Transition Met. Chem., 21, 23-27 (1996) Antitumour properties of metal(II) complexes 23

Synthesis, characterization and antitumour properties of metal(II) solid complexes with Morin Zhang Qi, Wang Liufang* and Liu Xiang State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 73000, P.R. China and State Key Laboratory of Structural Chemistry, Fujian Institute of Research on Structure Matter, Chinese Academy of Science, Fuzhou Fujian, 350002, P.R. China

Li Shuben and He Fengying State Key Laboratory of Oxo-Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, La:nzhou 730000, P.R. China

Summary

Six metal(II) complexes with Morin ML2-nH20 [L = Morin(2'-OH group deprotonated); M = Mn, Co, Ni, Cu, Zn, or Cd; n = 2 or 3-] have been synthesized and characterized by elemental analysis, molar conductance, i.r., ~H-n.m.r., t.g.-d.t.a and n.v.-vis, spectroscopic tech- niques and by fluorescence analysis. Comparative anti- tumour activities of Morin '2H20 and two complexes [ZnL2-3H20 and CuL2.2H20 ] were tested by in vitro screening. The results show that the inhibitory ratio of complexes against the tested tumour cells was higher than that of Morin.

Introduction

Morin (2', 3,4', 5, 7-pentahy'droxyflavone; Figure 1) has been used ir~ fluorometric analysis ofmetal ions because of its ability to form fluorescent compounds with metal ions in solution. However, solid complexes of metals with Morin have rarely been reported. As Morin itself exhibits antitumour activity ~1'2), and some transition metals play a vital role in a vast number of widely differing biological processes (3"4), the study of the synthesis of transition metal-Morin complexes and their antitumour activity is appealing. Six new transition metal(II) complexes of Morin have been prepared and characterized. The anti- turnout activities of two complexes [ZnLz-3H20 and CuLz.2HzO ] against different tumour cells [Hep-2, BHK-21; BEL-7402, HL-60, KB] were tested. The results showed that although different complexes had different effects on different turnout cells, the inhibitory ratios of the two complexes tested were more or less greater than that of Morin '2H20 itself. Of the two complexes, the antitumour effect of CuL2"2H20 against HL-60 was out- standing [ICso = 6.7 x 10- 5 M].

Experimental

AR grade chemicals were used.

Preparation of MeONa

Small, freshly cut, pieces of Na (2.5 g) were added to dry MeOH (100 cm 3) (AR and refractionated).

Preparation of EtONa

Small, freshly cut, pieces of Na (2.3 g) were added to dry EtOH (100 cm 3) (AR and redistilled).

* Author to whom all correspondence should be directed.

Preparation of complexes

In a 150cm 3 three-necked, round-bottomed flask pro- vided with an electromagnetic stirrer, a reflux condenser and a CaC12-guard tube were placed Morin-2H20 (0.010tool) and absolute EtOH (70cm3). The flask was then heated to 50~ on a water-bath. When the solid Morin '2H20 dissolved, EtONa (0.30cm 3) solution was added. After ca. 3-4 rain, the metal(II) acetate (0.007 tool) was added quickly and the solution was stirred and boiled under reflux for 8 h. The mixture was then cooled to room temperature and poured into H20 (700 cm3). The yellow precipitate, which formed, immediately, was set aside for 48 h, then filtered, washed thrice with 1:3 E tOH-H20 , then several times with H20 to free the filtrate from metal ion. The solid product was dried in vacuo for 48 h. Yield 85-91%. The complexes thus prepared and their yields are given in Table 1.

Characterization of the complexes

Metal contents of the complexes were estimated by titra- tion with EDTA. C and H were determined using a Carbo Erba 1106 elemental analyser. Molar conductances at room temperature were measured in 10 3 M DMSO sol- ution using a DSS-12 molar conductivity meter. The i.r. spectra were recorded on a Nicolet-170 SX F.T.-i.r. spec- trophotometer in KBr disc in the 4000-200 cm - ~ range. U.v.-vis. spectra of the complexes in EtOH, MeOH and MeOH-NaOMe were recorded on a Beckmann-Du7 spectrophotometer (ca. 10-4M solution), aH-n.m.r. spectra of the complexes in d6-DMSO were recorded on an FT-80A n.m.r, spectrometer. T.g.-d.t.a. measurements were made between room temperature and 600 ~ using a PCI-2-type t.g.-d.t.a, meter. Fluorescence spectra were recorded on a 1.c. 850-type ftuorometer.

