and schizonts, respectively. The percentage of

stage-specific inhibition of each treatment was calculated in com-

parison with drug-free control by microscopic counting of 3000

cells for each stage. Parasitized and non-parasitized cells were

counted by using Plasmodium auto count 0.1 software developed

by Ma et al.^[33]The parasites with pycnotic morphology were con-

sidered as nonviable cells.

Inhibition of hemoglobin uptake by 3g: Synchronized tropho-

zoite-stage (28 h post-invasion) P. falciparum 3D7 cultures (4% par-

asitemia, 2% hematocrit) were treated with the IC₉₀value of 3g

(3.5mm) for 15 h. The relative hemoglobin contents of treated and

untreated PRBCs were determined at A₅₄₀by following sorbitol-

mediated hemolysis. Identical amounts of treated and untreated

parasitized cultures (50 mL PCV) were subjected to sorbitol treat-

ment (50 mL PCV with 200 mL sorbitol, 10 min, 378C) to enable he-

molysis. Thereafter, samples were centrifuged (400 g, 5 min) and

supernatants were aspirated into fresh microfuge tubes. Further,

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the supernatant was centrifuged (2200 g, 7 min) to remove eryth-

rocyte ghosts. The supernatants were then transferred to wells of a

96-well plate, and absorbance of released hemoglobin was esti-

mated at l=540 nm by using an ELISA plate reader

weighed compounds were wetted with Tween 80 (2% final con-

centration) and were triturated in a mortar and pestle; then, 2%

hydroxypropyl methylcellulose in normal saline was slowly added

to make up a final volume of 1.1 mL. From this solution, each

mouse was administered 200 mL. Drug formulations were made on

each day of dosing. The control group was administered with

200 mL of vehicle solution [2% hydroxypropyl methylcellulose with

2% Tween 80 in normal saline]. All the mice groups were moni-

tored daily for their behavior. From the fifth day onward, thin

blood smears were prepared every alternate day till the end of the

experiment by using a drop of tail blood. Further, the blood

smears were stained with Giemsa and were evaluated under micro-

scope for percentage of parasitemia.

FACS analysis of inhibition of FITC–dextran endocytosis by

malaria parasite

1) Preparation of FITC–dextran-loaded resealed RBCs: The endo-

cytosis process of Plasmodium was measured by using parasites in-

fected into FITC–dextran-resealed RBCs. FITC–dextran-resealed

RBCs were obtained by incubating RBCs in four volumes of ice-

cold hypotonic buffer [sodium phosphate buffer (5 mm, pH 7.5),

Mg-ATP (1 mm) and FITC–dextran (50 mm)] for 10 min. NaCl was

added to a final concentration of 0.15m; incubation done for

45 min at 378C to allow resealing.^[34]The resealed cells were

washed with RPMI (2ꢂ) and were stored at 48C. The incorporation

of FITC–dextran into the RBCs was evident from the bright-green

fluorescent cells observed by fluorescence microscopy.

Abbreviations

CQ: chloroquine, Pf: Plasmodium falciparum, CYP: cytochrome

P450, SI: selectivity index, FBS: fetal bovine serum, PBS: phos-

phate-buffered saline, FCS: fetal calf serum, SGF: simulated gastric

fluid, SIF: simulated intestinal fluid, HRMS (ESI): high-resolution

electrospray ionization mass spectrometry, HPMC: hydroxypropyl

methylcellulose.

2) Isolation of late-stage parasites and incubation with FITC–

dextran-loaded resealed RBCs: Late-stage parasites isolated by

using 60% percol^[35]were incubated with resealed RBCs to facilitate

their invasion by merozoites.

3) Measurement of the effect of 3g on endocytosis: After inva-

sion, the 18 and 24 h cultures were incubated with or without 3g

(IC₉₀: 3.5 mm) for 6 h each. After 6 h of incubation, all the cultures

were subjected to saponin (0.025% w/v) lysis to release parasites

from RBCs. Further the released parasites were washed twice with

PBS and re-suspended in PBS for measurement of endocytosed

FITC–dextran by FACS analysis.

Acknowledgements

R.M. thanks the Council of Scientific & Industrial Research (CSIR)

for a research fellowship. The authors are thankful to the Analyti-

cal Department, Indian Institute of Integrative Medicine (IIIM) for

analytical support. The chemistry work was supported by the

CSIR 12^thFYP grant BSC-0205 and CSIR-Young Scientist Grant (P-

81–113). The authors declare no competing financial interest.

Pharmacokinetic analysis: The pharmacokinetic properties of 3g

were evaluated in male BALB/c mice following a single 10 mgkg^ꢁ1

oral dose or 1 mgkg^ꢁ1intravenous (i.v.) dose. For oral dose, 3g

was taken in normal saline containing Tween 80 and 0.5% (w/v)

methyl cellulose. The i.v. dose of 3g was prepared by taking it in a

mixture of DMSO/(solutol/absolute alcohol=1:1, v/v)/normal

saline=5:5:90 (v/v). Formulations were made on the same day of

dosing and were kept at room temperature. Prepared drug formu-

lations were administered to mice groups (12 mice per group) by

appropriate routes. After dosing, plasma samples were collected

from mice blood samples for 0–24 h. To extract the compounds

from the plasma sample, tert-butyl methyl ether (1.5 mL) was

added to plasma (50 mL), and the sample was vortexed for 5 min.

Then, the samples were centrifuged (14000 rpm, 5 min, 48C). After

centrifugation, the organic layer of the samples were separated

into a fresh tube and dried. The residues were reconstituted in ace-

tonitrile. Then, the samples were analyzed by LC–MS/MS by using

an Atlantis dC18 column [4.6ꢂ50 mm, 3 mm (Waters)] and a gradi-

ent of 0.2% formic acid and acetonitrile as the mobile phase. Fur-

ther, the results were analyzed for pharmacokinetic parameters by

using the WinNonlin 5.3 software package.

Keywords: alkaloids

·

antimalarial agents

·

fused-ring

systems · hemoglobin · parasites

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Manuscript received: August 27, 2018

Revised manuscript received: October 10, 2018

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Accepted manuscript online: October 24, 2018

Version of record online: && &&, 0000

&

ChemMedChem 2018, 13, 1 – 19

www.chemmedchem.org

18

ꢁ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

FULL PAPERS

Bug out: The discovery of orally bio-

available compound 3g possessing

promising in vitro and in vivo antimalar-

ial activity is reported. Compound 3g

targets the food vacuole of the parasite,

inhibits the uptake of host hemoglobin,

and also inhibits the basic endocytic

processes of the parasite. In a Plasmodi-

um berghei (ANKA)-infected mouse

model of malaria, mice treated with 3g

show suppression of parasitemia and in-

creased life span.

R. Mudududdla, D. Mohanakrishnan,

S. S. Bharate, R. A. Vishwakarma,

D. Sahal,* S. B. Bharate*

&& – &&

Orally Effective Aminoalkyl 10H-

Indolo[3,2-b]quinoline-11-carboxamide

Kills the Malaria Parasite by Inhibiting

Host Hemoglobin Uptake

ChemMedChem 2018, 13, 1 – 19

www.chemmedchem.org

19

ꢁ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

&

These are not the final page numbers! ÞÞ

Article Doi

Orally Effective Aminoalkyl 10H-Indolo[3,2-b]quinoline-11-carboxamide Kills the Malaria Parasite by Inhibiting Host Hemoglobin Uptake

DOI: 10.1002/cmdc.201800579

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Article abstract of DOI:10.1002/cmdc.201800579

Full text of DOI:10.1002/cmdc.201800579

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