PharmacoI. Thu. Vol. 76, Nos. 1-3, pp. 125-133,1997 Copyright 0 1997 Elsevier Science Inc.

ISSN 0163-7258197

$32.00

PI1 SO163-7258(97)WO95-8

ELSEVIER Associate Editor: D. Shugar

Antimalarial Activity of Synthetic Analogues of Distamycin

Paolo Lombardi * $ and Andrea Crisantit
*MENARINI RICERCHE, VIA TIT0 SPERI, 10, FOMEZIA, ROMA, ITALY +ISTITUTO DI PARASSITOLOGIA, UNlVERSlTzd

LASAPIENZA, P.LE ALDO MORO, 5,ROMA, ITALY

ABSTRACT. Malaria, one of the most serious diseases transmitted by arthropods, is largely present in tropical and even temperate zones in endemic or epidemic form. More than 40% of the worlds population lives in areas at risk for exposure, and the World Health Orga nization reports that approximately 300 million people are affected by the infection (mostly caused by the species Plasmodium fakipanrm), with 1-2 million deaths per year. These data, and the fact that malaria is becoming increasingly refractory to treatment through resistance of the parasite to antimalarial agents currently in use, e.g., chloroquine, emphasize the need to develop new drugs. The well-known antiparasitic activity of oligopyrrolamidine natural products, such as distamycin and netropsin, suggested the antimalarial evaluation of related compounds obtained by new chemical modifications. Besides possessing antiviral and antitumoural properties, distamycin exhibits interesting in vitro activity against P. @cigarurn. Unfortunately, the high toxicity associated with this product precludes its development as a drug. However, some synthetic analogues of distamycin proved to be highly active against chloroquine-sensitive and -resistant strains of P. falcipa~m, besides showing low toxicity in vitro. PHARMACOL. THER. 76( l-3):125-133, 1997. 0 1997 Elsevier Science Inc. KEY WORDS. CONTENTS 1. INTRODUCTION . . . . . . . . . . . . .
2. ANTIBIOTICPROPERTIES OF 125 Plasmodium fukiparum, malaria, distamycin, pytrole-amidine antibiotics.

OLIGOPEPTIDE-AMIDINE COMPOUNDS . . . . . . . . . . . . . . . 3. MALARIA, THE PROBLEM . . . . . . . .

125 128

4. NEW DISTAMYCINANAL~GUES . . 5. ANTIPROTOZOAL ACTIVITY . . . . 6. CONCLUSIONS AND PERSPECTIVE . ACKNOWLEDGEMENTS. . . . . . . . . REFERENCES . . . . . . . , . . . . . .

. . . . .

. . . . .

. . . . .

129 130 132 132 132

1. INTRODUCTION Distamycin (syn. distamycin A, stallimycin), et al., 1964, 1967), kikumycins a pyrrole-amiis a representacompounds, and antiviruses et al., with 1972; dine oligopeptide isolated from the mycelium of Saeptomyoccurring

with dA-dT-rich and Waring,

DNA sequences

(Lane et al., 1983; Fox

1984; Youngquist and Dervan, 1985; Neidle et studies of DNA complexes have demonstrated of adenine with distaof

al., 1987; Sapse et al., 1993). Crystallographic mycin and netropsin the importance and O(2)

ces distallicus (Arcamone which include netropsin, otic TAN-868 amidinomycin selectively (vaccinia, erentially

tive of a small group of naturally A, anthelvencins activities

A and B, the antibiantibiotic,

hydrogen bonds from the amide NH groups that bridge the strands to the exposed N(3) mine residues. Methylenes van der Waals nonbonded supported by NMR this interaction DNA structure with DNA-protein key biological transcription of thyin and ring CHs are involved

A and B, noformycin, (Hahn, 1975).

(Fig. l), and which exhibit the multiplication

viral, and antitumour inhibits

Distamycin

contacts with purine and pyrimet al., 1986). change Because in the In

of different (Verini

idine bases (Kopka et al., 1985). These findings have been studies (Klevit induces a conformational (Low et al., 1985), interactions (Gambari

herpes simplex, to dA-dT-rich

Rous sarcoma) sequences,

1976) by binding in the minor groove of duplex DNA prefthus interfering (Zimmer et al., sequences both replication dT)5 and (dA-dT),, tin, and transcription 1986). respectively,

it is likely to interfere et al., 1991).