Antitumour activity

In vitro screening: the colorimetry were used (5).

visual test method and MTT

Results and discussion

We assumed that Morin acted as a bidentate ligand and formed a mononuclear complex, where one ion is bound to two ligands. This assumption is in accord with elemen- tal analyses, and i.r. data, ~H-n.m.r. and u.v.-vis, spectros- copies. The results suggest that the complexes have the general formula [ML2"nH20-I, where M = m a n g a n - ese(II), cobalt(II), nickel(II), copper(II), zinc(II) or cadmium(II); L = Morin (2'-OH group deprotonated);

0340-4285 �9 1996 Chapman & Hall

24 Qi et al. Transition Met. Chem., 21, 23-27 (1996)

HO~OH

"U Y -o. OH O

Ring A (Band II) benzoyl system Ring B (Band I) cinnamoyl system

Figure 1. The structure of Morin and the divisions of bands I and II.

upon complexation, indicating that this group does not form metal-oxygen bonds with the metal ion by de- protonation of the 3-OH or 5-OH groups. However, the v (C--O--C) frequency ~9) decreased by ca. 15cm -1 to 1230cm-1 in the complexes, suggesting coodination of the ring oxygen with the metal(II) ion and of the 2'-OH group of the ligand after deprotonation to form the M - - O bond in the complexes. The v(O--H) frequency appears as a broad peak (from 3000 to 3600 cm 1) indica- ting the existence of water, which is also coincident with the results of thermal analysis. The Vring frequency de- creases by ca. 20cm -1 to 1550cm -I in the complexes. This red shift is due to increase of conjugative effect when the complexes are formed to give a new ring.

n = 2-3. The complexes are soluble in MeOH, EtOH and DMSO, slightly soluble in M%CO, but insoluble in H20 and CC14. Low molar conductances for the complexes in DMSO correspond to non-electrolytes ~7).

I.r. spectra

The i.r. spectra of the six complexes were similar; the main bands with tentative assignments are listed in Table 2. By comparing the spectra of the ligand (Morin'2HzO) with that of complexes, important information can be ob- tained: e.9. v(M--O) peaks appeared at 495-520 cm- 1(7), while the ligand exhibited no such bands, pr(H20) and pw(HzO) peaks appeared at 690-693 and 590-605 cm- 1, respectively, whereas the ligand also exhibited no such bands. This result proved the existence of coordinated water in the complexes and coincided with the thermal analysis results. The characteristic v (CzO) frequency of the ligand carboxyl group (1652 cm- 1)(8) changed slightly

U.v.-vis. spectra

The u.v.-vis, spectra of the six complexes and Morin.2H20 were recorded in EtOH. Two peaks appear at 357 (band I) and 263 (Band II) nm (Table 3) in the lig- and spectra. Band I (1~ is related to ring B (cinnamoyl system) and band II to ring A (benzoyl system) (Figure 1). After formation of the complexes, band II red shift moves slightly (ca. 0-13 nm); but band I red shift moves by ca. 50nm, a rather large shift, suggesting formation of the metal-oxygen bond in ring B (in cinnamoyl system).

In order to confirm the function of the proton of the 4'-OH group in forming the complexes, the u.v. spectra of the CuL 2'2H20 in both MeOH and MeONa medium were also studied. In MeOH solution, peaks at 415 and 270 nm indicate that the red shift of band II is small, while that of the band I very large (ca. 60 nm). Upon addition of three drops of MeONa to the MeOH solution, the u.v. spectra gave three peaks: 447, 323 and 287nm. It is apparent that after addition of MeONa in MeOH sol-

Table 1. Physical properties of the complexes prepared

Complex Colour Found (CMcd.) (%) Mol. A M C H M(II) wt. (f~- 1 cm 2 mol - 1)

MnL 2 .2H20 dark yellow 52.30 3.3 7.50 693.28 1.20 (52.0) (3.2) (7.92)

CoL~.3H20 dark yellow 50.8 3.3 8.4 715.39 1.63 (50.4) (3.4) (8.2)

NiL 2-3H20 grey-yellow 50.6 3.3 8.0 715.15 1.23 (50.4) (3.4) (8.2)

CuL 2'2H20 dark yellow 51.2 3.0 8.8 701.99 2.08 (51.3) (3.2) (9.1)

