Access to New Cytotoxic Quinone-Amino Acid Conjugates Linked through A Vinylic Spacer from 2-Acylnaphthoquinones and Methyl 3-Aminocrotonate

The reaction of 2-acetyl- and 2-benzoyl-1,4-naphthoquinone with (Z)-methyl 3-(hydroxymethyl)aminocrotonate proceeds through a formal [3+3] process to yield the corresponding 1,2-dihydrobenzisoquinolinequinones in 63% and 72% yield, respectively. The reactions of 2-acyl-1,4-naphthoquinone with enaminones, derived from diverse l- and d-amino acid methyl esters, produced the corresponding naphthoquinone amino acids conjugates bonded through a vinyl spacer in the yields range 40–71%. The presence of not-separable isomers of the naphthoquinone amino acids conjugates in the 1H- and 13C-NMR spectra is explained by the existence of conformational isomers generated by hindered rotation of the substituent bonded to the quinone double bond. These new naphthoquinone amino acids conjugates were screened in vitro on normal and cancer cell lines and showed moderate cytotoxic activities.

Older studies reported by Allen and Weiss on the behavior of 2-methoxycarbonyland 2-acetyl-1,4-benzoquinone in the Nenitzescu indol synthesis [18], demonstrated that the reaction of the former with ethyl 3-aminocrotonate yield a Michael-type adduct, namely ethyl 2-phenyl-3-aminocrotonate. However, 2-acetyl-1,4-benzoquinone reacts with ethyl 3-aminocrotonate to give the respective dihydroxiisoquinoline derivative through a formal [3+3] process. These facts reveal that ethyl 3-aminocrotonate behaves both as C-nucleophile and as C,N-ambident nucleophile depending upon the nature of the carbonyl substituent bonded at the quinone nucleus. Based on the behavior of 2-acetyl-1,4-benzoquinone with ethyl 3-aminocrotonate, which produces a dihydroxyisoquinoline derivative in a single step, we have reported a general synthetic procedure to prepare diverse cytotoxic isoquinolinequinone-containing compounds from 2-acyl-1,4-quinones and primary enaminones [10,19,22]. Within the framework of target chemotherapeutic agents, a number of studies on the synthesis of cytotoxic carbocyclic quinones linked to amino acid or dipeptide fragments have been reported [23][24][25][26][27]. In this context, we have recently undertaken the synthesis of highly cytotoxic isoquinolinequinone α-amino ester conjugates [28]. In the search for new potential cytotoxic quinones we were interested to evaluate the access to the 1,2-dihydrobenzisoquinolinequinone scaffold through a [3+3] process between acylnaphthoquinones and secondary enaminones derived from α-amino esters. As far as we know, there are no precedents in the literature regarding the assembling into an N-heterocyclic scaffold of two biologically relevant naphthoquinones and α-amino acid fragments, through this hypothetical strategy. Herein, we report the reaction of a number of acyl-1,4-naphthoquinones with secondary enaminones derived from aminoethanol and diverse methyl esters of L-and D-α-amino acids. As a result of this study we have developed a convenient access to novel 1,4-naphthoquinones linked to α-amino acid fragments via a vinyl spacer, endowed with in vitro cytotoxic activity on cancer cells.

