Crafting Carbazole-Based Vorinostat and Tubastatin-A-like Histone Deacetylase (HDAC) Inhibitors with Potent in Vitro and in Vivo Neuroactive Functions.

Small-molecule inhibitors of HDACs (HDACi) induce hyperacetylation of histone and nonhistone proteins and have emerged as potential therapeutic agents in most animal models tested. The established HDACi vorinostat and tubastatin-A alleviate neurodegenerative and behavioral conditions in animal models of neuropsychiatric disorders restoring the neurotrophic milieu. In spite of the neuroactive pharmacological role of HDACi (vorinostat and tubastatin-A), they are limited by efficacy and toxicity. Considering these limitations and concern, we have designed novel compounds 3-11 as potential HDACi based on the strategic crafting of the key pharmacophoric elements of vorinostat and tubastatin-A into architecting a single molecule. The molecules 3-11 were synthesized through a multistep reaction sequence starting from carbazole and were fully characterized by NMR and mass spectral analysis. The novel molecules 3-11 showed remarkable pan HDAC inhibition and the potential to increase the levels of acetyl H3 and acetyl tubulin. In addition, few novel HDAC inhibitors 4-8, 10, and 11 exhibited significant neurite outgrowth-promoting activity with no observable cytotoxic effects, and interestingly, compound 5 has shown comparably more neurite growth than the parent compounds vorinostat and tubastatin-A. Also, compound 5 was evaluated for possible mood-elevating effects in a chronic unpredictable stress model of Zebrafish. It showed potent anxiolytic and antidepressant-like effects in the novel tank test and social interaction test, respectively. Furthermore, the potent in vitro and in vivo neuroactive compound 5 has shown selectivity for class II over class I HDACs. Our results suggest that the novel carbazole-based HDAC inhibitors, crafted with vorinostat and tubastatin-A pharmacophoric moieties, have potent neurite outgrowth activity and potential to be developed as therapeutics to treat depression and related psychiatric disorders.

Introduction

Histone deacetylases (HDACs) are enzymes involved in the deacetylation of histone and nonhistone proteins and are implicated in diseases as diverse as cancer to the nervous system disorders.[1] Interestingly, small-molecule inhibitors of HDACs (HDACi) have shown therapeutic effects in preclinical models as well as in clinical observations;[2] the HDACi vorinostat (SAHA, suberoylanilide hydroxamic acid) and romidepsin (depsipeptide) have been approved for the treatment of cutaneous T-cell lymphoma.[3] In addition to their robust anticancer activity, HDACi is involved in diverse in vitro neuroactive functions such as neuroprotection,[4−7] neurogenesis,[8−11] neurite growth,[12−14] and in amelioration of conditions in rodent models of psychiatric and neurological disorders.[15−17] However, many of these HDACi have failed at various levels of preclinical and clinical trials for central nervous system (CNS) disorders, mostly limited by efficacy and nonspecific toxicity.[1] This necessitates the design and development of novel HDAC inhibitors or modulators with the intention of overcoming these limitations, which ultimately would lead to potential therapeutics for treating diverse neurological and psychiatric disorders.

Vorinostat is a highly effective pan class I and class II HDAC inhibitor[18,19] (Figure ). Mounting evidence shows vorinostat as a potent anticancer agent for monotherapy and also in combination with other agents in treating hematological and solid tumors.[3,18,20] Interestingly, it has been in the clinic for treating cutaneous T-cell lymphoma. In addition, vorinostat crosses the blood–brain barrier (BBB) and shows remarkable therapeutic effects in animal models of various neurological[21,22] and psychiatric disorders,[23,24] but with nontargeted side effects.[25] Tubastatin-A, a selective HDAC6 inhibitor, has been shown to provide neuroprotection in homocysteine-induced in vitro stress model.[26] It has also demonstrated therapeutic efficacy in rodent models of cognitive and neurodegenerative disorders.[27−30] In addition, tubastatin-A shows minimal toxic effects, unlike other HDACi, including vorinostat. However, its low BBB permeability and sparse distribution in brain parenchyma limit its potential to become a central nervous system (CNS) therapeutic (Figure ).

Figure 1

Structures of the FDA-approved drug vorinostat and tubastatin-A as HDAC inhibitors.

Considering the individual therapeutic benefits of HDACi vorinostat and tubastatin-A, and limitations in their use for developing a drug for the treatment of diverse neurological and psychiatric disorders, in particular depression, anxiety, and related mood disorders, we embarked upon the development of a novel HDACi. Here, we have crafted novel small molecules based on the hybridization of key pharmacophoric features of vorinostat and tubastatin-A, to get new molecules that would effectively inhibit the HDAC activity with potential in vitro and in vivo neuroactive properties and low toxicity, unlike the vorinostat. These active novel molecules were further screened in Zebrafish stress-induced anxiety and depression model for assessing their antidepressant and anxiolytic activities.

Results and Discussion

Design Strategy

In general, HDAC inhibitors consist of zinc-binding bidentate functional group (e.g., hydroxamic acid) and an alkyl chain or aromatic group as a linker and a cap group. It is evident from the literature that “cap group” plays a crucial role in selective inhibition of various HDAC isoforms. One such selective HDAC6 inhibitor reported was tubastatin-A. Its selectivity for HDAC6 could be due to a slightly wider carboline group in the “cap” region. We, therefore, envisaged that carbazole with a similar tricyclic architecture like carboline core could be a choice for the “cap group” for an investigative purpose. Further merging of carbazole unit derived from tubastatin-A with the pharmacophoric features of vorinostat could give envisaged diverse analogues 3–11 for their biological evaluation. Schematic representation of the design strategy for the generation of new molecules is depicted in Figure .

Figure 2

Schematic representation for design of new molecular entities 3–11.

The first set of designer molecules 3 and 4 (Figure ) have a carbazole cap with N-benzyl group (a pharmacophoric feature similar to tubastatin-A) and an alkyl linker with hydroxamic acid (a pharmacophoric feature similar to vorinostat). Molecule 5 is crafted by replacing aniline cap of vorinostat with carbazole, linking through C–C bond retaining all other pharmacophoric features (alkyl chain and hydroxamic acid). Molecules 6 and 7 (Figure ) have an additional oxime group in the cap region. Designer molecules 8 and 9 are analogues of 3 with additional hydroxamic acid replacing benzyl group, and 10 and 11 are N-alkylhydroxamic acid derivatives of carbazole. These designer molecules were varied in such a way as to obtain chemical entities to effectively inhibit HDAC activity for potential use in the investigation for neuroactive functions.

Figure 3

Structures of the designer compounds 3–11 used in the present study.

Chemistry

Initiating synthesis, the required key starting compounds 9-benzyl-9H-carbazole (12), ethyl 2-(9H-carbazol-9-yl)acetate, (13) and 9-tosyl-9H-carbazole (14) were prepared by reacting carbazole with benzyl bromide, ethyl bromoacetate, or p-toluenensulfonyl chloride in the presence of a base (Scheme ), using literature procedures. The products 12–14 were characterized by NMR and mass spectra analyses and were correlated with reported data.[31]

Scheme 1

Synthesis of Carbazole Derivatives 12–14

Having 9-benzyl-9H-carbazole (12) in hand, it was treated with anhydrides (gluteric, adipic, and suberic) in the presence of AlCl3 under Friedel–Crafts acylation conditions to form keto acids (15–17) in good yields (Scheme ). The keto group was further reduced using Wolff–Kishner reduction conditions (hydrazine hydride in the presence of KOH) to give reduced acid compounds 18 and 19. Esterification of acids with thionyl chloride in methanol followed by condensation with hydroxylamine hydrochloride resulted in 5-(9-benzyl-9H-carbazol-3-yl)-N-hydroxypentanamide (3) and 6-(9-benzyl-9H-carbazol-3-yl)-N-hydroxyhexanamide (4) in 92 and 89% yields, respectively. Similarly, 9-tosyl-9H-carbazole (14) gave 8-(9H-carbazol-3-yl)-N-hydroxyoctanamide 5 in 90% yield (Scheme ). Keto acids 16 and 17 were esterified with methanol in the presence of thionyl chloride and then reacted with hydroxylamine hydrochloride in the presence of sodium hydroxide to form 6-(9-benzyl-9H-carbazol-3-yl)-N-hydroxy-6-(hydroxyimino) hexanamide (6) and 8-(9-benzyl-9H-carbazol-3-yl)-N-hydroxy-8-(hydroxyimino)octanamide (7) in excellent yields (Scheme ). All of the new compounds 3–7 were fully characterized by their NMR and mass spectral analyses.

