Title of Invention: A METHOD OF SYNTHESIS OF CdL

COMPLEX WITH HIGH BIOLOGICAL ACTIVITY AGAINST AT LEAST CHLOROQUINE RESISTANT STRAIN OF THE

MALARIA PARASITE PLASMODIUM FALCIPARUM

RELATED ART

[I] Malaria annually kills more than one million people world-wide 90% of them in Africa. The eradication of malaria continues to be

[2] frustrated by the continued drug resistance of the malaria parasite. Hence, there is a great need to continue the search for more

[3] effective drugs in terms of activity and the cost. The use of metal complexes as pharmaceuticals has shown promise in recent

[4] year's particularly as anticancer agents and as contrast agents for magnetic resonance imaging. In the search for novel drugs

[5] against resistant parasites, the modification of existing drugs by coordination to metal centers has attracted considerable attention.

[6] However, the potential of metal complexes as antiparasitic agents has far been very little explored. As part of our research to

[7] develop metal complexes with potential antiprotozoal activities, we present the

synthesis and characterization and of metal

[8] complex of CdL ₂ with high biological activity against the chloroquine resistant strain of the plasmodium falciparum parasite.

BRIEF DESCRIPTION

[9] The metal complexes were synthesized and recrystallized. They were sent for spectroscopic measurements.

[10] The elemental analyses were performed by using an EA 1108 CHNS-0 instrument.

The proton NMR was recorded at ambient

[I I] temperature with Varian mercury (300 MHz) or Varian Unity Spectrometer (400 MHz) and TMS was used as an internal

[12] reference. The chemical shifts ( ) are given in parts per million relative to TMS ( =

0.00). The mass spectra were recorded by

[13] means of a low resolution mass spectroscopy apparatus. The infrared spectra were measured in solution using chloroform on a

[14] satellite Perkin-Elmer FT-IR spectrophotometer. The current invention presents a method of synthesis of a metal complex, CdL ₂ .

[15] The cobalt chloride hexahydrate, CoCl ₂ .6H ₂ 0 (0.264 g) was dissolved in water (5 .0 cm ³ ) to give a pink solution.

[16] The dithio containing ligand LH (0.50 g) was also dissolved in warm ethanol (80.0 cm ³ ) to give a pale yellow solution.

[17] The cobalt solution was added to the ligand solution and a dark-brown mixture was obtained.

[18] Addition of water resulted into the production of a brownish-orange precipitate.

[19] The precipitate was filtered off, washed with water, ethanol and ether and then dried at the water pump for 30 minutes.

[20] The yield obtained was 0.56 g.

[21] The complex was then recrystallized from acetone. Yield 0.45 g (80 %).

[22] Thiosemicarbazones and their corresponding thiosemicarbazides containing

2- acetylpyridine fragment have been found to show

[23] biological activity against malaria parasites, trypasomiasis, bacteria, and viruses. Our current findings indicate that the metal

[24] complexes containing the dithioester

3- [l-(2-pyridyl)ethylidene]hydrazinecarbodithioate have moderate potency against [25] falcipain-2 (FP-2) and falcipain-3 ( FP-3) cysteine protease enzymes from the

malaria parasite Plasmodium falciparum while

[26] they portray enormous potency against the chloroquine resistant strain (W2) of the parasite. This patent describes the synthesis,

[27] characterization and biological results of metal complexes containing deprotonated

3 - [ 1 - (2-pyridyl) ethylidene]

[28] hydrazinecarbodithioate ligand (Fig. 1).

[29] The metal complex were synthesized and recrystallized. The biological activities (nanomolar) of the metal complex against

[30] malaria parasites were tested and tabled as table 1 in figure 6 of the drawings. The metal potency was far much greater than the

[31] control drug with respect to W-2 . This observation is extremely important as malaria resistance against the chloroquine drug is a

[32] great challenge today. This metal complex may act as lead compounds for developing future malaria drugs . The potency of the

[33] metal complex is modest and less then that of the control drug with respect to FP-2 and FP-3 cysteine protease enzymes.

[34] The potency of cadmium is greatest with respect to W-2 compared to other metals as well as the control drug.

