do not intercalate into DNA and suggest a strong influence of ionic and H-bonding interactions, especially with the phosphate backbone, stabilizing the complex formation with single- and

double-stranded oligonucleotides. Thermal denaturation studies with three selected cryptolepine derivatives (3j, 3n and 3v) showed that these compounds stabilise the ds-DNA structure (Table 4.1).

This stabilizing effect is more pronounced for 3j and 3v, which show T_m values ranging from 5 to 12 ºC. Also, the observed magnitudes of Tm are consistent with association constants determined by spectroscopic titration, since both studies show that compound 3n has lower binding affinity to ds-DNA than 3j and 3v (Table 4.1 and Table 4.2).

B. Docking studies with double-stranded oligonucleotide

Molecular docking simulations indicated that 1 intercalates into 12-mer ds-DNA at nonalternated (CC)-(GG)-sites, being the complex stabilized mainly by - stacking interactions between the aromatic ring of the quinoline moiety of the cryptolepine and the aromatic rings of the consecutive guanine bases, in complete agreement with the X-ray diffraction studies.⁽²⁷⁷⁾

The docking simulation studies of cryptolepine derivatives 3 (Section 5.3) with the double-stranded d(GATCCTAGGATC)₂ oligonucleotide showed that these compounds do not intercalate into DNA structures, in the manner of parent compound 1, confirming the conclusions from the in vitro binding studies. A possible explanation for this change in DNA binding mode when a diamine side-chain is introduced at C11 of cryptolepine nucleus, is the observed increased molar volume of the derivatives when compared to 1 (Table 5.3), leading to the separation of the bases in the intercalation cavity, so breaking H-bonding between base pairs, prompting a structural rearrangement of the DNA helix (Appendix I). Most of the cryptolepine derivatives studied are predicted to bind preferentially to the minor groove, except for 3v and 3x which bind preferentially to the major groove. However, the binding stoichiometry of the cryptolepine derivative 3v:DNA complex from the Job plot was determined as 2:1 (Figure 4.1), indicating that cryptolepine derivatives can bind to the major and to the minor grooves of the same molecule of DNA, although only small differences in preference for minor or major grooves is evidenced by the complex formation energies (Table 5.4).

In an attempt to understand these observed structure-binding relationships, molecular properties of ten selected cryptolepine derivatives (3f, 3i-j, 3n, 3q-s and 3v-x) were studied by DFT (Table 5.3).

However, we must conclude that DFT studies alone were not able to consistently explain the differences in binding affinities observed in titration experiments of the compounds series presented in this work.

Incorporation of basic side-chain into the indoloquinoline scaffold shifts the binding mode from intercalation, as for cryptolepine, to interaction via electrostatic interactions, most likely to the phosphate backbone. Another major finding is that the novel cryptolepine derivatives also bind more strongly to double and single-stranded DNA than cryptolepine itself. Analysis of structure-binding affinity relationships revealed that linear alkyldiamine side-chains and a chlorine atom at position 3 of

the indoloquinoline moiety markedly increase the binding affinity to the double-stranded oligonucleotide. However, no correlation with antiplasmodial activity or cytotoxicity was found.

Overall, cryptolepine derivatives seems to accumulate inside the digestive vacuole of parasites and are able to complex with FPIX-OH at the same extent as chloroquine, which means that they can inhibit haemozoin formation. Additionally, cryptolepine derivatives are also able to complex with DNA structures, which could justify its antiplasmodial activity due to induced cytotoxicity. As such, these results suggest that the cryptolepine derivatives mode of action could probably be a combination of effects due to inhibition of haemozoin inside the digestive vacuole and cytotoxicity induced by interaction with DNA structure in the parasite nucleus.

6.3.2 Quindolones and Derivatives

In vitro Antiplasmodial Activity in human red blood cells and cytotoxicity

The synthesized quindolones (4, 91a and 91b) and derivatives derivatives 5, 94 and 95 were evaluated in vitro for their antiplasmodial activity in Human red blood cells infected with 1% P.

falciparum^(408-409) strains with the chloroquine resistance phenotype W2. Also, The in vitro cytotoxicity of quindolone 4 and of quindolone derivatives 5, 94 and 95a was evaluated using human hepatoma cell line HepG2 A16 (Dr. Virgílio do Rosário laboratory). Table 6.3 shows the in vitro antiplasmodial activity (IC₅₀) and cytotoxic activities (IC₅₀) against HepG2 A16 cells of 4 and derivatives, together with their selectivity index (cytotoxicity/antiplasmodial ratio).