Scheme 2

Synthesis of New Molecules 3 and 4

Scheme 3

Synthesis of New Molecule 5

Scheme 4

Synthesis of New Molecules 6 and 7

Ethyl 9H-carbazole-9-carboxylate (13) was treated with gluteric anhydride in the presence of aluminum chloride to give 5-(9-(2-ethoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoic acid (27). Esterification with methanol in the presence of thionyl chloride gave methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoate (28). Diester 28 was reacted with hydroxylamine hydrochloride in the presence of sodium hydride to produce trihydroxamide, N-hydroxy-5-(9-(2-(hydroxyamino)-2-oxoethyl)-9H-carbazol-3-yl)-5-(hydroxyimino)pentanamide (9) in 89% yield. Wolff–Kishner reduction of 27 gave 5-(9-(carboxymethyl)-9H-carbazol-3-yl)pentanoic acid (29). Esterification of diacid to form methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)pentanoate (30), followed by derivatization with hydroxylamine amine hydrochloride in presence of sodium hydroxide, gave N-hydroxy-5-(9-(2-(hydroxyamino)-2-oxoethyl)-9H-carbazol-3-yl)pentanamide (8) in 82% yield (Scheme ). Friedel–Crafts acylation of carbazole with glutaric anhydride in the presence of aluminum trichloride resulted 5-(9H-carbazol-9-yl)-5-oxopentanoic acid (31) in 65% yield. Methyl 5-(9H-carbazol-9-yl)-5-oxopentanoate (33), obtained after esterification of 31, was reacted with hydroxylamine hydrochloride in the presence of sodium hydroxide to give 5-(9H-carbazol-9-yl)-N-hydroxy-5-(hydroxyimino)pentanamide (10) in 90% yield. Similarly, 6-(9H-carbazol-9-yl)-N-hydroxy-6-(hydroxyimino)hexanamide (11) was also synthesized from carbazole in very good yield (Scheme ). All of the new products 8–11 were fully characterized by their NMR and mass spectral analyses. The purity of all final products 3–11 was determined to be >97% by analytical HPLC analysis.

Scheme 5

Synthesis of New Molecules 8 and 9

Scheme 6

Synthesis of New Molecules 10 and 11

Biology

HDACi vorinostat, tubastatin-A, and inspired derivatives 3–11 were screened for pan HDAC inhibition activity. Interestingly, these novel small molecules have shown potent pan HDAC inhibition (IC50 0.6–1.2 μM) similarly to vorinostat (IC50 0.8 μM) and tubastatin-A (IC50 1.32 μM) (Figure ).

Figure 4

IC50 values of novel HDACi (7, 6, 3, 4, 9, 5, 8, 10, and 11), vorinostat, and tubastatin-A. All of the novel small molecules have shown potent pan HDAC inhibition (IC50 # 0.6–1.2 μM) similarly to vorinostat (0.8 μM) and tubastatin-A (1.32 μM).

Furthermore, we validated the effect of novel small molecules 3–11 on acetyl H3 and acetyl tubulin levels, as HDACi are involved in diverse cellular functions by inhibiting HDACs and inducing hyperacetylation of histone and nonhistone proteins.[32,33] All of the novel compounds (3–11) at 10 μM concentration showed increased levels of acetyl H3 and acetyl tubulin levels in GL-261 cells; similar changes were observed in vorinostat- and tubastatin-A-treated cells (Figure A–C). These results support the finding that novel compounds 3–11 induce HDAC inhibitory activity and associated downstream changes, i.e., increased levels of acetyl H3 and acetyl tubulin.

Figure 5

Immunoblot data: (A) Acetyl H3, β-actin, acetyl tubulin, and tubulin levels in GL261 cells treated with no inhibitor, novel HDAC inhibitors (3–11), vorinostat, and tubaslatin A (at 10 μM concentration) for 2 h. The bar graph shows the densitometry values of acetyl H3/β-actin ratio (B) and acetyl tubulin/tubulin ratio (C); *p < 0.05 (one-way ANOVA using GraphPad Prism software) compared to no inhibitor treatment.

Interestingly, HDACi has been widely reported for diverse in vitro neuroactive functions (neurite outgrowth-promoting activity, neural differentiation, and neuroprotection).[34−39] Thus, all of the novel HDACi were screened for the neurite outgrowth activity in differentiated Neuro2A cells. Compounds 4, 5, 6, 7, 8, 10, and 11, as well as vorinostat and tubastatin-A, have shown significantly more neurite outgrowth promotion compared to no inhibitor (Vehicle) treatment group (Figure ). Interestingly, evaluation of optimum neuritogenic concentrations revealed that compound 5 showed remarkably more neurite outgrowth than vorinostat and tubastatin-A treatments (Figures and 7A,B). However, above the optimum neuritogenic concentration, novel small molecules (4, 5, 6, 7, 8, 10, and 11) and standard HDACi (vorinostat, tubastatin-A) have shown reduced neurite growth activity and viability. The potent neurite outgrowth activity of compound 5 compared to vorinostat and tubastatin-A, despite a similar level of pan HDAC inhibition and acetyl H3 and acetyl tubulin induction levels, led us to anticipate possible HDAC selectivity in compound 5.

Figure 6

Neurite outgrowth activity of novel HDAC inhibitors in Neuro2A cells. The bar graph shows the average neurite length induced by no inhibitor (vehicle-1% DMSO) and novel HDAC inhibitors (3–11). V (vorinostat) and T (Tubatatin-A) at different concentrations (10, 1, 0.1, and 0.01 μM). Neurites were measured using ImageJ software on bright-field images of Neuro2A cells, taken 48 h post-treatment. *p < 0.05, **p < 0.01, ***p < 0.00l (one-way ANOVA using GraphPad Prism software) compared to no inhibitor (vehicle)-treated cells.

Figure 7

Compound-induced optimum neurite outgrowth activity. (A) Immunofluorescence of Neuro2A cells with β III tubulin antibody shows neurite outgrowth induced by novel HDAC inhibitors (7—0.1 μM, 6—0.01 μM, 4—0.1 μM, 5—1 μM, 8—0.l μM, 10—0.1 μM, 11—0.1 μM), V (Vorirjostat—1 μM). T (Tubatarin-A—10 μM) and no inhibitor (Vehicle–1% DMSO). (B) The bar graph shows average neurite length of compounds (7—0.1 μM, 6—0.01 μM, 4—0.1 μM, 5—1 μM, 8—0.1 μM, 10—0.1 μM, 11—0.1 μM, V (vorinostat—1 μM), T (tubastatin-A—10 μM)) at optimized concentrations. **p < 0.01, ***p < 0.001 (one-way ANOVA using GraphPad Prism software) compared to no inhibitor (vehicle)-treated cells.

In addition, the potent neuritogenic compound 5 was assessed for its effect on cell viability (cytotoxicity). Compound 5 and the parent entities, i.e., vorinostat and tubastatin-A, were studied at different concentrations (10, 1.0, and 0.1 μM) on Neuro2A cell line for cell viability using MTT assay. Compound 5 (10 μM) does not show any observable toxicity unlike vorinostat-treated cells (Figure ).

Figure 8

Bar graph shows the percentage of viable Neuro2A cells after 72 h incubation with novel HDAC inhibitor (5), V (vorinostat), and T (tubastatin-A) at 10, 1, and 0.1 μM concentrations (assessed by MTT assay). *p < 0.05 (Student’s t-test).

