Synthesis, Chemical Characterization and Biological Screening for Cytotoxicity and Antitumor Activity of Organotin (IV) Derivatives of 3,4-Methylenedioxy 6-nitrophenylpropenoic Acid

Ahmad, Muhammad Sheeraz; Hussain, Mukhtiar; Hanif, Muhammad; Ali, Saqib; Mirza, Bushra

doi:10.3390/12102348

Open AccessArticle

Synthesis, Chemical Characterization and Biological Screening for Cytotoxicity and Antitumor Activity of Organotin (IV) Derivatives of 3,4-Methylenedioxy 6-nitrophenylpropenoic Acid

by

Muhammad Sheeraz Ahmad

¹,

Mukhtiar Hussain

²,

Muhammad Hanif

²,

Saqib Ali

² and

Bushra Mirza

^1,*

¹

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad Pakistan

²

Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan

^*

Author to whom correspondence should be addressed.

Molecules 2007, 12(10), 2348-2363; https://doi.org/10.3390/12102348

Submission received: 17 July 2007 / Revised: 9 August 2007 / Accepted: 10 August 2007 / Published: 25 October 2007

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Abstract

:

A series of mono-, di- and triorganotin compounds with general formulae [RSnL₂Cl], R = Bu (compound 3), [R₂SnL₂], where R = Me, Et, Bu, Oct (compounds 1, 2, 4 and 6) and [R₃SnL], where R = Bu, Cy and Ph (compounds 5, 7 and 8) and where L = 3,4-methylenedioxy-6-nitrophenylpropenoic acid have been prepared and characterized by elemental analysis, multinuclear (¹H-, ¹³C- and ¹¹⁹Sn-) NMR and mass spectrometry. The ligand and its respective organotin complexes were screened for cytotoxicity using the brine shrimp lethality assay and for antitumor activity using the crown gall tumor inhibition (potato disc) assay. The bioassay results support the conclusion that the biological activities of these synthetic compounds are in the following order: [RSnL₂Cl] < [R₂SnL₂] < [R₃SnL].

Keywords:

Antitumor; cytotoxicity; mass spectrometry; multinuclear NMR; organotin.

Introduction

Organotin (IV) complexes are extensively used as catalysts, stabilizers, biocides, antifouling agents and as wood preservatives [1,2,3,4,5]. Organotin (IV) derivatives of carboxylic acids are of special interest with regards to their methods of synthesis, structural elucidation and biological activity [6,7,8,9]. Generally these compounds can be well characterized by multinuclear NMR (¹H-, ¹³C- and ¹¹⁹Sn-) spectroscopy [10, 12]. In recent years organotin (IV) carboxylates have attracted much attention owing to their potential biocidal activity and cytotoxicity [5, 12]. In addition, among organometallic compounds there has been increased interest in organotin carboxylates due to their activity against various types of cancer cells as many of the known di- and triorganotin (IV) carboxylates display interesting antitumor activities [12,13,14,15].

Their structural diversity was also of interest as both diorganotin and triorganotin esters show rich and diverse structural chemistry, as cited in a recent review [8]. Keeping in mind this structural and biological diversity of organotin (IV) carboxylates, we have now synthesized some organotin (IV) carboxylates of 3,4-methylenedioxy-6-nitrophenylpropenoic acid (L). The structures of these compounds have been evaluated by various analytical techniques such as elemental analysis, multinuclear NMR (¹H-, ¹³C- and ¹¹⁹Sn-) and mass spectrometry. The ligand L and its organotin (IV) derivatives have been screened for cytotoxicity using the brine shrimp lethality assay and for antitumor activity with the crown gall tumor inhibition assay (potato disc assay).

Results and Discussion

Synthesis

Mono-, di- and triorganotin complexes were synthesized by refluxing stoichiometric amounts of the ligand acid, R^'COOH, with the corresponding oxides/hydroxides (Procedure I) or its sodium salt with the corresponding organotin chlorides in toluene (Procedure II), as summarized in equations I-V.

