Hydrolytic behaviour of mono- and dithiolato-bridged dinuclear arene ruthenium complexes and their interactions with biological ligands

The hydrolysis and the reactivity of two dinuclear p-cymene ruthenium monothiolato complexes, [(h-pMeC6H4Pr )2Ru2Cl2(m-Cl)(m-S-m-9-B10C2H11)] (1) and [(h -p-MeC6H4Pr )2Ru2Cl2(m-Cl)(m-SCH2-p-C6H4– NO2)] (2), and of two dinuclear p-cymene ruthenium dithiolato complexes, [(h -p-MeC6H4Pr )2Ru2(mSCH2CH2Ph)2Cl2] (3) and [(h -p-MeC6H4Pr )2Ru2(SCH2C6H4-p-OMe)2Cl2] (4) towards amino acids, nucleotides, and a single-stranded DNA dodecamer were studied using NMR and mass spectrometry. In aqueous solutions at 37 C, the monothiolato complexes 1 and 2 undergo rapid hydrolysis, irrespective of the pH value, the predominant species in D2O/acetone-d6 solution at equilibrium being the neutral hydroxo complexes [(h-p-MeC6H4Pr )2Ru2(OD)2(m-OD)(m-SR)]. The dithiolato complexes 3 and 4 are stable in water under acidic conditions, but undergo slow hydrolysis under neutral and basic conditions. In both cases, the cationic hydroxo complexes [(h-p-MeC6H4Pr )2Ru2(OD)(CD3CN)(m-SR)2] + are the only species observed in D2O/CD3CN at equilibrium. Surprisingly, no adducts are observed upon addition of an excess of L-methionine or L-histidine to the aqueous solutions of the complexes. Upon addition of an excess of L-cysteine, on the other hand, 1 and 2 form the unusual cationic trithiolato complexes [(h-pMeC6H4Pr )2Ru2{m-SCH2CH(NH2)COOH}2(m-SR)] + containing two bridging cysteinato ligands, while 3 and 4 yield cationic trithiolato complexes [(h-p-MeC6H4Pr )2Ru2[m-SCH2CH(NH2)COOH](m-SR)2] + containing one bridging cysteinato ligand. A representative of cationic trithiolato complexes containing a cysteinato bridge of this type, [(h-p-MeC6H4Pr )2Ru2[m-SCH2CH(NH2)COOH](m-SCH2-p-C6H4-Bu )2] + (6) could be synthesised from the dithiolato complex [(h-p-MeC6H4Pr )2Ru2(SCH2C6H4-p-Bu )2Cl2] (5), isolated as the tetrafluoroborate salt and fully characterised. Moreover, the monoand dithiolato complexes 1–4 are inert toward nucleotides and DNA, suggesting that DNA is not a target of cytotoxic thiolato-bridged arene ruthenium complexes. In contrast to the trithiolato complexes, monothiolato and dithiolato complexes hydrolyse and react with L-cysteine. These results may have important implications for the mode of action of thiolato-bridged dinuclear arene ruthenium drug candidates, and suggest that their modes of action are different to those of other arene ruthenium complexes.

Introduction
Ever since the serendipitous discovery of the anticancer properties of cisplatin in 1965 1 and its clinical use in 1978, 2 the research on platinum cytostatics has ourished. A plethora of analogous square-planar platinum(II) and octahedral platinum(IV) complexes was synthesised and studied in order to nd more selective and less toxic alternatives. 3 Despite immense efforts, only two additional platinum complexes were approved for global use in the clinic so far: the second-generation drug carboplatin was approved by the FDA in 1989 and the third-generation oxaliplatin in 2002. 4 Unfortunately, none of the three agents possess very high selectivity towards cancerous cells; their therapeutic application therefore causes severe side effects such as nephrotoxicity, 5-7 neurotoxicity 8,9 and nausea. 8,10 In order to overcome these side-effects, part of the research on metallodrugs has shied from platinum to ruthenium-based anticancer agents. Ruthenium compounds have in general lower systemic toxicity than platinum drugs, they can access a wide range of oxidation states under physiological conditions and have a very similar ligand exchange rate as platinum complexes. 11 The eld of anticancer ruthenium compounds was pioneered by Alessio, Sava and Keppler, who developed ruthenium(III) imidazole and indazole complexes, the imidazolium or indazolium salts of which (termed NAMI-A and KP1019, respectively) have already entered clinical trials. [12][13][14][15] The sodium analogue of KP1019, termed NKP-1339, is now on the edge of clinical application. 16 In 1992, Tocher and co-workers have observed a cytotoxicity enhancement by coordinating the anticancer agent metronidazole [1-b-(hydroxyethyl)-2-methyl-5-nitro-imidazole] to a benzene ruthenium dichlorido fragment. 17 Subsequently, it became obvious that water-soluble arene ruthenium complexes are in general cytotoxic, since they seem to have the right balance between lipophilicity and hydrophilicity, essential for cellular uptake. 18 The rst prototype arene ruthenium(II) complexes evaluated for anticancer properties in 2001 were [(h 6 -p-MeC 6 H 4 Pr i ) Ru(P-pta)Cl 2 ] (pta ¼ 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-C, from Dyson's laboratory 19 and the RAED family of compounds [(h 6 -C 6 H 5 Ph)Ru-(N,N-en)Cl] + as hexauorophosphate or chloride salt from Sadler's laboratory. 20 Importantly, although RAPTA-C only exhibits a low activity in vitro, it is very active in vivo, where it inhibits lung metastases in CBA mice. 21 Therefore, RAPTA-C can be considered as an antimetastatic agent like NAMI-A. 21,22 RAED compounds are highly cytotoxic to a range of cancer cell lines, 23 including cisplatin-resistant cell lines. An in vivo study established that RM175, [(h 6 -C 6 H 5 Ph)Ru(N,N-en)Cl]PF 6 , is active against MCa mammary carcinoma and causes metastasis reduction. 24 In addition to these two prototype compounds, a plethora of organometallic ruthenium(II) compounds have been prepared and their cytotoxicity to cancer cells examined. [25][26][27][28][29][30][31][32][33] In recent years, we have synthesised several series of dinuclear arene ruthenium thiolato-bridged complexes of the general formula [(h 6 -arene) 2 Ru 2 (m-SR) 3 ]Cl that are highly cytotoxic against ovarian cancer cell lines A2780 and A2780cisR, the most active derivative [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (m-S-p-C 6 H 4 Bu t ) 3 ]Cl (diruthenium-1) having an IC 50 value of 30 nM to both cell lines. [34][35][36][37][38][39] To the best of our knowledge, diruthenium-1 is the most cytotoxic arene ruthenium compound reported to date. A recent in vivo study has demonstrated that dinuclear arene ruthenium thiolato-bridged complexes have the potential to become anticancer chemotherapy agents, since diruthenium-1 signicantly prolongs the survival of tumour-bearing mice. 40 By varying the synthetic procedure, we succeeded in isolating neutral dithiolato complexes of the type [(h 6 -p-MeC 6 H 4 Pr i ) 2 -Ru 2 (m-SR) 2 Cl 2 ] 41 and subsequently also monothiolato complexes of the general formula [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 Cl 2 (m-Cl)(m-SR)], 42 intermediates in the formation of the cationic trithiolato complexes, thus opening new avenues for the ne-tuning the properties of dinuclear thiolato-bridged arene ruthenium complexes (Scheme 1). 43 Interestingly, the mono-and dithiolato analogues are systematically at least one order of magnitude less cytotoxic than their trithiolato counterparts. 36,38,39 The stability and reactivity studies of some trithiolato complexes with various amino acids, peptides, nucleotides and DNA revealed that the complexes are very stable in physiological conditions as well as under acidic and basic conditions and that they are particularly inert towards substitution. Only the sulphur-containing biomolecules cysteine (Cys) and the tripeptide glutathione (GSH) interact with the complexes. Unexpectedly, no adducts were identied, but Cys and GSH undergo a catalytic oxidation in the presence of the trithiolato complexes, forming cystine and GSSG. 35 Such a mode of action has already been proposed for arene ruthenium azopyridine iodo complexes, which are surprisingly cytotoxic despite their inertness to ligand substitution. 44 Although a decrease in the glutathione levels with increasing concentration of diruthenium-1 was observed in vitro and in vivo, 40 no direct correlation between their cytotoxicity and catalytic activity for the oxidation of glutathione was found. 36,38,39 We therefore assume that other mechanisms are mainly responsible for the high in vitro cytotoxicity of the trithiolato complexes.
In an effort to rationalise the relationship between reactivity and the in vitro cytotoxicity of thiolato-bridged arene ruthenium complexes, we set out to investigate the hydrolysis of two monothiolato complexes 1 and 2 and two dithiolato complexes 3 and 4 ( Fig. 1) as well as their interactions with selected amino acids, nucleotides and a single-stranded DNA dodecamer.