The dose (1 μM) at which compound 5 showed potent neuritogenic activity was used to check its effect on the levels of acetylation of histones H3, H4, and tubulin in Neuro2A cells. Compound 5 could induce a significant increase in acetylated H3 (Figure A,B), acetylated H4 (Figure A,C), and acetylated tubulin (Figure A,D) in Neuro2A cells compared to vehicle treatment.

Figure 9

Immunoblot data. (A) acetyl H3, acetyl H4, H3 pan, and acetyl tubulin. α tubulin levels in Neuro2A treated with vehicle (no inhibitor) and compound 5 (comp# 5). (B–D) The bar graphs show the densitometry values of immunoblot data. *p < 0.05 (Student’s t-test).

In light of the remarkable neuritogenic activity of novel HDACi compound 5, compared to vorinostat and tubastatin-A, and correlative evidence of the mood-elevating role of classic neurotrophic HDAC inhibitors,[40−42] we investigated the anxiolytic (in novel tank test) and antidepressant effects (in social interaction test) of compound 5 in Zebrafish chronic unpredictable stress (CUS) model[43] (Figure S72 A–D, S73 A–C). First of all, it was imperative to test whether the compound crosses the BBB. The intraperitoneal injection of the compound resulted in its crossing the BBB, as evident from the results shown in Figure S74. We also had obtained the prediction results for its possibility to cross the BBB, using in silico method, as shown in Figure S75.

In the novel tank test, our novel HDAC inhibitor compound 5 at doses 10 mg and 25 mg/kg showed significantly lower latency to upper zone in comparison to the vehicle-treated group, and a similar trend was observed in the fluoxetine (standard antidepressant used as positive control)[44,45]-treated group (p-value < 0.05) (Figure A). In addition, compound 5 also induced an increase in time spent in the top zone (Figure B) and in the number of crosses (Figure C) compared to the vehicle-treated group (p-value < 0.05), with a trend of decreased freezing duration (Figure D). Fluoxetine also induced an increase in time spent in the top zone and in the number of crosses. The fluoxetine-treated group also showed a trend in reduced freezing behavior (Figure B–D).

Figure 10

Novel tank test. (A–D) Compound 5 (10, 25, and 50 mg/kg)- and fluoxetine (15 mg/kg)-induced behavioral changes in the novel tank test. (A) Latency to upper zone (in seconds). (B) Total time spent in the upper zone (in seconds). (C) Number of crosses. (D) Freezing duration (in seconds). n = 15, *p < 0.05 compared to the control group (Student’s t-test), #p < 0.05 compared to CUS + vehicle group (one-way ANOVA using GraphPad Prism software).

Compound 5 was assessed at different doses (10, 25, and 50 mg/kg) for its antidepressant efficacy using the social interaction test. The compound-induced changes in latency to social interaction, total time spent in interaction zone, and freezing behavior were recorded. Like fluoxetine, compound 5 at 10 and 25 mg, but not at 50 mg dose, has shown remarkably less latency in interaction and more time spent in the interaction zone compared to the vehicle-treated group (p-value < 0.05) (Figure A,B). Furthermore, compound 5-treated animals have shown a trend of decreased freezing duration (change is not significant), similar to that observed in the fluoxetine-treated group (Figure ).

Figure 11

Social interaction test, (A–C) Compound 5 (10, 25, and 50 mg/kg)- and fluoxetine (15 mg/kg)-induced behavioral changes in social interaction test. (A) Latency to interaction (seconds). (B) Total interaction time (in seconds). (C) Freezing duration (in seconds). n = 15. *p < 0.05 compared to control group (Student’s t-test), #p < 0.05 compared with CUS + vehicle group (one-way ANOVA using GraphPad Prism software).

Further, the potent in vitro and in vivo neuroactive compound 5 was used to study the HDAC (Class I, Class IIa, and Class IIb) selectivity studies. The results show compound 5 selectivity for class II (class IIa-HDAC4 and class IIb- HDAC6) over class I (HDAC8). Thus, the neuroactive and class II selective compound 5 scaffold can be used for developing more selective and efficient neuroactive HDAC inhibitors (Figure ).

Figure 12

Bar graph showing the percentage of HDAC activity for HDAC8 (class I), HDAC4 (class IIa), and HDAC6 (class IIb) enzymes with different concentrations of compound 5 (10, 1, 0.1, 0.01, and 0.001 μM).

Conclusions

In conclusion, our results demonstrated the biological activity of novel small molecules 3–11 designed by crafting pharmacophoric functional groups of vorinostat and tubastatin-A into a single molecular entity. These carbazole-based new molecules were synthesized from carbazole in three to five steps, as depicted in scheme –. In addition, all of the new compounds 3–11 have shown the potential pan HDAC inhibition activity with increased levels of acetylated histone and tubulin. In addition, the most potent neuroactive compound 5 has shown selectivity for class II over class I HDACs. Furthermore, novel HDAC inhibitors 4, 5, 6, 7, 8, 10, and 11 exhibited potent neurite outgrowth activity, and interestingly, one of the novel HDAC inhibitors, 8-(9H-carbazol-3-yl)-N-hydroxyoctanamide (compound 5), has shown significantly more neurite outgrowth compared to vorinostat and tubastatin-A and also better antidepressant- and anxiolytic-like activity in chronic stress-induced Zebrafish model. However, further studies are required to assess the efficacy of this novel HDACi in rodent models and the toxicity, if any, after its long-term in vivo administration. Thus, these results suggest that the novel HDAC inhibitors crafted with vorinostat and tubastatin-A pharmacophoric moieties can be developed as potential therapeutics to treat anxiety, depression, and related psychiatric disorders.

Experimental Section

General Information

All of the reagents and solvents used were analytically pure. Carbazole was purchased from Sigma-Aldrich. The progress of chemical reactions was monitored by thin-layer chromatography (TLC) on precoated silica gel GF254 plates. The TLC plates were then visualized under UV illumination at 254 nm. For further visualization, the TLC plates were stained with phosphomolybdic acid and charred on a hot plate. Column chromatography was carried out using silica gel finer than 200 mesh. The columns were packed in hexane and equilibrated with appropriate solvent/solvent mixture prior to use. The Fisher–Johns melting point apparatus was used for measuring the melting points. Analytical HPLC (SPD-M20A, Shimadzu, Japan) was used for determining the purity of all compounds (>96%). Infrared (IR) spectra were recorded as neat liquids or KBr pellets, and the absorptions are reported in cm–1. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz (Varian, Palo Alto, CA) spectrometers in appropriate solvents. We used tetramethylsilane (TMS) as an internal standard; the chemical shifts are shown in δ scales. 13C NMR spectra were recorded on a 100 MHz spectrometer. Multiplicities of 1H NMR signals were designated as s (singlet), d (doublet), t (triplet), q (quartet), br (broad), m (multiplet, for unresolved lines), etc. High-resolution mass spectra were recorded using ESI-QTOF mass spectrometry.

General Procedure for the Synthesis of 15–17

A mixture of glutaric anhydride (21.40 mmol) and anhydrous AlCl3 (21.40 mmol) in dichloromethane (25 mL) was added slowly to a stirring solution of 9-benzyl-9H-carbazole 12 (5.0 g, 19.45 mmol) in dichloromethane (25 mL) at 0 °C and then refluxed for 3 h. After completion (TLC), it was cooled to 0 °C and excess AlCl3 was quenched with 1 N HCl, organic layer was separated, washed with water, dried over anhydride Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with ethyl acetate to give 5-(9-benzyl-9H-carbazol-3-yl)-5-oxopentanoic acid (15) as a semisolid (4.75 g, 62%). Similarly, adipic anhydride and suberic anhydride gave 6-(9-benzyl-9H-carbazol-3-yl)-6-oxohexanoic acid (16) and 8-(9-benzyl-9H-carbazol-3-yl)-8-oxooctanoic acid (17) in 60 and 55% yields, respectively.