RSn(OH)₂Cl + 2HL Molecules 12 02348 i001

RSn(Cl)L₂ + 2H₂O
R= n-Bu (3)

(I)

R₂SnO + 2HL Molecules 12 02348 i001

R₂SnL₂ + H₂O
R = n-Bu (4), n-Oct (6)

(II)

R₂SnCl₂ + 2NaL Molecules 12 02348 i001

R₂SnL₂ + 2NaCl
R = Me (1), Et (2)

(III)

R₃SnCl + NaL Molecules 12 02348 i001

R₃SnL + NaCl
R = n-Bu (5)

(IV)

R₃Sn(OH)+HL Molecules 12 02348 i001

R₃SnL+H₂O
R= Cy (7), Ph (8)

(V)

All the newly synthesized compounds are air stable crystalline solids, soluble in common organic solvents, and their physical data is summarized in Table 1. The structures of these compounds and coordination behavior of metal have been addressed by different analytical techniques such as elemental analysis, multinuclear NMR (¹H, ¹³C and ¹¹⁹Sn) and mass spectrometry.

Table 1. Physical data of 3,4-methylenedioxy-6-nitrophenylpropenoic acid (L) and its organotin (IV) derivatives.

**Table 1.** Physical data of 3,4-methylenedioxy-6-nitrophenylpropenoic acid (L) and its organotin (IV) derivatives.
Compound No.	Molecular Formula	Molecular Weight	M.P. (˚C)	Yield(%)	Elemental Analysis % Calculated (Found)
Compound No.	Molecular Formula	Molecular Weight	M.P. (˚C)	Yield(%)	C	H	N
1	C₂₂H₁₈O₁₂N₂Sn	622	237-240	86	42.51 (42.48)	2.90 (2.96)	4.51 (4.58)
2	C₂₄H₂₂O₁₂N₂Sn	650	176-178	67	44.35 (44.40)	3.46 (3.43)	4.31 (4.42)
3	C₂₄H₂₆O₁₂N₂SNCl	689.5	142-145	70	42.13 (42.24)	3.07 (3.15)	4.10 (4.04)
4	C₂₈H₃₀O₁₂N₂Sn	706	196-199	73	47.66 (47.74)	4.26 (4.16)	3.97 (4.01)
5	C₂₂H₃₃O₆NSn	527	143-146	89	50.19 (50.23)	6.27 (6.30)	2.66 (2.70)
6	C₃₆H₄₆O₁₂N₂Sn	818	151-153	75	52.88 (52.96)	5.63 (5.60)	3.43 (3.50)
7	C₂₈H₃₉O₆NSn	605	99-101	83	35.63 (35.69)	6.46 (6.50)	2.32 (2.36)
8	C₂₈H₂₁O₆NSn	587	207-210	80	57.34 (54.43)	3.58 (3.60)	2.39 (2.45)
Ligand	C₁₀H₇O₆N	237	278	80	50.63 (50.45)	2.95 (2.80)	5.90 (5.87)

¹H-NMR Spectroscopy

¹H-NMR spectra for the synthesized compounds and free acid have been recorded in CDCl₃ and DMSO solutions. They are reported in Table 2. ¹H resonance signals of the protons attached to the phenyl moieties of the ligand have been assigned by their distinct multiplicities, J-values and comparison with the results obtained from the incremental method [9]. The methyl protons of dimethyltin derivatives appear as sharp singlets at 0.89 ppm, with well-defined satellites; coupling constants are included in Table 2. The α-CH₂ protons of diethyltin (IV) compounds resonate as a quartet at 1.52 ppm and have well-resolved coupling constants, while β-CH₃ protons resonate as a triplet at 1.34 ppm with ³J[¹H,¹H] = 8.2 Hz. The phenyl moieties of the triphenyl- and diphenyltin (IV) derivatives mostly show a complex pattern and were assigned according to the literature [10]. Despite the complex pattern of the ¹H-NMR spectra of di- and tri-n-butyltin (IV) derivatives, assignments were possible: a clear triplet due to the terminal methyl group appears in the 0.91-0.92 ppm range [11,12a]. The α-CH₂ protons of the di-n-butyltin compound 4 appear as a triplet around 1.72 ppm, with ³J[¹H,¹H] = 8.0 Hz [12b]. The β-CH₂ and γ-CH₂ signals appear as multiplets and δ-CH₃ as a triplet at 0.91 with ³J[¹H,¹H] = 10.0 Hz. In the tri-n-butyltin (IV) complex, δ-CH₃ resonates as a triplet at 0.92 ppm with ³J[¹H,¹H] = 8.5 Hz, and γ-CH₂ and β-CH₂ protons appear as multiplets at 1.33-1.37 ppm and 1.61-1.65 ppm, respectively. The α-CH₂ appears as a triplet at 1.60 ppm. The methylene protons (CH₂) of the n-octyltin (IV) moiety exhibit a somewhat different behavior, compared with the n-butyl groups of the respective complexes. The α-CH₂, β-CH₂ protons give multiplet signals at 1.49-1.53 ppm, respectively, which are consistent with the values calculated by the incremental method [13]. The γ-CH₂ and γ′-CH₂ do not appear in the region. However, the δ′-CH₃ protons resonate as a triplet in the range of 0.65 ppm with ³J[¹H,¹H] = 7.0 Hz. The protons of the methylenedioxy moiety appear as singlets at 6.00-6.29 ppm and were seen in almost all the compounds. The ²J[¹¹⁹Sn-¹H] coupling constants and the C-Sn-C angles (Table 3) calculated from ²J[¹¹⁹Sn-¹H] values demonstrate that in the diorganotin (IV) derivatives, tin shows a coordination number greater that four, probably five or six in non coordinating solvents, while the triorganotin (IV) derivatives show distorted tetrahedral geometry [13, 14].