ZnLz.3H20 light yellow 49.8 3.5 8.9 721.85 1.56 (49.9) (3.4) (9.1)

CdL z.2H20 yellow-green 47.4 3.0 15.2 750.85 1.71 (48.0) (3.0) (15.0)

Table 2. I.r. data (cm- 1) of the complexes

Complex v(O--H) y(C~O) Fring(C~C ) v(C--O--C) pr(H20) pw(H20) v(M O)

Ligand (Morin" 2H20) 3477-3299 1652 1570 1245 - - - MnLa-2HzO 3365-3197 1648 1550 1234 690 603 498 CoL2.3H20 3219-3010 1652 1550 1234 691 592 5!6 NiLz.3HzO 3600-3030 1652 1549 1235 690 592 517 CuLz.2H20 3610-3060 1648 1554 1233 692 590 521 ZnL2.3H20 3219-3060 1658 1551 1230 692 605 518 CdLz.2H20 3600-3000 1649 1548 1228 692 603 500

Transition Met. Chem., 21, 23-27 (1996)

T a b l e 3. U.v. data of ligand and complexes

Complex Band II Band I 2max(nm) 2max(nm)

Ligand (Morin.2H20) 266.5 357.5 M n L 2 "2H20 269.0 411.5 CoL 2.3H20 266.9 411.0 NiL 2' 3 H 2 0 275.5 415.5 CuE 2 "2H20 277.5 420.5 ZnL z.3H20 267.0 409.0 CdL 2.2H20 266.5 409.0

ution, band I undergos a red shift (>30nm) but its strength does not weaken. This fact confirms that the 4'-OH group exists in the free state in the complexes (1~ (the new peak at 323 nm suggests that the 7-OH group also exists in free state).

1H-n.m.r. of ZnL2.2H20

The 1H-n.m.r. spectra of the zinc complex was studied using d6-DMSO as solvent and reference 11~ Morin.2H20:612.62 (1H, 5-OH); 610.55 (1H, 7-OH); 69.78 (1H, 3-OH); 67.07 (1H, H-6'); 66.28 (1H, H-5'); 66.20 (1H, H-8); 66.15 (1H, H-3'); 66.05 (1H, H-6); 63.0-3.7 (broad peak, H 2 0 ). ZnLz-2H20:611.85 (1H, 5-OH); 610.79 (1H, 7-OH); 69.68 (1H, 3-OH); 67.53 (1H, H-6'); 66.43 (1H, H-5'); 66.33 (1H, H-8); 66.24 (1H, H-3'); 66.15 (1H, H-6); 63.0-3.5 (broad peak, H 2 0 ). These data further indicate that the 5-OH, 7-OH and 3-OH group protons are also present in the complexes. All the ring protons shift to low field; among them the shift of 6'-OH is the biggest (6 = 0.46 p.p.m). This result is probably due to the fixation of ring B caused by the effect of coordination while the complex was being formed. Thus the 6'-H is close to the oxygen of the 3-OH group in space, forming a weak hydrogen bond.

Thermal stability of the complexes

The thermal behaviour of the six complexes was similar: a small broad endothermic peak appears on the d.t.a. curve (from 90 to 203 ~ and also a big exothermic peak (from 263 to 590 ~ corresponding to 88% weight loss. Not all of the complexes have a fixed m.p., but the water loss temperature for each complex is slightly different: if complexes contain three water molecules, the water loss

Antitumour properties of metal(II) complexes 25

- - o OH -7

.o Z7 ~176 I

? o . - - HO 0 - -

Figure 2. The possible structure of the complexes. M = M n " , Co It, NY, Cu", Zn It or Cdn; n = 0 or 1

temperature is below 100 ~ if the complexes contain two water molecules this temperature exceeds 110~ This fact suggests that the complexes contain two coordinated water molecules and one or no water of crystallization. Thermal data are listed in Table 4.

Fluorescence analysis

It is known that Morin exhibits a strong fluorescence (E x = 471 nm, E m = 519 nm), however the fluorescence is quenched completely after complex formation. This result suggests that the complexes are not planar.

Based on the above studies, we propose a possible structure for the complexes (Figure 2).