5-(9-Benzyl-9H-carbazol-3-yl)-5-oxopentanoic Acid (15)

1H NMR δ 8.77 (s, 1H), 8.70 (s, 1H), 8.16–7.99 (m, 2H), 7.45–7.20 (m, 6H), 7.10–7.03 (m, 2H), 5.50 (s, 2H), 3.08 (t, J = 6.7 Hz, 2H), 2.41(t, J = 6.7 Hz, 2H), 1.90–1.75 (m, 2H); MS (ESI) m/z 394 [M + Na]+.

6-(9-Benzyl-9H-carbazol-3-yl)-6-oxohexanoic Acid (16)

1H NMR (300 MHz, CDCl3): δ 8.77 (s, 1H), 8.71 (s, 1H), 8.13 (d, J = 7.5 Hz, 2H), 8.11–7.99 (m, 1H), 7.46–7.20 (m, 6H), 7.10–7.03 (m, 2H), 5.50 (s, 2H), 3.08 (t, J = 7.5 Hz, 2H), 2.42 (t, J = 6.0 Hz, 2H), 1.93–1.73 (m, 4H); MS (ESI) m/z 386 [M + H]+.

8-(9-Benzyl-9H-carbazol-3-yl)-8-oxooctanoic Acid (17)

1H NMR (300 MHz, CDCl3): δ 8.70 (s, 1H), 8.14 (d, J = 8.3 Hz, 1H), 8.02 (dd, J = 1.5, 6.7 Hz, 1H), 7.46–7.36 (m, 1H), 7.31–7.14 (m, 5H), 7.13–7.01 (m, 3H), 5.49 (s, 2H), 3.04 (t, J = 6.7 Hz, 2H), 2.34 (t, J = 6.7 Hz, 2H), 1.88–1.73 (m, 2H), 1.72–1.56 (m, 2H), 1.55–1.36 (m, 4H).

General Procedure for the Synthesis of 18–19

A mixture of keto acid 16–17 (1.0 mmol) and N2H4.H2O (8.0 mmol) in glycol (10 mL) was stirred at 100 °C. After 0.5 h, KOH (8.0 mmol) was added and refluxed further for 8 h. The reaction mixture was neutralized with 1 N HCl and diluted with EtOAc (20 mL), organic layer was washed with brine solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (1:2) to give 18–19 as a white solid.

5-(9-Benzyl-9H-carbazol-3-yl)pentanoic Acid (18)

Yield: 75%; mp: 88–90 °C; 1H NMR (300 MHz, CDCl3): δ 8.10 (d, J = 7.7 Hz, 1H), 7.91 (s, 1H), 7.42–7.36 (m, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.29–7.18 (m, 6H), 7.17–7.09 (m, 2H), 5.48 (s, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.40 (t, J = 7.0 Hz, 2H), 1.84–1.68 (m, 4H).

6-(9-Benzyl-9H-carbazol-3-yl)hexanoic Acid (19)

Yield: 80%; mp: 67–69 °C; 1H NMR (300 MHz, CDCl3): δ 8.04 (d, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.35–7.14 (m, 8H), 7.09 (d, J = 6.2 Hz, 2H), 5.48 (s, 2H), 2.78 (t, J = 7.3 Hz, 2H), 2.33 (t, J = 7.3 Hz, 2H), 1.82–1.63 (m, 4H), 1.53–1.36 (m, 2H); MS (ESI) m/z 372 (M + H)+.

General Procedure for the Synthesis of 20–21

To a solution of acid 18–19 (1.0 mmol) in methanol (6 mL) at 0 °C, SOCl2 (1.5 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (4:1) to give products 20–21 as a white solid, which were quickly used further in the next step.

Methyl 5-(9-Benzyl-9H-carbazol-3-yl)pentanoate (20)

Yield: 95%; 1H NMR (300 MHz, CDCl3): δ 8.72 (d, J = 1.5 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.05 (dd, J = 6.7, 1.5 Hz, 1H), 7.49–7.39 (m, 1H), 7.37–7.20 (m, 6H), 7.12–7.06 (m, 2H), 5.52 (s, 2H), 3.66 (s, 3H), 3.10 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 6.7 Hz, 2H), 1.92–1.72 (m, 4H); MS (ESI) m/z 372 (M + H)+.

Methyl 6-(9-Benzyl-9H-carbazol-3-yl)hexanoate (21)

Yield: 93%; 1H NMR (300 MHz, CDCl3): δ 8.71 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.02 (dd, J = 6.9, 1.5 Hz, 1H), 7.51–7.35 (m, 3H), 7.33–7.17 (m, 4H), 7.16–7.02 (m, 2H), 5.57 (s, 2H), 3.63 (s, 3H), 3.06 (t, J = 7.3 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.90–1.55 (m, 4H), 1.53–1.32 (m, 4H); MS (ESI) m/z 386 (M + H)+.

General Procedure for the Synthesis of 3–4

To a solution of ester 20–21 (0.1 mmol) and NH2OH·HCl (0.8 mmol) in MeOH at 0 °C, NaOH (1 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. Solvent was evaporated under reduced pressure, and crude reaction mixture was purified by silica gel column chromatography.

5-(9-Benzyl-9H-carbazol-3-yl)-N-hydroxypentanamide (3)

Yield: 92%; mp: 128–130 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.48 (s,1H), 8.03 (d,J = 6.9 Hz, 1H), 7.56 (s, 1H), 7.43–7.26 (m, 3H), 7.25–7.10 (m, 6H), 5.51 (s, 2H), 2.74 (t, J = 7.9 Hz, 2H), 1.97 (t, J = 7.9 Hz, 2H), 1.41–1.28 (m, 4H); 13C NMR (75 MHz, DMSO-d6): δ 159.6, 140.1, 138.4, 136.8, 132.5, 128.1, 126.7, 126.0, 125.8, 125.1, 122.3, 122.1, 119.6, 119.1, 118.4, 108.3, 108.1, 45.8,35.0, 31.2, 29.0, 24.6; IR (KBr): 3227, 2923, 1698, 1596, 1456, 1205, 1149, 950, 745 cm–1; MS (ESI) m/z 395 [M + Na]+; HRMS (ESI): Calcd for C24H24N2O2Na [M + Na]+: 395.1735, found: 395.1748.

6-(9-Benzyl-9H-carbazol-3-yl)-N-hydroxyhexanamide (4)

Yield: 89%%; mp:125-127 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.28 (s,1H), 8.05 (d,J = 7.8 Hz, 1H), 7.86 (s,1H), 7.40–7.09 (m, 10H), 5.52 (s,2H), 2.75 (t, J = 7.7 Hz, 2H), 2.01 (t, J = 7.3 Hz, 2H), 1.79–1.55 (m, 4H), 1.50–1.24 (m, 2H); 13C NMR (75 MHz, DMSO-d6): δ 169.1, 140.1, 138.4, 136.9, 132.7, 128.0, 126.7, 126.0, 125.9, 125.0, 122.2, 122.1, 119.6, 119.0, 118.3, 108.4, 108.2, 45.7, 35.1, 32.1, 31.3, 28.2, 24.8; IR (KBr): 3269, 2925, 2850, 1627, 1488, 1465, 1325,1207, 743 cm–1; MS (ESI) m/z 409 [M + Na]+; HRMS (ESI): Calcd for C25H26N2O2Na [M + Na]+: 409.1891, found: 409.1890.

General Procedure for the Synthesis of 8-Oxo-8-(9-tosyl-9H-carbazol-3-yl)octanoic Acid (22)

A mixture of suberic anhydride (1.1 mmol) and AlCl3 (1.1 mmol) in dichloromethane (25 mL) was added slowly to a stirring solution of 9-tosyl-9H-carbazole 14 (1.0 mmol) in dichloromethane (25 mL) at 0 °C and then refluxed for 3 h. After completion (TLC), it was cooled to 0 °C and excess AlCl3 was quenched with 1 N HCl, organic layer was separated, washed with water, dried over anhydride Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with ethyl acetate to give 8-oxo-8-(9-tosyl-9H-carbazol-3-yl)octanoic acid (22) as a white solid (66%).

