PharmacoI. Thu. Vol. 76, Nos. 1-3, pp. 125-133,1997

Copyright 0 1997 Elsevier Science Inc.

ELSEVIER

Associate Editor: D. Shugar

ISSN 0163-7258197 $32.00

PI1 SO163-7258(97)WO95-8

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 “LA SAPIENZA”, 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 world’s 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. Plasmodium fukiparum, malaria, distamycin, pytrole-amidine antibiotics.

CONTENTS

1. INTRODUCTION . . . . . . . . . . . . . 125

2. ANTIBIOTIC PROPERTIES OF OLIGOPEPTIDE-AMIDINE COMPOUNDS . . . . . . . . . . . . . . . 125

3. MALARIA, THE PROBLEM . . . . . . . . 128

1. INTRODUCTION

Distamycin (syn. distamycin A, stallimycin), a pyrrole-ami- dine oligopeptide isolated from the mycelium of Saeptomy- ces distallicus (Arcamone et al., 1964, 1967), is a representa- tive of a small group of naturally occurring compounds, which include netropsin, kikumycins A and B, the antibi- otic TAN-868 A, anthelvencins A and B, noformycin, and amidinomycin (Fig. l), and which exhibit antibiotic, anti- viral, and antitumour activities (Hahn, 1975). Distamycin selectively inhibits the multiplication of different viruses (vaccinia, herpes simplex, Rous sarcoma) (Verini et al., 1976) by binding in the minor groove of duplex DNA pref- erentially to dA-dT-rich sequences, thus interfering with both replication and transcription (Zimmer et al., 1972; Zimmer and Wahnert, 1986). These sequences are (dA- dT)5 and (dA-dT),, respectively, for netropsin and distamy- tin, and bear a high electrostatic negative potential that seems to play a key role in drug-DNA recognition and drug DNA affinity (Pullman, 1990).

Although the biochemical mechanism of action of dista- mycin and related compounds is not adequately understood (Denny, 1988), recent data suggest that these drugs are ca- pable of forming reversible complexes with a high degree of selectivity by making contact at points in the minor groove

K&rent address for corresponding author: lstituto Biochimico It&no G.

Lorenzini S.p.A., via di Fossignano, 2, 04011 Aprilia, Latina, Italy.

4. NEW DISTAMYCIN ANAL~GUES . . . . . 129 5. ANTIPROTOZOAL ACTIVITY . . . . . . . 130 6. CONCLUSIONS AND PERSPECTIVE . . . . 132 ACKNOWLEDGEMENTS. . . . . . . . . . . . 132 REFERENCES . . . . . . . , . . . . . . . . . 132

with dA-dT-rich DNA sequences (Lane et al., 1983; Fox and Waring, 1984; Youngquist and Dervan, 1985; Neidle et al., 1987; Sapse et al., 1993).

Crystallographic studies of DNA complexes with dista- mycin and netropsin have demonstrated the importance of hydrogen bonds from the amide NH groups that bridge the strands to the exposed N(3) of adenine and O(2) of thy- mine residues. Methylenes and ring CHs are involved in van der Waals nonbonded contacts with purine and pyrim- idine bases (Kopka et al., 1985). These findings have been supported by NMR studies (Klevit et al., 1986). Because this interaction induces a conformational change in the DNA structure (Low et al., 1985), it is likely to interfere with DNA-protein interactions (Gambari et al., 1991). In addition, should this occur in genomic regions involved in key biological activities, such as DNA replication and/or transcription of cellular genes, DNA binding of distamycin and related compounds is expected to contribute greatly to their overall cytotoxic effects.

2. ANTIBIOTIC PROPERTIES OF OLIGOPEPTIDE-AMIDINE COMPOUNDS

Netropsin (syn. sinanomycin, congocidine) originally was isolated from a strain of Streptomyces netropsis (Finlay et al., 1951) and Streptomyces chromogenus (Isono et al., 1955). The antibiotic exhibits an inhibitory effect on the growth

126

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 con- golense (hence the synonym), a parasite resistant to most chemotherapeutic agents active against the two related spe- cies 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 phaeochro- mogenes R-719 (Kikuchi et al., 1965).. They are effective against gram-positive and gram-negative microorganisms.

P. Lombardi and A. Crisanti

No activities against fungi, yeasts, viruses, or parasites have been reported. By contrast, the antibiotic TAN-868 A, iso- lated 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 (Tak- izawa 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 antihelm-

inthic agents against E: histolytica, Syphacia obplelata, Aspicu- laris tetraptera, Ascaris suum, and Thricuris suis. The antimi-

crobial effects and the structure-activity relationship of

*Ha

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

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

amidinomycin and synthetic stereoisomers have been dis- cussed (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 for-

mulated by Farmitalia (now Pharmacia & Upjohn, Nervi-

ano, 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).