Zimmer and Wahnert,

These

are (dAthat

addition, should this occur in genomic regions involved in activities, such as DNA replication is expected to contribute effects. and/or of cellular genes, DNA binding of distamycin greatly to

for netropsin and distamynegative potential and drug

and bear a high electrostatic 1990).

seems to play a key role in drug-DNA recognition DNA affinity (Pullman, Although (Denny, the biochemical mechanism

and related compounds their overall cytotoxic

of action of dista2. ANTIBIOTIC Netropsin PROPERTIES OF COMPOUNDS congocidine) originally was

mycin and related compounds

is not adequately understood

1988), recent data suggest that these drugs are caat points in the minor groove
G.

pable of forming reversible complexes with a high degree of selectivity by making contact

OLIGOPEPTIDE-AMIDINE (syn. sinanomycin,

isolated from a strain of Streptomyces netropsis (Finlay et al.,

K&rent address for corresponding author: lstituto Biochimico It&no Lorenzini S.p.A., via di Fossignano, 2, 04011 Aprilia, Latina, Italy.

1951) and Streptomyces chromogenus (Isono et al., 1955). The antibiotic exhibits an inhibitory effect on the growth

126

P. Lombardi and A. Crisanti No activities against fungi, yeasts, viruses, or parasites have been reported. By contrast, the antibiotic TAN-868 A, isolated from the culture broth of Streptomyces idiomorphus sp. nov., is active against bacteria, fungi, and a protozoan, and has cytotoxic activity against murine tumour cells (Takizawa et al., 1987). The anthelvencins, isolated from the strain Streptomyces wenezuelue (Probst et al., 1965), have only modest activity against a wide spectrum of bacteria and some fungi, but are effective as antiprotozoal and antihelminthic agents against E: histolytica, Syphacia obplelata, Aspicularis tetraptera, Ascaris suum, and Thricuris suis. The antimicrobial effects and the structure-activity relationship of

of several gram-positive and gram-negative microorgan, isms, myco-bacteria, and viruses (Schabel et al., 1953). Nevertheless, the major interest of this compound rests in its trypanocide action, particularly against Trypanosoma congolense (hence the synonym), a parasite resistant to most chemotherapeutic agents active against the two related species Trypanosoma brucei and Trypanosoma equiperdum (Cosar et al., 1952). The compound is an effective antiprotozoal against infections by Entamoeba histolytica. Kikumycins are antibiotics produced by the strain Streptomyces phaeochromogenes R-719 (Kikuchi et al., 1965).. They are effective against gram-positive and gram-negative microorganisms.

Kikumych A (R=H) and B (R=CH3)

*Ha

Antibiotic TAN-868 A

Anthehencin A (R=H) and B (R=CH3)

FIGURE 1. Structures of naturally occurring oligopeptide-amidine antibiotics.

Antimalarial

Activity of Disramycin and Analogues

FIGURE

2. Structures

of synthetic

N-formimidoyl

analogues (14)

and carbamoyl

analogues (S-7)

of distamycin.

128 3. MALAAZA, THEE2ROBLEM

P. Lombardi and A. Crisanti

amidinomycin and synthetic stereoisomers have been discussed (Sung et al., 1997). The somewhat high toxicity of the amidine oligopeptide antibiotics did not permit their clinical use, although a top ical preparation of distamycin, named HerperalB, was formulated by Farmitalia (now Pharmacia & Upjohn, Nerviano, Milan, Italy) as an antiherpetic, but was not launched on the market. More recently, the in vitro sensitivity of the malaria parasite P. falciparum to distamycin was reported independently by several groups (Mongelli and Spreafico, 1991; Lee and Inselburg, 1993; Ginsburg et al., 1993).

Malaria is one of the most serious diseases transmitted by arthropods, and is present largely in tropical and even temperate zones in endemic or epidemic form. More than 40% of the worlds population lives in areas at risk for exposure, and the World Health Organisation reports that approximately 300 million people are affected by the infection, with l-2 million deaths per year. Although malaria is no longer present in Europe, the number of cases diagnosed in the Western World is increasing as a consequence of extensive travel and immigration. Such cases of imported ma-

8
V

. 2HCI L V

"zN q;
2

.2HCI

2 HCI

vi, v
2N

FIGURE 3. Synthesis of oligopyrrole backbones. Reagents and conditions:

furan; iii, Peaminopropionitrile, diisopropylethylamine; iv, HCl, NH,;

i, HN03, acetic anhydride; ii, thionyl chloride, tetrahydrov, Hz, 10% Id/C; vi, 8, diisopropylethylamine.

Antimalarial Activity of Distamycin and Analogues laria are at risk of misdiagnosis sometimes female mosquitoes and delayed treatment, by drugs, hampering effective disease control.