Results and discussion
2.1. Stability of the monothiolato complexes 1 and 2 in aqueous solution Fig. 2 shows the 1D 1 H-NMR spectra of 1 dissolved in a D 2 O/ acetone-d 6 mixture (ratio 7 : 3) at pD 7, recorded at 37 C between 5 min and 24 h. Additional resonances appeared immediately aer sample preparation, indicating rapid hydrolysis, and equilibrium is reached aer about 1 h. Analysis of the doublets for the p-cymene ring protons suggests that three species are present in solution. One with resonances at d ¼ 5.53 and 5.80 ppm is identied as 1, and a second one with four resonances at d ¼ 5.11, 5.27, 5.62 and 5.72 ppm is attributed to a species that underwent chloro/aqua exchange. This species, which is the most abundant one present in solution aer 1 h, is assumed to be the neutral hydroxo complex [(h 6 -p-MeC 6 H 4 -Pr i ) 2 Ru 2 (OD) 2 (m-OD)(m-S-m-9-B 10 C 2 H 11 )] (Scheme S1 †). The high symmetrical nature of this fully hydrolysed complex is supported by the single 1 H-NMR resonances observed for the p-cymene protons in the high eld region of the 1 H-NMR spectrum (Fig. S1 †). For the hypothetical mixed hydroxo-chloro complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OD)(Cl)(m-OD)(m-S-m-9-B 10 C 2 H 11 )], two sets of resonances would be observed for these protons. The fact that four resonances are observed for the p-cymene ring protons is not surprising. The molecular structures of several dinuclear trithiolato-bridged ruthenium complexes, which are apparently highly symmetrical, show that the two ruthenium atoms adopt a pseudo-octahedral geometry. 36 Consequently, four doublets caused by the eight aromatic protons of the two p-cymene rings are oen present in the region between 4.90 and 6.00 ppm. 39 A third, minor species with resonances at d ¼ 5.50, 5.62, 5.73, 5.82, 5.96 and 6.00 ppm is also present in the mixture immediately aer sample preparation. A full characterisation was not attempted due to its very low concentration. However, since six pcymene ring proton resonances and four methyl resonances between 1.10 and 1.32 ppm in the high eld region of the 1 H-NMR spectrum (Fig. S1 †) are observed for this species, it may be assumed that it is a mixed hydroxo-chloro complex, for instance [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OD)(Cl)(m-OD)(m-S-m-9-B 10 C 2 H 11 )].
Similar results are obtained for 2, however, the hydrolysis is signicantly faster, and the equilibrium is already reached aer 5 min since no further changes in the NMR spectra are noticed subsequently (Fig. S2 †). In order to study the suppression of hydrolysis by chloride ions, the same series of NMR spectra of both complexes were recorded in 105 mM NaCl solution. No spectral changes are observed, indicating that aquation is inhibited and that the observed products are indeed hydrolysed species. These results indicate that 1 and 2, unless metabolised in a different way, e.g. by protein binding, should survive the conditions in the bloodstream and that the hydrolysis could take place inside the cells where the chloride concentration decreases to ca. Interestingly, for 2, a group of peaks centred at m/z ¼ 842.2 corresponding to the dithiolato complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 -Ru 2 Cl(m-SCH 2 -p-C 6 H 4 -NO 2 ) 2 ] is also visible the ESI-MS spectrum. The formation of a dithiolato complex during hydrolysis is not surprising, since equilibria between the thiolato complexes and the thiol have been evidenced. 42,43 The coordinated thiolato bridge can come off as thiol to be replaced by a thiolato bridge derived from another thiol, which leads to a mixture of thiolato complexes. These equilibria are governed mostly by the reactivity of the thiols and to a lesser extent by the choice of the solvent and reaction conditions. 42 Fig. 2 The p-cymene ring proton region of the 1 H NMR spectrum of [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 Cl 2 (m-Cl)(m-S-m-9-B 10 C 2 H 11 )] (1) dissolved in D 2 O/acetone-d 6 (ratio 7 : 3) , recorded between t ¼ 5 min and t ¼ 24 h at 37 C and at pD 7. The labels correspond to p-cymene ligands in different species present in solution. The species labeled * corresponds to complex 1, the species labeled > is presumably the fully hydrolysed species [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OD) 2 (m-OD)(m-S-m-9-B 10 C 2 H 11 )], while the species labeled with C has not been further characterised. cymene protons in the high eld region of the 1 H-NMR spectrum (Fig. S7 †). Two other minor species with resonances at d ¼ 5.63 and 5.76 ppm as well as at d ¼ 5.59 and 5.82 ppm can be observed in the mixture immediately aer sample preparation, but disappear gradually and are not visible aer 5 h. Due to their low concentration and transient character, further characterisation was not attempted. The stability of 3 in CD 3 CN was also evaluated; the NMR spectra show that 3 remains stable for at least 48 hours. Hydrolysis of 3 at pH 7 was supported by the ESI-MS spectrum, which shows a single group of peaks centred at m/z ¼ 808.17 corresponding to the hydrolysed complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OH)(CH 3 CN)(m-S-C 2 H 4 Ph) 2 ] + (Fig. S8 †).

Stability of the dithiolato complexes 3 and 4 in aqueous solution
The kinetic experiment was repeated at pD ¼ 11 aer addition of NaOD. Analysis of the doublets for the p-cymene ring protons show that, apart form 3 with broad resonances at d ¼ 5.41 and 5.54 ppm, the hydroxo-acetonitrile complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OD)(CD 3 CN)(m-S-C 2 H 4 Ph) 2 ] + with four resonances at d ¼ 5.28, 5.32, 5.34 and 5.41 ppm is present in the mixture. Intriguingly, the equilibrium is reached only aer 48 h (Fig. S9 †). The same experiment was repeated at pD 2 aer addition of DCl. Weak additional resonances can be detected directly aer sample preparation, suggesting a rapid hydrolysis or the presence of a second unknown species. However, the equilibrium is reached immediately, as no further changes are visible in the subsequent NMR spectra recorded between t ¼ 5 min and t ¼ 24 h (Fig. S10 †). Analysis of the 1 H NMR spectra shows that 3 is the major species present in the mixture.
In the case of 4, additional resonances also appeared immediately aer sample preparation, indicating rapid hydrolysis, and the equilibrium is reached rapidly aer about 2 h (Fig. 4). The species with two broad resonances at d ¼ 5. Intriguingly, as observed with 3, the equilibrium is not reached before 48 h (Fig. S12 †). The experiment repeated at pD 2 aer addition of DCl shows that weak additional resonances appeared directly aer sample preparation, indicating a rapid hydrolysis, but equilibrium is reached immediately. Analysis of 1 H NMR spectra shows that 4 is the major species present in the mixture (Fig. S13 †).

Reactivity of 1-4 with nucleotides and DNA
In general, arene ruthenium compounds have been shown to bind to DNA model compounds 45,46 via loss of the chlorido ligands, and loss of the arene was also observed. More importantly, by using a 14-mer oligonucleotide, it has not been possible to identify preferential binding sites, due to the multiple interactions that are available.
Upon addition of 3 eq. of dAMP and dGMP, no changes in the NMR spectra are observed for complexes 2 and 3. As an example, the 2D-1 H DOSY NMR spectrum of the mixture 3 : dGMP (ratio 1 : 3) recorded aer 24 h is shown in Fig. S14. † In the spectrum two species can be identied: one with the lowest diffusion coefficient being the hydrolysed derivative of 3, and the second species being free dGMP. Furthermore, inspection of the 1D 1 H and 2D-1 H DOSY NMR spectra shows a single H8 resonance at d 8.22 ppm for dGMP, suggestive of the absence of a coordination of N7 to the Ru atom.
In order to investigate possible reactions with DNA, the complexes 1-4 were incubated at 37 C together with the singlestranded DNA 20-mer (CGCGATCGCGGCGCTAGCGC) for 24 h. In analogy with platinum complexes and some other ruthenium compounds, 20,42,47,48 we assumed that complexes 1-4 could bind to the nucleophilic nitrogen of the nucleotides once they underwent hydrolysis. However, ESI mass spectroscopy spectra recorded in the negative and positive mode (Fig. S15 †) revealed that no adducts are formed between the mono-and dithiolato complexes and the single-stranded DNA 20-mer. Interestingly, we have previously shown that trithiolato complexes are also inert towards nucleotides and DNA. 35,36 As such, unlike other arene ruthenium complexes, these results suggest that DNA is not a target of thiolato-bridged arene ruthenium complexes and that they exert their cytotoxicity through other mechanisms.

Reactivity of 1-4 with aspartic acid, alanine and histidine
Most of the metallodrugs currently used in antitumoural therapy, such as carboplatin and cisplatin, are administered intravenously and can therefore encounter various reactive biomolecules in the bloodstream. 20,47 Interactions with peptides and serum proteins can have a pronounced inuence on the drug distribution, bioavailability and target or off-target effects. To investigate some of these possible interactions, the neutral amino acid Ala, the acidic amino acid Asp and the basic amino acid His were used. His was also chosen to investigate a possible binding between the imidazole ring and the ruthenium atom, since we and others already showed that imidazole rings strongly and rapidly interact with the ruthenium atom 49-54 and especially with arene ruthenium complexes. [55][56][57][58] Alanine usually does not react with ruthenium complexes, although a hexacationic arene ruthenium assembly possessing an oxalato linker slowly degrades in the presence of excess of Ala to give mononuclear ruthenium-alanine adducts. 53 Asp with a coordinating carboxyl side chains is known to form tridentate chelate complexes with ruthenium and other metal centres. 54,55,59 Upon addition of 3 eq. of Ala and Asp, no changes in the NMR spectra are observed for the four complexes 1-4 (Fig. 5, S16 and S17 †). As an example, the 2D-1 H DOSY NMR spectrum of the mixture 1 : Ala (ratio 1 : 3) recorded aer 24 h is shown in Fig. 5. In the spectrum four species can be clearly distinguished: two, with the lowest diffusion coefficient, being complex 1 and the hydrolysed derivative thereof, the third species being free Ala, and the fourth one acetone.  Furthermore, inspection of the 1D 1 H and 2D-1 H DOSY NMR spectra shows a single aH resonance and a single bCH 3 resonance for Ala, suggestive of the absence of a coordination of Ala to the Ru atom.
Upon addition of 3 eq. of His, surprisingly, both NMR and ESI mass spectrometry data strongly suggest that the mono-and dithiolato complexes 1-4 do not react with His ( Fig. 6 and S18 †). As an example, the 2D-1 H DOSY spectrum of the mixture 3 : His (ratio 1 : 3) recorded aer 24 h is shown in Fig. 6. In the spectrum two species can be clearly distinguished: one with the lowest diffusion coefficient being the hydrolysed derivative of complex 3, the second one being free His. Furthermore, inspection of the 1D 1 H and 2D-1 H DOSY NMR spectra shows for His a single aH resonance, two bCH 2 resonances for the two bH diastereotopic protons and especially only two CH resonances for the imidazole group, suggestive of the absence of a coordination of the imidazole ring of His to the Ru atom. The ESI-MS spectrum of the mixture 2 : His (ratio 1 : 3) recorded aer 24 h shown in Fig. 7 conrms the absence of a coordination of His to the Ru atom. The groups of peaks observed in the ESI-MS spectrum at m/z ¼ 692.2, 709.1, and 842.2, respectively, are identical to those observed aer the hydrolysis of 2 and correspond the monohydroxo complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (OH)(m-Cl)(m-SCH 2 -p-C 6 H 4 -NO 2 )], complex 2, and the dithiolato complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 Cl(m-SCH 2 -p-C 6 H 4 -NO 2 ) 2 ].