1H NMR (300 MHz, CDCl3): δ 8.18 (d, J = 8.3 Hz, 3H), 7.95 (d, J = 6.7 Hz, 3H), 7.49–7.40 (m, 2H), 7.33 (d, J = 7.5 Hz, 3H), 3.12 (t, J =7.5Hz, 2H), 2.43–2.31 (m, 5H), 1.75–1.62 (m, 2H), 1.56–1.47 (m, 2H), 1.36–1.22 (m, 4H); MS (ESI) m/z 478 [M + H]+.

General Procedure for the Synthesis of 8-(9H-Carbazol-3-yl)-8-oxooctanoic Acid (23)

A mixture of 8-oxo-8-(9-tosyl-9H-carbazol-3-yl)octanoic acid (22) (1.0 mmol) and N2H4.H2O (8.0 mmol) in glycol (10 mL) was stirred at 100 °C. After 0.5 h, KOH (8.0 mmol) was added and refluxed further for 8 h. The reaction mixture was neutralized with 1 N HCl, diluted with EtOAc (20 mL), organic layer was washed with brine solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (1:2) to give 23 as a white solid (62%).

1H NMR (300 MHz, CDCl3): δ 8.01 (d, J = 7.5 Hz, 1H), 7.81 (s, 1H), 7.45–7.34 (m, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.27–7.21 (m, 2H), 7.14 (t, J = 6.7 Hz, 1H), 2.76 (t, J = 7.5 Hz, 2H), 2.26 (t, J = 7.5 Hz, 2H), 1.83–1.47 (m, 4H), 1.42–1.31 (m, 6H); MS (ESI) m/z 332 [M + Na]+.

General Procedure for the Synthesis of 8-(9H-carbazol-3-yl)octanoate (24)

To a solution of 8-(9H-carbazol-3-yl)-8-oxooctanoic acid 23 (1.0 mmol) in methanol (6 mL) at 0 °C, SOCl2 (1.5 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (4:1) to give 8-(9H-carbazol-3-yl)octanoate 24 as a white solid (91%), which was quickly used in the next step.

1H NMR (300 MHz, CDCl3): δ 8.00 (d, J = 7.5 Hz, 1H), 7.82 (s, 1H), 7.45–7.28 (m, 2H), 7.27–7.19 (m, 2H), 7.15 (t, J = 6.7 Hz, 1H), 3.63 (s, 3H), 2.76 (t, J = 7.5 Hz, 2H), 2.26 (t, J =7.5 Hz, 2H), 1.81–1.52 (m, 4H), 1.47–1.25 (m, 6H); MS (ESI) m/z 324 [M + H]+.

Synthesis of 8-(9H-Carbazol-3-yl)-N-hydroxyoctanamide (5)

To a solution of 8-(9H-carbazol-3-yl)octanoate 24 (0.40 g, 1.23 mmol) and NH2OH·HCl (9.90 mmol) in MeOH at 0 °C, NaOH (10.23 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by silica gel column chromatography and quickly used in the next step.

Yield: 90%; mp: 140–142 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.69 (s, 1H), 10.25 (s, 1H), 8.05–7.93 (m,1H), 7.79 (d, J = 13.7 Hz, 1H), 7.44–7.36 (m, 2H), 7.33–7.26 (m, 1H), 7.18–7.04 (m, 2H), 2.73 (t, J = 7.3 Hz, 2H),1.96 (t, J = 7.3 Hz, 2H), 1.79–1.63 (m, 4H), 1.61–1.44 (m, 4H), 1.40–1.26 (m, 2H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 168.9, 140.5, 138.8, 132.4, 126.0, 125.0, 121.8, 121.7, 119.6, 119.0, 118.0, 108.3, 108.2, 35.2, 32.1, 31.7, 28.68, 28.61, 28.5, 25.0; IR (KBr) 3251, 2922, 2850, 1628, 1488, 1465, 1452, 1325, 1210, 1150, 744, 726 cm–1; MS (ESI) m/z 347 [M + Na]+.

General Procedure for the Synthesis of 25–26

To a solution of keto acid 16–17 (1.0 mmol) in methanol (6 mL) at 0 °C, SOCl2 (1.5 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane/ethyl acetate (4:1) to give products 25–26 as gummy material.

Methyl 6-(9-Benzyl-9H-carbazol-3-yl)-6-oxohexanoate (25)

Yield: 95%; 1H NMR (300 MHz, CDCl3): δ 8.72 (d, J = 1.5 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.05 (dd, J = 6.7, 1.5 Hz, 1H), 7.49–7.39 (m, 1H), 7.37–7.20 (m, 6H), 7.12–7.06 (m, 2H), 5.52 (s, 2H), 3.66 (s, 3H), 3.10 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 6.7 Hz, 2H), 1.92–1.72 (m, 4H); MS (ESI) m/z 400 [M + H]+.

Methyl 8-(9-Benzyl-9H-carbazol-3-yl)-8-oxooctanoate (26)

Yield: 93%; 1H NMR (300 MHz, CDCl3): δ 8.71 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.02 (dd, J = 6.9, 1.5 Hz, 1H), 7.51–7.35 (m, 3H), 7.33–7.17 (m, 4H), 7.16–7.02 (m, 2H), 5.57 (s, 2H), 3.63 (s, 3H), 3.06 (t, J = 7.3 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.90–1.55 (m, 4H), 1.53–1.32 (m, 4H).

General Procedure for the Synthesis of 6–7

To a solution of keto ester 25–26 (0.1 mmol) and NH2OH·HCl (0.8 mmol) in MeOH at 0 °C, NaOH (1 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by silica gel column chromatography, and quickly used in the next step.

6-(9-Benzyl-9H-carbazol-3-yl)-N-hydroxy-6-(hydroxyimino)hexanamide (6)

Yield: 93%; mp: 128–130 °C; 1H NMR (300 MHz, DMSO-d6): δ 8.33 (s, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 9.4 Hz, 1H), 7.45–7.30 (m, 3H), 7.29–7.16 (m, 4H), 7.11 (d, J = 7.7 Hz, 2H), 5.52 (s, 2H), 2.89 (t, J = 6.9 Hz, 2H), 2.10 (t, J = 6.9 Hz, 2H), 1.85–1.54 (m, 4H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 169.4, 157.2, 140.3, 140.1, 136.5, 128.2, 127.3, 126.9, 125.9, 125.3, 123.7, 122.4, 122.2, 119.9, 118.9, 117.6, 108.5, 108.2, 45.9, 32.0, 25.6, 25.2, 25.0; IR (KBr): 3233, 2925, 1627, 1495, 1466, 1345, 1267, 1212, 1028, 941, 723 cm–1; MS (ESI) m/z 438 [M + Na]+; HRMS (ESI): Calcd for C25H25N3O3Na [M + Na]+: 438.1793, found: 438.1776.

8-(9-Benzyl-9H-carbazol-3-yl)-N-hydroxy-8-(hydroxyimino)octanamide (7)

Yield: 95%; mp: 125–127 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.60 (s, 1H), 10.25 (s, 1H), 8.31 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 7.1 Hz, 1H), 7.48–7.35 (m, 3H), 7.27–7.11 (m, 6H), 5.60 (s, 2H), 2.83 (t, J = 7.1 Hz, 2H), 1.97 (t, J = 7.5 Hz, 2H), 1.70–1.49 (m, 4H), 1.44–1.20 (m, 4H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 168.9, 157.0, 140.9, 140.3, 140.1, 137.2, 128.2, 127.3, 127.0, 126.3, 125.7, 123.7, 122.2, 120.1, 119.0, 117.5, 108.9, 45.6, 32.1, 28.9, 28.3, 25.9, 25.1, 24.9; IR (KBr): 3234, 2925, 2855, 1629, 1598, 1495, 1466, 1337, 1265, 1039, 747, 728 cm–1; MS (ESI) m/z 466 (M + Na)+; HRMS (ESI): Calcd for C27H29N3O3Na [M + Na]+: 466.2106, found: 466.2106.