Antitumour activity

The inhibiting effects (tested by Lanzhou Turnout Re- search Institute) of ZnLz.3HzO and Mor in .2H20 against two tumour cells (Hep-2 and BHK-21) were studied, and the results are listed in Tables 5 and 6. From Table 5, it can be seen that when the concentration of the two compounds was 200/zg/0.1 cm 3, they all had a strong inhibitory effect against Hep-2.

When the concentration decreased to 100#g/0.1 cm 3, the ZnL 2. 3H20 still had a strong inhibitory effect, but the ligand (Morin. 2H20 ) did not. From Table 6, we note that when the concentrations of both the ligand and ZnL2"3H20 were 100#g/0.1cm 3, they all possessed a strong inhibitory effect against BHK-21. When the concentration was decreased to 10 #g/0.1 cm 3, ZnL 2.3H20 still possessed a strong inhibitory effect, but

T a b l e 4. Thermal data of the complexes

Complex H20 loss temp. (~ a Dec. temp. (~ Total wt. Residue T~ T 2 % T 3 T 4 loss (%)

Ligand (Morin-2H20) 89 185 285 538 - - MnL2'2H20 128 192 ,5.23 263 515 89.4 MnO CoL2"3H20 89 203 7.31 295 559 90.7 CoO NiL 2 ' 3 H 2 0 94 175 7.43 336 547 88.2 NiO CuL 2 "2H20 117 196 5.10 313 590 88.7 CuO ZnL 2"3H20 90 183 7.55 326 583 88.7 ZnO CdL2 "2H20 111 179 4.74 315 536 88.1 CdO

a2 or 3 mol of H2O lost.

26 Qi et al.

Table 5, Effect against Hep-2 a

Transition Met. Chem., 21, 2 3 - 2 7 (1996)

Compound [Sample(pg/0.1cm 3) Character ofpathological 200 i00 10 1.0 0.I changes

Ligand (Morin '2HzO) + + + + ZnL2 .3H20 + + + + + + + +

Single cell, round, shrinking Single cell, round, shrinking

~Using EtOH as solvent control, results after 48 h; each sample was tested four times at each concentration. +, Positive (pathological change cells ~> 50%), suggests that the compound has a killing effect to the turnout cells in the corresponding concentration. - , Negative (the tumour cells grow well), suggests that the compound has no killing effect to the tumour cells.

Table 6. Killing effect against BHK-21"

Compound [Sample (#g/0.1 cm 3) Character of pathological 200 100 10 1.0 0.1 changes

Ligand (Morin-2H20) + + + + + + + + ZnL2"3HaO + + + + + + + + + + + +

Single cell, round, shrinking Single cell, round, shrinking

aUsing EtOH as solvent control, results after 48 h; each sample was tested four times at each concentration. +, Positive (pathological change cells >~ 50%), suggests that the compound has an inhibitory effect on the turnout cells at the corresponding concentration. -., Negative (the turnout cells grow weI1), suggests that the compound has no inhibitory effect on the lumour cells.

Table 7. Effect against BEL-7402 (using D M S O as solvent control)

Compound [Compound] O D data Statistical Inhibiting ICs0 Evaluation (M) (x _+ SD) result a ratio (%) (M)

Ligand (Morin-2H20)

C u L z 3 H 2 0

10- v 0.809 + 0.015 * * -- 11.34 10 -6 0.783 • 0.015 * * - 7 . 7 9 10 -5 0.695 + 0.066 4.29 10 4 0.617_+ 0.129 15.08

10 -7 0.722 _+ 0.093 0.57 10 -6 0.677 _+ 0.041 6.72 10 -s 0.660 + 0.127 9.02 10 -~ 0.376 + 0.094 * * 48.18

"*~,greatdif~renceexists between test group and control, P <0.01. ~> t • 10 4, - , n o effect; ~<l x 10 -4, +,weak effect; ~<l x i0 -~, + +,obvious effect; ~< 1 x 10 6, + + +, strong effect.

