Design, synthesis and biological evaluation of six dinuclear platinum(II) complexes

Accepted Manuscript Design, synthesis and biological evaluation of six dinuclear platinum(II) complexes Congtao Yu, Chuanzhu Gao, Linkui Bai, Qinghua Liu, Zhuxin Zhang, Yingjie Zhang, Bo Yang, Chunli Li, Peng Dong, Xiaojun Sun, Yunxu Qian PII: DOI: Reference:

S0960-894X(16)31362-2 http://dx.doi.org/10.1016/j.bmcl.2016.12.084 BMCL 24573

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

4 October 2016 14 December 2016 29 December 2016

Please cite this article as: Yu, C., Gao, C., Bai, L., Liu, Q., Zhang, Z., Zhang, Y., Yang, B., Li, C., Dong, P., Sun, X., Qian, Y., Design, synthesis and biological evaluation of six dinuclear platinum(II) complexes, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.084

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Design, synthesis and biological evaluation of six dinuclear platinum(II) complexes Congtao Yua, Chuanzhu Gao*a, Linkui Baia, Qinghua Liua, Zhuxin Zhanga, Yingjie Zhangcd, Bo Yanga, Chunli Liab, Peng Dongcd, Xiaojun Sunab, Yunxu Qiana *Correspondence to C.Z Gao, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China E-mail: [email protected] a Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China b Department of Cardiovascular Medicine, Wenshan People’s Hospital, Wenshan 663000, China c Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China d Engineering Laboratory for Advanced Battery and Material of Yunnan Province, Kunming 650500, China Abstract: Six dinuclear platinum(II) complexes with a chiral tetradentate ligand, (1R,1’R,2R,2’R)-N1,N1’-(1,4-phenylenebis(methylene)) dicyclohexane-1,2-diamine, have been designed, synthesized and characterized. In vitro cytotoxicity evaluation of these metal complexes against human A549, HCT-116, MCF-7 and HepG-2 cell lines have been carried out. All compounds showed antitumor activity to HepG-2, HCT-116 and A549. Particularly, compounds A1 and A2 exhibited significant better activity than other four compounds and A2 even showed comparable cytotoxicity to cisplatin against HepG-2 cell line. Keywords: dinuclear platinum(II) complexes; cytotoxicity; anticancer; DNA insertion group

Cisplatin, which was found to own great anticancer activity by Rosenberg B in 1965, has been widely used in treatment of many types of cancer such as ovarian, cervical, bladder and lung.1,2 The advanced platinum-based anticancer drugs such as carboplatin, oxaliplatin which own similar molecular structures to cisplatin have shown much more activities against some kinds of cancer and/or fewer side effects.3,4 However, the cross-resistances and severe side effects of clinical approved platinum drugs obstruct them from wider applications.5 Therefore, developing platinum complexes with less side

effects, better curative effects and a broader anticancer spectrum is an area of focus for anticancer drugs research. Multinuclear platinum complexes, which have distinct ways to bind DNA compared with cisplatin analogues, have shown bright prospects in the cisplatin resistance cancers therapy.6 They could bind to DNA forming multifunctional intrastrand cross-linking adducts and prevent cancer cells from growing. For instance, BBR3464 (Figure 1), a trinuclear platinum complex based on structure of cisplatin possessing 1,6-diaminohexyl chains as bridges to connect metal ions, was once in Phase II clinical trials for the treatment of patients with melanoma, pancreatic, lung, ovarian and gastric tumors.7 However, the lack of in vivo activity against certain types of cancer and severe dose-limiting side effects such as diarrhea and vomiting hindered its clinical approval and further applications.8,9 Intercalative binding is a kind of noncovalent stacking interaction resulting from the insertion of planar aromatic rings into the grooves of the DNA double helix structure.10 Polynuclear platinum complexes with intercalative bindings are capable to intercalate between DNA bases while the metal coordinates directly to DNA bases. Intercalative interactions between metal complexes and DNA could lead to DNA functional changes, such as inhibitions of transcription, replication and DNA repair processes, which make intercalators potent mutagens.11 Several active dinuclear platinum compounds with intercalative bindings have been screened for their antitumor potency.12-14 For example, a benzene-bridged dinuclear platinum(II) complex (Figure. 1) has shown potential to exert cytotoxicity on several human tumor cells.