Results and Discussion
The reactions of the required acylnaphthoquinones 2a-d with the enaminones derived from methyl aminocrotonate 3 ( Figure 2) were carried out by means of a one pot procedure, where the electrophiles are in situ generated from their corresponding acylnaphthohydroquinones 1a-d with silver (I) oxide. Quinones 2a,b were firstly selected to get preliminary insights into their reactivity patterns toward a secondary enaminone such as 4a (Scheme 1). Compound 4a was prepared in 81% yield by transamination reaction of methyl 3-aminocrotonate 3 with 2-aminoethanol in methanol, at room temperature. The reactions of acylquinones 2a and 2b with enaminone 4a were carried out at room temperature in dichloromethane (DCM) to produce the corresponding benzisoquinolinequinones 5a and 5b in 63 and 72% yield, respectively (Scheme 1). The results confirmed the behavior of the secondary enaminone 4a to react as a C,N-bidentate nucleophile with the electrophiles 2a and 2b to give the respective [3+3] products 5a and 5b. Scheme 1. Reaction of acylquinones 2a,b with enaminones 3a.
Then, we examined the reaction of quinones 2a,b with enaminone 4b, prepared by reaction of 3 with L-alanine methyl ester ( Table 1). The treatment of 2a with 4b in methanol, at room temperature provided a complex mixture of products, as was observed by thin layer chromatography (TLC). Interestingly, in the case of quinone 2b, the treatment with 4b yield the napthoquinone amino ester conjugate 6 (Scheme 2). This compound was isolated by column chromatography as a 1:1 mixture of two isomers as was evidenced by their 1 H-and 13 C-NMR spectra. Structural characterization of 6 was complemented by infrared spectroscopy (IR), bidimensional nuclear magnetic resonance (2D-NMR) and high resolution mass spectroscopy (HRMS). To the best of our knowledge, the sole precedent regarding the synthesis of quinone amino acid conjugates bonded through a vinyl spacer such as 6 was reported by Bittner et al., employing transamination reactions of diethylamino naphthoquinonic enaminone intermediates with amino acid derivatives [29]. To further evaluate the scope of this interesting one-step formation of an α-amino acid conjugated to a 1,4-napthoquinone core via a vinyl spacer such as 6, a variety of α-amino acid-derived enaminones 4c-g were prepared from aminocrotonate 3 and a representative number of L-and D-α-amino acid methyl esters ( Table 1). The structures of enaminones 4a-g were established by IR, 1 H-NMR, 13 C-NMR and HRMS. The Z configuration was assigned for the alkenyl portion of these compounds on the basis of 2D-NMR experiments performed on 4a,b,g. Acylnaphthoquinones 2b-d were reacted with the N-substituted aminocrotonate methyl esters 4c-g to give the corresponding napthoquinone amino ester conjugates 7-14 in moderate to good yields ( Table 2). The structures of the new products 7-14 were determined by IR, 1 H-and 13 C-NMR and HRMS. As was observed for compound 6, the 1 H-and 13 C-NMR spectra of compounds 7-14 revealed that these compounds coexist as two not separable isomers in nearly 1:1 ratio. The Z configuration for 7-14 was assigned for the alkenyl portion of these compounds on the basis of 2D-NMR experiments performed in compounds 6-8.
It is noteworthy that compounds 6-14 exhibit homogeneous properties in terms of their melting points and TLC. Based on these facts, it is probable that compounds 6-14 exist as a mixture of two conformational isomers arising from hindered internal molecular rotation. Inspection of one minimal energy conformation of compounds 6, represented in ball and stick mode, shows that rotation of the substituents linked to the quinone double bond is strongly hindered, in particular about the 3-2' C-C bond ( Figure 3).  To provide further proofs of the existence of conformational isomerism in the members of the series 6-14, compounds 7 and 9 were subject to 1 H-NMR coalescence experiments in DMSO-d 6 at temperatures over 25.6 • C. In the 1 H-NMR spectrum of 7 the signals of the amine protons appear downfield as two doublets at δ 10.09 and 10.20. As the temperature increased, the two signals broadened, and coalesced at~348 K. In the case of compound 9, it was observed that the signals of the amine and methine protons at δ 9.38/9.43 and 4.65/4.76 coalesced at~378 K. Therefore, these rather high coalescence temperatures are consistent with the doubling of signals observed in the 1 H-and 13 C-NMR spectra of compounds 6-14 at room temperature.
The results of the reactions of acylquinones 2b-d with 4b-g revealed that the secondary enaminones behave either as C-unidentate or as a C,N-bidentate nucleophile depending upon the structure of the nitrogen substituents bonded at the enaminone C,C double bond. It is reasonable to assume that the [3+3] process, observed in the reactions of quinones 2a,b with enaminone 4a, proceed through a Michael adduct intermediate that undergoes a 6-exo trig closure. Based on this scenario, the lack of cyclisation of the Michael adduct intermediates derived from the amino esters-enaminones 4b-g, may be attributed to steric factors.
The series of naphthoquinone amino ester conjugates 6-14 were evaluated in vitro for their cytotoxic activity against normal human lung fibroblast (MRC-5) and three human cancer cells lines: human gastric adenocarcinoma (AGS), human lung cancer (SK-MES-1) and human bladder carcinoma (J82), in 72 h drugs exposure assays ( Table 3). The cytotoxic activity of the new compounds was measured using conventional microculture tetrazolium reduction assays [30]. Cytotoxic activities of the compounds are expressed in terms of IC 50 . Etoposide, a clinically used anticancer agent, was taken as a positive control. The cytotoxic activity data are summarized in Table 3.  Table 3 shows moderate cytotoxic activities for 6-14, in the range IC 50 = 4.5-53.4 µM, lower than those displayed by the drug etoposide. Compound 14 appears as the most potent member of the series on human lung and bladder carcinoma cell lines (IC 50 : 5.5 and 4.5 µM) and with cytotoxicity ten times lower than the drug etoposide on normal human lung fibroblasts cells.