P. Lombardi and A. Crisanti

3. MALAAZA, THEE2ROBLEM

Malaria is one of the most serious diseases transmitted by

arthropods, and is present largely in tropical and even tem- perate zones in endemic or epidemic form. More than 40%

of the world’s population lives in areas at risk for exposure, and the World Health Organisation reports that approxi-

mately 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 exten- sive travel and immigration. Such cases of “imported ma-

8 V

"zN .

2HCI L V ‘q; .2HCI

2

2 HCI

vi, v

“2N

FIGURE 3. Synthesis of oligopyrrole backbones. Reagents and conditions: i, HN03, acetic anhydride; ii, thionyl chloride, tetrahydro-

furan; iii, Peaminopropionitrile, diisopropylethylamine; iv, HCl, NH,; v, Hz, 10% I’d/C; vi, 8, diisopropylethylamine.

Antimalarial Activity of Distamycin and Analogues

laria” are at risk of misdiagnosis and delayed treatment, sometimes with fatal results. The disease is transmitted by

female mosquitoes of the genus Anopheks, and it is caused by minute parasitic protozoa of the genus Plasmodium, which infect human and insect hosts alternately. The parasites in- fect a human while the mosquito is feeding on blood. In hu- mans, the parasites multiply dramatically, first in the liver, then in the blood, where they undergo different stages of development. Other mosquitoes become infected by feed- ing from an infected human and spread the disease. Four species of malaria parasites infect humans. Three, P. wiuax, P. ovale, and P. m&r& , may cause severe illness, but are rarely fatal, albeit debilitating. The fourth, P. fulcipurum, causes much more serious and progressive illness, leading to coma and death within a few days. The development of P. falciparum in the blood differs from that of the other species of human malaria. Infected erythrocytes develop on their surface knobs that contain a histidine-rich protein secreted by the parasite. This interacts with a component of the en- dothelial cell membrane, causing the red blood cell to ad- here. This process, called sequestration, is particularly intense in the brain, causing a measurable reduction in brain blood flow (Knell, 1991).

For a long time, chloroquine has been the mainstay of antimalarial chemotherapy, due to its efficacy and low tox- icity. Because of the rapid spreading of chloroquine-resis- tant malaria since the early 1960s in most tropical areas, re- sulting from the remarkable adaptability of P. falciparum and the use of antimalarials for prophylaxis and for inade- quate routine treatment of undiagnosed fevers in endemic areas, the chemotherapy of malaria since then has had to rely on the use of comparably less efficient and more toxic

129

drugs, hampering effective disease control. The search for active agents whose structure and mechanism of action dif- fer from that of drugs currently in use, such as quinine,

chloroquine and derivatives, the sulphonamides, and the most recent artemisinine and derivatives, might facilitate the development of a novel therapeutic arsenal to control the spread of resistant strains of P. falciparum.

4. NEW DISTAMYCIN ANALOGUES

The finding of the antiprotozoal activity of distamycin has its rationale in the observation that the genome of P. f&i- parum (but not of other species affecting different verte- 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 para- site 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 in- hibitory to the parasite than to its human host. Neverthe- less, the low therapeutic index of distamycin, due to general toxicity caused by degradation products, may once more pre- vent its development as a drug. We have reviewed the results of screening against I’. falciparum of synthetic analogues of distamycin that eventually led to identification of a com- pound that may merit specific clinical trials as an antiproto- zoal agent (Lombardi et al., 1994; Alfieri et al., 1997).

In the course of investigations aimed at developing new chemotherapeutic agents endowed with high specificity of action and favorable therapeutic index, we synthesized the distamycin analogues l-7 (Fig. 2) in which the N-formyl group, a typical structural feature of distamycin, is replaced

FIGURE4. Synthesis of the car- bamoyl unit 9. Reagents and

conditions: i, NaOH; ii, toluene,

reflwc; iii, oleum H,SO,; iv, thio- nyl chloride; v, N&OH, vi,

NaOH, HCl; vii, trichloroacetyl

chloride, methylene chloride; viii,

AICI,, dichloromethylmethyl-

ether; ix, KMnO,; x, metha-

nol, triethylamine.

