Reagent grade chemicals were used without further purification. Ethyl cyanoacetate (S. D. Fine Chem Ltd, India) and o-phenylenediamine (Koch Light Laboratories Ltd., England) was used. The metal contents of the complexes were determined by complexometric titrations against EDTA. Carbon, hydrogen, and nitrogen contents were determined by using a Carlo-Erba Strumentazione (Italy) CHN analyzer. Molar conductivities in DMSO (10⁻³ M) at room temperature (26°C) were measured using an Elico conductivity bridge having platinum electrodes. Magnetic moments were determined by a Faraday balance. The IR spectra of ligand and its metal complexes were recorded on a Nicolet 170 SX FT-IR spectrometer in the range 400–4000 cm⁻¹ using KBr discs. The EPR spectrum of the Gd(III) complex was recorded on a Varian E-4X band spectrophotometer. ¹H NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer using DMSO-d₆ as solvent. UV-visible spectra were measured on a Hitachi 2001 spectrophotometer using dimethylsulfoxide (DMSO) as solvent. Thermogravimetry (TG) and differential thermal analysis (DTA) measurements were made in N₂ atmosphere between 20 and 1000°C using a Perkin-Elmer (Pyris Diamond) analyzer. Lanthanide nitrates were prepared by dissolving the corresponding oxide (99.99%, Indian Rare Earths Ltd, India) in 50% HNO₃, followed by the evaporation of the excess acid.

Two local varieties of wheat (DWR-195 and GW-349, developed at UAS, Dharwad) were selected for the experiment. The solution of compounds (10⁻⁶ M) was prepared in methanol due to its insolubility in water. The compounds to be tested consisted of La(BIA) complex, HBIA ligand, La(NO₃)₃, methanol, and water (control) assigned as groups 1, 2, 3, 4, and 5, respectively. The periods of treatment varied from 2, 5, 8, and 11 minutes assigned as T1, T2, T3, and T4, respectively.

The seeds were soaked in the respective solutions for the above-mentioned time periods and then placed between germination papers (46 cm × 29 cm in size) previously soaked with water in two rows. They were then rolled carefully ensuring no excess pressure was placed on seeds, wrapped in a sheet of polythene film to reduce surface evaporation, and placed in a germination chamber. On the fifth day after germination, the germination count was taken. Ten seedlings (out of 25 taken) were randomly selected and measured for their coleoptile and root length. Three replications were carried out for each observation.

The statistical evaluation of the results was conducted with use of SPSS-(statistical package for social science; Windows version 6.0) packed program. One-way analysis of variance (ANOVA) was used to analyze the results. The level of statistical significance was set at P < .05.

In case of variety GW-349 (Table 1), there was no significant difference between all the groups at T1 and T2. In case of T3, groups 3 and 4 showed significant decrease in germination percentage (GP) compared to the control (P < .05). At T4, group 5 showed significant increase in GP compared to groups 2, 3, and 4. Group 1 showed significant increase in GP compared to groups 3 and 4.

In case of variety DWR-195 (Table 2), at T1, T2, and T3 there was no significant difference in GP in case of all the groups. At T4, group 1 seeds showed significant increase in GP compared to group 5(control).

In case of variety GW-349 (Table 3), at T1, group 1 showed significant increase in root length (RL) compared to groups 2, 4, and 5(control). At T2, T3, and T4 there was no significant differences between the groups.

In case of variety DWR-195 (Table 4), at T1, T2, and T4, group 1 shows significant increase in RL compared to groups 2, 3, 4, and 5(control), while at T3 group 1 does not show any significant differences between the groups.

In case of variety GW-349 (Table 5), group 1 showed increased shoot lengths (SL) compared to groups 2, 4, and 5 at T1, T2, and T3 while group 3 showed significant difference compared to group 1. At T2 and T3, group 1 showed increased SL compared to group 3. However, at T3 and T4, group 1 showed no significant increase in SL compared to control, and compared to group 2 at T4. Group 1 showed significant increase in SL compared to groups 3 and 4 at T4.

In case of variety DWR-195 (Table 6), group 1 showed significant increase in SL for the treatment periods T1, T2, T3, and T4 compared to groups 2, 3, 4, and control. However group 3 showed significant increase in SL compared to group 1 at T3 while at T4 there was no significant difference between group 1 and control.

The complexes are nonhygroscopic, off-white in color, and have 3 : 2 (M : L) stoichiometry (Table 7). The complexes are almost soluble in methanol, ethanol and completely soluble in DMF and DMSO whereas they are insoluble in acetone, chloroform, benzene, and ethyl acetate. The molar conductivity values of the complexes in 10⁻³ M DMSO (Table 7) are in the range 4.4−9.6 Ohm⁻¹ cm² mol⁻¹ indicating their nonelectrolytic nature [18].

Magnetic moment values indicate the absence of metal-metal interaction and the noninvolvement of 4f electrons in bonding.

Nitrogen and sulfur were estimated by the Kjeldhal's and Messenger's methods, respectively. Silicon was determined gravimetrically as SiO₂. Molecular weights were determined by the Rast camphor method (freezing point depression method) using resublimed camphor (MP 178°C).

The conductance measurements were carried out in dry dimethylformamide (DMF) at room temperature using a systronics conductivity bridge (model 305) in conjunction with a cell having a cell constant of 1.0.

The electronic spectra were recorded on a Perkin Elmer UV visible spectrophotometer in the range 200–600 nm, using dry methanol as the solvent.

Infrared spectra were recorded on a Nicolet Magna FT-IR 550 spectrophotometer in KBr pellets.

Multinuclear magnetic resonance spectra (¹H, ¹³C, and ²⁹Si) were recorded on an FX 90 Q JEOL spectrometer operating at 90 MHz.

¹H NMR spectra were recorded in deuterated methanol at 89.55 MHz using tetramethylsilane (TMS) as an internal standard.

¹³C NMR spectra were recorded in dry methanol using TMS as the internal standard at 22.49 MHz.

²⁹Si NMR spectra were recorded at 17.75 MHz using deuterated dimethylsulphoxide (DMSO-d₆) as the solvent.

The sulphonamide imine was prepared by the condensation of 2-acetylfuran with sulphathiazole in equimolar ratio in absolute alcohol. The contents were refluxed for 3–4 hours and the solid which separated out was filtered off, recrystallized from the same solvent (ethanol), and dried in vacuo. The physical properties and microanalysis of this sulphonamide imine are recorded in Table 1.

For the synthesis of the complexes, first the sodium salt of the ligand was prepared by dissolving sodium metal (0.04–0.07 g) in 30 mL of methanol. Now to the weighed amount of organosilicon chlorides in 1 : 1 (0.38–0.51 g) or 1 : 2 molar ratios (0.11–0.29) in 20 mL methanol, the above prepared sodium salt of the ligand was added. The solution was refluxed for a period of 15–17 hours. The white precipitate of sodium chloride, formed during the course of the reaction, was removed by filtration and the filtrate was dried under reduced pressure. The resulting product was repeatedly washed with a mixture of methanol and n-hexane (1 : 1 v/v) and then finally dried for 3–4 hours. The purity was further checked by TLC using silica gel G. The details of these reactions and the analyses of the resulting products are recorded in Table 1.