129 The search for of action dif-

with fatal results. The disease is transmitted

active agents whose structure and mechanism fer from that of drugs currently chloroquine most recent

of the genus Anopheks, and it is caused The parasites infirst in the liver, stages of by feedP. wiuax, infected

in use, such as quinine,

by minute parasitic protozoa of the genus Plasmodium, which infect human and insect hosts alternately. mans, the parasites multiply dramatically, development. Other mosquitoes become fect a human while the mosquito is feeding on blood. In huthen in the blood, where they undergo different ing from an infected P. ovale, and P. m&r&

and derivatives, the sulphonamides, and the artemisinine and derivatives, might facilitate of a novel therapeutic arsenal to control

the development

the spread of resistant strains of P. falciparum.

human and spread the disease. Four

4. NEW

DISTAMYCIN

ANALOGUES activity of distamycin has that the genome of P. f&idifferent verte-

species of malaria parasites infect humans. Three, rarely fatal, albeit debilitating. The fourth,

The finding of the antiprotozoal its rationale in the observation

, may cause severe illness, but are

P. fulcipurum, of P.

parum (but not of other species affecting

causes much more serious and progressive illness, leading to coma and death within a few days. The development of human malaria. Infected surface knobs that contain dothelial cell membrane, erythrocytes a histidine-rich falciparum in the blood differs from that of the other species develop on their protein secreted of the en-

brates) is extremely rich (82%) in dA-dT base pairs (Weber, 1987, 1988), while that of the human host consists of 59% dA-dT. Moreover, during the erythrocytic stage, the parasite has a growth rate, and therefore, rate of DNA synthesis, that is more like malignant than normal human cells. Hence, a ligand possessing a specific dA-dT bias could be more inhibitory to the parasite than to its human host. Nevertheless, the low therapeutic index of distamycin, due to general toxicity caused by degradation products, may once more prevent its development as a drug. We have reviewed the results of screening distamycin against I. falciparum of synthetic that eventually led to identification analogues of of a com-

by the parasite. This interacts with a component

causing the red blood cell to adin brain blood

here. This process, called sequestration, is particularly intense in the brain, causing a measurable reduction flow (Knell, antimalarial 1991). has been the mainstay of chemotherapy, due to its efficacy and low toxFor a long time, chloroquine

icity. Because of the rapid spreading of chloroquine-resistant malaria since the early 1960s in most tropical areas, resulting from the remarkable and the use of antimalarials quate routine treatment areas, the chemotherapy adaptability for prophylaxis of P. falciparum and for inadefevers in endemic and more toxic

pound that may merit specific clinical trials as an antiprotozoal agent (Lombardi et al., 1994; Alfieri et al., 1997). In the course of investigations chemotherapeutic distamycin action and favorable therapeutic analogues l-7 aimed at developing new of agents endowed with high specificity

of undiagnosed less efficient

index, we synthesized the is replaced

of malaria since then has had to rely

(Fig. 2) in which the N-formyl

on the use of comparably

group, a typical structural feature of distamycin,

2 HC3XOOCH3 + CH3NHOH.HCI 1_

. ciq 3 q+
3

FIGURE4. Synthesis of the carbamoyl unit 9. Reagents and

conditions: i, NaOH; ii, toluene, reflwc; iii, oleum H,SO,; iv, thionyl chloride; v, N&OH, vi, NaOH, HCl; vii, trichloroacetyl chloride, methylene chloride; viii, AICI,, dichloromethylmethylether; ix, KMnO,; x, methanol, triethylamine.
H2 iv

... 111 1

H I \
COOCH3

/
=Q

\ COOH AH3 v
vi

X c;y

ix

Q
I\
!Hs

vii _I_) /\ Q\ coccl3

Vlll

...
I -A

t X
\ cocci3

AH3

AH3

130

P. Lombardi and A. C&anti are known (Di Pietro et al., 1996; Alfieri et al., 1997). For compounds 5 and 6, studies have been made of their binding to oligodeoxynucleotide duplexes by two-dimensional, high resolution lH NMR. Both compounds bind strongly to the central dA,dT, sequence of the dodecamer d(CGCAAATTTGCG)z, and 6, particularly, gives rise to a single, stable complex with 2:l stoichiometry (Animati et al., 1995). Extensive DNase I foot-printing experiments revealed that the carbamoyl analogues 5-7 bind to dA-dT-rich sequences and that 5 gives weaker footprints than 6 and 7, whereas few significant differences can be seen between the latter two (Alfieri et al., 1997).