Table 2. ¹H-NMR data of 3,4-methylenedioxy-6-nitrophenylpropenoic acid and its organotin (IV) derivatives ^{a, b}

**Table 2.** ¹H-NMR data of 3,4-methylenedioxy-6-nitrophenylpropenoic acid and its organotin (IV) derivatives ^{a, b}
¹H No	Compound
¹H No	Ligand	1	2	3	4	5	6	7	8
2	7.09 (s)	7.12 (s)	7.00 (s)	7.10 (s)	7.09 (s)	7.01 (s)	7.02 (s)	7.03 (s)	6.96 (s)
5	7.52 (s)	7.58(s)	7.54 (s)	7.58 (s)	7.52 (s)	7.51 (s)	7.33 (s)	7.51(s)	7.78 (s)
7	6.29 (s)	6.24 (s)	6.12 (s)	6.00 (s)	6.11 (s)	6.14 (s)	5.98 (s)	6.14 (s)	6.11 (s)
8	7.82 (d,15.9)	7.96 (d,15.0)	8.17 (d,16.5)	7.95 (d,15.0)	7.99 (d, 15.0)	8.00 (d,15.0)	7.82 (d,16.0)	8.02 (d, 15.5)	7.82 (d,16.0)
9	6.50 (d,15.6)	6.47 (d,15.0)	6.33 (d,15.0)	6.47 (d,15.0)	6.30 (d, 15.0)	6.31 (d,15.0)	6.17 (d,15.5)	6.31 (d, 15.5)	6.30 (d,15.0)
α	−	0.89 (s)	1.52 (q, 10.0)	−	1.72 (t, 8.0)	1.6	1.49-1.53 (m)	−	−
β	−	−	1.34 (t, 8.2)	−	1.34-1.37 (m)	1.61-1.65 (m)	1.49-1.53 (m)	1.26-1.76 (m)	7.35-7.49 (m)
γ	−	−	−	−	1.34-1.37 (m)	1.33-1.37 (m)	−	1.26-1.76 (m)	7.35-7.49 (m)
δ	−	−	−	−	0.91 (t, 10.0)	0.92 (t, 8.5)	−	1.26-1.76 (m)	7.35-7.49 (m)
γ-γ′	−	−	−	−	−	−	1.15 (bs)	−	−
δ'	−	−	−	−	−	−	0.65 (t, 7.0)	−	−

^a Chemical shifts (δ) in ppm. ²J[¹¹⁹Sn, ¹H]; ⁿ[¹H, ¹H] in Hz where n= 3,4 are listed in square brackets and parenthesis, respectively. Multiplicity is given s=singlet, bs = broad signal, d = doublet, dd=doublet of doublet, q = quartet, m = multiplet; ^b Numbering in accordance with Figure 1, Figure 2, Figure 3 and Figure 4.

Figure 1. ¹H- and ¹³C-NMR numbering scheme of the ligand L.