Reactivity of 1-4 with cysteine and methionine
Reactions with the sulphur-containing amino acids Cys and Met appear to play key roles in the biological chemistry of platinum(II), platinum(IV) and ruthenium(III) anticancer agents, and were shown to form tridentate chelates with arene ruthenium complexes. 60 The nucleophilicity of methionine is less than that of cysteine, but still it is known to react with electrophilic sites. 61,62 Upon addition of 4 eq. of Met, NMR data strongly suggest that no reaction takes place between the mono-and dithiolato complexes 1-4 and Met. As an example, 1D 1 H NMR spectra recorded at t ¼ 10 min and t ¼ 24 h and the 2D-1 H DOSY spectrum of the mixture 3 : Met recorded aer 48 h are shown in Fig. S19. † Two species can be identied in the spectrum; one with the lowest diffusion coefficient being the hydrolysed derivative of complex 3, the second one being free Met. Moreover, inspection of the 1D 1 H and 2D-1 H DOSY NMR spectra shows a single aH resonance and especially a single dCH 3 resonance for Met at d 2.15 ppm, suggesting that there is no coordination of the methionine sulphur atom to the Ru atom, because otherwise a characteristic high eld shi for the dCH 3 group would be expected as observed in such complexes. 63 However, upon addition of 4 eq. of Cys to the complexes 1-4, the spectra reveal that Cys reacts rapidly with the four complexes. In all cases, additional resonances appear immediately aer sample preparation, indicating a rapid reaction, and equilibrium is reached rapidly aer 2 h. As an example, the 2D-1 H DOSY NMR spectrum of the mixture 1 : Cys (ratio 1 : 3) recorded aer 24 h is shown in Fig. 8. Three species can be clearly identied: the rst one with the lowest diffusion coefficient is attributed to an "adduct" containing cysteine and the dithiolato complex, the second species to free Cys, and the third one to acetone. A more in-depth analysis of the 1 H-and the 2D-1 H DOSY NMR spectra shows two distinct aH resonances (highlighted with a circle) at d 3.7 and 3.8 ppm, respectively, suggesting the presence of two   types of cysteine, which is cross-conrmed by their different diffusion coefficient. The aH resonance at d 3.8 ppm has clearly the same diffusion coefficient than the p-cymene proton resonances, although it appears slightly out of alignment because of the close proximity of the intense water resonance at 4.64 ppm. The ESI-MS spectrum shown in Fig. 9 exhibits a group of peaks centred at m/z ¼ 887.21, which corresponds to a diruthenium trithiolato complex containing one cysteinato bridge, [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (m-SCH 2 CH(NH 2 )COOH)(m-S-m-9-B 10 C 2 H 11 ) 2 ] + . Similar results were obtained with complexes 2-4, the formation of the corresponding cysteinato-containing trithiolato complexes as the only species present in the mixtures aer 24 h is conrmed by both NMR and ESI-MS data (Fig. S20-S24 †). Interestingly, for both monothiolato complexes The in situ formation of cysteinato-containing trithiolato complexes is somewhat surprising, since our previous attempts to synthesise such complexes by standard methods were unsuccessful; only degradation of the arene ruthenium unit was observed. On the other hand, such complexes are conceivable in the light of the equilibria between the thiolato complexes and the thiol. 42,43 For trithiolato complexes, the equilibria between the thiolato complexes and the thiol do not yield cysteinatocontaining trithiolato complexes but catalyse the oxidation of Cys to give cystine. 35,43 Sadler et al. had observed the formation of dinuclear biphenyl ruthenium complexes with cysteinato bridges from the reaction of [(h 6 -C 6 H 5 Ph)RuCl(en)] + with Cys 61 and the formation of dinuclear p-cymene ruthenium trithiolato complexes by reacting [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 Cl(en)] + with 2-mercaptobenzanilide in aqueous solution at pH ¼ 5.3. 64 We therefore decided to modify the synthetic conditions and attempted the synthesis and isolation of cysteinato-containing trithiolato complexes by mimicking the conditions of the NMR experiments. The dithiolato complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 (m-SCH 2 C 6 H 4 -p-Bu t ) 2 Cl 2 ] (5) 41 was dissolved in a small amount of methanol and an aqueous solution of cysteine was added dropwise in large excess. The resulting mixture was stirred at ambient temperature for two days, and a gradual colour change from orange to yellow was observed. The resulting product was unstable at elevated temperature; it also degraded rapidly in purely organic solutions. The product was therefore puried by size exclusion chromatography on Sephadex® LH20 with a MeOH/H 2 O (9 : 1) mixture, evaporated at room temperature and dried in vacuo. Spectroscopic methods and elemental analysis proved the compound to be the tetrauoroborate salt of the cysteinato-containing trithiolato complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 -Ru 2 (m-SCH 2 C 6 H 4 -p-Bu t ) 2 {m-SCH 2 CH(NH 2 )COOH}] + (6) (Scheme 2).
Because of its low stability, it was not possible to obtain crystals suitable for X-rays analysis. Synthesis of the other cysteinato-containing trithiolato complexes starting from the dithiolato precursors 3 and 4 was also attempted, but the resulting complexes degraded rapidly and could not be isolated in analytically pure form. The synthesis of the cysteinatocontaining trithiolato complexes starting from the monothiolato precursors 1 and 2 was not attempted.
To the best of our knowledge, together with the two diruthenium complexes obtained fortuitously by Sadler et al., 61 complex 6 is only the second example of a dinuclear arene ruthenium complex containing a cysteinato bridge. Since intracellular biological environment contain millimolar amounts of the tripeptide glutathione (g-Glu-Cys-Gly), such complexes could be of considerable signicance inside cells. This is currently being further investigated.

Conclusions
As reported for many other arene ruthenium complexes, our results show that the presence of chlorido ligands makes monoand dithiolato complexes 1-4 less stable than their trithiolato counterparts, which do not have Ru-Cl bonds. The complexes 1-4 hydrolyse readily, though slowly in comparison with other arene ruthenium complexes like RAPTA-C and the RAED family of complexes. Our results also suggest that complexes 1-4 remain relatively inert towards model biomolecules and DNA,  as adducts were only observed upon addition of Cys. Interestingly, unusual cationic dinuclear trithiolato complexes from which the chlorido ligands had been removed, such as [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 {m-SCH 2 CH(NH 2 )COOH}(m-SR) 2 ] + containing one bridging cysteinato ligand and [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 {m-SCH 2 CH(NH 2 )COOH} 2 (m-SR)]] + containing two bridging cysteinato ligands are observed.
It can be assumed that the propensity of mono-and dithiolato complexes to hydrolyse and to react with sulphurcontaining molecules explains why they are at least an order of magnitude less cytotoxic than trithiolato complexes. As already demonstrated for mononuclear Ru(II)-arene complexes with O,O-bound alkoxycarbonylmethyl-3-hydroxy-2(1H)-pyridones, 65 our results suggest that there is a negative reactivity-in vitro cytotoxicity relationship for dinuclear thiolato-bridged arene ruthenium complexes: the more reactive mono-and dithiolato complexes are signicantly less cytotoxic compared to their trithiolato counterparts. The high stability of dinuclear arene ruthenium trithiolato complexes allows them to accumulate inside the cells of a tumour, 40 where they can reach their molecular target and deploy their anticancer activity. The monothiolato and dithiolato complexes, on the other hand, can potentially bind to proteins and other molecules containing sulydryl groups, which may impede their accumulation in cancer cells in sufficient concentration. However, the prominent examples of the arene ruthenium RAPTA complexes, which are reactive and only exhibit a low activity in vitro, but are very active in vivo, prompt us to further evaluate mono-and dithiolato complexes in in vivo and preclinical models.

Experimental section
4.1. Materials and methods 4.1.1. Chemicals. Analytical grade chemicals obtained from commercial suppliers were used as received. L-alanine, Laspartic acid, L-cysteine and L-histidine were purchased from Acros. GSH, Sephadex® LH20 and NaOD and DCl solutions were obtained from Sigma-Aldrich. The single-stranded DNA dodecamer (CGCGATCGCG) 2 was purchased from Microsynth. Complexes 1-4 and 5 were prepared according to previously published methods. 42 All reactions were rst monitored by 1D 1 H NMR spectroscopy. Two-dimensional homonuclear 1 H-1 H and DOSY experiments were performed aer 24 h of incubation at 37 C. All experiments were carried out in aqueous solutions. The pD values of the solutions were obtained by use of a glass electrode and by addition of 0.41 to the pH meter reading. 66,67 Oxygen was not excluded from the solutions.
4.1.2. NMR spectroscopy. NMR data were acquired at 37 C using a Bruker Avance II 400 MHz NMR spectrometer equipped with a 5 mm broadband dual channel probehead (Smartprobe®) and a Bruker Avance II 500 MHz NMR spectrometer equipped with an inverse triple channel ( 1 H, 13 C, 31 P) z-gradient 1.7 mm microprobehead. Both probeheads are equipped with z-gradient accessory. One-dimensional 1 H NMR data were measured with 256 transients into 32 k data points over the width of 12 ppm using a classic presaturation to eliminate the water resonance. A relaxation delay of 2 s was applied between the transients. Two-dimensional 1 H-1 H COSY data were acquired over a frequency width of 12 ppm in both F 2 and F 1 into 2 k complex data points in F 2 (acquisition time 213 or 170 ms) using 128 t 1 increments. 32 transients were recorded per t 1 increment. The water resonance was suppressed by means of a presaturation routine.
Two-dimensional 1 H diffusion-ordered spectroscopy (DOSY) NMR data were acquired with a standard longitudinal encodedecode pulsed-eld-gradient stimulated echo sequence containing bipolar gradients. 68 Experimental parameters were D ¼ 100 ms (diffusion delay), s ¼ 1 ms (gradient recovery delay) and T ¼ 5 ms (eddy current recovery delay). For each data set, 4 k complex points were collected, and the gradient dimension was sampled by means of 32 experiments in which the gradient strength was linearly increased from 1.0 to 50.8 G cm À1 . The gradient duration d/2 was adjusted to observe a near-complete signal loss at 50.8 G cm À1 . A 2 s recycle delay was used between scans for the data shown. 32 transients were recorded per experiments. For each data set, the spectral axis was processed with an exponential function (3-5 Hz line broadening), and a Fourier transform was applied to obtain 8 k real points. The DOSY reconstruction was realised with 8 k complex points in the detection dimension and with 128 points in the diffusion dimension. All NMR data were processed using TopSpin (Version 3.2, Bruker, Switzerland) and Dynamics Center (Version 2.3.1, Bruker, Switzerland).
4.1.3. Electrospray ionisation mass spectrometry. Massspectrometric (MS) analyses were performed with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientic, Bremen, Germany) equipped with a nanoelectrospray ion source. The samples were analysed in the positive or negative ion mode with a voltage of +700 V applied to the glass emitter (New Objective, Woburn, MA, USA). The tube lens was at +150 V and the transfer capillary was held at 200 C. Spectra were acquired in Fourier transform mass spectrometry mode over an m/z range from 100 to 2000 with a resolution of 100 000 at m/z 400. Calibration of the instrument was performed with ProteoMass LTQ/ FT-Hybrid electrospray ionisation (ESI) positive mode calibration mix (Supelco Analytical, Bellefonte, PA, USA). The Xcalibur soware package (Version 2.0.7, Thermo Fisher Scientic) was used for instrument control and data processing. The dithiolato complex [(h 6 -p-MeC 6 H 4 Pr i ) 2 Ru 2 Cl 2 (m-SC 6 H 4 -p-Bu t ) 2 ] (5) (0.056 mmol) was dissolved in 25 mL of methanol. To this solution, a solution of cysteine (10 eq., 0.560 mmol) in distilled water (15 mL) was added dropwise. This mixture was stirred at room temperature for two days. Then 10 equivalents of NaBF 4 were then added and the suspension was stirred overnight. The solution obtained aer ltration was concentrated by applying a vacuum at room temperature; the light orange solid that precipitated during the evaporation was washed

Stability of 1-4 in aqueous solution
The stability of complexes 1-4 in aqueous solutions was studied by dissolving 1 mg of the complexes in the mixture of an appropriate deuterated solvent and water (D 2 O/acetone-d 6 7 : 3 for 1, D 2 O/acetone-d 6 6 : 4 for 2 and D 2 O/CD 3 CN 7 : 3 for 3 and 4). The pD of the solutions was varied by adding NaOD, phosphate buffer or DCl. The solutions were subsequently transferred to 1.7 mm NMR tubes and 1D 1 H-NMR experiments were recorded at 37 C aer 5 min, 30 min, 1 h, 2 h, 3 h, and every two hours until 24 h. An additional spectrum was also systematically recorded aer 48 or 72 h.