[35] The metal complex CdL ₂ containing the deprotonated dithioester L- have been synthesized and characterized by elemental

[36] analysis, mass spectrometry, proton NMR and Fourier transform IR. The ligand LH undergoes tautomerism which can readily

[37] get ionized to generate a deprotonated ligand Both LH and L are potentially

tridentate via the pyridine ring nitrogen, the

[38] methine nitrogen ( -nitrogen) and the sulphur (mercapto sulphur) atom . Figure 2 shows the deprotonation process and mode

[39] coordination of L-. The analytical data and molecular masses of the complexes are given in Figure 5, Table 1.

[40] This information is consistent with the formulation of the synthesized complex as

ML ₂ (M = Cd )

[41] The x-ray single crystal structure analysis was done for CdL ₂ complex. The structure is a distorted octahedral geometry and

[42] indicates that the L behaves as a tridentate ligand (NNS). [ilO] It is quite clear that the fragmentation of the complexes involved

[43] the bound deprotonated ligand The main decomposition points are indicated in

Fig. 3 as 1, 2, 3, 4 and 5. The coupling of the

[44] pyridine hydrogen rings according to figure 4 . The results of the biological activities of the metal complexes against malaria

[45] parasites are shown in Figure 6, Table 1. The metal complex was tested against two cysteine protease enzymes falcipain-2

[46] (FP-2) and falcipain-3 (FP-3) as well as the chloroquine-resistant strain from the malaria parasite Plasmodium falciparum.

[47] The following activity sequences can be discerned.

[48] FP-2: CONTROL >Cd

[49] FP-3: CONTROL >Cd

[50] W-2 Cd > CONTROL

[51] Although the metals were bound to the same ligand, L , their activities differed dramatically. CdL ₂ complex yielded a nanomolar

[52] strength ratio of 45,620 against FP-2 and 31,370 against FP-3 and 14,4 against W2 and 172,4 against W-2 .

[53] It is quite clear from our work that keeping the ligand constant and varying the

central metal atom, affects the biological activity of

[54] the complex. It is also well known that a change in molecular structure may influence its biological activity dramatically.

[55] The biological activity may either remain the same, decrease, increase or disappear completely. This has been observed in

[56] thiosemicarbazones and thiosemicarbazides in the malaria studies. For instance, the

2-acetylpyridine moiety in

[57] thiosemicarbazones has been found to be crucial in promoting the biological activity against malaria parasites and Trypanosoma

[58] rhodesiense and so was the presence of the sulphur atom. The modifications at the pyridine nitrogen and/or the terminal nitrogen

[59] (N4) of the thiosemicarbazone chain also affected the biological activity against malaria, trypanosomiasis, and Herpes Simplex

[60] Virus. The molecular geometry is also crucial in determining the biological activity in metal complexes.

[61] This is illustrated by cis-[PtCl ₂ (NH ₃ ) ₂ ] (Cisplatin) is biologically active and used as a drug against cancer whereas the trans

[62] isomer is biologically inactive against cancer. Dissociative mechanism of the CI ligands was advanced to explain the anti-tumor

[63] activity in cis-[PtCl ₂ (NH ₃ ) ₂ ] complex. In this mechanism one of the CI ligand is

replaced by water to form [Cl(H ₃ N) ₂ Pt(OH ₂ )] ⁺

[64] complex. Then the platinum aquo complex reacts further with a DNA 'molecule' of the cancerous cell to form the new complex

[65] [Cl(H ₃ N) ₂ Pt(DNA)] ⁺ and in so doing terminates or minimizes the cancerous growth.

The DNA molecule binds the platinum

[66] metal via the guanine moiety. Green and Berg also observed that the retroviral nu- cleocapsid from the Rauscher murine leukemia

[67] binds to metal ions, in particular, it has a higher affinity ²⁶ for Co ²⁺ and Zn ²⁺ In this case the nucleocapsid behaves as a 'ligand'

[68] for the metal ions. It is also very interesting to note that complexation mechanism has been advanced to explain the antimalarial

[69] activity of chloroquine. It does this by binding the heme fragments and thereby

preventing the crucial polymerization process of

[70] the parasite. This ultimately leads to the death of the parasite. In this case the

chloroquine molecule acts as a ligand to bind the

[71] biological heme fragment. Circular dichroism studies of [MLC1] (M = Pd, Pt, L = methyl- 3 - [2-pyridylmethylene]