Table 3. (C−Sn−C) Angles (^o) Based on NMR Parameters of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid

**Table 3.** (C−Sn−C) Angles (^o) Based on NMR Parameters of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid
Compound No.	¹J[¹¹⁹Sn, ¹³C] (Hz)	²J[¹¹⁹Sn, ¹H] (Hz)	Angle (^o)
Compound No.	¹J[¹¹⁹Sn, ¹³C] (Hz)	²J[¹¹⁹Sn, ¹H] (Hz)	¹J	²J
1	-	78	-	128.4
2	-	72	-	121.8
3	-	-	-	-
4	-	-	-	-
5	360	74	108.33	123.9
6	-	-	-	-
7	-	-	-	-
8	-	-	-	-

¹³C-NMR Spectroscopy

¹³C-NMR data recorded in CDCl₃ and DMSO solutions of the ligand and its respective mono-, di- and triorganotin (IV) derivatives are given in Table 4. The ¹³C-NMR spectral data for the R groups attached to the tin atom where R = Me, Et, n-Bu, n-Oct, Ph and Cy were assigned by comparison with related analogues as model compounds, combined with the ⁿJ[¹¹⁹Sn,¹³C] coupling constants [15, 18]. The assignment of the ¹³C- resonance associated with the carboxylate ligands is based: (I) on comparison with the results obtained from incremental methods [14], and (II) on comparison with the resonance values available in the literature [19]. The positions of the phenyl and olefinic carbon signals undergo minor variation in the complexes, compared to those observed in the free acid and its sodium salt. The carboxylate carbon shifts to a lower field region almost in all the complexes, indicating participation of the carboxyl group (COO) in coordination to tin (IV) [20].

Table 4. ¹³C- and ¹¹⁹Sn -NMR data^a,b of 3,4-methylenedioxy-6-nitrophenylpropenoic acid and its organotin (IV) derivatives. ^{a, b}

**Table 4.** ¹³C- and ¹¹⁹Sn -NMR data^a,b of 3,4-methylenedioxy-6-nitrophenylpropenoic acid and its organotin (IV) derivatives. ^{a, b}
¹³CNo	Ligand	1	2	3	4	5	6	7	8
1	125.78	126.36	126.96	127.11	127.86	127.93	127.25	128.07	127.53
2	107.19	105.93	107.64	107.65	107.46	107.37	107.68	107.39	107.40
3	151.73	153.10	152.05	152.62	151.90	151.90	153.09	151.87	151.94
4	148.73	150.18	149.24	148.78	148.65	148..63	150..29	148.55	148.87
5	106.90	104.86	105.18	105.01	105.60	105.56	105.75	105.56	105.63
6	138.87	141.35	142.67	143.81	138.96	139.03	141.52	138.78	140.88
7	103.74	104.30	103.06	103.45	103.26	103.25	104.68	103.20	103.20
8	142.98	141.25	143.92	143.81	143.13	143.06	144.34	143.07	143.10
9	122.67	123.10	120.78	124.54	125.82	124.54	122.49	124.85	122.77
10	167.03	187.87	175.39	186.84	171.62	170.90	174.75	170.70	171.96
α	-	4.34	19.17	22.70	22.70	15.24 [340, 360]	25.43	14.11	130.22
β	-	-	14.11	25.57	26.37	26.79	23.27	29.37	137.13
γ	-	-	-	29.19	26.83	27.04	33.56	31.95	128.97
δ	-	-	-	14.11	13.67	13.64	29.72	28.85	128.97
α′	-	-	-	-	-	-	31.34	-	-
β′	-	-	-	-	-	-	30.29	-	-
γ′	-	-	-	-	-	-	32.51	-	-
δ′	-	-	-	-	-	-	25.90	-	-
¹¹⁹ Sn	-	-112.63	-150.68	-150.03	-156.43	+116.10	-175.00	45.64	-112.45

^a Chemical shifts (δ) in ppm. ⁿJ[^117/119Sn, ¹³C]; ⁿJ[¹¹⁹Sn, ¹³C]; in Hz are listed in square brackets; ^b Numbering in accordance with Figure 1, Figure 2, Figure 3 and Figure 4.

Figure 2. Numbering scheme for groups attached to tin.

Figure 3. Structural diagrams of triorganotin (IV) Compounds.