Interactions of 1-4 with amino acids
1 mg of the respective complex was dissolved in the appropriate aqueous solution (vide supra). When the hydrolysis reactions had reached a steady state, three to four equivalents of amino acid (Ala, Asp, Cys, Met or His) were added. The solutions were then transferred into NMR tubes and 1D 1 H-NMR experiments were measured at 37 C aer 5 min, 30 min, 1 h, 2 h, 3 h and every two hours until 24 h. An additional spectrum was also systematically recorded aer 48 or 72 h. ESI-MS, 2D 1 H-1 H COSY and 2D 1 H-DOSY NMR spectra were recorded aer the last 1D 1 H-NMR experiment. Between the measurements, the tubes were stored in the incubator at 37 C.

Interactions of 1-4 with the single stranded DNA dodecamer
Interaction of complexes with the single-stranded DNA dodecamer (CGCGATCGCGGCGCTAGCGC) was studied by dissolving the complexes in appropriate aqueous solution (vide supra), adding 1 mg of DNA fragment (M w ¼ 6119.2) and incubating the mixture at 37 C. MS measurements were performed aer 2 h and 24 h of incubation.


Design and synthesis of novel diamide derivatives of glycine as antihyperglycemic agents

ABSTRACT DPP-4 inhibition is one of the most extensively explored approaches for the management of type 2 diabetes (T2D). Most DPP-4 inhibitors in the market contain a proline mimetic active pharmacophore. Herein, we report the design, synthesis, and preliminary evaluation of a series of novel diamide derivatives of glycine, devoid of the proline mimic, for the treatment of T2D. As predicted from in silico studies, the diamide derivatives of glycine exhibited comparable DPP-4 inhibition with the standard as confirmed by the preliminary in vitro studies. Compound 6b was found to be the most potent (IC50 94.82 nM) DPP-4 inhibitor among all the molecules synthesized in the series. GRAPHICAL ABSTRACT

Introduction
Diabetes mellitus is a chronic metabolic disorder symptomatically characterized by hyperglycemia or high blood sugar. [1] The onset of hyperglycemic events can occur because of either absence of insulin secretion (type 1 diabetes) or lack of insulin secretion and insulin resistance both (T2D). In recent years, diabetes has become a severe and increasingly prevalent disease due to urbanization and lifestyle changes. [2] About 415 million people worldwide suffer from diabetes and this is projected to increase to 642 million by 2040. [3] Type 2 diabetes (T2D) is the major contributor to the overall diabetic population. T2D is linked to aging, hypertension, and obesity and often remains undetected until the microvascular or macrovascular complications begin to show. [4] Long-term hyperglycemia is associated with adverse cardiovascular complications, leading to high mortality rates. [5] Most of the modern therapeutic interventions are directed toward decreasing the hyperglycemic events within the patients. A major drawback of this first-line therapy is the induction of hypoglycemia and weight gain. To overcome these side effects, targets involving glucose-dependent insulin secretion (GDIS) have gained importance. Glucagon-like peptide-1 (GLP-1), an incretin hormone, plays an important role in GDIS. [6] GLP-1 acts as a stimulator of endogenous insulin release, while inhibiting the glucagon secretion in glucose-dependent manner, thereby reducing the risk of hypoglycemia. [7][8][9] However, the GLP-1(7-36)amide is rapidly degraded, by a serine protease, dipeptidyl aminopeptidase-4 (DPP-4, EC 3.4.14.5), into its inactive form, GLP-1(9-36)amide, with no therapeutic effects. [10,11] Hence DPP-4 is involved in pathogenesis of T2D and is an attractive target for therapeutic intervention. Thus, inhibition of DPP-4 has gained importance, as it results in increased half-life of endogenous GLP-1, thereby improving glucose exclusion in diabetics. [12,13] The DPP-4 inhibitors tested in animal models and in clinical trials have clearly demonstrated therapeutic potential for treatment of T2D (Fig. 1).
The DPP-4 acts by cleaving the N-terminal dipeptides with L-proline or L-alanine at the penultimate position. [14][15][16][17] Several classes of DPP-4 inhibitors have been reported from many laboratories and most of them are derived from aor β-amino acids mimicking the N-terminal dipeptide residues of the incretin hormones. In view of all these studies and with the aim of identifying it for DPP-4 inhibition, studies were carried out in our laboratory by preparing diamide derivatives of glycine (Fig. 2).

Results and discussion
A series of novel, non-proline mimetic, diamide derivatives of glycine 6a-k were designed by substituting 1-(phenylsulfonyl)piperidine-3-carboxamide at the P2 site and various secondary or tertiary amides at the P1 site. From the in silico studies it was predicted that the binding affinity of the diamide derivatives of glycine (6b ¼ À 8.5 kcal/mol) for the DPP-4 enzyme was comparable with that of the marketed drug (Vildagliptin ¼ À 6.7 kcal/mol) and hence was expected to show comparable potency. To synthesize diamide derivatives of glycine, commercially available boc-protected glycine 1 was at first reacted with amine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP) to yield various C-substituted amide derivatives of glycine 2a-k as shown in Scheme I.
However, when dicyclohexyl carbodiimide (DCC) was used in place of EDCI and HOBt for the reaction, a very strong ester bond was formed between the carboxylate of glycine and carbodiimide carbon, which rendered it unreactive for further reaction with the amines, and it was isolated by column chromatography as a nonpolar compound. Also, EDCI, in itself, could not sufficiently activate the carboxylic acid group of glycine and thus addition of HOBt was necessary for this coupling reaction. On the other hand, reaction of piperidine-3-carboxylic acid with benzene sulfonyl chloride, in the presence of sodium carbonate as a base, in a mixture of dichloromethane (DCM)/water (1:1) yielded 1-(phenylsulfonyl) piperidine-3-carboxylic acid 5 on acidification. Infrared (IR) spectrum of 5 showed bands at 1693 and 1352 cm À 1 for the carbonyl group of carboxylic acid and sulfonamide group respectively while the 1 H NMR showed a multiplet from δ 7.56 to 7.79 for the five aromatic protons and a broad singlet at δ 8.98, indicating the proton of the carboxylic acid group and thereby confirming the formation of 5. Compounds 2a-k were converted into their salts 3a-k by addition of 10% trifluoroacetic acid (TFA) in DCM. The salts 3a-k, on further reaction with acid 5 in the presence of peptide coupling agents EDCI, HOBt, and DMAP, gave the desired diamide derivatives of glycine 6a-k as shown in Scheme 1. The structures of 6a-k were confirmed by their IR, 1 H NMR, 13 C NMR, and ESI-MS analyses. While the IR spectra of compounds 6a-k exhibited one strong band at ∼3300 cm À 1 for the -NH (amide), two strong bands between 1730 and 1600 cm À 1 for amide carbonyls and a strong band ∼1350 cm À 1 for the sulfonamide group were found. In the 1 H NMR of 6a-k, a multiplet at δ ∼ 4.00 for the methylene group of glycine is observed due to the interactions with the neighboring amide group protons. The IR spectrum of compound 6b exhibited five strong bands: two bands at 3329, 3303 cm À 1 for the two -NH (amide), another two bands 1679 and 1644 cm À 1 for amide carbonyls, and a band at 1355 cm À 1 for the sulfonamide group. In the 1 H NMR spectrum of 6b, the methylene group of glycine exhibited a multiplet at δ 3.84-3.86 due to the interactions with the neighboring amide group protons and a multiplet from δ 7.36 to 7.74 representing the aromatic protons. In the 13 C NMR spectrum two peaks at 168.26 and 173.16 for the carbonyl carbons of the amide groups, six peaks from 24.14 to 48.63 for the piperidyl and glycyl carbons, and eight peaks ranging from 121.10 to 138.25 for the aromatic carbons confirmed the formation of 6b, which was also supported by its ESI-MS spectrum with a peak at m/z 435.9 for [M þ H] þ . The molecular docking studies were performed by selecting binding site residues of the A chain of DPP-4 (PDB ID: 3W2T) at a distance of 4.5 Å from NVP-LAF237. [18] To carry out the docking studies, chain A of 3W2T was considered for analysis. The molecular docking was carried out using AutoDock (version 4.2) for predicting the mode of binding. [19] The binding affinity of the most potent compound in the series, 6b, for DPP-4 enzyme was calculated to be À 8.5 kcal/mol. The list of residues interacting with the ligand were also obtained from AutoDock Tools. [19] Pymol [20] was used to visualize the structures (the protein and the docked compound 6b) and align the ligands to highlight the interactions as seen in Fig. 3.
Several potent diamide and sulfonamide derivatives with peptidomimetics pharmacophore (proline mimic) as DPP-4 inhibitors have been reported but the aim of this study was to recognize the DPP-4 inhibition potential of this new series of nonproline mimetic, diamide derivatives of glycine. [21,22] Preliminary in vitro DPP-4 inhibition assay was performed to screen test compounds 6a-k and to study the effect of different amides at the P1 site, for the DPP-4 enzyme inhibition potential. The IC 50 values were then determined for those compounds that exhibited greater than 50% enzyme inhibition at 3 µM concentration, as shown in Table 1.
Among the substituted secondary amide analogs 6a-f (at the P1 site), the effect of fluoro, chloro, and methyl substituents on the benzamide was studied and it was observed that the p-chloro benzamide derivative 6b (IC 50 94.82 nM) was six-fold more potent than p-fluoro benzamide derivative 6a (IC 50 573.74 nM) and about three-fold more potent than m-fluoro benzamide derivative 6d (IC 50 205.40 nM), but p-methyl benzamide derivative 6c was relatively inactive. Also, 6d was found to be twice as potent as 6a but a similar trend was not observed in 6e, 6f, and 6b. While among the substituted tertiary amide analogs (at the P1 site) 6g-k, the simplest tertiary amide, N,N-dimethyl amide derivative 6g (IC 50 188.97 nM), was three times more potent than N-methyl benzamide derivative 6k (IC 50 592.56 nM) and twice as potent as the tetrahydroisoquinoline amide derivative 6j (IC 50 444.60 nM). Cyclic aliphatic amide derivatives 6h and 6i were reported inactive. Thus the calculated IC 50 values, obtained from the in vitro analysis of the diamide derivatives of glycine 6a-k, clearly demonstrated 6b (IC 50 94.82) as the most potent DPP-4 inhibitor.