[72] hydrazinecarbodithioate ion ) with DNA also indicate that an adduct is formed

between the two moieties. Biological activities of

[73] certain thiosemicarbazone ligand complexes were found to be less active against malaria parasites than other ligands. On the

[74] other hand, it was observed that metal complexes of pyridoxal semicarbazones, thiosemicarbazones and

[75] isothiosemicarbazones were more biologically active than the others ligands. POSSIBLE MECHANISM OF THE BIOLOGICAL ACTIVITY OF CdL2 COMPLEX

FP-2 or FP-3

Hemoglobin ► 'Heme' fragments + peptides

^ML 2 ? LM ⁺ + LT

I interactions with the 'Heme' fragment

LM ⁺ + 'Heme' _► [ LM-Heme f complex

L ^" + 'Heme' ► ²

ML ₂ + 'Heme' ► 'Heme' - ML ₂ complex

Scheme 1 . The Interactions of the Ligand rkietal complex fragments J-ML ⁺ with the Heme fragment.

Interactions with FP-2 cysteine protease enzyme

LM ⁺ + FP-2 > [ LM-FP-2 f complex l_- _{+ Fp} _2 _► [ L-FP-2r complex

ML ₂ + FP-2 ► FP-2 - ML2 complex

Scheme 2. The Interactions of the Ligandrbetal complex fragments J-ML ⁺ with

FP-2 protease enzyme.

Interactions with FP-3 cysteine protease enzyme

LM ⁺ + FP-3 _► [ LM-FP-3 f complex l_- _{+ F} p. _{3 >} [ L-FP-3r complex

ML ₂ + FP-3 FP-3 - Mli complex

Scheme 3. The interaction of FP-3 protease enzyme with the Ligand L and metal complex fragments, ML ₂ and ML ⁺ .

Interactions with W-2

+ W-2 _► [ LM-W-2 ] ⁺ complex

_{+ w} _2 _► [ L-W-2]- complex

_{+ ►} W-2 - ML ₂ complex

Scheme 4. The interaction of W-2 with the LigandalQd metal

complex fragments, ML ₂ and ML ⁺

Interactions with WE-2

LM ⁺ + WE-2 [ LM-WE-2 ] ⁺ complex

_^ [ L-WE-2] ^" complex

L- + WE-2

Ml_ 2 + WE-2 WE-2 - ML ₂ complex

Scheme 5. The interaction of WE-2 with the Ligandabd

metal complex fragments, ML ₂ and ML ⁺ .

[77] In view of the information about the activity of chloroquine against malaria parasite and that of cis-platin complex,

[78] cis-[PtCl ₂ (NH ₃ )2] against cancer, we have proposed the following possible

schemes 1-5 to explain the activity of our metal

[79] complexes, ML ₂ on malaria cysteine protease enzymes FP-2 and FP-3 as well as the chloroquine resistant strain W-2.

[80] Since the metal complex ML ₂ is rather bulky, it is plausible to suggest a dissociative mechanism resulting into the formation of

[81] ML ⁺ and L fragments . A similar mechanism was put forward to explain the activity of cis-[PtCl ₂ (NH ₃ ) ₂ ] in cancer

[82] chemotherapy. L is a deprotonated dithio ligand shown in Figure 2 [i3] . The ML ⁺ fragment consists of a metal atom with a three

[83] coordination . This is also shown in Figure 2. The x-ray crystal structure of CdL ₂ was taken. It shows the cadmium atom in a

[84] six-coordination configuration with the ligand acting as a tridentate NNS System.

The corresponding atoms of the NNS

[85] ligands are trans to each other in a distorted manner. That is, the sulphur atoms, the pyridine ring nitrogen's and the imine

[86] nitrogen's. It is likely that the ligand binds in the same manner for the Cd(II)

complexes. The fragmentation patterns of the

[87] selected complexes are summarized in Figure 7. The Mass Spectrum was listed in

Figure 8 and proton NMR of the metal

[88] complex was tabled in Figure 9. The infrared spectra of the complexes ML ₂ (M =

Cd) are tabled in Figure 10.