Figure 4. Structural diagram of diorganotin (IV) Compounds.

¹¹⁹Sn-NMR Spectroscopy

The possibility of detecting the presence of coordinatively different organotin (IV) moieties was explored by acquisition of ¹¹⁹Sn-NMR spectra for all the investigated compounds. The ¹¹⁹Sn- chemical shifts usually cover a range, quoted relative to tetramethyltin, with increasing coordination number of tin producing a large upfield shift for δ(¹¹⁹Sn). The ¹¹⁹Sn-NMR spectra were recorded in CDCl₃ solutions (a non-coordinating solvent). It is reported [20, 21] that in alkyltin carboxylates, tetra- coordinated tin has δ(¹¹⁹Sn) values ranging from about +200 to -60 ppm, and penta-coordinated tin from -200 to -400 ppm. A single resonance peak of ¹¹⁹Sn chemical shift values obtained for the triorganotin (IV) derivatives lies in the range expected for a tetrahedral geometry. ¹¹⁹Sn chemical shift values of diorganotin compounds were observed in the -112.63 to -175.0 ppm range. It appears from the shift values that a weakly hexacoordinated tin generates a deformed octahedron or a skewed trapezoidal bipyramid with four strong and two somewhat weaker bonds, as indicated by the upfield ¹¹⁹Sn chemical shift values [14, 16, 20, 21].

In order to gain further information about the possible coordination geometries in solution, close examination of the ¹J[¹¹⁹Sn,¹³C] and ²J[¹¹⁹Sn,1H] coupling constants was undertaken as indicators of structural details. As reported by Nadvornik et al. [13, 22a], ²J [¹¹⁹Sn, ¹H] in n-Bu₃Sn (IV) carboxylates changes very little with a change in coordination number, being the smallest among such couplings. In the case of diorganotin compounds 1 and 2, the ²J[¹¹⁹Sn,¹H] values are 78 and 72 Hz, respectively, which indicate that tin displays hypervalent behavior (valence higher than four) in these compounds. .¹J[¹¹⁹Sn,¹³C] is, on the other hand, quite useful for making predictions about the geometry around the tin atom. In case of triorganotin (IV) carboxylates, ¹J[¹¹⁹Sn,¹³C] values ranging between 327 and 387 Hz for tetra-coordinated compounds and between 442 and 509 Hz for penta-coordinated ones were observed [22b]. For the tri-n-butyltin (IV) derivative the ¹J[¹¹⁹Sn,¹³C] values were 360 Hz and based on the Holeček and Lycka equation [20, 21], corresponds to a quasi-tetrahedral geometry in CDCl₃ solution. The geometric data calculated, as just described, are consistent with tetrahedral geometries for the triorganotin (IV) species [20], i.e. monomers in solution. Unfortunately, ¹J[¹¹⁹Sn,¹³C] values are not observed in any compound, but from the ¹¹⁹Sn values and ²J[¹¹⁹Sn,¹H], it can be predicted that diorganotin (IV) species, possess skew-trapezoidal bipyramidal geometries with the lower apparent coordination number arising from the asymmetric coordination mode of the carboxylate ligand [23,24].

Mass Spectrometry

The mass spectra of all the synthesized compounds 1-8 were determined using the electrospray ionization (ESI) technique. The molecular ion [M]⁺ peak is not usually observed in case of organometallic compounds while using the conventional electron ionization (EI) or chemical ionization (CI) mass spectral techniques. However in case of ESI, not only the molecular ion peak [M]⁺, but also peaks due to aggregation of fragments with [M]⁺ or [M+H]⁺ are encountered [25]. These fragment aggregates have molecular weights which is greater than [M]⁺ (Table 5).

The 1^st order +ve ESI-MS mode is more informative than the –ve mode because it can also provide information about the polymeric species and hence it is the more preferred for organotin species [26]. The neutral loses observed in ESI-MS are attributed to loss of CO_2, methane, ethane, butane and butene. The presence of more than one tin isotope in a molecule causes a wide distribution of the total signal among the many fragment aggregates, which decreases the relative intensity of the centroid peak. The presence or absence of a tin atom in individual ion can easily be recognized on the basis of the 10 characteristic natural tin isotopes, with the most abundant being the ¹²⁰Sn isotope.