Conclusion
A novel series of diamide derivatives of glycine devoid of a proline mimic has been designed and synthesized, and its application as DPP-4 enzyme inhibitor for treatment of T2D has been evaluated. Compound 6b (IC 50 ¼ 94.82 nM) was found to be the most potent compound in the series, which is in agreement with the molecular docking studies. An extension of this approach to other scaffolds is in progress in our laboratory.

Chemistry
Reagent-grade chemicals and solvents were purchased from commercial supplier and used after purification. Thin-layer chromatography (TLC) was performed on silica-gel F254 plates (Merck). Merck silica gel (60-120 mesh) was used for column chromatographic purification. All reactions were carried out in a nitrogen atmosphere. Melting points are uncorrected and were measured in open capillary tubes, using a Rolex melting-point apparatus. IR spectra were recorded as KBr pellets on a Perkin-Elmer RX 1 spectrometer and the wave numbers are reported in cm À 1 . 1 H NMR and 13 C NMR spectral data were recorded on Advance Bruker 400 spectrometer ( 1 H 400 MHz/ 13 C 100 MHz) with CDCl 3 or DMSO-d 6 as solvent and tetramethylsilane (TMS) as internal standard and reported in δ (ppm). J values are in hertz (Hz). Mass spectra were determined by ESI-MS, using a Shimadzu LCMS 2020 apparatus. Elemental analyses were recorded on ThermoFinnigan Flash 11-12 series EA. All reactions were carried out under a nitrogen atmosphere. Purity of all the tested compounds was greater than 97%.

Inhibitory activity
In vitro enzyme (DPP-4) inhibitory activity was determined using fluorescence based assay. Gly-ProAminomethylcoumarin (AMC) was used as a substrate to measure DPP-4 activity. Cleavage of the peptide bond by DPP-4 releases the free AMC group, resulting in fluorescence that is analysed using an excitation wavelength of 350-360 nm and emission wavelength of 450-465 nm. Human recombinant DPP-4 enzyme was procured from Enzo Life Science (batch no. BML-SE434-9091), substrate H-Gly-Pro-AMC was procured from Enzo Life Science (batch no. BML-P189-9091), and assay buffer of pH 7.8 was used in the assay. DPP-4 activity was measured by mixing reagents in a 96-well plate (order of addition of reagents: assay buffer, enzyme, solvent/inhibitor, and finally substrate). Both the enzyme and 96-well plate were incubated for 30 min and the resulting fluorescence was measured using Spectra Max Fluorometer (Molecular Devices, Sunnyvale CA) by exciting at 360 nm and emission at 460 nm with the excitation filter at 360 nm and emission filter at 460 nm at sensitivity of 45.

1-(Phenylsulfonyl)piperidine-3-carboxylic acid (5)
Benzene sulfonyl chloride (1.1 mmol) was added to a mixture of piperidine-3-carboxylic acid 4 (1.0 mmol) and sodium carbonate (3.0 mmol) in 25 mL DCM/water (1:1), and the reaction mixture stirred at room temperature for 16 h or until the completion of reaction, as monitored by TLC. On completion of reaction, the reaction mixture was washed with petroleum ether (20 mL) and then acidified with concentreated HCl, to pH 2. The white solid thus separated was filtered, washed with water several times, and then dried to yield the desired product as white solid. Yield

General procedure for the preparation of compounds 6a-k
Compounds 2a-k were deprotected by stirring in 10% trifluoroacetic acid (TFA) in DCM. On completion of the reaction after an hour or as monitored by TLC, the solvent was evaporated under reduced pressure and once again the product was dissolved in DCM to give a solution of compounds 3a-k. To a solution of compound 5 (1.0 mmol) in DCM (20 mL), EDCI (1.5 mmol), HOBt (1.0 mmol), and DMAP (1.0 mmol) were added at 0-5°C, followed by the solution of compound 3a-k in DCM (5 mL), and the reaction mixture was then stirred at room temperature for 10 h or until the completion of the reaction as detected by TLC. After completion of the reaction, it was washed with water (2 � 20 mL) and brine (1 � 10 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give the crude product, which was then purified by column chromatography using silica gel, employing ethylacetate/petroleum ether (70:30) as eluent to give pure product 6a-k as a white solid.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Synthesis and Biological Evaluation of Novel Aromatic Imide-Polyamine Conjugates

Three types of conjugates in which aromatic imide scaffolds were coupled to diverse amine/polyamine motifs were synthesized, and their antitumor activities were evaluated in vitro and in vivo. Results showed that the conjugate 11e of 1,8-naphthilimide with spermine had pronounced effects on inhibiting tumor cell proliferation and inducing tumor cell apoptosis via ROS-mediated mitochondrial pathway. The in vivo assays on three H22 tumor transplant models revealed that compound 11e exerted potent ability in preventing lung cancer metastasis and extending lifespan. Furthermore, the efficacy of 11e in inhibiting tumor growth and improving body weight index were better than that of positive control, amonafide. Our study demonstrates that compound 11e is a valuable lead compound for further investigation.

Introduction
The development of effective anticancer agent continues to be a formidable challenge in medicine. Aromatic imides provide an excellent template for new drug development and have drawn special attention of medicinal chemists. Some phthalimides such as thalidomide and lenalidomide ( Figure 1) are now in clinical use [1,2]. Thalidomide, initially introduced for treatment of multiple myeloma (MM) because of its anti-angiogenic properties, has shown remarkable activity alone and in combination with other drugs in patients across all stages of the disease. Lenalidomide has been shown to benefit patients with multiple myeloma, myelodysplastic syndromes, and lymphoma [3][4][5].
Naphthalimides are also a class of intriguing pharmacophores [6,7]. Although many naphthalimides and their synthetic analogs have reached clinical trials, most of them were abandoned because of various adverse effects. For example amonafide ( Figure 1) had shown potent anticancer activity. However, due to unexpected central neurotoxicity and limited efficiency, further application in the clinical trial was abandoned [8]. In order to improve the tumor selectivity, efficiency, and safety, a broad variety of novel naphthalimide derivatives by modified amonafide at different positions have been reported [9,10]. Currently, extensive researches have emphasized the fact that polyamine conjugates acting as promising antitumor candidates are becoming increasingly important in the polyamine field [11][12][13][14][15]. F14512, an epipodophyllotoxin-spermine conjugate, is perhaps the most promising compound which is presently being evaluated in phase I trials [16][17][18][19]. Our work demonstrated that derivatives incorporating an aromatic imide skeleton or connecting to a polyamine linker such as spermidine or homospermidine, exhibit marked cell selectivity and lung cancer metastasis inhibition . These findings stimulated us to search for more promising imide scaffolds suitable for polyamine modification. We report herein three kinds of aromatic imide (Figure 1), which are coupled with diverse polyamine motifs ( Figure 2). In addition to the routine in vitro evaluation, in vivo experiments are designed to provide more data to justify their future drug development.

Chemistry
The acetyl-substituted naphthalimide-polyamine compounds 5d-f were synthesized according to Scheme 1. Intermediates 2 and 3 were prepared by a modified previously reported procedure [23,24]. The respective N-Boc protected intermediates 4d-f were obtained by the condensation of R1NH2 (6, the Boc protected polyamines were prepared by a modified previously reported procedure) [25] (as shown in Figure 2) with 4-acetyl-1,8-naphthalic anhydride (3). After purification by flash column chromatography, these intermediates were mixed with 4 M HCl in EtOH at room temperature to obtain the target compounds 5d-f as their hydrochloride salts with a yield between 70%-80% [22].
Compounds 11a-b, e-f were synthesized following a four-step procedure as shown in Scheme 1. Compound 8 obtained by reacting 1,8-naphthalic anhydride with 1,3-propanediamine reacted with chloroacetyl chloride to give intermediate 9. Compounds 10a-b, e-f were easily formed by the reaction of 9 and R1NH2 in acetonitrile. After purification by flash column chromatography, these intermediates were mixed with 4 M HCl at room temperature to obtain the target compounds 11a-b, e-f as their corresponding hydrochloride salts with a yield between 70%-80%. Currently, extensive researches have emphasized the fact that polyamine conjugates acting as promising antitumor candidates are becoming increasingly important in the polyamine field [11][12][13][14][15]. F14512, an epipodophyllotoxin-spermine conjugate, is perhaps the most promising compound which is presently being evaluated in phase I trials [16][17][18][19]. Our work demonstrated that derivatives incorporating an aromatic imide skeleton or connecting to a polyamine linker such as spermidine or homospermidine, exhibit marked cell selectivity and lung cancer metastasis inhibition . These findings stimulated us to search for more promising imide scaffolds suitable for polyamine modification. We report herein three kinds of aromatic imide (Figure 1), which are coupled with diverse polyamine motifs ( Figure 2). In addition to the routine in vitro evaluation, in vivo experiments are designed to provide more data to justify their future drug development.  Currently, extensive researches have emphasized the fact that polyamine conjugates acting as promising antitumor candidates are becoming increasingly important in the polyamine field [11][12][13][14][15]. F14512, an epipodophyllotoxin-spermine conjugate, is perhaps the most promising compound which is presently being evaluated in phase I trials [16][17][18][19]. Our work demonstrated that derivatives incorporating an aromatic imide skeleton or connecting to a polyamine linker such as spermidine or homospermidine, exhibit marked cell selectivity and lung cancer metastasis inhibition . These findings stimulated us to search for more promising imide scaffolds suitable for polyamine modification. We report herein three kinds of aromatic imide (Figure 1), which are coupled with diverse polyamine motifs ( Figure 2). In addition to the routine in vitro evaluation, in vivo experiments are designed to provide more data to justify their future drug development.

Chemistry
The acetyl-substituted naphthalimide-polyamine compounds 5d-f were synthesized according to Scheme 1. Intermediates 2 and 3 were prepared by a modified previously reported procedure [23,24]. The respective N-Boc protected intermediates 4d-f were obtained by the condensation of R1NH2 (6, the Boc protected polyamines were prepared by a modified previously reported procedure) [25] (as shown in Figure 2) with 4-acetyl-1,8-naphthalic anhydride (3). After purification by flash column chromatography, these intermediates were mixed with 4 M HCl in EtOH at room temperature to obtain the target compounds 5d-f as their hydrochloride salts with a yield between 70%-80% [22].
Compounds 11a-b, e-f were synthesized following a four-step procedure as shown in Scheme 1. Compound 8 obtained by reacting 1,8-naphthalic anhydride with 1,3-propanediamine reacted with chloroacetyl chloride to give intermediate 9. Compounds 10a-b, e-f were easily formed by the reaction of 9 and R1NH2 in acetonitrile. After purification by flash column chromatography, these intermediates were mixed with 4 M HCl at room temperature to obtain the target compounds 11a-b, e-f as their corresponding hydrochloride salts with a yield between 70%-80%.