The electronic spectra of the sulphonamide imine and its 1 : 1 and 1 : 2 organosilicon(IV) complexes have been recorded. The spectrum of the ligand shows a broad band at 370 nm which can be assigned to the n-π* transitions of the azomethine group. This band shows a blue shift in the silicon complexes appearing at 351, 353, 359, and 355, 362 nm for 1 : 1 and 1 : 2 derivatives, respectively, due to the polarisation within the >C=N chromophore caused due to formation of covalent silicon–nitrogen bond. The bands at 255 and 285 nm are due to π–π* transitions, within the benzene ring and (>C=N) band of the azomethine group, respectively. The K band π–π* showed a red shift due to the overlap of the central metal d-orbital with the p-orbital of the donor atom which causes an increase in conjugation and the B-bands undergo a hypsochromic shift in the complexes [21], see Table 2.

The assignments of characteristic IR frequencies for the resulting complexes may be discussed as follows.

The IR spectra of these derivatives do not show any band in the region 3400-3150 cm⁻¹ which could be assigned to νNH. This clearly indicates the deprotonation of the ligand as a result of complexation with the silicon atom. A sharp band at 1628 cm⁻¹ due to ν(>C=N) frequency of the free azomethine group in the ligand shifts to the lower frequency (ca 15 cm⁻¹) in the silicon complexes and indicating thereby the coordination of the azomethine nitrogen to the silicon atom. A shift of this frequency to the higher and lower wave number side as well as the “no change” has also been reported in the literature [16].

In dimethylsilicon(IV) complexes, a band at ca 1420 cm⁻¹ has been ascribed to the asymmetric deformation vibrations of (CH₃−Si) group, whereas the band at ca 1270 cm⁻¹ has been ascribed to the symmetric deformation mode of (CH₃−Si) group. New bands are observed in the spectra of the complexes at ca 570–582 cm⁻¹ due to the ν(Si ← N) vibrations. These remain absent in the spectrum of the ligand. A band due to ν(Si−Cl) at ca 423 and 439 cm⁻¹ is observed in 1 : 1 diorganosilicon(IV) derivatives. It has been reported [16] that the cis form of such complexes gives rise to two ν(Si ← N ) bands, whereas in the transform only one IR active ν(Si ← N) band is observed. The presence of only one ν(Si ← N) band in the present case suggests that the complexes exist in the transform, see Table 3.

The proton magnetic resonance spectral data of sulphonamide imine and its corresponding silicon complexes have been recorded in DMSO-d₆. The chemical shift values relative to the TMS peak are listed in Table 4.

The broad signal due to the −NH proton in the ligand disappears in the case of silicon complexes showing the coordination of silicon to nitrogen after the deprotonation of the functional group. The azomethine proton signal due to methyl proton (H3−C|=N) appears at δ 2.10 ppm in the ligand. The downfield shift of this position in the spectra of the complexes substantiates the coordination of azomethine nitrogen to the silicon atom. The additional signal in the region δ (1.01 and 1.13 ppm) in Me₂SiCl(2-Ac-F-St) and Me₂Si(2-Ac-F-St)₂ types of complexes are due to Me₂Si group.

The ligand shows a complex pattern in the region δ 8.10–6.92 ppm for the aromatic protons and this is observed in the region δ 8.78–6.95 ppm in the spectra of the organosilicon(IV) complexes. This shifting also supports the coordination through the nitrogen atom.

The conclusions drawn from the UV, IR, and ¹H NMR spectra are concurrent with the ¹³C NMR spectral data regarding the confirmation of the proposed structure. ¹³C NMR spectra of the ligand and its silicon complexes were also recorded in dry DMSO. The shifting of the signals due to carbon attached to the azomethine nitrogen in the spectra of the complexes further supports the involvement of this group in complexation [15]. Data are recorded in Table 5.

The ²⁹Si NMR spectra of Me₂SiCl(2-Ac-F-St), Ph₂SiCl(2-Ac-F-St), and Ph₃Si(2-Ac-F-St) give sharp signals at δ-91 to δ-98 ppm and the spectra of Me₂Si(2-Ac-F-St)₂ and Ph₂Si(2-Ac-F-St)₂ give sharp signals at δ-128 to δ-110 ppm, which clearly indicates the penta- and hexa-coordinated environment, respectively, around the silicon atom. Though, the exact geometries of these complexes can be suggested on the basis of X-ray crystal structure; inspite of our best efforts we could not develop a suitable crystal for the X-ray studies. Hence, X-ray data could not be included in the present paper.

Thus, on the basis of the above spectral features, as well as the analytical data, the penta-coordinated trigonal bipyramidal and hexa-coordinated octahedral geometries shown in Figure 1 have been suggested for the organosilicon(IV) complexes.

Like plant cells, fungi also possess cell walls but they cannot perform photosynthesis, moulds spoil food, damage potato, and crop plants (corn and wheat). They also cause rotting of clothes, shoes, and wooden materials. Some fungi cause diseases like athlete's foot and ring worm.

The antifungal activities were evaluated against Macrophomina phaseolina, Aspergillus niger, Fusarium oxysporum, and Alternaria alternata by agar plate technique [22]. The compounds were dissolved in 25, 50, and 100 ppm concentrations in methanol and then mixed with the medium. The linear growth of the fungus was obtained by measuring the diameter of the colony after 96 hours. The inhibition percentage was calculated as 100 (D_fc − D_ft)/D_fc, where D_fc and D_ft are the diameters of the fungus colony in the control and the test plates, respectively.

Of all the microorganisms, bacteria are the most abundant. They generally reproduce quite fast, such as P cepacicola which reproduces itself every 9.5 minutes. However, some bacteria are very slow growing, such as those that cause tuberculosis and leprosy. This makes early diagnosis of these diseases rather difficult. The most common bacteria used for scientific research is E coli. Its normal living place is the lower human intestine (COLON).

Bactericidal activities were evaluated by the paper disc plate method [23]. The nutrient agar medium (peptone, beef extract, NaCl, and agar-agar) and 5 mm diameter paper discs (Whatman No. 1) were used. The compounds were dissolved in methanol in 500 and 1000 ppm concentrations. The filter paper discs were soaked in different solutions of the compounds, dried, and then placed in the petri plates previously seeded with the test organisms (P cepacicola, E coli, K aerogenous, and S aureus). The plates were incubated for 24–30 hours at 28±2°C and the inhibition zone around each disc was measured.

The free ligand and its respective metal chelates were screened against selected fungi and bacteria to assess their potential as antimicrobial agents. The results are quite promising. The antimicrobial data reveal that the complexes are superior than the free ligands. The enhanced activity of the silicon chelates may be ascribed to the increased lipophilic nature of these complexes arising due to the chelation [24]. The toxicity increased as the concentration was increased. Further, the results of bioactivity were compared with the conventional fungicide, Bavistin, and the conventional bactericide, Streptomycin, taken as standards in either case.

In fungicide activity, most of the organosilicon(IV) complexes were able to inhibit and kill the pathogens at 50 ppm concentration, whilst 100 ppm concentration proved invariably fatal. None of the fungi was able to withstand this concentration. In bactericidal activity, the complexes exhibited remarkable potential in inhibiting the growth of pathogens. Many of the complexes were found to be even more toxic than the standard. Thus, it can be postulated that further intensive studies of these complexes in this direction as well as in agriculture could lead to the interesting results.