Figure 1. Structures of BBR3464 and a benzene-bridged dinuclear platinum(II) complex.

Considering the favourable anticancer activities and relatively mild side effects of oxaliplatin, we have designed, synthesized six dinuclear platinum(II) complexes based on the structure of oxaliplatin. The structure of trans-(1R,2R)-diaminocyclohexane which has been believed to play an important role in the success of oxaliplatin was retained while a benzene-bridge was introduced as DNA insertion group to enhance the ability of DNA-bindings (Figure. 2).15,16 Six different anions were selected as leaving groups to adjust lipid/water partition coefficient.

Figure. 2 Dinuclear platinum complexes based on oxaliplatin with a benzene-bridge

For the synthesis of the ligand (HL), mono-Boc protecting DACH (1) was used as the starting material and prepared according to a procedure reported.17. Detailed processes are as followed. Reaction of 1 with p-phthalaldehyde offered a Schiff base (2) which was then reduced by NaBH4 to give intermediate 3, then 3 was treated with HCl/EtOAc to remove the Boc group, leading to 4 of HL hydrochloride, which was finally neutralized by aqueous Na2CO3 solution to give free HL (Scheme 1).

Scheme 1. Preparation of ligand HL. Reagents and conditions: (a) p-phthalaldehyde, toluene, refluxing and water separating for 2h; (b) NaBH4; (c) HCl–EtOAc, Et2O; (d) Na2CO3.

When preparing the targeted platinum complexes, we first prepared the important intermediate [PtLI]18, which was then used to react with AgNO3 and related sodium dicarboxylates, respectively, to afford compounds A1–A6 (Scheme 2).19

Scheme 2. Synthesis of dinuclear complexes A1-A6

Intermediate [PtLI] and targeted dinuclear platinum complexes have been characterized by IR, 1H NMR, ESI-MS spectra. In the IR spectra, the amino group participation in binding with Pt(II) was confirmed by the examination of υNH2/υNH and δNH2/δNH frequencies, which were shifted to lower frequencies comparing with the free amino group, due to Pt(II)-NH2/Pt(II)-NH coordinations. Carboxylate anions binding with Pt(II) were confirmed by the examination of the C=O absorptions shifting from free carboxylic acids near 1700 cm-1 to bands near 1633-1598 cm-1. The complexes showed [M-H]-, [M+H]+, [M+Na]+ or [M+K]+ corresponding to their formula weights and relative fragment peaks in their ESI mass spectra. The 1H NMR spectral of all prepared

complexes in Figure 3 were all consistent with their corresponding protons both in the chemical shifts and in the number of protons.

H2N

NH

O O

Pt O

O

O

HN

NH

O NH2

O O

Cl

Cl

H2N

O (CH2)6

NH

HN

NH2

O

O O

O

HN

A5

O Pt

O

O (H2C)8

O (CH2)8

H2N

O

HN

O NH2

NH

NH2

O

O

O O

Pt

Pt

O O

(CH2)8 O

A4

O

Pt O

NH2

(H2C)8 O

Pt

A3

H2N

HN

NH

O

Pt

O

O (H2C)6

O Pt

O

(CH2)6 O O

O

Pt O

O

A2

(H2C)6 O

NH

O

O

A1

H2N

Cl

Pt

Pt

O

O

H2N

O

Cl

O Pt

O O

HN

NH2

A6

Figure 3. Molecular structure of the resulting dinuclear platinum(II) complexes

Some platinum anticancer drugs, such as cisplatin, are limited due to their poor aqueous solubility. Thus, the aqueous solubility of all compounds was measured at 25℃. Compared with cisplatin whose aqueous solubility is only 1 mg/mL, compounds A1 and A2 have been improved, with aqueous solubility 6.3 and 9.7 mg/mL.