either by a N-formimidoyl moiety (14) or by a carbamoyl group (S-7). More precisely, compounds l-3 bear an additional positively charged amidine group at the N-terminus, whereas compound 4 bears a primary amide group at the Cterminus and an amidine group at the N-terminus. Moreover, compounds 1, 3,6, and 7 present a different number of pyrrole-derived units with respect to distamycin. The synthesis of compounds l-7 entails the preparation of the oligopyrrole backbones with the required number of pyrrolecarboxamido units, which are obtained from 8 by an iterative process (Fig. 3), and the preparation of the carbamoylpyrrole unit 9 (Fig. 4). The oligopyrrole intermediates are then condensed with formamidine hydrochloride or ethyl formamidate to give N-formimidoyl analogues l-4 (Fig. 5, top) or with 9, activated as acyl chloride or with standard condensing agents, to yield carbamoyl analogues 5-7 (Fig. 5, bottom). Data concerning DNA affinity, as well as antiherpetic activity and cytotoxicity of the above described compounds.

5. ANTIPROTOZOAL ACTIVITY
The antiprotozoal activities of distamycin, and of compounds 1-7, against the chloroquine-sensitive IT04 strain of P. falcipurum were measured by incubating unsynchronized cultures of the parasite in erythrocytes with the drugs

b?N

AcjNH2 .2HCl % n
n=3
i t

I
3

-&NH2

.2HCI

HzN
I I

-<.
NH n

2HCI

I
-f,
n

PH2 .2HCl
NH

l-3

n = 1,3,4

HCI

5-7

n = 2,3,4

FIGURE 5. Synthesis of N-formimidoyl analogues (14) and carbamoyl analogues (S-7) of distamycin. Reagents and Conditions: i, NaOH, methanol; ii, formamidine hydrochloride, NaHCO,, methanol, reflex or ethyl formamidate hydrochloride, NaHC03, ethanol; iii, the acyl chloride of 9, diisopropylethylamine.

Antimalarial

Activity

of Distamycin

and Analogues

131
and Calf Thymus DNA-Binding Properties of Distamycin and Distamycin

TABLE

1. Antiprotozoal,

Antiviral,

Cytotoxic

Activities,

Analogues (l-7) IQ, Compound

Distamycin N-formimodoyl analogues 1 2 3 4 Carbamoyl analogues 5 6 7

($4
HSV-1 11.2 HF Hep-2 32.9 I<, (M-1 26.5

DNA-binding Parameters
x 106)
rb yDD

P. fakiparum
0.7-1.3

(M-1 28.0

x 105)

0.106

24 1.45 0.3 1.16

>400 26.1 26.0 >400

>400 17.3 130.0 >400

8.22 94.3 20.0

(no binding) 0.130 0.215 0.057

10.7 203.0 11.4

1 .OO-1.32 0.4-0.7 0.25

>lOO 27 8.6

>lOO >400 >400

2.27 3.47 8.35

0.118 0.123 0.083

2.68 4.27 6.93 hind-

required to reduce a given biological effect by 50%; K_ intrinsic binding constant; r,,, stoichiometric Q0, inhibition dose, i.e., the drug concentration ing ratio, i.e., number of molecules bound per nucleotide base; K,,,, apparent binding constant, i.e., the product K,> X rh. HSV, herpes simplex virus.

for 20 hr. The frequency of parasitized cells in cultures was determined by microscopic examination of Giemsa-stained fields were culsmears. The data collected separate experiments examined represent average values from 3

carbamoyl analogues 5-7, chemical

most likely related to their higher (Alfieri et al.,

stability compared with distamycin

1997), and their remarkable antiprotozoal activity prompted further biological I. f&pawn percent inhibition assays against the chloroquine-resistant 2 lists comparative parasitemia and values of chloroquine, (IT04) distamycin, (Kl) strain. Table

in which 50 microscope

for each culture, and were plotted as dose-effect

curves from which ID50 values were estimated. Control thylsulphoxide) In Table or medium. activities

tures were treated with the same quantity of solvent (dime1, the antimalarial of the compounds

carbamoyl analogues 5-7 chloroquine-sensitive

at three decreasing doses against and -resistant strains.