Chemistry
The acetyl-substituted naphthalimide-polyamine compounds 5d-f were synthesized according to Scheme 1. Intermediates 2 and 3 were prepared by a modified previously reported procedure [23,24]. The respective N-Boc protected intermediates 4d-f were obtained by the condensation of R 1 NH 2 (6, the Boc protected polyamines were prepared by a modified previously reported procedure) [25] (as shown in Figure 2) with 4-acetyl-1,8-naphthalic anhydride (3). After purification by flash column chromatography, these intermediates were mixed with 4 M HCl in EtOH at room temperature to obtain the target compounds 5d-f as their hydrochloride salts with a yield between 70% and 80% [22].
Compounds 11a-b, e-f were synthesized following a four-step procedure as shown in Scheme 1. Compound 8 obtained by reacting 1,8-naphthalic anhydride with 1,3-propanediamine reacted with chloroacetyl chloride to give intermediate 9. Compounds 10a-b, e-f were easily formed by the reaction of 9 and R 1 NH 2 in acetonitrile. After purification by flash column chromatography, these intermediates were mixed with 4 M HCl at room temperature to obtain the target compounds 11a-b, e-f as their corresponding hydrochloride salts with a yield between 70% and 80%. The synthesis of target compounds 17b-d are described as in Scheme 2. Intermediates 12 and 13 were prepared by a modified previously reported procedure [26,27]. Compound 13 was N-alkylated by commercial dibromoalkane to yield intermediate 14 in good yield. Subsequently, compound 14 was condensed with corresponding amines R1NH2, after the completion of the reaction, the solvent was removed by rotary evaporation yielding a residue. This residue, without further purification, was N-protected to form 15b-d using excess di-tert-butyl dicarbonate (Boc2O). Suzuki coupling of 15b-d with phenylboronic acid catalyzed by PdCl2 gave intermediates 16b-d [28]. From these compounds, Boc groups in the polyamine skeleton were subsequently removed with 4 M HCl in EtOH at room temperature to provide target compounds 17b-d as hydrochloride salts [29].    Table 1. The results showed that the naphthalimide-polyamine conjugates 5d-f exhibited antitumor activities as potent as previously reported naphthalimide-polyamine conjugates [2,21]. Compounds 11a-b, e-f exhibited mixed results, compound 11e, which displayed improved potency in comparison to other analogues in the series, was of comparable potency to amonafide. Surprisingly, the 5-phenylphthalimidepolyamines 17b-d were not active. The synthesis of target compounds 17b-d are described as in Scheme 2. Intermediates 12 and 13 were prepared by a modified previously reported procedure [26,27]. Compound 13 was N-alkylated by commercial dibromoalkane to yield intermediate 14 in good yield. Subsequently, compound 14 was condensed with corresponding amines R 1 NH 2 , after the completion of the reaction, the solvent was removed by rotary evaporation yielding a residue. This residue, without further purification, was N-protected to form 15b-d using excess di-tert-butyl dicarbonate (Boc 2 O). Suzuki coupling of 15b-d with phenylboronic acid catalyzed by PdCl 2 gave intermediates 16b-d [28]. From these compounds, Boc groups in the polyamine skeleton were subsequently removed with 4 M HCl in EtOH at room temperature to provide target compounds 17b-d as hydrochloride salts [29].  The synthesis of target compounds 17b-d are described as in Scheme 2. Intermediates 12 and 13 were prepared by a modified previously reported procedure [26,27]. Compound 13 was N-alkylated by commercial dibromoalkane to yield intermediate 14 in good yield. Subsequently, compound 14 was condensed with corresponding amines R1NH2, after the completion of the reaction, the solvent was removed by rotary evaporation yielding a residue. This residue, without further purification, was N-protected to form 15b-d using excess di-tert-butyl dicarbonate (Boc2O). Suzuki coupling of 15b-d with phenylboronic acid catalyzed by PdCl2 gave intermediates 16b-d [28]. From these compounds, Boc groups in the polyamine skeleton were subsequently removed with 4 M HCl in EtOH at room temperature to provide target compounds 17b-d as hydrochloride salts [29].     Table 1. The results showed that the naphthalimide-polyamine conjugates 5d-f exhibited antitumor activities as potent as previously reported naphthalimide-polyamine conjugates [2,21]. Compounds 11a-b, e-f exhibited mixed results, compound 11e, which displayed improved potency in comparison to other analogues in the series, was of comparable potency to amonafide. Surprisingly, the 5-phenylphthalimidepolyamines 17b-d were not active. Scheme 2. Synthetic protocol of target compounds 17b-d (R 1 = N-Boc protected polyamine chains; R 2 = deprotected polyamine chains).

Cytotoxicity in Human Tumor Cell Lines
The inhibitory activity of target compounds toward five tumor cells (HCT-116: human colorectal cancer cell line; K562: human leukemia cell line; HepG2: human hepatoma cell line; MDA-MB-231: human breast cancer cell line; QSG-7701: normal hepatocyte cell line) were measured by the traditional MTT ({3-(4,5-dimethylthiazol-2-yl)}-2,5-diphenyl-tetrazolium bromide) test. Amonafide was tested as a reference compound. Structures and biological results of target compounds are shown in Table 1. The results showed that the naphthalimide-polyamine conjugates 5d-f exhibited antitumor activities as potent as previously reported naphthalimide-polyamine conjugates [2,21]. Compounds 11a-b, e-f exhibited mixed results, compound 11e, which displayed improved potency in comparison to other analogues in the series, was of comparable potency to amonafide. Surprisingly, the 5-phenylphthalimide-polyamines 17b-d were not active.
a IC50 values represent the concentration causing 50% growth inhibition. They were determined by the linear regression method. Each sample is the mean of three independent experiments. b Not determined. Amo = Amonafide.

Apoptosis
Previous studies revealed that most naphthalimide-polyamine conjugates may trigger tumor cell death via an apoptotic process [30,31]. To appraise if novel naphthalimide-polyamine conjugates induce cell apoptosis, morphological study was applied after HepG2 cells were stained with AO/EB. The percentage of apoptotic cells was calculated after observing a total of 300 cells and as shown in Figure 3A,C, obvious apoptosis induced by 11e was observed in a dose-dependent manner [32]. To furnish more credible information about the apoptosis of HepG2 cells induced by compound 11e, we used the high content screening (HCS) technique to detect related apoptosis indicators. Mitochondria are vital cell organelles in the process of transmitting apoptosis signals, whereas the mitochondrial membrane potential (MMP) is a critical parameter related to mitochondrial function in cells. As a MMP specific cationic fluorescent dye, Rh123 is used to measure the change of MMP, which accumulates in healthy mitochondria with intact membrane potential and is absent in the depolarized mitochondria [22]. Lysosomes, as cellular targets in cancer therapy, participate in various cell death profiles such as apoptosis and senescence [9,33,34]. Thus, the lysosomal mass/pH (Lys/pH) variation was detected by a basic fluorescent probe Lyso-Tracker Red which selectively accumulates in acidic lysosomes during cytotoxicity occurs. After treatment with compound 11e for 48 h, the green fluorescence of Rh123 in cytoplasm was significantly decreased in a dose-dependent manner, indicating that the destroyed mitochondria lost their mitochondrial transmembrane potential ( Figure 3A,D). Meanwhile, the remarkable accruement of red fluorescence in cells treated with compound 11e implied the dose-related occurrence of lysosomes impairment ( Figure 3B,D).
It is known that polyamine conjugates may produce reactive oxygen species (ROS) [22]. The elevated ROS could bring corresponding influences on MMP loss and LYS/pH destabilization, which generally activates the cell death. Therefore, the ROS generation was detected by the DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) experiment. Indeed, the apparently increased ROS was observed in a dose-dependent pattern as shown in Figure 3B,D.
a IC 50 values represent the concentration causing 50% growth inhibition. They were determined by the linear regression method. Each sample is the mean of three independent experiments. b Not determined. Amo = Amonafide.

Apoptosis
Previous studies revealed that most naphthalimide-polyamine conjugates may trigger tumor cell death via an apoptotic process [30,31]. To appraise if novel naphthalimide-polyamine conjugates induce cell apoptosis, morphological study was applied after HepG2 cells were stained with AO/EB. The percentage of apoptotic cells was calculated after observing a total of 300 cells and as shown in Figure 3A,C, obvious apoptosis induced by 11e was observed in a dose-dependent manner [32]. To furnish more credible information about the apoptosis of HepG2 cells induced by compound 11e, we used the high content screening (HCS) technique to detect related apoptosis indicators. Mitochondria are vital cell organelles in the process of transmitting apoptosis signals, whereas the mitochondrial membrane potential (MMP) is a critical parameter related to mitochondrial function in cells. As a MMP specific cationic fluorescent dye, Rh123 is used to measure the change of MMP, which accumulates in healthy mitochondria with intact membrane potential and is absent in the depolarized mitochondria [22]. Lysosomes, as cellular targets in cancer therapy, participate in various cell death profiles such as apoptosis and senescence [9,33,34]. Thus, the lysosomal mass/pH (Lys/pH) variation was detected by a basic fluorescent probe Lyso-Tracker Red which selectively accumulates in acidic lysosomes during cytotoxicity occurs. After treatment with compound 11e for 48 h, the green fluorescence of Rh123 in cytoplasm was significantly decreased in a dose-dependent manner, indicating that the destroyed mitochondria lost their mitochondrial transmembrane potential ( Figure 3A,D). Meanwhile, the remarkable accruement of red fluorescence in cells treated with compound 11e implied the dose-related occurrence of lysosomes impairment ( Figure 3B,D).
It is known that polyamine conjugates may produce reactive oxygen species (ROS) [22]. The elevated ROS could bring corresponding influences on MMP loss and LYS/pH destabilization, which generally activates the cell death. Therefore, the ROS generation was detected by the DCFH-DA (2 ,7 -dichlorodihydrofluorescein diacetate) experiment. Indeed, the apparently increased ROS was observed in a dose-dependent pattern as shown in Figure 3B,D.