Development of the concept of pest management and their implementation have led to a greater appreciation of the need for a wide range of tactics for nematode control. The objective of nematode control is to improve growth and yield of plants, which can be achieved through a reduction of the nematode population in soil or in plants, or through a reduction of their damage. Chemical method can be used to control nematodes [25]. M incognita produce galls on the roots of many host plants and responsible for 44.87 percent of yield loss in brinjal [26].

First of all we applied different concentrations (25, 50, and 100) in ppm of complexes and ligand on root-knot nematode M incognita spp. in a step-by-step [27] procedure. For experiment, egg masses were separated from heavily infected brinjal roots and washed under running water. After cutting the roots, one percent of sodium hypochlorite solution was added, shaked, and then sieved through 150 and 400 sieves. Then the eggs of nematode were counted and replicated three times. At this experiment, temperature range was 30 ± 2°C.

Maximum hatching was recorded in control. All the metal complexes are more toxic than the ligand and all bimolar complexes are more active than unimolar organosilicon derivatives. Dimethylsilicon(IV) complexes are less hazardous than diphenylsilicon(IV) complexes. The activity increases with increasing the concentration of the solutions.

Much smaller amounts of the nonfumigant and fumigant nematicides are needed in plant protection against nematode because the indirect hematostatic effects of non-fumigant nematicides resulting from impairment of neuromuscular activity, interfere with movement, feeding, invasion, development, reproduction, fecundity, and hatching of nematodes which are considered more important than their direct killing action.

Many insects cause injury to economic plants by feeding on them externally: by chewing their leaves or other part: In order to raise more food, man has devised methods to alter normal population growth of many insect pests by reducing their chance for survival. To control the insect pests, the man since long has been employing various strategies which include mechanical, physical, chemical, and biological methods [28].

Some insecticides are physical poisons causing asphyxiation, some are protoplasmic poisons, a few are respiratory poisons, but the majority of them are nerve poisons. The action of insecticides upsets the normal behaviour and actions of the target organisms.

Ovicidal, larvicidal, pupicidal, and adulticidal results are shown in Tables 9, 10, 11, and 12. The data indicated the same observations as were observed in nematicidal activity.

Synthetic calanolides

(+)-calanolide A (Scheme 1), (+)-[10R, 11S, 12S]-10,11-trans-dihydro-12-hydroxy-6,6,10,11-tetramethyl-4-propyl-2H,6H-benzo[1,2-b:3,4-b′:5,6-b″]tripyran-2-one, is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) with potent activity against HIV-1 [2–4]. The compound was first isolated from a tropical tree (Calophyllum lanigerum) in Malaysia [4]. Due to low availability of naturally occurring (+)-calanolide A, a total synthesis of this polycyclic coumarin was developed to provide material for preclinical and clinical research [4, 5]. Structural biology studies and enzyme kinetic experiments bear out the unique anti-HIV properties of calanolide A. In particular, calanolide A is active against viral isolates with the Y181C amino acid mutation in the reverse transcriptase of HIV-1. This is a commonly observed mutation identified in both laboratory and clinical viral isolates and is associated with high-level resistance to most other NNRTIs. However, viral isolates that contain multiple AZT-resistant mutations and the Y181C mutation are actually hypersensitive to the antiviral activity of calanolide A. When tested in vitro in combination with a range of nucleoside analogues, protease inhibitors and NNRTIs, calanolide A demonstrates additive to synergistic anti-HIV activity.

The anti-HIV agent (+/−)-calanolide A has been synthesized [5–7] in a five-step approach starting with phloroglucinol, which includes Pechmann reaction, Friedel-Crafts acylation, chromenylation with 4,4-dimethoxy-2-methylbutan-2-ol, cyclization, and Luche reduction. Cyclization of chromene to chromanon was achieved by employing either acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine or PPTS. Luche reduction of chromanone at lower temperature preferably yielded (+/−)-calanolide A. The synthetic (+/−)-calanolide A has been chromatographically resolved into its optically active forms, (+)- and (−)-calanolide A (Scheme 2). The anti-HIV activities for synthetic (+/−)-calanolide A, as well as resultant (+)- and (−)-calanolide A, have been determined. Only (+)-calanolide A accounted for anti-HIV activity, which was similar to the data reported for the natural product, and (−)-calanolide A was inactive.

In order to examine the structure-activity relationships of the trans-10,11-dimethyldihydropyran-12-ol ring (designated ring C), a series of structural analogues were prepared and evaluated using cell cytopathicity assay (XTT) [14]. Removal of the 10-methyl group resulted in decreased activity, with only one epimer exhibiting anti-HIV activity. Substituting the 10-methyl group with an ethyl chain maintained anti-HIV activity, with only a 4-fold reduction in potency relative to racemic calanolide A. Substitution of the 10-methyl group with an isopropyl moiety completely eliminated the anti-HIV activity. Addition of an extra methyl group at either the 10- or 11-position maintained the basic stereochemical features of the parent calanolide system while removing the chirality at the respective carbon, but resulted in decreased activity relative to calanolide A. In the above examples, analogues containing a cis relationship between the 10- and 11-alkyl moieties were completely devoid of activity. Synthetic intermediates in which the 12-hydroxyl group was in the ketone oxidation state exhibited suppressing anti-HIV activity, with EC₅₀ values only 5-fold less potent than that of calanolide A for both the 10,11-cis and -trans series. These ketones represent the first derivatives in the calanolide series to exhibit anti-HIV activity while not containing a 12-hydroxyl group. Analogues which showed anti-HIV activity in the CEM-SS cytoprotection assay were further confirmed to be inhibitors of HIV-1 reverse transcriptase. The synthesis of calanolide A has been reported in [15–17].

The delta 7,8 olefinic linkages within (+)-calanolide A and (−)-calanolide B (Scheme 3) were catalytically reduced to determine impact on the anti-HIV activity of the parent compounds [18]. In addition, a series of structure modifications of the C-12 hydroxyl group in (−)-calanolide B was made to investigate the importance of that substituent to the HIV-1 inhibitory activity of these coumarins. A total of 14 analogues were isolated or prepared and compared to (+)-calanolide A and (−)-calanolide B in the National Cancer Institute. While none of the compounds showed activity superior to the unmodified leads, some structure-activity requirements were apparent from the relative anti-HIV potencies of the various analogues [19–22]. The synthesis of the isomers of calanolide A has been reported in [23, 24].

NMR spectra of synthetic structures corresponding to those initially reported for natural compounds calanolide C and calanolide D showed some subtle differences from those of the natural products. Further analysis has resulted in revision of the structures of the natural compounds, now renamed as pseudocalanolides C and D. The absolute stereochemistry of pseudocalanolide C was established as [6S, 7S, 8R] using the modified Mosher's method [25].