With the resistant strain, both 6 and 7 exhibit a remarkably high inhibition of parasitemia at a dose at which chloroquine and the other compounds tested appear almost ineffective. analogue 7, by The superior activity of the 5-ring carbamoyl

are shown and compared with the respective IDS0 values for their cytotoxicity, viral cytopathic expressed as cell proliferation in Hep2 cells, activity, expressed as reduction of strain HF). The calf bound per the effect on the same cell line infected with parameters, rb (number expressed as K, (intrinof molecules affinity constant, and for their antiherpetic

relative to the 4-ring homolog 6, was further confirmed were synchronized (Lambros and Vanderberg, Data collected

Herpes simplex (herpes simples virus-l, thymus DNA-binding sic binding constant), nucleotide

assaying the two compounds in cultures where the parasites 1979) to the same stage of development. at a single dose

base), and KaPP (apparent

of 1 kg/mL, but at decreasing times (8, 6, 4, 2 hr) of exposure to the drug, indicate for compound 7 a faster inhibiting action. Finally, a comparative compound evaluation of chloroquine against the and the carbamoyl 7 for activities

product K,, * r,,), which is considered as a measure of the binding affinity, are also included. The general picture that emerges from an analysis of the biological tin and the related synthetic larial than antiherpetic affinity further confirm activities is that distamycompounds are better antima-

agents. The data for DNA-binding that the binding process is domiand van der Waals contacts, analogues 5-7 have a series, by the number of pyrroleTABLE2. Antiprotozoal Activity of Distamycin and of Carbamoyl Distamycin Analogues (S-7) against ChloroquineSensitive (IT04) and Chloroquine-Resistant (Kl) Strains of P. fakiparum
IT04 5 Compound Chloroquine Distamycin 5 6 7 100 100 94 100 100 1 (kg/mL) 100 87 54.7 86 100 85 8 11 20.2 64.7 100 100 100 100 100 0.2 5 Kl 1 ( w/mL) 76 78 53.1 94 100 2.7 12 1.5 53 71 0.2

nated by hydrogen bonding hence, within homologous carboxamido

units. The carbamoyl

lower affinity for calf thymus DNA than distamycin, indicating that the formylamino group plays a specific role in stabilizing the DNA complex, similar to that of the N-formimidoyl group of analogues 2-4. Nonetheless, the most striking result forthcoming from Table 1 is that the antiprotozoal activity against P. fulcipurum of all the listed compounds units of backbone, irrespective of the type of funcstrictly follows the number of pyrrolecarboxamido the oligopeptide

tional groups present on the termini of the molecule, of their antiviral and cytotoxic activities, or of their calf thymus DNA-binding properties. The very low cytotoxicity of the

Values are expressed as percent parasitemia inhibition, controls, evaluated after 20 hr of exposure to the drug.

with respect to

132

P. Lombardi and A. Crisanti

,I
.a !3 .x P

3-

I I

..
*.

chloroquine *. l.
l.

0 - - - - -0
n

i i

0 -.

l.

*..

compound 7

*.

\\

0..
W

-I -.

L O

*c

-.0

FIGURE 6. Comparative evaluation of the activities of chloroquine and carbamoyl diitamycin analogue 7 against the chloroquine-resistant Kl strain of P. fakiparum. Dose effect curves at 4-hr exposure to the drugs. Effect is reported as absolute values of parasitemia (percent of infected cells). M, medium; DMSO, dimethylsulphoxide.

IM

I DMSO

1 0.2

1 1

PglmL
carbamoyl rapid analogue 7. This compound antiparasitic action on the shows a chloroaf-

chloroquine-resistant in synchronized

strain of P. fakiparum was carried out curves that this

the 5ring potent ter

cultures. Data plotted as dose-effect

and

at a 4-hr exposure to the drugs (Fig. 6) demonstrate tent action than chloroquine. 6. CONCLUSIONS Malaria still constitutes AND PERSPECTIVES

quine-resistant

strain of P. falci~urum (100% clinical importance,

inhibition

time is sufficient for compound 7 to exert in vitro a more po-

4 hr

at 1 FglmL) experiments

and greatly improved the therapeutic which warrants ad7 represents the agents, and in uioo. Compound

index of potential ditional lead compound

for a new class of antimalarial

a serious medical problem worldthis disease. Chloroand

possibly a good candidate for further development.

Acknowledgements-Financial support for rhts research from A. Menarini (Industrie Farmaceutiche Riunite), Bristol-Myers Squibb (Italy), and Istituto Mobiliare Italian0 (Grant N 53658) is gratefully acknowledged. The authors acknowledge the contributions of F. Animati, F. Arcamone, G. Di
Pietro, G. Giannini, P. Feliceh, E. lafrate, I. Reckmann, and C. Rossi.

wide, despite strong efforts to control prophylactic

quine has been the drug of choice both for treatment bility. Quinine,

use, and because of low cost and good tolerarare and toxic, has been in use only in spemeflohas led and to are expensive and also have very

cialized centers. The new and effective antimalarials quine and halofantrine to the increasing emergence serious side effects. Moreover, development misuse of chloroquine of cross resistance

of new resistant strains of P. fulcipurum, which hopes for ready develfor the

limit their use. This and diminishing

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