Antitumor Activity In Vivo
Several of our in vitro observations reported here demonstrate that 11e has the ability to interfere with the proliferation of tumor cells. However, few in vivo studies provided limited messages for rational design of the conjugates in comparison to the plentiful in vitro data. Indeed, there are significant discrepancies between in vitro and in vivo experiments in many cases. In vivo trials, based on preliminary in vitro screens, provide more valuable messages for further drug development. To further evaluate the antitumor activity of 11e in vivo, we chose three H22 (mice hepatoma cell line) tumor transplant models: solid tumor (tumor growth inhibition evaluation), ascites tumor (live time evaluation), and pulmonary metastasis tumor (tumor metastasis evaluation). As shown in Figure 4A,B and Table 2 after treatment of mice bearing hepatoma xenografts (H22 mouse hepatocellular carcinoma model) with 1 mg/kg of 11e once every day, obvious tumor weight suppression by 70.92% was observed compared to the negative control group that treated with normal saline. Mice bearing hepatoma xenografts were also treated with 5 mg/kg of amonafide and the tumor weight suppression by 43.79% was observed compared to the negative control group. Thus, tumor growth inhibitory rates in mice treated with 11e and amonafide were dramatically increased, and the in vitro antitumor effect of 11e was also obviously stronger than that of amonafide.

Antitumor Activity In Vivo
Several of our in vitro observations reported here demonstrate that 11e has the ability to interfere with the proliferation of tumor cells. However, few in vivo studies provided limited messages for rational design of the conjugates in comparison to the plentiful in vitro data. Indeed, there are significant discrepancies between in vitro and in vivo experiments in many cases. In vivo trials, based on preliminary in vitro screens, provide more valuable messages for further drug development. To further evaluate the antitumor activity of 11e in vivo, we chose three H22 (mice hepatoma cell line) tumor transplant models: solid tumor (tumor growth inhibition evaluation), ascites tumor (live time evaluation), and pulmonary metastasis tumor (tumor metastasis evaluation). As shown in Figure 4A,B and Table 2 after treatment of mice bearing hepatoma xenografts (H22 mouse hepatocellular carcinoma model) with 1 mg/kg of 11e once every day, obvious tumor weight suppression by 70.92% was observed compared to the negative control group that treated with normal saline. Mice bearing hepatoma xenografts were also treated with 5 mg/kg of amonafide and the tumor weight suppression by 43.79% was observed compared to the negative control group. Thus, tumor growth inhibitory rates in mice treated with 11e and amonafide were dramatically increased, and the in vitro antitumor effect of 11e was also obviously stronger than that of amonafide. Metastasis is the process by which tumors spread from the primary organ in which they arise to other sites in the body. In most cases, tumors that stay confined to one organ will not be fatal, tumors that have spread to other organs have a greater potential for causing harm. To test whether 11e was effective against an established lung metastasis, we injected H22 cells intravenously into 24 Kunming mice (5 × 10 6 cells each mouse). To ensure that all mice bore actively growing lung tumors before the drug treatment, pulmonary cancer was allowed to develop for 10 days. Mice were treated with 11e, amonafide and normal saline for 7 consecutive days, respectively. Mice treated with 11e displayed few metastases (62.42%), whereas all mice treated with normal saline had an extensive tumor burden in the lungs. In contrast, amonafide moderately decreased lung metastasis nodus numbers (40.7%) ( Figure 4C,D; Table 3). These results supported that 11e inhibited the tumor metastasis lighter than amonafide.
Impacts of 11e on survival time in H22-bearing mice were evaluated by measuring the extension of the lifespan. The median survival time (MST) in control mice was (12.75 ± 2.5) days. After the 11e or amonafide i.v. administrated, the life span was increased by 2.41-fold ((30.75 ± 4.4) days) or 1.53-fold (19.5 ± 3.6 days) compared with that of the control group, respectively ( Figure 4E). Metastasis is the process by which tumors spread from the primary organ in which they arise to other sites in the body. In most cases, tumors that stay confined to one organ will not be fatal, tumors that have spread to other organs have a greater potential for causing harm. To test whether 11e was effective against an established lung metastasis, we injected H22 cells intravenously into 24 Kunming mice (5 × 10 6 cells each mouse). To ensure that all mice bore actively growing lung tumors before the drug treatment, pulmonary cancer was allowed to develop for 10 days. Mice were treated with 11e, amonafide and normal saline for 7 consecutive days, respectively. Mice treated with 11e displayed few metastases (62.42%), whereas all mice treated with normal saline had an extensive tumor burden in the lungs. In contrast, amonafide moderately decreased lung metastasis nodus numbers (40.7%) ( Figure 4C,D; Table 3). These results supported that 11e inhibited the tumor metastasis lighter than amonafide.
Impacts of 11e on survival time in H22-bearing mice were evaluated by measuring the extension of the lifespan. The median survival time (MST) in control mice was (12.75 ± 2.5) days. After the 11e or amonafide i.v. administrated, the life span was increased by 2.41-fold ((30.75 ± 4.4) days) or 1.53-fold (19.5 ± 3.6 days) compared with that of the control group, respectively ( Figure 4E).

Systemic Toxicity
It is reported that amonafide can result in severe adverse effects and which greatly limited its clinical application [35]. In both cell and animal models, some polyamine conjugates such as F14512 has been confirmed to possess favorable cell or tissue selectivity [36], which is expected to reduce the

Systemic Toxicity
It is reported that amonafide can result in severe adverse effects and which greatly limited its clinical application [35]. In both cell and animal models, some polyamine conjugates such as F14512 has been confirmed to possess favorable cell or tissue selectivity [36], which is expected to reduce the adverse effects of parent drugs. However, there are few reports about the systemic toxicology of naphthalimide-polyamine conjugates to support the above hypothesis. Consequently, we tested the systemic toxicity of 11e (1 mg/kg) with amonafide (5 mg/kg) in tumor-bearing mice for 7 consecutive days (intravenous injection). At the same time, the body weight was monitored to detect the dose-limited toxicity. On day 8, these mice were killed by cervical vertebra, and the organs and tissues, including samples of heart, liver, lung, kidney, spleen and thymus, were taken for the inspection of systemic toxicity. There was no weight loss in the 11e-treated mice, on the contrary, the weight increased slightly. Whereas treatment with amonafide was associated with some degree of weight loss, indicating possibly dose-related toxicity ( Figure 5A). Heart, kidney and lung had no obvious index value changes in the examination of both compounds ( Figure 5C). The liver and spleen index numbers had no significant variation after 11e treatment compared to controls, but amonafide treatment obviously decreased these index values, implying that 11e had no influence on liver and immune function, at the same time suggesting that amonafide injured liver function and caused damage to immune function in some extent ( Figure 5B,C). In short, 11e performed better than amonafide in the experiments of the toxicological profile at the doses that exert antitumor activity in vivo. adverse effects of parent drugs. However, there are few reports about the systemic toxicology of naphthalimide-polyamine conjugates to support the above hypothesis. Consequently, we tested the systemic toxicity of 11e (1 mg/kg) with amonafide (5 mg/kg) in tumor-bearing mice for 7 consecutive days (intravenous injection). At the same time, the body weight was monitored to detect the dose-limited toxicity. On day 8, these mice were killed by cervical vertebra, and the organs and tissues, including samples of heart, liver, lung, kidney, spleen and thymus, were taken for the inspection of systemic toxicity. There was no weight loss in the 11e-treated mice, on the contrary, the weight increased slightly. Whereas treatment with amonafide was associated with some degree of weight loss, indicating possibly dose-related toxicity ( Figure 5A). Heart, kidney and lung had no obvious index value changes in the examination of both compounds ( Figure 5C). The liver and spleen index numbers had no significant variation after 11e treatment compared to controls, but amonafide treatment obviously decreased these index values, implying that 11e had no influence on liver and immune function, at the same time suggesting that amonafide injured liver function and caused damage to immune function in some extent ( Figure 5B,C). In short, 11e performed better than amonafide in the experiments of the toxicological profile at the doses that exert antitumor activity in vivo. The organ indexes of immune organ (spleen) were evaluated after treatment with 11e, amonafide, and normal saline (n = 3, x ± SD *** p < 0.001); (C) The organ indexes (heart, liver, kidney and lung) were evaluated after treatment with 11e, amonafide, and normal saline (n = 3, x ± s ** p < 0.01).

ADMET Prediction
The in silico prediction of physicochemical, ADME and toxicity properties of compounds 11e was calculated using the ACD/Labs Percepta Platform 23 (License#58830) and results were compared to those obtained for amonafide. As demonstrated in Table 4, the druglikeness of compound 11e was similar to amonafide except rotatable bonds [37,38]. Regardless of the poor human intestinal absorption (HIA), compound 11e showed excellent solubility and general permeable based on predicted permeability across Caco-2 monolayers (Pe). The drug safety profile of these compounds was also projected using Program ACD/Percepta based on probabilistic predictors. The inhibition of hERG (human Ether-a-gogo-Related Gene) and the mutagenic profile were calculated and the results converted in the so called classification scores ( Table 5). As depicted in Table 4, compound 11e was predicted as undefined (score > 0.33 and <0.67) in HLM (human liver microsomes), hERG and mutagenic, which were similar to amonafide. The changes of body weight of mice treated with 11e, amonafide, and normal saline; (B) The organ indexes of immune organ (spleen) were evaluated after treatment with 11e, amonafide, and normal saline (n = 3, x ± SD *** p < 0.001); (C) The organ indexes (heart, liver, kidney and lung) were evaluated after treatment with 11e, amonafide, and normal saline (n = 3, x ± s ** p < 0.01).

ADMET Prediction
The in silico prediction of physicochemical, ADME and toxicity properties of compounds 11e was calculated using the ACD/Labs Percepta Platform 23 (License#58830) and results were compared to those obtained for amonafide. As demonstrated in Table 4, the druglikeness of compound 11e was similar to amonafide except rotatable bonds [37,38]. Regardless of the poor human intestinal absorption (HIA), compound 11e showed excellent solubility and general permeable based on predicted permeability across Caco-2 monolayers (Pe). The drug safety profile of these compounds was also projected using Program ACD/Percepta based on probabilistic predictors. The inhibition of hERG (human Ether-a-go-go-Related Gene) and the mutagenic profile were calculated and the results converted in the so called classification scores ( Table 5). As depicted in Table 4, compound 11e was predicted as undefined (score > 0.33 and <0.67) in HLM (human liver microsomes), hERG and mutagenic, which were similar to amonafide.

General Information
All solvents and reagents were purchased from the suppliers and used without further purification. All NMR spectra were recorded on an AV-400 model spectrometer (Bruker BioSpin, Zürich, Switzerland) in D 2 O or CDCl 3 , Chemical shifts for 1 H-NMR spectra are reported in parts per million to residual solvent protons. ESI-MS spectra were recorded on an ESQUIRE-LC mass spectrometer (Agilent, Palo Alto, CA, USA). The target compounds with the purity being higher than 95% were analyzed using combustion analysis, Elemental analyses were performed on a Gmbe VarioEL elemental instrument (Elementar ,Langenselbold, Germany), and results were within 0.4% of the theoretical values.