The base peak in these compounds whether di- or triorganotin (IV) compounds derives from the different fragmentation patterns. All the investigated compounds were found to possess a base peak resulting from the loss of the ligand acid (HL), the R group or the ligand moiety itself.

Table 5. Mass fragmentation patterns of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid.

**Table 5.** Mass fragmentation patterns of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid.
Fragment, m/z (%)
1	2	3	4	5	6	7	8
[C₂₃H₂₀N₂O₁₂Sn]⁺ 636 (8.5)	[C₂₂H₁₆N₂O₁₂Sn]⁺ 620 (3.5)	[C₂₄H₁₉ClN₂O₁₂Sn]⁺ 682.5 (2.5)	[C₃₂H₃₇N₂O₁₂Sn]⁺ 761(16.5)	[C₃₀H₅₀NO₆Sn]⁺ 640(7.3)	[C₄₀H₅₄N₂O₁₂Sn]⁺ 874 (9.5)	[C₂₈H₃₉NO₆Sn]⁺ 605 (2.5)	[C₂₂H₁₅NO₆Sn]⁺ 509(14.9)
[C₂₂H₁₆N₂O₁₂Sn]⁺ 620 (17.3)	[C₁₅H₁₂NO₈Sn]⁺ 454 (41.6)	[C₂₀H₁₀ClN₂O₁₂Sn]⁺ 625.5 (11.3)	[C₂₇H₂₆N₂O₁₂Sn]⁺ 690 (7.6)	[C₂₂H₃₁NO₆Sn]⁺ 525 (6.4)	[C₃₄H₄₀N₂O₁₂Sn]⁺ 788 (16.2)	[C₂₇H₃₆NO₄Sn]⁺ 558 (7.3)	[C₂₁H₁₃NO₄Sn]⁺ 463 (3.3)
[C₂₁H₁₅N₂O₁₀Sn]⁺ 575 (3.4)	[C₁₀H₁₃O₄Sn]⁺ 317 (17.1)	[C₁₄H₁₄Cl NO₆Sn]⁺ 447.5 (32.7)	[C₂₄H₁₉N₂O₁₂Sn]⁺ 647 (19.9)	[C₁₈H₂₃NO₆Sn]⁺ 469 (23.6)	[C₂₈H₂₈N₂O₁₀Sn]⁺ 672 (26.3)	[C₂₂H₂₇NO₆Sn]⁺ 521 (33.4)	[C₁₅H₈NO₄Sn]⁺ 386 (27.1)
[C₁₉H₉N₂O₁₀Sn]⁺ 545 (37.7)	[Sn]⁺ 120 (3.7)	[C₅H₁₁Sn]⁺ 191 (41.5)	[C₈H₁₇N₂O₁₂Sn]⁺ 453 (25.2)	[C₁₄H₁₄NO₆Sn]⁺ 412 (12.7)	[C₁₈H₂₂NO₆Sn]⁺ 468 (8.7)	[C₁₆H₁₆NO₆Sn]⁺ 438 (17.1)	[C₉H₆O₂Sn]⁺ 266 (72.4)
[C₁₁H₆NO₆Sn]⁺ 368 (60.9)	[C₄H₉]⁺ 57 (100)	[C₄H₈Sn]⁺ 176 (6.9)	[C₄H₇Sn]⁺ 175 (79.7)	[C₇H₁₀O₂Sn]⁺ 246 (40.7)	[C₉H₁₆O₂Sn]⁺ 276 (100)	[C₉H₁₂O₂Sn]⁺ 272 (63.4)	[C₁₁H₆NO₆Sn]⁺ 368 (60.9)
[C₄H₄O₂Sn]⁺ 204 (100)	-	[C₃H₈Sn]⁺ 164 (18.8)	[C₂H₃Sn]⁺ 145 (100)	[C₄H₉]⁺ 57 (100)	[C₄H₉]⁺ 57 (76.3)	[C₄H₆O₂Sn]⁺ 206 (100)	[C₆H₅]⁺ 77 (100)
-	-	[C₄H₉]⁺ 57 (100)	-	-	-	-	-

Biological Activity

All the synthesized compounds and their acid ligand were screened for their antitumor activity and cytotoxicity using standard in vitro biocidal screening tests.