2 HC3XOOCH3 .

+

CH3NHOH.HCI 1_ ciq q+

3 3

1

. . . 111

H2

=Q

iv H

/ \ I \ COOH v

AH3 vi X COOCH3

c;y

9

t

ix X . . .

Q I\ vii

_I_) /\

Q\

Vlll I \

coccl3

!Hs AH3

-A cocci3

AH3

130

either by a N-formimidoyl moiety (14) or by a carbamoyl group (S-7). More precisely, compounds l-3 bear an addi- tional positively charged amidine group at the N-terminus, whereas compound 4 bears a primary amide group at the C- terminus and an amidine group at the N-terminus. More- over, 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 car-

bamoylpyrrole unit 9 (Fig. 4). The oligopyrrole intermedi-

ates 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.

b?N

HzN

t i

AcjNH2 %

.2HCl

n

n=3

-<. 2HCI NH

n

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 bind- ing to oligodeoxynucleotide duplexes by two-dimensional, high resolution lH NMR. Both compounds bind strongly to the central dA,dT, sequence of the dodecamer d(CG- CAAATTTGCG)z, and 6, particularly, gives rise to a sin-

gle, stable complex with 2:l stoichiometry (Animati et al., 1995). Extensive DNase I foot-printing experiments re-

vealed 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).

5. ANTIPROTOZOAL ACTIVITY

The antiprotozoal activities of distamycin, and of com- pounds 1-7, against the chloroquine-sensitive IT04 strain of P. falcipurum were measured by incubating unsynchro- nized cultures of the parasite in erythrocytes with the drugs

HCI

5-7 n = 2,3,4

4

l-3

: I I I I

-&NH2

B .2HCI

3

PH2 -f, .2HCl

NH

n

n = 1,3,4

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

TABLE 1. Antiprotozoal, Antiviral, Cytotoxic Activities, and Calf Thymus DNA-Binding Properties of Distamycin and Distamycin Analogues (l-7)

IQ, ($4 DNA-binding Parameters

Compound P. fakiparum HSV-1 HF Hep-2 I<, (M-1 x 106) rb yDD (M-1 x 105)

Distamycin

N-formimodoyl

analogues 1

2

3

4

Carbamoyl

analogues

5

6

7

0.7-1.3 11.2 32.9

24 >400 >400

1.45 26.1 17.3

0.3 26.0 130.0

1.16 >400 >400

1 .OO-1.32 >lOO >lOO

0.4-0.7 27 >400

0.25 8.6 >400

26.5

8.22

94.3

20.0

2.27

3.47

8.35

0.106

(no binding)

0.130

0.215

0.057

0.118

0.123

0.083

28.0

10.7

203.0

11.4

2.68

4.27

6.93

Q0, inhibition dose, i.e., the drug concentration required to reduce a given biological effect by 50%; K_ intrinsic binding constant; r,,, stoichiometric hind- 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 smears. The data collected represent average values from 3 separate experiments in which 50 microscope fields were examined for each culture, and were plotted as dose-effect curves from which ID50 values were estimated. Control cul- tures were treated with the same quantity of solvent (dime- thylsulphoxide) or medium.

In Table 1, the antimalarial activities of the compounds are shown and compared with the respective IDS0 values for their cytotoxicity, expressed as cell proliferation in Hep2 cells, and for their antiherpetic activity, expressed as reduction of viral cytopathic effect on the same cell line infected with Herpes simplex (herpes simples virus-l, strain HF). The calf thymus DNA-binding parameters, expressed as K, (intrin- sic binding constant), rb (number of molecules bound per nucleotide base), and KaPP (apparent affinity constant, the product K,, * r,,), which is considered as a measure of the bind- ing affinity, are also included. The general picture that emerges from an analysis of the biological activities is that distamy- tin and the related synthetic compounds are better antima- larial than antiherpetic agents. The data for DNA-binding affinity further confirm that the binding process is domi- nated by hydrogen bonding and van der Waals contacts, hence, within homologous series, by the number of pyrrole- carboxamido units. The carbamoyl analogues 5-7 have a lower affinity for calf thymus DNA than distamycin, indicat- ing that the formylamino group plays a specific role in stabi- lizing the DNA complex, similar to that of the N-formimidoyl group of analogues 2-4. Nonetheless, the most striking re- sult forthcoming from Table 1 is that the antiprotozoal ac- tivity against P. fulcipurum of all the listed compounds strictly follows the number of pyrrolecarboxamido units of the oligopeptide backbone, irrespective of the type of func- 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