General Procedure for the Synthesis of 5d-f
A mixture of 4-acetyl-1,8-naphthalic anhydride 3 (2 mmol) and polyamine 6 (2 mmol) (as shown in Figure 2) in EtOH (30 mL) was heated at refluxing temperature, and the reaction process was monitored by TLC. After completion, the reaction mixture was cooled to room temperature and concentrated under vacuum to give an oily residue, which was purified by chromatography on silica gel using petroleum ether/ethyl acetate mixture as eluent to obtain the Boc protected intermediates 4d-f. The respective N-Boc protected intermediates 4d-f (1.2 mmol) were dissolved in EtOH (20 mL) and stirred at 0 • C for 10 min. Then 4 M HCl was added dropwise at 0 • C. The reaction mixture was stirred at room temperature overnight. The solution typically gave a white solid as precipitate. The solid was filtered, washed several times with absolute ethanol, and target compounds 5d-f were obtained [19].  166.21, 165.60, 140.89, 134.59, 133.39, 132.11, 131.39, 130.69,  128.68, 128.48, 125.27, 122.21, 49.63, 49.44, 42.27

General Procedure for the Synthesis of 11a-b, e-f
To a solution of intermediate (8) (5 mmol) and anhydrous K 2 CO 3 (7.5 mmol) in acetonitrile (50 mL), chloroacetyl chloride (6 mmol) in acetonitrile (5 mL) was added at room temperature. The mixture was stirred at room temperature overnight, then compound 9 (1.52 g) was collected by filtration and washed with water (three times with each 10 mL).
A mixture of 9 (1 mmol), anhydrous K 2 CO 3 (1.5 mmol), a catalytic amount of KI, in DMF (5 mL) and acetonitrile (15 mL) was stirred at room temperature for 0.5 h, then 1 equivalent of Boc protected polyamine 6 was added and the mixture was stirred overnight. After completion, the reaction mixture was concentrated under vacuum to give an oily residue. The residue was then dissolved in CH 2 Cl 2 (25 mL) and washed with aqueous Na 2 CO 3 (25 mL). The organic layer was separated, dried with anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum. The residue was purified by chromatography on silica gel using CH 2 Cl 2 /CH 3 OH (100:3 to 100:5) mixture as eluent to yield compounds 10a-b, e-f. The respective N-Boc protected intermediates 10 (1.0 mmol) were dissolved in EtOH (10 mL) and stirred at 0 • C for 10 min. Then 4 M HCl was added dropwise at 0 • C. The reaction mixture was stirred at room temperature overnight. The solution typically gave a white solid as precipitate. The solid was filtered, washed several times with absolute ethanol, and dried under vacuum to give the pure target compounds 11a-b, e-f.

Synthesis of Compounds 14a-b
Intermediates 12, 13 were prepared by a modified previously reported procedure [26,27]. Compound 13 (1.13 g, 5 mmol) added portion wise to 1,3-dibromopropane or 1,4-dibromobutane (10 mmol) in acetone (30 mL). The solution was refluxed for 12 h; after cooling and filtration, the solvent was removed under vacuo to give a solid. The latter was recrystallized from ethanol to afford compound 14a-b.

Synthesis of Compounds 15b-d
Intermediates 14 (1 mmol), Boc protected polyamine (1.2 mmol), anhydrous K 2 CO 3 (1.5 mmol) were added to the flask in the presence of KI (0.01 mmol) and CH 3 CN (30 mL). The resulting mixture was heated at 45 • C for 12 h, then the solvent was evaporated in vacuo, water was added to the residue, and extracted with CH 2 Cl 2 . The organic layer was dried over Na 2 SO 4 and evaporated in vacuo. The residue was dissolved in CH 3 OH and a solution of di-tert-butyl dicarbonate (1.5 mmol) in methanol (5 mL) was added dropwise at 0 • C then the mixture was stirred for an additional 1 h at 0 • C. The temperature was allowed to gradually rise to room temperature, and the reaction was stirred overnight. The mixture was evaporated in vacuo. The residue was dissolved in CH 2 Cl 2 and washed with water several times. The organic layer was separated, dried with anhydrous Na 2 SO 4 , filtered, and concentrated. The residue could be purified by flash column chromatography on silica gel.

Synthesis of Compounds 17b-d
A 50 mL round bottomed flask equipped with a magnetic stirring bar was charged with pure 15 (2 mmol), toluene (20 mL), phenylboronic acid (2 mmol), K 2 CO 3 (3 mmol), PdCl 2 (0.03 mmol) and tetrabutylammonium bromide (0.03 mmol). The mixture was vigorously stirred for 6 h at the boiling temperature. After reaction completion, the reaction mixture was filtered and evaporated to dryness, and the residue was subjected to column chromatography to obtain the intermediates 16b-d. The respective N-Boc protected intermediates 16b-d (1.2 mmol) were dissolved in EtOH (20 mL) and stirred at 0 • C for 10 min. Then, 4 M HCl was added dropwise at 0 • C The reaction was stirred at room temperature overnight. The solution typically gave a white solid as a precipitate. The solid was filtered, washed several times with absolute ethanol, and dried under vacuum to give the pure target compounds 17b-d.

Cytotoxicity against Cancer Cell Lines
The antiproliferative ability of compounds was evaluated in HCT-116 cells, QSG-7701, MDA-MB-231 cells, K562 and HepG2 cells by the conversion of MTT to a purple formazan precipitate as previously described [39]. Briefly, exponentially growing HCT-116 cells, QSG-7701, MDA-MB-231 cells and HepG2 cells were seeded in 96-well plates (5 × 10 3 cells to each well) and allowed to attach overnight. After 24 h, the cells were treated with various concentrations (1, 5, 10, 30, and 50 µM) of samples for 48 h, and then MTT (100 µL, 1 mg/mL) was added. After incubation for 4 h at 37 • C, the MTT solution was removed and the crystals of viable cells were dissolved with DMSO (150 µL) in each well; 5000/well exponentially growing K562 were seeded in 96-well plates and treated with indicated concentrations (1, 5, 10, 30, and 50 µM) of samples for 48 h, and then MTT (10 mg/mL, 10 µL) was added. After incubation for 4 h at 37 • C the crystals of viable cells were dissolved overnight with SDS (sodium dodecylsulfonate, 10%, 100 µL) in each well. The absorbance spectra were measured on an ELISA Processor II Microplate Reader (Thermo Scientific, Waltham, MA, USA) at a wavelength of 570 nm. The percentage of cytotoxicity was defined with treated and untreated cell lines. The 50% antitumor activity dose (IC 50 ) was defined as the concentration of samples that reduced the absorbance of the treated cells by 50%.

Cellular Apoptotic Evaluation
HepG2 cells were seeded in 96 well plates (6 × 10 3 cells/well), cultured for 24 h to obtain a confluent monolayer and then treated with various concentrations of tested compounds for 48 h. Cells were incubated with Acridine Orange (50 µM)/ethidium bromide (50 µM) for 30 min, then washed with PBS to remove unbound dyes. Images were obtained on the High Content Screening (HCS, ArrayScan, Thermo Scientific) reader using the Target Activation BioApplication software (Thermo Scientific).
HepG2 cells were seeded in 96 well plates (6 × 10 3 cells/well), cultured for 24 h to obtain a confluent monolayer, and then treated with various concentrations of tested compounds for 48 h. Cells were incubated with Rh123 (0.25 nM), or DCFH-DA (10 µM), or Lyso-Tracker Red (50 nM ) in the dark at 37 • C for 30 min. for the measurement of mitochondrial membrane potential (MMP), or reactive oxygen species (ROS) or lysosomal mass/pH staining, respectively. After being washed with PBS to remove excess dyes, cells were further stained with the nuclear dye Hoechst 33,342 (10 µM) in the dark at 37 • C for 20 min. Images were acquired on the ArrayScan HCS reader using the Target Activation BioApplication software.

Subcutaneous Xenograft of H22 Cells in Kunming Mice
All animal care and experimentation conformed to the Guide for the Care and Use of Laboratory Animals published by Henan University (HUSOM2016-162). For solid tumor development, 30 male Kunming mice (Laboratory Animal Center of Henan, Zhengzhou, China) aged 5 weeks (weighing 18-22 g) were injected subcutaneously with 2 × 10 6 H22 cells. On the eighth day after inoculation, the mice were randomly divided into three groups (control, 11e group, and amonafide group) and then were administered by caudal vein injection 11e (1 mg/kg) or amonafide (5 mg/kg, as positive control) or physiologic saline for 7 consecutive days. On day 16, the mice were killed by cervical vertebra dislocation, and solid tumors were removed and weighed. The inhibitory rate was calculated as follows: inhibitory rate (%) = [(A − B)/A] × 100, where A was the mean tumor weight of the control group and B was that of the drug treated or positive group.

H22 Cells Lung Metastasis Models
H22 cells (5 × 10 6 cells per mouse) for Kunming mice were injected intravenous (i.v.) through the tail vein for tumor passive metastasis. To ensure all mice bore actively growing lung tumors before the drug treatment, pulmonary metastasis was allowed to develop for 10 days. On day 11, mice were injected intravenously with 11e (1 mg/kg), amonafide (5 mg/kg, positive, control), and normal saline (negative control) (n = 8) for 7 consecutive days. On day 18, mice were killed by cervical vertebra dislocation, and lungs were removed and weighed, and then fixed in Bouin's fluid. After the lungs were fixed, lung metastases nodus was numbered.

Survival Time in Mice Bearing H22 Cells
For calculation of the survival time, 30 male Kunming mice were inoculated via intraperitoneal (i.p.) injection with 1 × 10 6 H22 cells/mouse on day 0 and were randomly divided into three groups. The treatment with 11e (1 mg/kg), amonafide (5 mg/kg), or physiologic saline by caudal vein injection was started 24 h after inoculation for 7 consecutive days. The median survival time (MST) for each group was observed, and the antitumor activity of the drug was evaluated by measuring the increase of the lifespan. The extended lifespan rate was calculated as follows: extend rate (%) = (MST treated group/MST control group) × 100.

Conclusions
Three series of aromatic imide modified polyamines were synthesized, and their induced growth inhibition of multiple cancer cell lines in vitro was determined using the MTT assay. We found that both the imide scaffold and the polyamine motif affect the biological activities of the designed compounds. One naphthalene imide-polyamine conjugate potently inhibited the growth of multiple cancer cell lines as the reference drug amonafide. The impacts of 11e on cell morphology, mitochondrial transmembrane potential, and lysosomes indicated that 11e may impair tumor cells via apoptosis. The present study identified compound 11e induces apoptosis via a ROS mediated mitochondrial pathway in the preliminary study of cell death profile. Further in vivo experiments on H22 tumor transplant models showed that 11e prevented the lung cancer metastasis and extended lifespan compared to amonafide. Encouragingly, 11e inhibited tumor growth and improved body weight more efficiently than amonafide, which strongly corroborated that compound 11e is worthy of further investigation as a new lead compound for cancer. Collectively, the present report may provide new inspiration for tackling critical issues ranging from naphthalimide/polyamine-based drug design to antitumor drug screening.