General Procedure for the Synthesis of 5-(9-(2-Ethoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoic Acid (27)

A mixture of glutaric anhydride (21.73 mmol) and AlCl3 (21.73 mmol) in dichloromethane (25 mL) was slowly added to a stirring solution of ethyl 2-(9H-carbazol-9-yl)acetate 13 (5 g, 19.76 mmol) in dichloromethane (25 mL) at 0 °C and then refluxed for 3 h. After completion (TLC), the reaction was cooled to 0 °C and excess AlCl3 was quenched with 1 N HCl, organic layer was separated, washed with water, dried over anhydride Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with ethyl acetate to give 5-(9-(2-ethoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoic acid (27) as a colorless semisolid (4.35 g, 60%). 1H NMR (300 MHz, DMSO-d6): δ 8.80 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H), 8.09 (dd, J = 7.1, 1.5 Hz, 1H), 7.58–7.45 (m, 3H), 7.35–7.26 (m, 1H), 5.27 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.18 (t, J = 7.1 Hz, 2H), 2.39 (t, J = 7.1 Hz, 2H), 2.06–1.94 (m, 2H), 1.27 (t, J = 6.9 Hz, 3H); MS (ESI) m/z 368 [M + H]+.

General Procedure for the Synthesis of Methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoate (28)

To a solution of 5-(9-(2-ethoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoic acid 26 (0.5 g, 1.36 mmol) in methanol (6 mL) at 0 °C, SOCl2 (1.63 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane/ethyl acetate (4:1) to give product 28 as a white solid (80%). 1H NMR (300 MHz, CDCl3): δ 8.74 (d, J = 1.4 Hz, 1H), 8.17–8.11 (m, 2H), 7.54–7.48 (m, 1H), 7.38–7.31 (m, 3H), 5.03 (s, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 3.18 (t, J = 7.1 Hz, 2H), 2.50 (t, J = 7.1 Hz, 2H), 2.19–2.11 (m, 2H); MS (ESI) m/z 390 [M + Na]+.

General Procedure for the Synthesis of N-Hydroxy-5-(9-(2-(hydroxyamino)-2-oxoethyl)-9H-carbazol-3-yl)-5-(hydroxyimino)pentanamide (9)

To a solution of methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoate 28 (0.30g, 0.81 mmol) and NH2OH·HCl (6.53 mmol) in MeOH at 0 °C, NaOH (8.17 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by silica gel column chromatography.

Yield: 89%; mp: 135–137 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.94 (br s, 1H), 10.63 (br s, 1H), 10.33 (br s, 1H), 8.35 (s, 1H), 8.09 (d, J = 7.5 Hz, 1H), 8.00–7.86 (m, 1H), 7.57–7.45 (m, 1H), 7.44–7.34 (m, 2H), 7.23–7.10 (m, 1H), 4.86 (s, 2H), 2.88 (t, J = 7.3 Hz, 2H), 2.12 (t, J = 7.1 Hz, 2H), 2.00 (s, 2H), 1.96–1.79 (m, 2H); 13C NMR (75 MHz, DMSO-d6): δ 169.0, 164.2, 156.8, 140.9, 140.8, 127.2, 125.9, 123.6, 122.4, 122.2, 120.3, 119.3, 117.8, 109.6, 109.4, 43.5, 24.9, 22.4, 14.1; IR (KBr): 3208, 2920, 1665, 1493, 1327, 1208, 1047, 964, 748 cm–1; MS (ESI) m/z 407 [M + Na]+; HRMS (ESI): Calcd for C19H20N4O5Na [M + Na]+: 407.1331, found: 407.1334.

General Procedure for the Synthesis of 5-(9-(carboxymethyl)-9H-carbazol-3-yl)pentanoic Acid (29)

A mixture of 5-(9-(2-ethoxy-2-oxoethyl)-9H-carbazol-3-yl)-5-oxopentanoic acid 27 (2 g, 5.44 mmol) and N2H4.H2O (43.59 mmol) in glycol (10 mL) was stirred at 100 °C. After 0.5 h, KOH (43.59 mmol) was added and refluxed further for 8 h. The reaction mixture was neutralized with 1 N HCl, diluted with EtOAc (20 mL), organic layer was washed with brine solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (1:2) to give 29 as a white solid (70%).1H NMR (500 MHz, DMSO-d6): δ 8.00 (t, J =7.9 Hz, 1H), 7.84 (s, 1H), 7.51–7.41 (m, 1H), 7.40–7.34 (m, 2H), 7.33–7.28 (m, 1H), 7.20–7.08 (m, 1H), 4.99 (s, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.24 (t, J = 6.9 Hz, 2H), 1.78–1.58 (m, 4H).

General Procedure for the Synthesis of Methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl) Pentanoate (30)

To a solution of 5-(9-(carboxymethyl)-9H-carbazol-3-yl) pentanoic acid 29 (1.0 g, 3.07 mmol) in methanol (6 ml) at 0 °C, SOCl2 (3.69 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (4:1) to give methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)pentanoate 30 as a white solid (80%).

1H NMR (300 MHz, CDCl3): δ 8.06 (d, J = 7.5 Hz, 1H), 7.88 (s, 1H), 7.55–7.20 (m, 5H), 4.97 (s, 2H), 3.70 (s, 3H), 3.66 (s, 3H), 2.80 (t, J = 7.9 Hz, 2H), 2.36 (t, J = 6.6 Hz, 2H), 1.87–1.65 (m, 4H); MS (ESI) m/z 376 [M + Na]+.

General Procedure for the Synthesis of N-hydroxy-5-(9-(2-(hydroxyamino)-2-oxoethyl)-9H-carbazol-3-yl)pentanamide (8)

To a solution of methyl 5-(9-(2-methoxy-2-oxoethyl)-9H-carbazol-3-yl)pentanoate 30 (0.60 g, 1.69 mmol) and NH2OH·HCl (13.59 mmol) in MeOH at 0 °C, NaOH (13.59 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by silica gel column chromatography to give N-hydroxy-5-(9-(2-(hydroxyamino)-2-oxoethyl)-9H-carbazol-3-yl)pentanamide (8).

Yield: 82%; mp: 135–137 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.78 (br s, 1H), 10.31 (br s, 1H), 7.97 (t, J = 5.4 Hz, 1H), 7.81 (d, J = 10.9 Hz, 1H), 7.55–7.30 (m, 3H), 7.21–7.04 (m, 2H), 4.84 (s, 2H), 2.76 (t, J = 5.6 Hz, 2H), 2.00 (t, J = 9.2 Hz, 2H), 1.79–1.61 (m, 2H), 1.36–1.20 (m, 2H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 174.1, 167.7, 142.7, 140.7, 128.4, 126.1, 125.6, 122.4, 122.0, 120.8, 120.1, 119.9, 109.0, 108.4, 43.9, 36.8, 32.8, 19.3, 13.7; IR (KBr) 3214, 2922, 2183, 1664, 1491, 1463, 1326, 1084, 748 cm–1; MS (ESI) m/z 378 [M + Na]+; HRMS (ESI): Calcd for C19H21N3O4Na [M + Na]+: 378.1429, found: 378.1420.

General Procedure for the Synthesis of 31–32

A mixture of glutaric anhydride (6.50 mmol) and AlCl3 (6.50 mmol) in dichloromethane (25 mL) was added slowly to a stirring solution of carbazole (1 g, 5.98 mmol) in dichloromethane (25 mL) at 0 °C and then refluxed for 3 h. After completion (TLC), the reaction mixture was cooled to 0 °C and excess AlCl3 was quenched with 1 N HCl, organic layer was separated, washed with water, dried over anhydride Na2SO4, and evaporated under reduced pressure. Crude residue was purified over silica gel column chromatography and eluted with ethyl acetate to give 5-(9H-carbazol-9-yl)-5-oxopentanoic acid (31) as a white solid (1.09 g, 65%). Similarly, adipic anhydride gave 6-(9H-carbazol-9-yl)-6-oxohexanoic acid (32) in 60% yield and quickly used further in the next step.