Table 1. Cytotoxicity of the targeted compounds against four tumor cell lines

IC50(µM)a

solubility

a

25℃ （mg/ml）

Complex

6.3 9.7 1.1 0.9 1.2 0.7 17.0 7.0 1.0

HEPG-2b

A549c

HCT-116d

MCF-7e

A1

5.9

6.5

6.1

16.1

A2 A3 A4 A5 A6 carboplatin oxaliplatin cisplatin

0.9 23.6 35.0 69.1 86.0 8.2 not tested 1.1

2.7 17.0 26.9 56.8 71.2 10.5 not tested 1.9

3.2 11.8 22.2 78.6 66.3 not tested 3.6 not tested

13.2 36.5 43.0 ＞100 ＞100 13.3 not tested 1.2

All IC50 values (drug concentration giving 50% survival) calculated based on at least three separated

experiments. b c

Human non-small-cell lung cancer cell.

d e

Human hepatocellular carcinoma cell. Human colorectal cancer cell.

Human breast carcinoma cell.

In vitro cytotoxicity of targeted dinuclear platinum complexes has been tested by MTT assay, using A549, HepG-2, HCT-116 and MCF-7 cell lines. The IC50 values of these compounds as well as positive controls, cisplatin, carboplatin and oxaliplatin, are given in Table 1. The in vitro results showed that all six dinuclear complexes exhibited certain cytotoxicities towards A549, HepG-2, HCT-116 cell lines. A5 and A6 didn’t display sensitive activity (IC50>100µM) against MCF-7 cell line as well as A1-A4 also showed lower cytotoxicities against MCF-7 than other three cell lines. This kind of selective cytotoxicities to different cancer cells could have been caused by the similar carrier ligand structures of A1-A6. As an obvious rule, these complexes exhibited different cytotoxicities which were closely related to aqueous solubility by MTT assay except A5. Compounds A1 and A2 displayed better aqueous solubility as well as better cytotoxicities against the four tested cancer cell lines than other four targeted platinum complexes. The cytotoxicities of compounds A1 and A2 are significantly higher than that of carboplatin against HepG-2 and A549 cell lines. Even compared to cisplatin, A2 demonstrated comparability in HepG-2 cancer cell line. What’ more, A2 showed improving cytotoxicities than oxaliplatin against HCT-116 cell line. Compounds A5 and