The three chromanone derivatives (+)-, (−)-, and (+/−)-12-oxocalanolide A (Scheme 4) were evaluated for in vitro antiviral activities against HIV and simian immunodeficiency virus (SIV). The compounds were determined to be inhibitors of HIV-1 reverse transcriptase (RT) and exhibited activity against a variety of viruses selected for resistance to other HIV-1 non-nucleoside RT inhibitors. They are the first reported calanolide analogues capable of inhibiting SIV [26].

Synthetic DCK analogues

Numerous plant-derived compounds have been evaluated for inhibitory effects against HIV replication, and some coumarins have been found to inhibit different stages in the HIV replication cycle. The review article [27] describes recent progress in the discovery, structure modification, and structure-activity relationship studies of potent anti-HIV coumarin derivatives. A dicamphanoyl-khellactone (DCK) analogue, which was discovered and developed in author's laboratory, and calanolide A are currently in preclinical studies and clinical trials, respectively.

Through a bioactivity-directed search for plant-derived, naturally occurring compounds, the lead compound sukudorfin was isolated from the fruit of Lomatium suksdorfii and its structure was identified. Sukudorfin (Scheme 5) inhibited HIV-1 replication in H9 lymphocytes (the EC₅₀ and the therapeutic index—TIs for some coumarins are reviewed in Table 1). The discovery of suksdorfin led to the synthetic compounds and the most promising lead compound was 3,4-di-O-(s)-(−)-camphanoyl-(3′R, 4′R)-(+)-cis-khellactone (or DCK) Scheme 6, which showed extremely potent activity [1].

Forty two dihydroseselins based on the structure of suksdorfin were synthesized in order to evaluate their anti-HIV activity [8]. These synthetic derivatives include 3′,4′-di-O-acyl- and 3′- or 4′-O-acyl-cis-dihydroseselins and 3′,4′-trans-dihydroseselins with O-acyl and/or O-alkyl groups at the 3′ and 4′ positions. Two 4′-azido and three 4′-alkylamido derivatives were also prepared. By using optically pure reagents, three pairs of diastereoisomers were synthesized and separated as optically pure compounds. Together with the above synthetic derivatives, seselin Scheme 7 and (+/−)-cis-, (+)-cis-, and (+/−)-trans-dihydroseselin-3′,4′-diol were also tested for anti-HIV activity in vitro. An optically pure compound, 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone, showed potent inhibitory activity and remarkable selectivity against HIV replication. The EC₅₀ value and in vitro therapeutic index (TI) of 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone are better than those shown by AZT (Table 1). In addition, compound 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone is also active against HIV replication in a monocytic cell line and in peripheral blood mononuclear cells (PBMCs). In vitro assay indicated that, like compound suksdorfin, compound 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone is not an inhibitor of HIV-1 reverse transcriptase. Moreover, the anti-HIV activity of 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone is stereoselective as its three diastereoisomers are at least 10,000 times less active. Since other synthetic dihydroseselin derivatives with different substituents or without any substituents are inactive or are active only at much higher concentration, the antiviral potency of 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone could be associated with the camphanoyl moieties of its structure. Therefore, compound 3′,4′-di-O-(−)-camphanoyl-(+)-cis-khellactone represents a unique coumarin structure with promising anti-HIV activity.

DCK lactam analogues were synthesized [9] and evaluated in H9 cells. 4-methyl-DCK lactam exhibited potent anti-HIV activity (Table 1).

A series of disubstituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogues were synthesized [10] and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. 5-methoxy-4-methyl DCK was the most promising compound. As seen in Table 1, its parameters were much better than those of lead compound DCK. Another six disubstituted DCK analogues were more potent than AZT but less active than DCK. Conformational analysis suggested that resonance of the coumarin system is an essential structural feature for potent anti-HIV activity. Steric compression of C(4) and C(5) substituents of the coumarin moiety can reduce the overall planarity and thus resonance of the coumarin nucleus, resulting in a decrease or lack of anti-HIV activity.

To explore the structural requirements of (+)-cis-khellactone derivatives as novel anti-HIV agents, 24 monosubstituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) derivatives were synthesized in enantiometrically pure form [12]. These compounds included four isomeric monomethoxy analogues, four isomeric monomethyl analogues, four 4-alkyl/aryl-substituted analogues, and twelve 4-methyl-(+)-cis-khellactone derivatives with varying 3′,4′-substituents. These (+)-cis-khellactone derivatives were screened in acutely infected H9 lymphocytes. The results demonstrated that the (3′R, 4′R)-(+)-cis-khellactone skeleton, two (S)-(−)-camphanoyl groups at the 3′- and 4′-positions, and a methyl group on the coumarin ring, except at the 6-position, were optimal structural moieties for anti-HIV activity. EC₅₀ and TI values for 3-methyl-, 4-methyl-, and 5-methyl-3′,4′-di-O-(S)-camphanoyl-(3′R, 4′R)-(+)-cis-khellactone are shown in Table 1. Furthermore, 4-methyl-, and 5-methyl-3′,4′-di-O-(S)-camphanoyl-(3′R, 4′R)-(+)-cis-khellactone also showed potent inhibitory activity in CEM-SS cell line, and most monosubstituted DCK analogues were less toxic than DCK.

Six 3-substituted 3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives were synthesized from 3-methyl DCK. 3-hydroxymethyl DCK exhibited potent anti-HIV activity in H9 lymphocytes shown in Table 1. These values are similar to those of DCK and better than those of AZT [13].

In additional structure-activity-relationship (SAR) studies [1], the carbonyl oxygen of DCK was replaced with a sulfur atom. This bioisostere was less potent but also less cytotoxic than DCK. The 4-methyl, -propyl, and -benzyl analogues were also prepared; the order of activity was methyl > H > propyl > benzyl. 4-methyl-3′,4′-di-O-(−)-camphonyl-(+)-cis-khelthiolactone exhibited extremely potent anti-HIV activity (Table 1).

To enhance the water solubility and oral bioavailability of DCK analogues, 12 new mono- and disubstituted (3′R, 4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogues were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes [11]. 3-hydroxymethyl-4-methyl-DCK exhibited significant anti-HIV activity in H9 lymphocytes and even less in primary peripheral blood mononuclear cells (Table 1). Although this compound was not as potent as 4-methyl-DCK and 3-bromomethyl-4-methyl-DCK, it provides increased water solubility and possible linkage to other moieties. Of particular note, 3-hydroxymethyl-4-methyl-DCK exhibits moderate oral bioavailability (15%) when administered as a carboxymethylcellulose suspension to rats, whereas 4-methyl-DCK is not orally bioavailable in the same formulation. Further studies on mechanism of action suggest that 3-hydroxymethyl-4-methyl-DCK inhibits the production of double-stranded viral DNA from the single-stranded DNA intermediate. In addition, 3-bromomethyl-4-methyl-DCK is the most potent compound in this series of new analogues with EC₅₀ and TI values shown in Table 1. Thus, further modification at the 3-position of the coumarin ring can improve the potency of new DCK analogues.

Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5′ end of various oligonucleotides (ODNs). The interaction of these compounds with HIV-1 RT was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives. Some compounds activated the enzyme at relatively low concentrations (0.1–0.5 μM), followed by inhibition of the activity at higher concentrations (5–20 μM), whereas others behave just as inhibitors. Because the presence of some coumarin or chromone derivatives conjugated to ODNs enhanced the interaction with RT, Martyanov et al [28] analyzed the capacity of such ODN derivatives to be used as primers. The introduction of a chromone derivative, the 2-[3-(aminopropyl)amino]-8-isopropyl-5- methyl-4-oxo-4H-1-benzopyran-3-carbaldehyde], and a coumarin derivative, the 1-(3-aminopropoxy)-2-ethyl-3H-naphto [2,1-b] pyran-3-one, into the 5′ end of a noncomplementary ODN allowed these compounds to be used as primers. In the case of complementary primers, the presence of conjugated derivatives enhanced the affinity with Km values that were two to three orders of magnitude lower than that of a complementary primer of the same length. After addition of a ddT-unit to the 3′-terminal end of the ODN, some of these primers became very effective inhibitors of RT with Ki values in the nanomolar range. Martyanov et al [29] have carried out a comparison of Km and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5′-end pyranone derivatives was two to three orders of magnitude higher (Km = 3–15 nM) than that of d(pT)6 (Km = 12.6 mM). ODNs noncomplementary to poly(A) template were not elongated by RT. However, derivatives of d(CAGGTG) containing the 5′-terminal chromone and coumarin related groups were efficient primers showing Km (30–300 nM) and Vmax (75%–93%) values comparable with that for d(pT)10 (800 nM; 100%). The [d(CAGGTG)]ddT ODN derivatives were effective inhibitors of RT. The primer function of derivatives of noncomplementary ODNs appears to be due to the additional interactions of their 5′-terminal groups with the enzyme tRNA-binding site.

Toddacoumaquinone is a coumarin-naphthoquinone dimer, and it was synthesized through Diels-Alder reaction between 8-(1-acetoxy-3-methyl-1, 3-butadienyl)-5, 7-dime-thoxycoumarin and 2-methoxy-1, 4-benzoquinone [30]. The activities of Toddacoumaquinone against several viruses were examined. A weak activity (EC₅₀ = 10 μgr/mL) was observed against Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), but no activity was seen against HIV-1.

A new series of 3,5-bis(arylidene)-4-piperidones, as chal- cone analogues carrying variety of aryl and heteroaryl groups, pyrazolo[4,3-c]pyridines, pyridolo[4,3-c]pyrimi-dines, and pyrido[4,3-c]pyridines, carrying an arylidene moiety, and a series of pyrano[3,2-c]pyridines, as flavone and coumarin isosteres, were synthesized [31] and screened for their in vitro antiviral and antitumor activities at the National Cancer Institute. Several compounds proved to be active against HSV-1, while other compounds showed moderate activity against HIV-1. The pyrano[3,2-c]pyridines heterocyclic system proved to be the most active antitumors among the investigated heterocycles.

A single dose of coumarin derivatives, warfarin (Scheme 8), 4-hydroxycoumarin (Scheme 9), and umbelliferone (Scheme 10), added at the time of inoculation either by free virus or by contact with U1 monocytes exhibited a dose-dependent inhibitory effect on viral replication in target MOLT-4 lymphocytes observable even at 5 days after infection [32]. In addition, marked decrease of HIV-1 p24 release and reduction in RT activity was observed when chronically HIV-infected ACH-2 lymphocytes were treated with coumarins (ED₅₀ range 10⁻⁶−10⁻⁹ mol/L). However, the intracellular composition of HIV-1 core proteins in drug-exposed cells was not modified. Results suggest that although no complete inhibition of viral production has been observed in vitro, this class of drugs may present potential interest as antiviral agents.

The structures of a large number of HIV-1 integrase inhibitors have in common two aryl units separated by a central linker. Frequently, at least one of these aryl moieties must contain 1,2-dihydroxy substituents in order to exhibit high inhibitory potency. The ability of o-dihydroxy-containing species to undergo in situ oxidation to reactive quinones presents a potential limitation to the utility of such compounds. The report of tetrameric 4-hydroxycoumarin-derived inhibitor provided a lead example of an inhibitor which does not contain the catechol moiety. Tetrameric 4-hydroxycoumarin-derived inhibitor represents a large, highly complex, yet symmetrical molecule. It was the purpose of the study [33] to determine the critical components of tetrameric 4-hydroxycoumarin-derived inhibitor, and if possible to simplify its structure while maintaining potency. In the study, dissection of tetrameric 4-hydroxycoumarin-derived inhibitor (IC₅₀ = 1.5 μM) into its constituent parts showed that the minimum active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. However, in the simplest case in which the central linker aryl unit consisted of a phenyl ring (IC₅₀ = 43 μM), a significant reduction in potency resulted by removing two of the original four coumarin units. Replacement of this central phenyl ring by more extended aromatic systems having higher lipophilicity improved potency, as did the addition of 7-hydroxy substituents to the coumarin rings. Combining these latter two modifications resulted in compounds such as 3,3′-(2-naphthalenomethylene)bis[4,7-dihydroxycoumarin] (IC₅₀ = 4.2 μM) which exhibited nearly the full potency of the parent tetramer, yet were structurally much simpler.

The most important method that can be applied to develop new anti-AIDS compounds is computer-assisted drug design (CADD). The used method involves traditional or classic QSAR and 3D QSAR. In the traditional approach to QSAR, the chemical structure can be described with experimental and theoretical steric, electronic, and hydrophobic parameters. 3D QSAR methods were developed as an alternative to traditional QSAR to describe molecules. The results indicated that the flavonoids with hydroxyl groups at C-5 and C-7 in the A-ring, and with a C-2-C-3 double bond were the most potent HIV growth inhibitors [33]. According to the above information on structure-activity relationships, structure modification methods can be also used for flavonoid lead compounds, which are derived from plants with possible anti-HIV activity. As a potential target, the heteroatom in position 1 of the C-ring of the flavonoid compounds has been considered. Therefore, similar or even new biological activities could be anticipated when the oxygen of bioactive flavonoids is replaced by another atom such as nitrogen or sulfur, which lines up closely with oxygen in the periodic table. Thus, a series of 5,6,7,8-subsituted-2-phenylthio-chromen-4-ones has been synthesized and evaluated for anti-HIV activity [34]. Among them, one new compound was the most active (IC₅₀ value of 0.65 μM) against HIV in acutely infected H9 lymphocytes, and had a TI of approximately 5. A systematic series of chemically modified coumarin dimers has been synthesized and tested for their inhibitory activity against HIV-1 integrase. Mao et al [35] observed that modified coumarin dimers containing hydrophobic moiety on the linker display potent inhibitory activities.

HIV-1 protease has been identified as a significant target enzyme in AIDS research. While numerous peptide-derived inhibitors have been described, the identification of a nonpeptide inhibitor remains an important goal. Using an HIV-1 protease mass screening technique, 4-hydroxy-3-(3-phenoxypropyl)-2H-1-benzopyran-2-one was identified as a nonpeptide competitive inhibitor of the enzyme [36]. Employing a Monte Carlo-based docking procedure, the coumarin was docked in the active site of the enzyme, revealing a binding mode that was later confirmed by the X-ray crystal analysis. Several analogues were prepared to test the binding interactions and improve the overall binding affinity. The most active compound in the study was 4,7-dihydroxy-3-[4-(2-methoxyphenyl)butyl]-2H-1-benzo-pyran-2-one (Scheme 11).