5-(9H-Carbazol-9-yl)-5-oxopentanoic Acid (31)

1H NMR (300 MHz, CDCl3): δ 8.20 (d, J = 8.1 Hz, 2H), 7.95 (d, J = 7.7 Hz, 2H), 7.51–7.40 (m, 2H), 7.39–7.29 (m, 2H), 3.25 (t, J = 6.9 Hz, 2H), 2.64 (t, J = 6.9 Hz, 2H), 2.34–2.20 (m, 2H); MS (ESI) m/z 282 [M + H]+.

6-(9H-Carbazol-9-yl)-6-oxohexanoic Acid (32)

1H NMR (300 MHz, DMSO-d6): δ 8.25 (d, J = 7.5 Hz, 1H), 8.13–7.98 (m, 2H), 7.55–7.33 (m, 4H), 7.20 (t, J = 7.5 Hz, 1H), 3.11 (t, J = 7.3 Hz, 2H), 2.32 (t, J = 7.1 Hz, 2H), 1.87–1.60 (m, 4H); MS (ESI) m/z 318 [M + Na]+.

General Procedure for the Synthesis of 33–34

To a solution of keto acid 31–32 (0.1 mmol) in methanol (6 mL) at 0 °C, SOCl2 (0.12 mmol) was added dropwise and refluxed for 3 h. The reaction mixture was neutralized with NaHCO3 solution and diluted with CHCl3 (25 mL). The organic phase was washed with brine solution, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was purified over silica gel column chromatography and eluted with hexane:ethyl acetate (4:1) to give product 33–34 as white gummy materials.

Methyl 5-(9H-Carbazol-9-yl)-5-oxopentanoate (33)

Yield: 85%; 1H NMR (300 MHz, CDCl3): δ 8.71 (d, J = 18.1 Hz, 3H), 8.16–8.00 (m, 3H), 7.48–7.38 (m, 2H), 3.65 (s, 3H), 3.06 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.88–1.73 (m, 2H); MS (ESI) m/z 296 [M + H]+.

Methyl 6-(9H-Carbazol-9-yl)-6-oxohexanoate (34)

Yield: 87%; 1H NMR (300 MHz, CDCl3): δ 8.78–8.55 (m, 3H), 8.15–8.07 (m, 2H), 7.45–7.36 (m, 3H), 3.67 (s, 3H), 3.11 (t, J = 7.3 Hz, 2H), 2.40 (t, J = 7.1 Hz, 2H), 1.92–1.68 (m, 4H); MS (ESI) m/z 310 [M + H]+.

General Procedure for the Synthesis of 10–11

To a solution of ester compounds 33–34 (0.1 mmol) and NH2OH·HCl (8 mmol) in MeOH at 0 °C, NaOH (8 mmol) in MeOH was added and stirred for 8 h at room temperature. After completion of the reaction (TLC), the reaction mixture was neutralized with 1 N HCl and diluted with EtOAc. The organic phase was washed with brine solution and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified by silica gel column chromatography.

5-(9H-Carbazol-9-yl)-N-hydroxy-5-(hydroxyimino)pentanamide (10)

Yield: 90%; mp: 113–115 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6): δ 11.03 (br s, 1H), 10.70 (br s, 1H), 10.40 (br s, 1H), 8.01 (d, J = 7.7 Hz, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.50–7.25 (m, 2H), 7.20–7.12 (m, 2H), 2.97 (t, J = 7.9 Hz, 2H), 2.17 (t, J = 6.9 Hz, 2H), 2.02–1.90 (m, 2H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 168.6, 157.6, 139.0, 136.2, 125.2, 123.6, 122.9, 121.9, 120.7, 119.6, 118.7, 118.1, 117.5, 111.1, 32.1, 28.9, 23.9; IR (KBr) 3370, 2924, 1644, 1455, 1417, 1319, 1268, 1227, 749 cm–1; MS (ESI) m/z 334 [M + Na]+; HRMS (ESI): Calcd for C17H17N3O3Na [M + Na]+: 334.1167, found: 334.1176.

6-(9H-Carbazol-9-yl)-N-hydroxy-6-(hydroxyimino)hexanamide (11)

Yield: 92%; mp: 130–132 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.05 (br s, 1H), 10.66 (br s, 1H), 10.29 (br s, 1H), 8.55 (s,2H), 8.24 (s, 1H), 8.07 (m, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.65–7.35 (m, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.13 (t, J = 7.1 Hz, 1H), 2.86 (t, J = 6.7 Hz, 2H), 2.01 (t, J = 6.7 Hz, 2H), 1.81–1.43 (m, 4H); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 169.0, 157.0, 139.9, 139.7, 126.6, 125.1, 123.3, 122.4, 122.1, 119.7, 118.3, 117.3, 110.7, 110.4, 32.1, 25.7, 25.3, 24.9; IR (KBr) 3387, 2924, 1629, 1461, 1337, 1243, 753 cm–1; MS (ESI) m/z 348 [M + Na]+; HRMS (ESI): Calcd for C18H19N3O3Na [M + Na]+: 348.1324, found: 348.1327.

Cell Culture Maintenance

Neuro2A (mouse neuroblastoma) and GL-261 (mouse glioblastoma) lines were obtained from the American Type Culture Collection (ATCC). Neuro2A and GL-261 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Gibco), penicillin/streptomycin (1×), Non-Essential Amino Acids Solution (1×), and sodium pyruvate (1×) at 37 °C in a humidified atmosphere supplemented with 5% CO2.

HDAC Inhibition Assay

The HDAC inhibitory activity was measured using Epigenase HDAC activity/inhibition direct assay colorimetric kit from Epigentek (catalog- P4034). The nuclear extract of GL261 cells was used as an enriched source of HDAC enzyme. The protocol was followed according to the user guide provided with the kit. Vorinostat (SAHA) and tubastatin-A have been used as a positive control. All of the compounds were screened for HDACi activity at 10, 1, 0.1, and 0.01 μM concentrations, and IC50 values are calculated for all of the small molecules.

HDACi Selectivity Assay

Human cervical cancer, HeLA, cells were grown in DMEM supplemented with 1× Pen-strep solution and 10% FBS and incubated at 37 °C. The cells were subcultured twice a week. The cells were harvested when 80% confluent and lysed in IP lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1× Protease Inhibitor Cocktail) to isolate total proteins. Antibody (2 μg) (HDAC8, HDAC4, and HDAC6) is incubated with 60 μL of Dyna beads for 30 min on rotation. Then, 100 μg of total protein was added to the bead–antibody complex and incubated for 4 h at 4 °C on rotation. The beads were washed with lysis buffer and used as enzyme source for the activity assay. For HDAC activity assay,[46] the bead-bound enzyme was incubated with HDAC substrate, Fluor-de-lys (Enzo Life Sciences), and different concentrations (10, 1, 0.1, 0.01, and 0.001 μM) of compound 5 for 15 min, followed by addition of developer solution according to the manufacturer’s protocol (Enzo Life Sciences), and the fluorescence was measured at 360 and 460 nm excitation and emission wavelengths, respectively. The % HDAC enzyme activity is calculated as a ratio to vehicle-treated control.

Neurite Outgrowth Assay

Neurite outgrowth activity assay was performed as reported.[47] Briefly, mouse neuroblastoma cells Neuro2A were seeded at 8000 cells/cm2 in six-well plates. After 24 h, the cells were induced to differentiate using serum-deprived media (DMEM + 1% FBS). After 6 h, the cells were incubated with different concentrations of compounds and were observed for neurite outgrowth activity 48 h after the treatment. Bright-field images of Neuro2A cells treated with different concentrations of compounds were used to measure the neurite outgrowth activity. The cells were then fixed with 4% paraformaldehyde in 1× PBS in preparation for the immunocytochemical reaction. To determine how neurite outgrowth was affected by different concentrations of compounds, the average neurite length was measured (using ImageJ software) on 60 neurons from six microscopic fields randomly captured as two images per well, from three independent experiments.