General
All solvents and reagents were purchased from different companies such as Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany) and were used as supplied. Melting points were determined on a Stuart Scientific SMP3 (Bibby Sterilin Ltd., Staffordshire, UK) apparatus and are uncorrected. The IR spectra were recorded on a FT IR Bruker spectrophotometer; (model Vector 22 Bruker, Rheinstetten, Germany), using KBr disks, and the wave numbers are given in cm −1 . 1 H-and 13 C-NMR spectra were recorded on Bruker Avance-400 instrument (Bruker, Ettlingen, Germany) in CDCl 3 at 400 and 100 MHz, respectively. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane and the coupling constants (J) are reported in Hertz. Data for 1 H-NMR spectra are reported as follows: s = singlet, br s = broad singlet, d = doublet, m = multiplet and the coupling constants (J) in Hz. Bidimensional NMR techniques (HMBC and NOESY) and distortionless enhancement by polarisation transfer (DEPT) were used for signal and E/Z configuration assignments. The 1 H-NMR coalescence experiments were recorded in DMSO-d 6 solutions on the Bruker spectrometer operating at 400 MHz equipped with the 5 mm PAQNP probe. HRMS-ESI were carried out by using a Thermo Scientific Exactive Plus Orbitrap spectrometer with a constant nebulizer temperature of 250 • C. The experiments were performed in positive ion mode, with a scan range of m/z 100-300. All fragment ions were assigned by accurate mass measurements at high resolution (resolving power: 140,000 FWHM). The samples were infused directly into the electrospray ionization source (ESI) using a syringe pump at flow rates of 5 µL min −1 . Optical rotations were obtained for CHCl 3 solutions in a Polarimeter instrument (Optical Activity Ltd., Cambridgeshire, UK) in a 1 dm cell and their concentrations are expressed in g per mL. Silica gel Merck 60 (70-230 mesh, from Merck, Darmstadt, Germany) was used for preparative column chromatography and TLC aluminum foil 60F254 for analytical thin layer chromatography (TLC). After completion of the reaction as indicated by TLC, the solvent was removed under reduced pressure and the residue was purified by chromatography over silica gel (CH 2 Cl 2 ) to yield pure enaminone 4a (112 mg, 0.70 mmol, 81%) as yellow oil; IR ν max : 3340, 2947 and 1636 cm

Preparation of Enaminone-Amino Acid Derivatives. General Procedure
Suspensions of methyl 3-aminocrotonate 3 (1 equiv.), L-or D-α-amino acid methyl esters hydrochloride (1.2 equiv.) and NaOAc (1.2 equiv.) in methanol (15 mL) were stirred at room temperature until completion of the reaction as indicated by TLC. The solvents were removed under reduced pressure and the residues purified by column chromatography over silica gel (CH 2 Cl 2 ) to yield the corresponding enaminones 4b-g.

Conclusions
In summary, we have studied the reactivity of a number of 2-acylnaphthoquinones with secondary enaminones derived from 2-aminoethanol and α-amino acid methyl esters. The reactions provided access to 1,2-dihydrobenzisoquinolinequinones 5a and 5b and a variety of napthoquinone-amino ester conjugates 6-14. The main novelty of the study concerns the facile access, and with high atom economy, to a new scaffold containing the biological relevant naphthoquinone and amino acid fragments, bonded through a vinyl spacer. The preliminary results on the biological evaluation of conjugates 6-14 showed interesting in vitro cytotoxic activity on cancer cells.