The screening of the HIV-1 protease (PR) inhibitory activity (IC₅₀) of various substituted 3-phenyl-4-hydroxy- coumarins, 3-benzyl-4-hydroxycoumarins, 3-phenoxy-4-hy-droxy-coumarins, 3-benzenesulfonyl-4-hydroxycoumarins, and 3-(7-coumarinyloxy)-4-hydroxycoumarins was performed [37]. The data indicate the importance of substituents at positions 5 and 7 of the coumarin ring on the inhibitory potency of the HIV-1-PR.

The interaction of novel series of synthetic inhibitors with various serine proteases (leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators, and plasmin) and an aspartic protease (HIV-1 protease) were studied [38]. Various aspects were analyzed: mechanism of action, structure-activity relationships, and in some cases, molecular modeling, and biological evaluation. Functionalized cyclopeptides and N-aryl azetidin-2-ones behaved as suicide substrates acting specifically on trypsin-like proteases (thrombin or urokinase) and elastases, respectively. Novel hydrazinopeptides acted as reversible inhibitors of elastases. Coumarin derivatives inactivated very efficiently chymotrypsin-like proteases (k(inact)/K(I) = 760,000 M⁻¹. s⁻¹). Inhibitors of HIV-1 protease acting either as inactivators or dimerization inhibitors are under investigation. The inhibitors described above are useful for elucidating the biological roles of the target enzymes and constitute potential drugs.

All the reagents 2-aminobenzothiazol (Farak berlin, Germany), salicylaldehyde, KBH₄ (Lancaster), Calf thymus DNA, guanine, adenine (Sigma), (CH₃)₂SnCl₂ (Fluka), CuCl₂ · 2H₂O, and ZnCl₂ (anhydrous) (Merck) were used without further purification. Microanalyses were performed by a Carlo Erba Analyzer Model 1108. Molar conductance was determined at room temperature by a Digisun electronic conductivity bridge. IR spectra (Nujol mull) (200−4000 cm⁻¹) were recorded on a Shimadzu 8201 PC spectrophotometer. ¹H and ¹³C NMR spectra were recorded by Bruker DRX-300 spectrometer. Mass spectra were obtained on a Jeol SX-102 (FAB) spectrometer. EPR spectra were recorded on a Varian E112 spectrometer at X-band frequency (9.1 GHz) at liquid nitrogen temperature (LNT).

Cyclic voltammetry was carried out at CH instrument electrochemical analyzer. High purity H₂O and DMSO (95 : 5) was employed for the cyclic voltammetric studies with 0.4 M KNO₃ as a supporting electrolyte. A three electrode configuration was used comprising of a Pt disk working electrode, Pt wire counter electrode, and Ag/AgCl as reference electrode. Kinetic studies were carried out with a Cintra 5 UV-Vis spectrometer attached to an online data analyzer on which absorption spectra were evaluated. All experiments involving the interaction of the complex (5) with guanine, adenine, and calf thymus DNA were conducted in buffer (9.2 pH), doubly distilled water, and Tris buffer (7.5 pH), respectively. The progress of the reaction was monitored by measuring absorbance changes at 269 nm (λ_max of complex (5) + guanine), 260 nm (λ_max of adenine), and 260 nm (λ_max of CT-DNA), respectively. Pseudo-first-order rate constants, k_obs, were determined by linear least squares regression method.

To a solution of 2-aminobenzothiazol (5 g, 0.033 mol) in 50 mL of methanol was added (3.49 g, 0.033 mol) salicylaldehyde. The reaction mixture was refluxed for 3 hours. Yellow precipitate appears immediately on cooling, which was separated by filtration, recrystallized from methanol, and dried in vacuo over fused CaCl₂. Yield 7.0 g (82%) mp 120 ± 2°C (found: C, 66.16; H, 3.90; N, 10.98. C₁₄H₁₀N₂SO%) requires C, 66.14; H, 3.93; N, 11.02. IR/cm⁻¹ (Nujol mull): 1608_vs (C=N), 1286 (C−OH), 753(C−S). δ_H (300 MHz, DMSO, TMS) 7.57-6.55 (ArH), 7.90-7.82 (HC=N), 10.12 (OH). δ_c 129-124 (ArC), 165 (HC=N), 152 (C−S).

To a solution of Schiff base (4.7 g, 0.018 mol) in 100 mL dry DMF was added KBH₄ (0.5 g, 0.009 mol). This reaction mixture was refluxed for circa. 10 hours in a closed assembly fitted to monitor the evolution of H₂ gas. During the course of refluxing the reaction mixture, the solution changes colour slowly from dark yellow to colorless and later turns brown. After 10 hours, the evolution of hydrogen gas ceased and light brown colored product appeared in the reaction flask. The product was filtered and washed twice with toluene and dried in vacuo over fused CaCl₂. Yield 3.6 g (69%) mp 260 ± 1°C (found: C, 64.88; H, 3.81; N, 10.78. C₂₈H₂₀N₄S₂O₂B requires C, 64.86; H, 3.86; N, 10.81%). Mass spectrum (FAB⁺): m/z 518. IR/cm⁻¹ (Nujol mull): 1595_vs (C=N), 1391 (B−O), 2395 (B−H). ∧_M (CH₃OH): 102 Ω⁻¹ cm² mol⁻¹ (1 : 1 electrolyte). δ_H (300 MHz, DMSO, TMS) 7.55-6.52 (ArH), 7.99-7.97 (HC=N), 0.72 (B−H). δ_c 129-124 (ArC), 165 (HC=N), 114 (C−N), 153 (C−S).

The borate ligand (1.03 g, 0.002 mol) in 50 mL methanol was treated with CuCl₂· 2H₂O (0.340 g, 0.002 mol). The reaction mixture was stirred for 2 hours and then allowed to stand overnight in refrigerator. A brown product separates out, which was isolated by filtration under vacuum. It was washed thoroughly with hexane and dried in vacuo over fused CaCl₂. Yield 0.57 g (54%) mp 205 ± 3°C (found: C, 53.00; H, 3.46; N, 8.84. C₂₈H₂₂N₄S₂O₃BCuCl requires C, 52.99; H, 3.47; N, 8.83%). IR/cm⁻¹ (Nujol mull): 1598_vs (C=N), 1419 (B−O), 2400 (B−H), 391 (Cu−O), 310 (Cu−Cl).

This was synthesized by a procedure similar to that described for complex [C₂₈H₂₂N₄S₂O₃BCuCl] (3). Yield 0.6 g (57%) mp 220 ± 2°C (found: C, 52.84; H, 3.47; N, 8.78. C₂₉H₂₄N₄S₂O₃BZnCl requires C, 52.83; H 3.45; N, 8.80%). IR/Cm⁻¹ (Nujol mull): 1596_vs (C=N), 1423 (B−O), 2394 (B−H), 400 (Zn−O), 321 (Zn−Cl). δ_H (300 MHz, DMSO, TMS) 7.55-6.45 (ArH), 7.94–7.97 (HC=N), 0.54 (B−H). δ_c 127-125 (ArC), 165 (HC=N), 115(C−N), 151(C−S).

