Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.


Introduction
Carbazole alkaloids natural products are mostly isolated from higher plants of Rutaceae family and major components of the Clausena genus [1,2]. With the isolation of carbazole core from coal tar in 1872 [3] and the description of the antimicrobial murrayanine in 1965 [4], the interest on these alkaloids began. Since then, natural-occurring carbazole alkaloids have been reported to exhibit a broad pharmacological profile, including activities such as antitumor (i.e., heptaphylline 1, Figure 1) [5], 7-methoxy-heptaphylline (2) [6], 2-hydroxy-7-methoxy-9H-carbazole-3-carbaldehyde or 7-methoxy-mukonal (3) [7]), antiplasmodial (i.e., compounds 1 [8] and 3 [7]), antiplatelet aggregation, and vasorelaxing (i.e., clausine E (4) [9]), antibacterial (i.e., clausamine B (5), clausine F (6) [10], and Inhibitors of tumor cell lines have been associated to several mechanisms, one of which being through the p53 pathway. The tumor suppressor protein p53 is a transcription factor that plays a key role in the prevention of cancer development, mainly due to Inhibitors of tumor cell lines have been associated to several mechanisms, one of which being through the p53 pathway. The tumor suppressor protein p53 is a transcription factor that plays a key role in the prevention of cancer development, mainly due to its major role in cellular events such as apoptosis, cell cycle progression, and DNA repair [20,21]. However, over 50% of p53 proteins present missense mutations, generating a defective protein in high levels in cells due to the impairment of MDM2 (murine doble minute 2) mediated negative feedback, which is responsible for p53 degradation. p53 Protein is known as the guardian of the genome because one of the most important p53 functions is the ability to activate apoptosis and the disruption of this process can be correlated with tumor progression and chemoresistance [22]. In tumor cells, the restoration of p53 function has shown to be highly effective against tumor cells, thus reactivating mutant p53 has been a goal in anticancer drug development [23]. Some small molecules in the group of carbazole alkaloids have been reported to reactivate mutant p53 by restoration the wild-type (wt) structure/function [24,25]. For example, PhiKan083 (15), an amino derivative of the carbazole, emerged from an in silico screening [26] and was reported as a small molecule for restoration of wild-type like p53 conformation by targeting Y220 mutation [26,27]. This derivative 15 established electrostatic and hydrogen bonding interactions with residues of Y220 which gave additional stability to Y220 mutant p53. This particular mutation creates a druggable surface crevice and PhiKan083 (15) binds to this crevice and stabilizes the structure of this mutant p53 [27,28]. Up to date, none of the natural isolated carbazole alkaloids or their chemical modified ana was reported to have effect on p53 mutants. Herein, a series of semi-synthetic aminocarbazoles was synthesized from naturally-occurring heptaphylline (1), and their tumor cell growth inhibition and potential activity on p53 were studied.

Semi-Synthesis of Aminocarbazole Alkaloids by Direct Reductive Amination
The reaction of carbonyl groups, aldehydes, or ketones with amines in the present of reducing agents to give corresponding amines, known as reductive amination (of carbonyl compounds) or reductive alkylation (of amine compounds) is one of the most useful and important methods in the synthesis of different kind of amines as well as a powerful reaction to obtain drug candidates [29]. The choice and understanding of the reducing agent are essential for the selection of the reaction conditions. Sodium triacetoxyborohydride [NaBH(OAc) 3 , STAB] was reported as the most powerful reducing agent in direct reductive amination due to its stability and safety.
Reductive aminations of 1, 2, and 3 were performed in an one-pot conversion of their carbonyl group in the present of STAB with two different solvents-dried tetrahydrofuran (THF) or dried 1,2-dichloroethane (DCE) with selected amines precursors present in inhibitors of p53:MDM2 interaction. The reaction mixtures were stirred under nitrogen gas until no further developments to yield aminocarbazole alkaloids derivatives 1a-1e, 2a-2f, and 3a (Table 1). Products were treated with different work-up procedures before purification, as described in the experimental section.
Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction with secondary amines yield tertiary amines (entry 1-2, 6-7 and 11-12), via enamine intermediates. The final products were categorized into 3 groups, alkylated linear aminocarbazoles, compounds 1a, 2a, and 3a, heterocyclic aminocarbazoles, compounds 1b, 2b, and 2f, and halogenated aminocarbazoles, compounds 1c-1e and 2c-2e. The reactions mostly showed no further development between 3-10 days. All the reactions required long reaction times due to the steric hindered of the hydroxyl at position 2 and/or prenyl group at position 1. Aminocarbazoles modified from 2, compounds 2a-2f, and from 3, compound 3a, required longer reaction time than those derived from 1, compounds 1a-1e, in both solvent conditions. These longer times should be related to the effect of the methoxy electron donating group at position 7. The reductive amination with primary amines was faster than with secondary amines (entry 4-6 and 8-10). Reactions performed in DCE required shorter times and produced higher yields (3-5 days, 34-90%) compared to those performed in THF (3-10 days, 13-51%), and these results are in agreement with previous reports [30]. All the compounds were confirmed by one-and two-dimensional NMR and high-resolution mass spectrometry. The chemical shift of protons and carbons of 1, 2, and 3 were accordance to the literature [31,32]. The analysis of (+) HRMS-ESI, 1 H, 13 C NMR, HSQC, HMBC, and X-ray crystallographic data (in case of compound 1b) revealed the success of the reductive amination to produce amine derivatives. Compounds derived from 1 and 2, showed the proton H-1 signal as a doublet (d) with chemical shift δ values of c.a 3.50-3.68 ppm while proton H-3 signal appeared as a singlet with δ values c.a 3.74-3.83 ppm. The proton signal of one of the methyl groups of the prenyl substituent appeared as a singlet at c.a 1.90 ppm while another methyl signal was presented as a narrow doublet at c.a 1.76 ppm due to the correlation to the H-1". Aminocarbazoles derived from 1, compounds 1c, 1d, 1e, and compounds derived from 2, compounds 2c, 2d, and 2e, presenting secondary amine moieties showed the chemical shift of proton H-5 with δ values c.a 4.14 and 4.05 ppm, respectively appearing as singlets (see in experimental section). We also summary the key protons of amine derivatives obtained from substrate 1 and 2 as shown in Figure 2.   Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction  Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction with secondary amines yield tertiary amines (entry 1-2, 6-7 and 11-12), via enamine in-  Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction with secondary amines yield tertiary amines (entry 1-2, 6-7 and 11-12), via enamine in-  Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction with secondary amines yield tertiary amines (entry 1-2, 6-7 and 11-12), via enamine in-