carbamoyl analogues 5-7, most likely related to their higher chemical stability compared with distamycin (Alfieri et al., 1997), and their remarkable antiprotozoal activity prompted further biological assays against the chloroquine-resistant I’. f&pawn strain. Table 2 lists comparative parasitemia percent inhibition values of chloroquine, distamycin, and carbamoyl analogues 5-7 at three decreasing doses against chloroquine-sensitive (IT04) and -resistant (Kl) 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. The superior activity of the 5-ring carbamoyl analogue 7, relative to the 4-ring homolog 6, was further confirmed by assaying the two compounds in cultures where the parasites were synchronized (Lambros and Vanderberg, 1979) to the same stage of development. Data collected at a single dose of 1 kg/mL, but at decreasing times (8, 6, 4, 2 hr) of expo- sure to the drug, indicate for compound 7 a faster inhibiting action. Finally, a comparative evaluation of chloroquine and the carbamoyl compound 7 for activities against the

TABLE2. Antiprotozoal Activity of Distamycin and of Carbamoyl Distamycin Analogues (S-7) against Chloroquine- Sensitive (IT04) and Chloroquine-Resistant (Kl) Strains of

P. fakiparum

Compound

IT04 Kl

5 1 0.2 5 1 0.2

(kg/mL) ( w/mL)

Chloroquine 100 100 85 100 76 2.7

Distamycin 100 87 8 100 78 12

5 94 54.7 11 100 53.1 1.5

6 100 86 20.2 100 94 53

7 100 100 64.7 100 100 71

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

132 P. Lombardi and A. Crisanti

,I FIGURE 6. Comparative eval-

.a I

uation of the activities of chlo-

3- !3 I

roquine and carbamoyl diitamycin . . analogue 7 against the chloro-

.x - *. chloroquine 0 - - - - -0

P 2 ’

*. l . l . compound 7 n - n

\\

quine-resistant Kl strain of P. fakiparum. Dose effect curves

i 0 -. at 4-hr exposure to the drugs. l . Effect is reported as absolute

i *. ‘.

*. values of parasitemia (percent

‘3 ‘0..

of infected cells). M, medium;

-I DMSO, dimethylsulphoxide. W -. *c

L -.

0

O I- -

I 1 1 I 1

M DMSO 0.2 1 5 PglmL

chloroquine-resistant strain of P. fakiparum was carried out

in synchronized cultures. Data plotted as dose-effect curves at a 4-hr exposure to the drugs (Fig. 6) demonstrate that this time is sufficient for compound 7 to exert in vitro a more po- tent action than chloroquine.

6. CONCLUSIONS AND PERSPECTIVES

Malaria still constitutes a serious medical problem world- wide, despite strong efforts to control this disease. Chloro- quine has been the drug of choice both for treatment and prophylactic use, and because of low cost and good tolera- bility. Quinine, rare and toxic, has been in use only in spe- cialized centers. The new and effective antimalarials meflo- quine and halofantrine are expensive and also have very serious side effects. Moreover, misuse of chloroquine has led to the increasing development of cross resistance and to emergence of new resistant strains of P. fulcipurum, which limit their use. This and diminishing hopes for ready devel- opment of a malaria vaccine dictate that the control of this disease will have to rely again on chemotherapy for the foreseeable future. Many potentially interesting new targets have been identified in P. fulci~arum in recent years (Mur- ray and Perkins, 1996), and a great part of strategies in ma- laria control are now focused on how to overcome the prob- lem of resistance (Rasoanaivo et al., 1996).

The use of distamycin as an antiviral or anticancer drug has been abandoned due to its high toxicity. For the same reason, the antiparasitic properties exhibited by some oli- gopeptide-amidine antibiotics have been hurriedly neglected, hampering any perspective of development. The selectivity against malarial parasites suggests that distamycin should be now considered as a novel antimalarial with a new mecha- nism of action. The selective antimalarial effect of distamy- tin has been greatly improved by chemical modifications, which eventually led to the synthesis and identification of

the 5ring carbamoyl analogue 7. This compound shows a potent and rapid antiparasitic action on the chloro- quine-resistant strain of P. falci~urum (100% inhibition af- ter 4 hr at 1 FglmL) and greatly improved the therapeutic index of potential clinical importance, which warrants ad- ditional experiments in uioo. Compound 7 represents the lead compound for a new class of antimalarial agents, and possibly a good candidate for further development.

Acknowledgements-Financial support for rhts research from A. Menarini (Industrie Farmaceutiche Riunite), Bristol-Myers Squibb (Italy), and Isti- tuto 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.

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