Table 8. Effect against HL60 (using D M S O as solvent control)

Compound [Compound] O D data Statistical Inhibiting ICso Evaluation (M) (x _+ SD) result" ratio (%) (M)

Ligand (Morin-2HzO)

CuL 2-3H20

10- 7 0.461 _+ 0.217 * 35.35 10 -6 0.446 _+ 0.081 * * 37.59 10 - s 0.483 • 0.045 * * 32.38 10 4 0.347_+0.013 * * 51.34

lO 7 0.477 i 0 . 0 3 1 * * 33.14 10 6 0.403_+ 0.019 * * 43.54 10- s 0.440 + 0.042 * * 38.43 10 -4 0.327 + 0.047 * * 54.19

6.7 x 10 -s +

"*, Obvious difference exists between test group and control, P < 0.05; * ~', great difference exists between test group and control, P < 0.0 l. ~> 1 x 10 - 4, - , no effect; ~< 1 x 10-'*, +, weak effect; ~< 1 • 10-~, + +, obvious effect; ,%< 1 x 10-~, + + +, strong effect.

the l igand d id not. These resul ts sugges t tha t Z n L 2 . 3 H 2 0 possesses a be t t e r i nh ib i t o ry effect aga ins t H e p - 2 and B H K - 2 1 t h a n tha t of M o r i n ' 2 H 2 0 , espec ia l ly t o w a r d s B H K - 2 1 .

T h e inh ib i t ing effects ( tested by the S ta te K e y L a b o r a - to ry of Bei j ing N a t u r e a n d Bionics Med ic ine ) of C u L 2 - 2 H z O and M o r i n . 2 H 2 0 aga ins t th ree t u m o u r cells (Bei-7402, H L - 6 0 a n d KB) were tes ted a n d the resul ts a re

Transition Met. Chem., 21, 23 27 (1996)

Table 9. Effect against KB (using DMSO as solvent control)

Ant i tumour properties of metal(II) complexes 27

Compound [Complex] OD data Statistical Inhibiting ICso Evaluation (M) (X • SD) result" ratio (%) (M)

Ligand (Morin'2H20)

CuL2'3H20

10 -~ 0.335 • 0.009 -- 1,722 10 - 6 0.283 • 0.027 , 13.982 10- ~ 0.289 +_ 0.036 12.158 10 -4 0.224 + 0.013 ** 31.991

10- 7 0.334 ___ 0.026 - 1.520 10 - 6 0.270 ___ 0.049 , 17.933 10 -s 0.267 ___ 0.019 ** 18.769 10 - 4 0.137 • 0.032 ** 58.359

+

a,, Obvious difference exists between test group and control, P < 0.05; **, great difference exists between test group and control, P < 0.01. >~ l x lO 4, - , no effect; ~< 1 x 10 -*, +, weak effect; ~< 1 x 10 5, + +, obvious effect; ~< 1 x 10 -6, § + +, strong effect.

listed in Tables 7-9, respectively. As can be seen from these tables, the inhibition of C u L a . 2 H 2 0 against KB is weak, but M o r i n . 2 H 2 0 gives no inhibiting effects. The inhibiting effect of C u L 2 ' 2 H 2 0 against HL-60 is relative- ly obvious, the ICso = 6.7 x 10 5 M, but the inhibiting effects of both C u L 2 . 2 H 2 0 and M o r i n - 2 H 2 0 against BEL-7402 are not so obvious.

Acknowledgement

This project was supported by the NSFC, Gansu.

References

(11Zhou Xiufang and Zheng Rongliang, J. Lanzhou University (Nat. Sci.), 27, 101 (1991).

(21A. J. Alldrick, J. Flynn and I. R. Rowland, Murat Res., 163, 225 (1986).

(3) M. N. Hughes, The Inorganic Chemistry of Biological Pro- cesses, Wiley Interscience, London, 1972.

(4)K. Fridbory, K. K. Kannan, A. Liljas, J. Lundin, B. Stran- dberg, R. Strandberg, B. Tilander and G. Wrien, J. Molec. Biol., 25, 505 (1967).

(S)L. M. Mosmmann, J. Immuno Meth., 65, 55 (1983). (61W. Greary, J. Coord. Chem. Rev., 7, 81 (1971). (7/Kazuo Nakamoto, Infrared and Raman Spectra of Inorganic

and Coordination Compounds, 3rd Edit., Wiley Interscience, New York, 1978.

(8)L. H. Briggs and L. D. Colebrook, Spectrochimica Acta, 18, 39 (1962).

(91Chen Yaozu, Organic Analysis, Advanced Education Pub- lishers, Beijing, China, 1983.

II~ R. Markham and T.J. Mabry in J. B. Harborne et al., (Eds), The Flavonoids, Chapman & Hall, London, p. 45.

(11)Wang Xiankai, Medicinal Chemistry of Natural Products, People Hygiene Publishers, Beijing, China, 1988.

(Received 15 March 1995) T M C 3468