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Molecules 2021, 26, 1637 4 of 16 Generally, in the present of STAB, the reactions of 1, 2 and 3 with primary amines yield secondary amines (entry 3-5 and 8-10), via imine intermediates, and the reaction with secondary amines yield tertiary amines (entry 1-2, 6-7 and 11-12), via enamine intermediates. The final products were categorized into 3 groups, alkylated linear ami- nocarbazoles, compounds 1a, 2a, and 3a, heterocyclic aminocarbazoles, compounds 1b  Compound 1b was obtained as in a crystal form in the mixture of methanol and ethyl acetate. The X-ray crystallographic representation of compounds 1b is presented in Figure 3. The Ortep diagram confirmed the structure of 1b, and the analyses of HSQC and HMBC provided the correlations of compound 1b depicted (Figure 3). The key proton H-4 showed correlations with C-3 , C-2, and C-9a while H-5 showed correlations with C-7 and C-8a. The proton of the indole group H-9 showed correlations with C-8 and C-9a while proton H-3 showed correlations with C-4 , C-2, and C-4.

Evaluation of Aminocarbazoles Potential Activation of p53 Using a Yeast-Based Screening Assay
Compounds containing a carbazole scaffold have been identified and tested against a certain mutation of p53 and showed to stabilize the mutant in which the carbazole ring system is sandwiched in hydrophobic side chains [26]. In this work, to identify small molecules of aminocarbazoles that could restore p53 pathway signalling, three aminocabazoles derived from heptaphylline (1b-d) and the natural compound 1 were tested for their ability to activate wt or mutant p53, using a previously developed yeast-based screening assay [33]. Among the tested compounds in cells lines, four were selected due to their highest growth inhibitory activity in tumor cells. In the yeast assay, yeast cells expressing wt p53 present a marked growth inhibition, which is reduced or abolished in case of mutant p53. Compounds able to activate wt p53 or to restore the wild-type-like activity to mutant p53 will increase the growth inhibition induced by expression of the human protein in yeast [33]. Yeast cells expressing mutant or wt p53, and control yeast (transformed with the empty vector) were treated with 10 µM of each compound and its impact on yeast growth inhibition was evaluated. All the compounds tested were able to reactivate at least two of the mutant p53 forms studied, increasing their yeast growth inhibitory effect (Table 3). Among the compounds tested, only compound 1b was able to also activate

Evaluation of Aminocarbazoles Potential Activation of p53 Using a Yeast-Based Screening Assay
Compounds containing a carbazole scaffold have been identified and tested against a certain mutation of p53 and showed to stabilize the mutant in which the carbazole ring system is sandwiched in hydrophobic side chains [26]. In this work, to identify small molecules of aminocarbazoles that could restore p53 pathway signalling, three aminocabazoles derived from heptaphylline (1b-d) and the natural compound 1 were tested for their ability to activate wt or mutant p53, using a previously developed yeastbased screening assay [33]. Among the tested compounds in cells lines, four were selected due to their highest growth inhibitory activity in tumor cells. In the yeast assay, yeast cells expressing wt p53 present a marked growth inhibition, which is reduced or abolished in case of mutant p53. Compounds able to activate wt p53 or to restore the wild-type-like activity to mutant p53 will increase the growth inhibition induced by expression of the human protein in yeast [33]. Yeast cells expressing mutant or wt p53, and control yeast (transformed with the empty vector) were treated with 10 µM of each compound and its impact on yeast growth inhibition was evaluated. All the compounds tested were able to reactivate at least two of the mutant p53 forms studied, increasing their yeast growth inhibitory effect (Table 3). Among the compounds tested, only compound 1b was able to also activate wt p53 in yeast. It is of note that compounds 2 and 3 were cytotoxic in control yeast, and therefore these natural products were excluded from the assay. Table 3. Effect of heptaphylline and amine derivatives 1b-1d on the growth of yeast cells expressing wild-type (wt) or mutant p53. Percentage of p53 reactivation induced by heptaphylline derivatives. Data were normalized to the percentage of wtp53 growth inhibitory effect in yeast cells. Yeast expressing human mutant p53 or wt p53 were treated for 42 h with the indicated compound. Results correspond to the percentage of wt p53-induced growth inhibition re-established by compounds in yeast expressing mutant p53. Data are mean ± SEM of 3-6 independent experiments. Dashes represent a reactivation effect lower than 30%.