To a solution of C₂₈H₂₂N₄S₂O₃BCuCl (0.634 g, 0.001 mol) in 40 mL DMF was added (CH₃)₂SnCl₂ (0.438 g, 0.002 mol) in 1 : 2 molar ratio. The reaction mixture was refluxed for 48 hours on a water bath. A dark brown precipitate appears, which was isolated, filtered off, washed with hexane, and dried in vacuo over fused CaCl₂. Yield 0.52 g (49%) mp (dec.) 340 ± 2°C (found: C, 35.84; H, 3.16; N, 5.20. C₃₂H₃₈N₄S₂O₇BCuSn₂Cl₅ requires C, 35.82; H, 3.17; N, 5.22%). IR/Cm⁻¹ (KBr): 1521 (C=N), 1420 (B−O), 2400 (B−H), 390 (Cu−O), 311 (Cu−Cl), 462 (Sn−Cl), 420 (Sn−N) 546 (Sn−C). (Scheme 1).

This was synthesized by a procedure similar to that described for complex [C₃₂H₃₄N₄S₂O₃BCuSn₂Cl₅] (5). Yield 0.50 g (59%) mp (dec.) 300 ± 3°C (found: C, 35.77; H, 3.16; N, 5.20. C₃₃H₄₀N₄S₂O₇BZnSn₂Cl₅ requires C, 35.75; H, 3.16; N, 5.21%). IR/Cm⁻¹ (Nujol mull): 1524 (C=N), 1421 (B−O), 2400 (B−H), 402 (Zn−O), 320 (Zn−Cl), 460 (Sn−Cl), 428 (Sn−N), 549 (Sn−C). δ_H (300 MHz, DMSO, TMS) 7.67-6.62 (ArH), 9.84-8.40 (HC=N), 0.55 (B−H), 1.21 (CH₃). δ_c 130-124 (ArC), 168 (HC=N), 118 (C−N), 155 (C−S) 39.8 (Sn−C).

The IR spectrum of the ligand shows prominent stretching vibration at 2372–2400 cm⁻¹ region due to ν(B−H). The BH stretch generally appears as a single peak in the regions 2400–2500 cm⁻¹, but the presence of both ¹⁰B and ¹¹B in natural boron results in the splitting of bands [22, 23]. Other characteristic frequencies due to the presence of the ligand appear at 1595 cm⁻¹, 1449 cm⁻¹, and 752 cm⁻¹ assigned to ν(C=N), ν(C−N), and ν(C−S) vibrations, respectively [24–26]. The formation of borate is authenticated by the appearance of ν(B−O) band at 1391 cm⁻¹ [27, 28], which is further confirmed by absence of ν(O−H) stretching vibration at 3422 cm⁻¹ which was present in the Schiff base [29]. The ν(B−O) band, however, shifts to higher frequencies (28 cm⁻¹) in the complexes indicating the participation of oxygen of borate in the formation of complexes.

In the far IR spectra of the monometallic complexes, sharp absorption bands appearing at 390–402 cm⁻¹, 311–320 cm⁻¹ are assigned to ν(M−O) and ν(M−Cl) vibration, respectively [30, 31]. The bimetallic complexes show absorption bands at 420–428, 546–549, and 460–462 cm⁻¹ assigned to ν(Sn−N), ν(Sn−C), and ν(Sn−Cl), respectively, confirming the coordination of tin(IV) center to azomethine nitrogen and chlorine atoms [32, 33].

The electronic absorption spectra of the borate ligand and complexes reveal three strong bands in 40 000–25 000 cm⁻¹ region which are attributed to intraligand and charge transfer transitions.

The complex (3) exhibits a broad and low energy band at 16 528 cm⁻¹ which is attributed to d-d transition (²B_1g →²A_1g), typical for Cu^II in square planar environment. The absorption spectrum of the (5) complex exhibits two MLCT bands at 34 013 cm⁻¹ and 30 303 cm⁻¹ and a broad band at 17 361 cm⁻¹ attributed to d-d transition which is typical for copper(II) complex in square planar geometry [34]. Although there is a shift in the d-d absorption band of the (5) complex in comparison to the absorption band observed for the (3) complex which is attributed to the presence of Sn(IV) metal ion, the environment around the copper center does not alter much, it retains square planar geometry.

The solid state X-band EPR spectrum of the Cu(II) complex recorded at LNT (77 K) was found to be anisotropic with only two peaks with “g” values g_⊥ = 2.037, g_∥ = 2.195, and g_av = 2.089, respectively. The parameter g_av was obtained according to the equation [(g_av) = 1/3 (g_∥ + 2g_⊥)] and is in good agreement with corresponding anisotropy in square planar environment. The existence of g_∥ > g_⊥ suggested that the unpaired electron is localized in d_x²−y² orbital of the Cu^II ion with “3d⁹” configuration, that is, (eg)⁴, (a₁g)² (b₂g)² (b₁g)¹, which is the characteristic of the square planar geometry [35].

The g values are related to the axial symmetry parameter G by the Hathaway [36, 37] expression G = (g_∥ − 2)/(g_⊥ − 2). According to Hathaway, if the value of G is greater than four, the exchange interaction is negligible, whereas when the value of G is less than four, a considerable exchange interaction is indicated in the complex. In the complex (5), the G value obtained was 5.27 which indicates that exchange interactions are absent.

¹H NMR spectra are particularly useful to confirm the formation of borate ligand. The absence of phenolic −OH resonance peak at 10.12 ppm in the ligand clearly indicates the formation of borate by the removal of hydrogen gas [38] and the presence of BH₂ signal at 0.72 ppm [39]. The other signals due to N=CH proton and aromatic ring proton appear at 7.99-7.97 and 7.55-6.52 ppm, respectively. In the complexes (4) and (6), there is a slight upfield shift in BH₂ signal due to the presence of metal ions in the proximity of BH₂ protons. A new signal appears in the complex (6) at 1.21 ppm due to methyl protons of the dimethyltin moiety.

¹³C NMR spectra of ligand have been recorded in DMSO and carbon resonance signals appears at 129-124, 165, 153, and 114 ppm assigned to aromatic phenyl ring carbons, HC=N, C−S, and C−N groups, respectively. Upon complexation, there is a slight shift in aromatic ring carbon resonance due to the coordination of metal to the oxygen atom of phenyl ring. Furthermore, complex (6) registers a new signal at 39.8 ppm attributed to −Sn−CH₃ carbons due to which CH=N carbon resonance gets altered slightly, this is also an indication of coordination of azomethine nitrogen to the diorganotindichloride. Other carbon signals remain unaltered in the complexes.