From the data in Table 6.3 it can be concluded that introduction of an alkylamine side chain (95) is essential for antiplasmodial activity as unsubstituted quindolones 4, 91a and 91b are inactive (IC₅₀ ranging from 8 to >10 µM). The introduction of a second alkylamine side chain further increased antiplasmodial activity, with derivatives 5 and 94 showing IC₅₀ values between 51 and 539 nM.

Analyzing the influence of chlorine substitution on antiplasmodial activity of bis-alkylated derivatives 5 and 94, it can be clearly seen that halogenation increases antiplasmodial activity of N,O- derivatives 94, whereas mono or di-halogenation of N,N- derivatives 5 has no effect on antiplasmodial activity.

Improved antiplasmodial activity with introduction of chlorine atoms in quinolines have been referred but the reason to justify that observation was never clearly found.⁽⁴¹²⁾ As a consequence of the structure-activity relationships discussed above, the dihalogenated and bis-alkylated quindolone derivative 94c emerges as the more active compound of the series with an IC₅₀ of 51 nM, more active than chloroquine (Table 6.1) Also, monoalkylated derivatives 95a-c are weakly (IC50 of 2.6-1.3 µM) or moderated active (IC₅₀ of 0.55 µM). Chlorine atoms in both aromatic rings also improved antiplasmodial activity, being the monohalogenated derivative 95b two-times more active than 95a and the dihalogenated derivative 95c even more active than 95b. Cytotoxicity was determined for the hepatic cell line HepG2 A16 and IC₅₀ values of quindolone 4 and derivatives 5, 94 and 95a range from 4.3 to 20 µM (Table 6.3).

Introduction of alkylamine side chains slightly increased cytotoxicity but no structure-cytotoxicity

pattern was observed for quindolone derivatives. The low cytotoxicity of alkylamine-quindolone derivatives indicates selectivity for erythrocytic stages of malaria parasite, as shown by the selective index (SI, Table 6.3), determined as the cytotocity IC₅₀/antiplasmodial IC₅₀ ratio, higher than 10 for all bis-alkylated quindolone derivatives 5 and 94. The most active compound 94c is also the most selective one, being 100-folds more toxic to the parasite than to the human hepatic cells HepG2.

Probing the site of action at cellular level

Investigation of the proposed mechanism of antiplasmodial activity of quindolone derivatives was performed by determining equilibrium binding constants (Kass) with hematin monomer (FPIX-OH), by UV-visible titration carried out in pH 5.5,(146, 361-362, 418) and by docking studies with haematin dimer.

Antiplasmodial potential targets - Haem A. In vitro binding to haematin monomer

Association constantsshown in Table 4.4 were determined from the fitting of the experimental data to a binding model with stoichiometry of one quindolone molecule to one FPIX-OH, according to the binding stoichiometry determined with Job’s methodology (Section 4.3.1). K_ass values for quindolones 4, 91a and 91b and derivatives 5, 94 and 95 ranges from 0.074 and 0.14 x10⁶ M^-1, comparable to K_ass value determined in our assay for chloroquine (Table 4.3). From this experiment it can also be concluded that binding affinity of quindolone derivatives (5, 94 and 95) to FPIX-OH monomer is only due to their aromatic tetracyclic structure as their K_ass are not significantly different from those of parent quindolones 4. Additionally, chlorine atoms in the aromatic structure of quindolone seems to not affect the π-π interactions with porphyrin, since no significant differences was found between K_ass.

B. Docking studies with double-stranded oligonucleotide

In fact, the docking studies with haemozoin showed that the formation of H-bond between the protonated terminal nitrogen and the oxygen of the carboxylate group (Fe-COO) occurs. The formation of the hydrogen bond can modulate the fitting of both aromatic nucleus. The two carbon side chain length, has a distance between the terminal amine in the side chain and the quinolinic nitrogen (N⁵) of 6.94 and 7.37 Å in derivative 5 and 94, respectively, which is small compared with the ca. 8.5 Å of distance between the center of the porphyrin rin (Fe³⁺) and the oxygen of the carboxylate. These differences obligate the aromatic ring of the quindolone derivatives to stacks on the edge of the porphyrin (Figure 5.11 and Appendix J), when compared with its counterparts 4, 91a and 91b (Figure 5.10 and Appendix J).