Mutant
Interestingly, in the yeast-screening assay, compound 1d demonstrated the most promising activity in mutations involving codon 245 of p53, namely G245D and G245S. In fact, through the antiproliferation assay, compound 1d displayed its greatest antiproliferative activity in LS-1034 expressing mutant p53 G245S.

Isolation
The root bark of Clausena harmandiana (Pierre) Guillaumin (Rutaceae) was collected in Khon Kaen province, Thailand, in June 2016. Authentication was identified by comparison with the herbarium specimen at the Faculty of Science, Khon Kaen University. The identified voucher specimen (KKU No. 21145) was deposited at Faculty of Pharmaceutical Sciences, Khon Kaen Univerisity, Thailand. The root barks (2.29 kg) were air-dried, ground, and sequentially extracted at room temperature for overnight with dichloromethane (4 times). The extracts were evaporated in vacuo to obtain crude dichloromethane extract (140 g). The crude dichloromethane was isolated by open column chromatography on silica gel 60 and subsequently eluted with a gradient of n-hexane and ethyl acetate (EtOAc) to give 1 (310 mg; 1.4 × 10 −2 of dry weight), 2 (340 mg; 1.5 × 10 −2 of dry weight), and 3 (170 mg; 0.7 × 10 −2 of dry weight). All isolated compounds were structurally elucidated by comparison with the authentic samples, which were identical in all respects [34].

Purity Determination by HPLC-DAD
The HPLC system consisted of Shimadzu LC-20AD pump, equipped with a Shimadzu DGV-20A5 degasser, a Rheodyne 7725i injector fitted with a 20 µL loop, and a SPD-M20A DAD detector (Kyoto, Japan). Data acquisition was performed using Shimadzu LCMS Lab Solutions software, version 3.50 SP2. The column used in this study was ACE-C18 (150 × 4.6 mm I.D., particle size 5 µm) manufactured by Advanced Chromatography Technologies Ltd. (Aberdeen, Scotland, UK). The mobile phase composition was water and methanol (2:8 v/v; 0.1% triethylamine), all were HPLC grade solvents obtained from Merck Life Science S.L.U. (Darmstadt, Germany). The flow rate was 1.0 mL/min and the UV detection wavelength was 312 nm. Analyses were performed at 27 • C in an isocratic mode. Peak purity index was determined by total peak UV-Vis spectra between 210-800 nm with a step of 4 nm. The percentage is indicated at each compound and detailed data is given in Supplementary Material.  1a, 1b, 2a, 2b, 2f, and 3a the crudes were extracted with CHCl 3 (3 × 50 mL), then solid phase extraction (SPE) through cation exchange cartridge Discovery ® DSC-SCX (Supelco, Bellefonte, Philadelphia, PA, USA) using 1% NH 3 in CH 3 OH. The basic fractions were purified on flash column using Hexane:EtOAc; 7:3. For compounds 1c, 1d, 1e and 2c, 2d, 2e, after reaction, the crudes extracts were treated with 5% of NaOH in CHCl 3 (3 × 50 mL) to remove excess STAB, the organic phases were treated with 5M HCl in CHCl 3 to remove excess amines. Then, the aqueous phases were treated with 20% of NaOH in CHCl 3 . The combination of organic phases was subjected to SPE through cation exchange cartridge Discovery ® DSC-SCX using 1% NH 3 in CH 3 OH. The basic fractions were purified on flash column using n-hexane:EtOAc 7:3.

Conclusions
A series of new semi-synthetic aminocarbazoles derived from carbazoles natural products was successfully obtained and evaluated regarding the in vitro tumor growth inhibition activity and potential ability to activate p53 of the compounds. The results revealed a modest tumor growth inhibitory activity and no selectivity to the p53 pathway, in human tumor cells for the natural products heptaphylline (1), 7-methoxy-heptaphylline (2), and 7-methoxy-mukonal (3). Despite this, the results obtained indicate that aminocarbazole semi-synthetic derivatives, particularly 3-(p-fluoro)aminoheptaphylline (1d), may represent a promising starting point to search for new mutant p53-reactivating agents with promising application in cancer therapy.
Supplementary Materials: The following are available online, Figure S1. 1