Cytotoxicity

Bioactive compounds are often toxic to shrimp larvae. Hence, shrimp larvae have been extensively used as rapid and simple preliminary test for cytotoxicity [11a, b]. All the synthesized compounds and ligand were screened for cytotoxicity by brine-shrimp bioassay lethality method [11b] and the results are summarized in Table 6. The LD₅₀data shows that all the compounds even the ligand acid are toxic with LD₅₀ values in the range 0.26 to 581.23 μg/mL for organotin derivatives and 975.24 μg/mL for the ligand L Tri-organotin derivatives are more toxic than di-organotin derivatives, mono-organotin derivatives and the ligand acid [27]. Compound 3 (a monoorganotin derivative) has been found to be the least toxic and compound 5 (a triorganotin derivative) was the most toxic of all the synthesized organotin derivatives [4, 5].

Table 6. Cytotoxicity data of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid.

**Table 6.** Cytotoxicity data of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid.
Compound No	LD₅₀µg/mL
1	-
2	130.98
3	581.23
4	33.39
5	0.26
6	191.64
7	0.40
8	0.56
Ligand	975.24

Against brine shrimps (in vitro); Data is based on mean value of 3 replicates of each for 1000, 100 and 10 μg/mL concentrations; - = activity not determined.

Antitumor Activity

For antitumor activity, the crown gall tumor inhibition assay (potato disc assay) [11b] was performed for all the synthesized compounds using Agrobacterium tumefaciens (strain At10). Crown gall is a neoplasmic disease of plants induced by Agrobacterium tumefaciens due to its Ti (tumor inducing) plasmid [11b]. Since the mechanism of tumor induction is similar to that seen in animals, this assay was used to evaluate the antitumor activity of these compounds. All the compounds showed significant levels of tumor inhibition, as shown in Table 7. Activity observed for synthesized organotin series was more than that of their parent acid, and triorganotin derivatives showed more tumor inhibition than mono- and diorganotin derivatives. Furthermore, compounds 2, 5 and 8 showed 100% tumor inhibition. The bioassays results reflect the conclusion that the activity are in the following order: [RSnL₂Cl] < [R₂SnL₂] < [R₃SnL]. Further, it has been observed that the activities of these mono-, di and triorganotin (IV) complexes are comparable with those reported earlier [2, 4, 5, 27].

Table 7. Antitumor activities of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid ^{a, b, c, d}.

**Table 7.** Antitumor activities of organotin (IV) derivatives of 3,4-methylenedioxy-6-nitrophenylpropenoic acid ^{a, b, c, d}.
Compound No.	Average number of tumors ± SE	% inhibition of tumors
1	2.8±0.89	62.66
2	0.0±0.0	100
3	3.9±1.02	48
4	1.0±0.36	86.66
5	0.0±0.0	100
6	5.3±0.84	29.33
7	4.8±1.13	36
8	0.0±0.0	100
Ligand	4.02±1.04	44
(-)ive control	7.5±1.13	-

^a) Potato disc antitumor assay, concentration: 1000 μg/mL in DMSO; ^b) More than 20% tumor inhibition is deemed significant; ^c) Percent Inhibition of tumors = [100 ─ (Number of tumors for sample/Number of tumors for control)] × 100; ^d) Data represents mean value of 15 replicates.

Experimental

General

Melting points were determined in a capillary tube using a MPD Mitamura Riken Kogyo (Japan) electrothermal melting point apparatus. Multinuclear NMR (¹H-, ¹³C- and ¹¹⁹Sn) spectra were recorded on a Bruker ARX 300 MHz-FT-NMR spectrometer using CDCl₃ as an internal reference for [δ ¹H (CDCl₃) = 7.25 and δ ¹³C (CDCl₃) = 77.0]. ¹¹⁹Sn-NMR spectra were obtained with Me₄Sn as an external reference [(¹¹⁹Sn) = 37.290665] [10]. Chemical shifts (δ) are given in ppm and coupling constants J are given in Hz. The multiplicities of signals in ¹H-NMR are given with chemical shifts; (s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet). Mass spectral data (ESI mode) were taken on a LCQ Finnigan MAT ion trap (USA). The m/z values were evaluated assuming that H = 1, C = 12, N = 14, O = 16, Cl = 35.5 and Sn = 120.