A6 showed close aqueous solubility to A3 and A4, but showed obvious lower cytotoxicities. One explanation could be that the huge cycloalkane structure in the leaving group of A5 and A6 hindered the aquation and reduced the DNA binding capacity of A5 and A6. In addition to suiltable aqueous solubility, another important reason that dinuclear platinum complexes A1 and A2, especially A2, showed inspiring in vitro cytotoxicities against selective cell lines may be relavent to the introducing of intercalative binding group of benzene. In conclusion, with a chiral tetradentate ligand, six dinuclear platinum complexes which owned several carboxylates as leaving groups have been designed, synthesized and antitumor bioactivity evalueted. All compounds showed positive antitumor activity to HepG-2, HCT-116 and A549 cell lines. Compound A2 which takes ClCH2COO- as a leaving group not only showed better antitumor activity against the four tested cancer cell lines than carboplatin, but also showed a comparable activity against HCT-116 and HepG-2 to oxaliplatin and cisplatin. Consequently, the obtained dinuclear platinum complexes A1 and A2, especially compound A2, may be deserved for further investigation. Acnowledgements This work is supported by National Natural Science Foundation of China (21361014, 21302074, 21362016). References and notes 1. Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V. H. NATURE 1969, 222, 385. 2. Kelland, L. NAT REV CANCER 2007, 7, 573. 3. Lebwohl, D.; Canetta, R. EUR J CANCER 1998, 34, 1522. 4. Seymour, M. T.; Thompson, L. C.; Wasan, H. S.; Middleton, G.; Brewster, A. E.; Shepherd, S. F.; O'Mahony, M. S.; Maughan, T. S.; Parmar, M.; Langley, R. E. The Lancet 2011, 377, 1749. 5. Kelland, L. NAT REV CANCER 2007, 7, 573. 6. Mangrum, J. B.; Farrell, N. P. CHEM COMMUN 2010, 46, 6640. 7. Manzotti, C.; Pratesi, G.; Menta, E.; Di Domenico, R.; Cavalletti, E.; Fiebig, H. H.; Kelland, L. R.; Farrell, N.; Polizzi, D.; Supino, R. CLIN CANCER RES 2000, 6, 2626. 8. Jodrell, D. I.; Evans, T.; Steward, W.; Cameron, D.; Prendiville, J.; Aschele, C.; Noberasco, C.; Lind, M.; Carmichael, J.; Dobbs, N. EUR J CANCER 2004, 40, 1872. 9. Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. DALTON T 2010, 39, 8113. 10. Wang, A. H. J.; Nathans, J.; Marel, G. V. D.; Boom, J. H. V.; Rich, A. NATURE 1978, 276, 471. 11. Liu, H. K.; Sadler, P. J. ACCOUNTS CHEM RES 2011, 44, 349. 12. Zerzankova, L.; Kostrhunova, H.; Vojtiskova, M.; Novakova, O.; Suchankova, T.; Lin, M.; Guo, Z.; Kasparkova, J.; Brabec, V. BIOCHEM PHARMACOL 2010, 80, 344. 13. Komeda, S.; Lutz, M.; Spek, A. L.; Chikuma, M.; Reedijk, J. INORG CHEM 2000, 39, 4230.

14. Komeda, S.; Ohishi, H.; Yamane, H.; Harikawa, M.; Sakaguchi, K. I.; Chikuma, M. Journal of the Chemical Society Dalton Transactions 1999, 1, 2959. 15. Tyagi, P.; Gahlot, P.; Kakkar, R. POLYHEDRON 2008, 27, 3567. 16. Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.; Keppler, B. K. DALTON T 2008, 14, 183. 17. Woo, L. D.; Hyun Joon, H.; Koo, L. W. SYNTHETIC COMMUN 2007, 37, 737. 18. Synthesis of complex [PtLI]: To a stirred aqueous solution of KI (160mmol), K2PtCl4 (20mmol) in water (20ml) was added. The blended solution was heated at 80 °C rapidly and then cooled to 15 °C. The mixture was stirred at 15 °C for 30min under a nitrogen atmosphere to generate a black suspension of K2PtI4. Then an aqueous solution (20ml) of HL (20mmol) was added dropwise under stirring in the dark at 25 °C. After 24 h, the dark yellow precipitate was collected by filtration, washed sequentially with water, ethanol and ether, and then dried in vacuum. Data for [PtLI]: Yield: 96%, dark yellow solid. IR(ν, cm-1): 3208s(br), 2936vs(br), 2853s, 1570m, 1447s, 1170m, 1126m, 1068m, 973m, 896w, 841m, 796w, 703w, 506w, 446m. 1H NMR (DMSO/TMS): δ 0.78-3.01(m, 20H of 2DACH)，4.47-5.01(m, 4H of 2CH2C6H4), 6.31-7.15(m, 6H of NH2 and NH), 8.13(s, 4H of C6H4). ESI-MS: m/z [M-I]+=1100.8(100%), [M+Na]+=1250.7(20%). 19. Synthesis of complexes: D1, D2, D3, D4 and D5.