Withal, the molecular docking simulations with haemozoin dimer also showed improved ligand binding (GoldScore, Table 5.6) with the introduction of chlorines in the aromatic nucleus. As already showed with 4-aminoquinolines, the presence of organic chlorine atoms can probably favour the

formation Coulombic interaction with the porphyrin ring, such as Cl···CH₃, and thus stabilize the complex with haemozoin.⁽⁷⁷⁾

Taken together, and even though no correlation was found between the antiplasmodial activity with binding constants (K_ass) and docking fitness values (GoldScore), the results suggest that alkylamine quindolone derivatives 5, 94 and 95 may share with chloroquine the same antiplasmodial mechanism of action, i.e., by inhibition of haemozoin formation, as far as they reach the target inside digestive vacuole. In fact, the increased antiplasmodial activity when compared with their counterparts 4, 91a and 91b is probably due to the increased accumulation in the parasite digestive vacuole, ^{(18, 130)} since derivatives with two side chains 5 and 94, would probably accumulate more efficiently in the DV than 95 (one side chain) and are around c.a ten-fold more active against the P. falciparum W2 strain.

Furthermore, the experimental result with FPIX-OH and docking studies with haemozoin dimer suggest that the antiplasmodial activity does not results entirely from association with haematin, but can also be due to the inhibition of haemozoin crystal growth by a capping effect on the crystal surface.(77, 127-128, 419) Table 6.3 - In vitro antiplasmodial activity (IC50) against P. falciparum W2 strains, cytotoxicity activity (IC50) against HepG2 A16 and selectivity index (SI) of quindolones 4, 91a and 91b and derivatives 5, 94 and 95.

4 or 91 5

a)The IC50 values are calculated from experiments carried out in triplicate and compared with CQ,^b)Selectivity Index (IC50HepG2 A16/IC ^W2).

6.4 Conclusions

The main objective of this project was to synthesise novel derivatives of the antimalarial natural compound cryptolepine as part of a larger project of development of safer and potent antimalarial drugs, at an affordable cost. The proposed approach was to synthesise new cryptolepine derivatives (3) with an ionisable diamine side chain at position C11 of the alkaloid scaffold, which was expected to improve the antimalarial activity and the selectivity against Plasmodium through an increase in the concentration of the compounds inside the acidic vacuole of the intraerythrocytic parasite. Additionally, we decided to start a second line of research based on the indolo[3,2-b]quinoline chemical structure 4 Derivatives 5 were designed based on the same rationale.

Cryptolepine and quindolones derivatives were synthesized from a common synthetic methodology that allowed us to achieve the indolo[3,2-b]quinolones intermediates (4 and 91) for their synthesis.^(13-14) Introduction of alkyldiamine and alkylamine side chains in cryptolepine and quindolone aromatic nucleus was achieved in general with good yields and with high degree of purity ( 95 %).

Cryptolepine derivatives 3 showed strong antiplasmodial activity against both CQ-resistant and CQ-sensitive P. falciparum strains when compared with the parent compound, and those with IC₅₀ values below 100 nM share the following features:

i. they all contain basic amine side chains;

ii. they all form complexes with FPIX-OH;

iii. they all are predicted to accumulate in digestive vacuole in the same extend as chloroquine;

iv. they all form strong complexes with double-stranded oligonucleotides.

Maximum potency, against Plasmodium sp. was achieved with propyl and butyl side-chains, while branched linkers seem to be poorly tolerated. Introduction of the basic terminal amine are predicted to increase accumulation inside the acid digestive vacuole of the parasite, improving the antiplasmodial activity. However, the antiplasmodial activity of these cryptolepine derivatives does not result entirely from their association with haematin and binding to the haemozoin crystal cannot be excluded. The cytotoxicity profile was also improved, with compound containing the conformationally restricted piperidine side-chain, 3n, presenting a selectivity index value of ca 1400, i.e. a 1000-fold increase when compared with the parent compound 1. This study revealed also that these cryptolepine derivatives are active against the blood-stages, but not to the liver-stages, of the parasite. Localization of 3n inside blood stages of parasite suggests that the mechanism of action for these compounds besides inhibition of haemozoin crystal growth can also be due to interaction with DNA structures.

In the indolo[3,2-b]quindolin-11-one skeleton the introduction of two alkylamine side chains generally improves antiplasmodial activity, probably by increasing accumulation in acidic digestive vacuole. On the other hand, substitution by chlorine atoms at positions 3 and 7 of quindolone skeleton improves antiplasmodial activity for N,O- bis-alkylamine derivatives but not for N,N-di-substitued ones, what is an interesting finding that deserves further investigation. Additionally, antiplasmodial activity of bis-alkylamine quindolone derivatives cannot be entirely justified by their affinity to haematin monomer. Taken together, these results indicate that the quindolone nucleus is a suitable scaffold for the design of active and selective compounds targeting parasite haem detoxification pathway and bis-alkylamine quindolones are a novel chemotype with potential for development as antimalarial agents.

Overall, cryptolepine and quindolone derivatives containing basic side-chains have been confirmed as promising lead compounds for the development of new agents active against the blood stages of P. falciparum malaria with improved activity and increased selectivity for the Plasmodium sp.