The electrochemical behavior of the complex (5) has been examined by cyclic voltammetry to study the metallointeraction. The cyclic voltammogram of the complex (5) in absence of guanine, adenine, and calf thymus DNA was recorded in DMSO/H₂O (5 : 95) at a scan rate of 0.1 Vs⁻¹ in the potential range −1.2 to 1.6 V versus Ag/AgCl electrode. It exhibits a well-defined quasireversible redox wave Cu^II/Cu^I attributed to one electron transfer process with E_p value at −0.615 V and −0.519 V (Figure 1, curve a). For this couple, the difference between the cathodic and anodic peak potentials ΔE_p is of the order 96 mV, a somewhat large peak to peak separations in comparison to Nernstian value (59 mV) observed for one electron transfer couple. Large peak width for one electron couple Cu^II→ Cu^I is fairly common observation and is due to the reorganization of the coordination sphere during the electron transfer process [40, 41]. The ratio of anodic to cathodic peak currents I_pa/I_pc is less then the unity (0.3). The criteria for reversibility of the process is satisfied as on increasing the scan rate; the voltammogram does not show any significant change, and current is proportional to V^1/2 [42].

The electrochemical behavior of the complex (5) in the presence of the guanine, adenine, and calf thymus DNA in DMSO/buffer, DMSO/H₂O, and DMSO/Tris buffer (5 : 95), respectively, are presented in Table 1.

The CV trace of the complex (5) in the presence of guanine shows a dramatic change in electrode potential E_p values, while the cathodic peak potential shifts to −0.669 V (in comparison to solution without guanine E_p = − 0.615 V), a positive shift of −0.054 V is observed. However, the anodic peak disappear completely (Figure 1, curve b) indicative of strong binding of the complex to guanine base.

The cyclic voltammogram of the complex (5) in the presence of adenine shows a slight shift in formal potential values (in comparison to the solution in absence of adenine) (Figure 2, curve c). Although it corresponds fairly well with quasireversible one electron redox couple, peak potential does not show any significant change (0.002 and 0.003 V). The CV trace of adenine bound complex clearly suggests that the binding of the complex (5) to adenine is possible but the degree of binding is much lower in comparison to guanine.

However, the cyclic voltammogram of the complex (5) in the presence of calf thymus DNA (Figure 3, curve d) shows a significant shift in electrode potential value; cathodic and anodic peak potentials both shift to positive values −0.642 V and −0.561 V, while for the solution of the complex (5) in the absence of calf thymus DNA, electrode potential values are −0.615, −0.561 V as depicted in Figure 1, curve a, indicating that both the copper(II) and copper(I) forms interact with the calf thymus DNA to the same extent and suggest strong binding with calf thymus DNA [43]. Employing a square redox scheme, the net shift in E_1/2 has been estimated from the ratio of equilibrium constants for the binding of Cu^II and Cu^I complexes to calf thymus DNA using the following equation: (1) where E^1/2 and E^1/2′ are formal potentials of the Cu(II)/Cu(I) couple in the free and bound forms, respectively. The ratios of binding constants of K₂₊ and K₁₊ were corresponding to binding constants for the Cu(II)/Cu(I) species to DNA, respectively [44]. The ratio of binding constants of 1+ and 2+ species was less than 1 (0.670 for calf thymus DNA), which provides an evidence for the preferential stabilization of Cu(II) species.

The interaction of the complex (5) with guanine, adenine, and calf thymus DNA in DMSO/buffer, DMSO/H₂O, and DMSO/Tris buffer (5 : 95) was studied spectrophotometrically at 25°C under pseudo-first-order conditions.

The electronic absorption spectrum of free guanine exhibits two characteristic bands at 244 nm and 273 nm. On addition of the complex (5), the UV band at 273 nm increased in intensity and shifted to 269 nm (a shift of 4 nm is observed), as shown in Figure 4. The binding of guanine with the complex (5) results in blue shift and increase in intensity which is attributed to “hyperchromism.” Hyperchromism was due to the breakage of intermolecular hydrogen bonds when bound to DNA and is consistent with many earlier reports for copper complexes [45, 46].

Kinetics of guanine binding to the complex (5) was studied at 269 nm (λ_max of the complex (5) + guanine) under pseudo-first-order condition keeping the concentration of complex constant (c = 1 × 10⁻³ M) and varying the concentration of guanine (c = 10 − 16 × 10⁻³ M) at different time intervals (Figure 5(a)). The rate constants k_obs were determined by the linear least squares regression method. An exponential log(Aα − A₀) of absorbance against time plots gave a straight line indicative of pseudo-first-order reaction upto 80% completion of the reaction (Figure 6).

The electronic absorption spectrum of adenine shows a characteristic UV band at 260 nm. On addition of the complex (5), there is no significant shift in wavelength and a slight increase in absorbance. Although a slight hyperchromism is observed, but the degree of hyperchromism is insignificant in comparison to the binding of guanine to the complex (5), which shows relatively weak interaction with adenine base.

Kinetics of adenine binding to the complex (5) was carried out at 260 nm (λ_max of complex (5) + adenine) under pseudo-first-order conditions. Figure 5(b) shows time scan plot of interaction of adenine with complex (5) depicting a small change in absorbance intensity. The rate constant k_obs values were plotted by linear least squares regression method (Figure 6).

The interaction of the complex (5) with calf thymus DNA was carried out to obtain detailed information concerning the magnitude of the kinetic influence from the DNA environment as a function of position of guanine N₇ within calf thymus DNA.

The interaction of the complex (5) to the calf thymus DNA was carried out at 260 nm (λ_max of calf thymus DNA) under pseudo-first-order conditions (Figure 5(c)). On addition of the complex (5) to the calf thymus DNA, absorption spectra reveal a sharp change in absorption intensity with a red shift of 3 nm. At different time intervals, the absorption maxima increases in intensity indicating “hyperchromic effect” with calf thymus DNA.

The kinetics is further studied by observed pseudo-first-order rate constants k_obs value, as they can be directly compared and used as a measure of the kinetic influence from surrounding DNA [47]. The observed rate constant 1.77 s⁻¹ for guanine bound complex is of large magnitude in comparison to k_obs 1.52 s⁻¹ value for calf thymus DNA bound complex. The rate constant for adenine bound complex is 0.94 s⁻¹, which is much slow in comparison to guanine and calf thymus DNA. The complex shows preference for guanine over adenine due to two effects. When adenine binds, only a weak hydrogen bond is formed between the chloride ligand of the complex and H₂N−C₆ group of adenine; secondly, a significantly stronger molecular orbital interaction is identified in guanine in comparison to adenine. The presence of the electron withdrawing oxo group at the C₆ position of the purine ring lowers the energy of the lone-pair orbital at N₇ of purine base. The guanine molecular orbital has an energy of −6.877 eV, whereas −6.675 eV is obtained for adenine [48]. These studies have been demonstrated in a recent article of interaction of cisplatin with purine bases by Lippard et al [49]. Our investigations show that the complex C₃₂H₃₄N₄S₂O₃BCuSn₂Cl₅ is strongly bound to calf thymus DNA via different modes. Cu(II) prefers to bind strongly to N₇ of guanine base while the Sn^IV atom binds to the phosphate group [50]. Moreover, the affinity of Sn^IV with dinegative phosphate group is very strong due to its hard Lewis acidic nature. The binding to guanine is also kinetically preferred and supported by large k_obs value (1.77 s⁻¹) for guanine bound complex.

Thus in conclusion, the complex (5) may first bind with phosphate group of calf thymus DNA, neutralize the negative charge of calf thymus DNA phosphate group, and cause contraction and conformation change of calf thymus DNA which is clearly evidenced by the „overall” hyperchromic effect observed in the absorption spectra.