To a stirred suspension of [PtLI] (0.5mmol) in solvent (50ml of water and 50ml of EtOH), a solution of AgNO3 (4.0mmol) in water (20 ml) was added. Under a nitrogen atmosphere, the mixture was heated at 50 °C for 12 h in the dark and the resulting AgI deposit was removed by filtration. Then corresponding sodium carboxylate (2.0mmol) aqueous solution was added to the filtrate. The filtrate was evaporated to nearly dryness and some white solids precipitated, which were washed with water and ethanol for several times, and dried in vacuum. Data for A1: Yield: 34%, wihte solid. IR(ν, cm-1): 3218vs(br), 2933s, 2858s, 1601m, 1468s, 1361w, 1180w, 1140w, 1073m, 1031m, 981m, 903m, 837m, 816m, 562m. 1H NMR (DMSO/TMS): δ , 2.56-2.78(m, 12H of 4CH3)，0.98-3.03(m, 20H of 2DACH)，4.36-5.02(m, 4H of 2CH2C6H4), 6.26-7.09(m, 6H of NH2 and NH), 8.06(s, 4H of C6H4). ESI-MS: m/z [M+1]+=957(100%), [M+K]+= 995(30%). Data for A2: Yield: 36%, wihte solid. IR(ν, cm-1): 3256vs(br), 2934s, 2856s, 1598m, 1460s, 1176w, 1144w, 1069m, 1028m, 976m, 900m, 828m, 809m, 558m. 1H NMR (DMSO/TMS): 1.01-2.98(m, 20H of 2DACH)，δ, 4.36-4.51(m, 8H of 4ClCH2)，4.30-5.01(m, 4H of 2CH2C6H4), 6.33-7.06(m, 6H of NH2 and NH), 7.98(s, 4H of C6H4). ESI-MS: m/z [M-H]-=1093(100%). Data for A3: Yield: 36%, wihte solid. IR(ν, cm-1): 3276vs(br), 2936s, 2860s, 1633m, 1492s, 1361w, 1151w, 1092m, 1038m, 983m, 921m, 856m, 813m, 572m. 1H NMR (DMSO/TMS): δ , 0.89-2.93(m, 60H of 4COO(CH2)6CH3), 1.01-2.96(m, 20H of 2DACH), 4.31-4.96(m, 4H of 2CH2C6H4), 6.20-7.01(m, 6H of NH2 and NH), 7.98(s, 4H of C6H4). ESI-MS: m/z [M-H]-= 1291(100%). Data for A4: Yield: 33%, wihte solid. IR(ν, cm-1): 3253vs(br), 2938s, 2862s, 1630m, 1489s, 1337w, 1149w, 1096m, 1030m, 982m, 933m, 857m, 806m, 568m. 1H NMR (DMSO/TMS): δ , 0.86-2.91(m, 76H of 4COO(CH2)8CH3), 1.03-2.89(m, 20H of 2DACH), 4.29-4.88(m, 4H of 2CH2C6H4), 6.22-6.98(m, 6H of NH2 and NH), 7.91(s, 4H of C6H4). ESI-MS: m/z [M+H]+= 1405(100%), [M+Na]+= 1427(30%). Data for A5: Yield: 31%, wihte solid. IR(ν, cm-1): 3218vs(br), 2930s, 2858s, 1603m, 1477, 1321w, 1139w, 1081m, 1032m, 973m, 926m, 793m, 552m. 1H NMR (DMSO/TMS): δ, 0.91-2.68(m, 36H of

4COOCH(CH2)4), 1.01-2.92(m, 20H of 2DACH), 4.26-4.91(m, 4H of 2CH2C6H4), 6.19-6.83(m, 6H of NH2 and NH), 7.96(s, 4H of C6H4). ESI-MS: m/z [M+H]+= 1173(100%), [M+Na]+= 1195(40%). Data for A6: Yield: 32%, wihte solid. IR(ν, cm-1): 3206vs(br), 2933s, 2859s, 1616m, 1501s, 1326w, 1083m, 1036m, 986m, 931m, 865m, 801m, 558m. 1H NMR (DMSO/TMS): δ , 0.86-2.52(m, 44H of 4COOCH(CH2)5), 0.96-2.83(m, 20H of 2DACH), 4.26-4.83(m, 4H of 2CH2C6H4), 6.18-6.79(m, 6H of NH2 and NH), 7.92(s, 4H of C6H4). ESI-MS: m/z [M+H]+= 1229(100%), [M+K]+= 1267(55%).