[89] The spectra are mainly due to the functional groups of the deprotonated ligand L shown in Figs 2 and 3. The key functional

[90] groups are C=S, C=N, C=N (Py), C-H, C-C, C-S and N-N. The molecular mass peaks for the complex, ML ₂ (M = and Cd) were

[91] readily discerned according to Fig.5. The reaction equation between the metal salt and the ligand can simply be represented by

[92] the equations:

[93] MC1 ₂ + 2LH→ ML ₂ + 2 HC1, (M = Cd)

[94] The degree of M-L bond strength will could affect bond dissociation and hence the degree of biological activity .

[95] In addition, other factors such the lability and the size of the metal atom could

influence the biological activity.

[96] For instance, Cd (II)> Mn(II)>Zn(II)>Co(II)>Ni(II) in size. This more or less

parallels the order for complex reactivity of ML ₂ with

[97] W-2. The dramatic variation in the biological activity of the complexes implies a direct participation of the metal atom. Hence, it is

[98] more plausible to assume that ML ⁺ fragment probably exerts more influence in the biological activity than the ligand L , and ML ₂

[99] complex . In conclusion, a lot more extensive work is needed to clearly understand the factors and mechanisms that influence

[100] the biological activity of the ligand, L and its corresponding metal complex, ML ₂ .

The proposed possible mechanisms by which

[101] the metal complexes affect the parasite are summarized in Schemes 1 to 5 and

condensed in Scheme 6. The malaria parasite

[102] decomposes human hemoglobin to produce free heme fragments and peptides in its food vacuole. The proteins are utilized by

[103] the parasite for its growth and replication. The heme acts as a parasite waste and is thus toxic to the parasite. Its toxicity is

[104] thought to occur by the heme lysing the membranes and producing reactive oxygen intermediates (ROI) and interfering with

[105] other biochemical processes. The parasite neutralizes the toxicity of the heme by converting it into a hemazoin polymer also

[106] known as the malarial pigment through a process called biocrystallization. The action of chloroquine drug is its interference with

[107] these processes. Chloroquine enters the food vacuole of the parasite due to its

enabling environment. The enabling

[108] environment includes the parasite transporters that assist in the uptake of

chloroquine, the existence of a specific parasite

[109] receptor for binding chloroquine and acidity of the food vacuole that promotes the protonation of the chloroquine nitrogen atoms.

[110] A postulated mechanism by which this activity occurs is through the formation of a complex with the heme and hence

[111] preventing it from forming a non-poisonous hemozoin The complex formed between the heme and chloroquine is

[112] poisonous to the parasite. This results into the death of the parasite. The mechanism we have proposed in schemes 1 to 5

[113] involve the formation of complexes between the complex ML ₂ , the fragments ML ⁺ and the ligand L on one hand with the

[114] parasite enzymes FP-2 and FP-3 , the heme, as well as the chloroquine resistant strain

W-2 and its enzymes represented by

[115] WE-2 on the other. The complexes so formed will ultimately poison the parasite leading to its death .

BRIEF EXPLINATION OF DRAWINGS

[116] Figure 1. Refers to the synthesis, characterization and biological results of met- alcomplex containing deprotonated 3-[l-(2-pyridyl)

ethylidene]hydrazinecarbodithioate ligand (Fig. 1).

[117] Figure 2. Refers to the deprotonation process and mode of of coordination of 1-. [118] Figure 3. Refers to positions where fragmentations can occur.

[119] Figure 4. Refers to the coupling of the pyridine hydrogens.

[120] Figure 5. Refers to the analytical data of and molecular mass of the complex

characterized.

[121] FIGURE6. Refers to the Biological Activity of the Metal Complex CdL ₂ against the

Malaria Parasite, Plasmodium Falciparum.

[122] FIGURE 7. Refers to the Mass Spectrum Fragmentation Patterns of the Metal [123] Complex CdL ₂

[124] FIGURE 8. Refers to the Mass Spectrum of CdL ₂

[125] FIGURE 9. Refers to the Infrared Spectra of CdL ₂

[126] FIGURE 10. Refers to the HNMR of the CdL ₂