Cell Viability Assay

Neuro2A cells were seeded at a concentration of 10 000 cells/cm2 in 96-well plates. After 24 h, the cells were induced to differentiate with serum deprivation (1% FBS) and incubated with different concentrations of compounds for 72 h. The percentage of cell viability was measured by MTT assay, as described previously by us.[47] MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma-Aldrich) was added to the cells in the culture medium at a concentration of 5 mg/mL and incubated for 2 h at 37 °C. Formazan crystals were solubilized in DMSO, and the absorbance was measured at 562 nm. Data were analyzed as relative activity in comparison to the vehicle treatment group.

Immunocytochemistry

Neuro2A cells, incubated with different concentrations of compounds, were processed for the immunostaining following our earlier protocol.[47] Briefly, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and permeabilization was done with 0.5% Triton X-100 and 0.05% Tween 20 in 1× PBS, followed by incubation in blocking buffer (2% bovine serum albumin + 0.1% Triton X-100 in 1× PBS) for 2 h at room temperature. Primary antibody against β-III tubulin (1:200, Millipore) was used. Samples were incubated with the primary antibody in blocking buffer overnight at 4 °C. The samples were washed with PBST (PBS with 0.1% Tween 20) and incubated with goat antimouse IgG conjugated to antirabbit cy5 (1:1000). Images were captured using a MoticAE31 microscope.

Immunoblotting

Cells in wells were incubated with compounds, vehicle, with and without inhibitor treatment, washed with 1× PBS, and harvested in 1× Laemmli buffer. Protein estimation was done with the amido black method, and an equal amount of protein was loaded onto 12% SDS-PAGE gel, followed by transfer onto the PVDF membrane. The blocking was done for 1 h at room temperature, followed by incubation with primary antibodies acetyl tubulin (1:1000), tubulin (1:2000), acetyl H3 (1:1000) (Millipore), and actin (1:5000) (Sigma) at 4 °C overnight. Incubation with secondary antibody antirabbit (1:5000), antimouse (1:10 000) was done at room temperature for 2 h. The blots were developed with Vilber Lourmat chemdoc instrument using super signal west dura luminal/enhancer solution (Thermo).

Animals and Housing

Adult Zebrafish (Danio rerio) were bred and raised in captivity. All of the animals were raised in large tanks with a natural daylight/dark cycle and two feedings until they arrived in the laboratory. In the fish laboratory, the animals were acclimatized to the experimental room conditions by maintaining them at 28 ± 2 °C, 14/10 h light/dark cycle, three feedings, and constant aeration. After the habituation period, the animals were grouped as control and test sets, and the test animals were subjected to two different stressors per day for a period of 7 days. Fish which exhibited stress-induced phenotypic changes, assessed by novel tank test and social interaction test, were treated intraperitoneal with compound 5 at 10, 25, and 50 mg/kg, with fluoxetine at 15 mg/kg, for about 3 days. Following behavioral testing (before and after drug treatment), all of the fish were euthanized and the brain was immediately dissected out for further analysis. All animal procedures were approved by the Institutional Animal Ethics Committee (IAEC/IICT/Protocol No. 26/2016).

Chronic Unpredictable Stress (CUS) Paradigm in Zebrafish

CUS paradigm was performed with appropriate minor modifications to our previously published protocol.[43] Briefly, for a period of 7 days, the fish were subjected to a variety of chronic stressors, two stressors per day (forenoon and afternoon), such as restraint stress (RS), heat stress (HS), cold stress (CS), social isolation (SI), overcrowding (OC), predator stress (PS), dorsal body exposure (DBE), tank change (TC), chasing (C), and alarm pheromone stress (APS). In restraint stress (RS), each animal was restrained for an hour in a 2 mL micro-centrifuge tube with perforations at both the ends for free water flow; for heat stress (HS) and cold stress (CS), the animals were transferred to new tanks maintained at 33 and 23 °C, respectively, for 30 min; social isolation (SI) was given in separate beakers for 60 min; overcrowding (OC) with 10 animals in a 250 mL beaker containing only 150 mL of water for 60 min; in predator stress (PS), the test animals were exposed to predator (cichlid fish) encounters (chases and attacks) for 1 min; in dorsal body exposure (DBE), the animals were housed in tanks with the low water level to expose the animal’s dorsal body for 2 min; in tank change (TC) stress, fish were transferred from one tank to another about six consecutive times; in chasing (C), fish were chased with a net for 8 min; in alarm pheromone stress (APS), the test fish was exposed to water containing the washing of epidermal cells from euthanized/sliced Zebrafish, for 30 min. To avoid habituation to stressors, unpredictability was maintained by changing the time and sequence of stressors daily during the 7-day stress paradigm. Aeration and temperature were controlled during the presentation of each stressor, except during heating and cooling stress. The nonstressed control group was maintained in the same room during the 7-day stress period.

Behavior Tests

The events were first recorded by a video camera (Sony Handycam) and later scored manually by two independent blinded observers and analyzed together.

Novel Tank Test (NTT)

NTT was performed as our previously reported protocol.[43] Briefly, Zebrafish were placed individually in a narrow 15 × 12 × 25 cm3 tank with a water depth of 18 cm divided into three equal, virtually horizontal sections and demarcated by a line on the outside of the tank wall. In the 2 min novel tank test, the time spent by the fish in different levels of the tank (bottom, middle, or upper level) was measured to assess the level of anxiety. A preference for the bottom and less frequent venturing into the middle and upper levels of the tank is suggestive of increased anxiety. Similarly, longer latency to enter the middle and upper levels, greater numbers of freezing bouts and longer durations in freezing mode indicates the anxious phenotype, as does the increased locomotor activity (number of crosses in the swim area).

Social Interaction Test

Depression has been well reported to affect the social behavior of the subjects exposed to chronic stressful conditions, as reported in mice.[48] The social avoidance behavior of the stressed Zebrafish at the end of the stress paradigm was assessed by the social interaction test based on the approach and interaction fervor with an unfamiliar conspecific target (pink Zebrafish) as described by us.[49] Briefly, experimental Zebrafish were introduced into the tank with the target in the interaction box for 120 s and a conspecific pink Zebrafish served as a target in the interaction zone. The entire test was video recorded with a Sony Handycam Camcorder 200E in HD mode and later analyzed using manual scoring. The socializing ability of the fish was individually assessed by interaction parameters like latency, interaction time, and freezing duration.

Tissue Extraction and Sample Preparation for Thin-Layer Chromatography (TLC)

To know the ability of compound 5 to cross the blood–brain barrier (BBB), Zebrafish were divided into two groups. The first group was treated with compound 5 at 25 mg/kg dose, and the other was treated with DMSO (vehicle). For the detection of compound 5 in the brain samples, a protocol reported by Woo et al. with minor modifications was adapted.[50] Zebrafish from both the groups were anesthetized by placing them on ice, and complete brain was taken out and freeze-dried in liquid nitrogen. To these samples, HPLC-grade ethyl acetate was added, finely ground, and subjected to sonication, followed by centrifugation (10 000 rpm for 20 min) and incubation for 20 min to allow the separation of brain tissues and their ethyl acetate extracts. Later, the supernatants were collected and filtered through 0.22 μ filters. The filtered extracts (E1, E2) were vacuum-dried by rota evaporation and dissolved in HPLC-grade methanol. On the other hand, compound 5 was also dissolved in HPLC-grade methanol to use as a standard (S). The TLC profiling was performed with a solvent system of methanol:chloroform (5:95) with S, E1, and E2. After running on TLC, observations were made by iodine staining and visualization under a UV transilluminator.

Statistical Analysis

The results were expressed as mean ± SEM from three independent experiments. Data were subjected by either two-tailed paired Student’s t-test or ANOVA, followed by Tukey’s post hoc analysis, depending on the number of samples to compare, using GraphPad Prism software. A value of p < 0.05 was considered statistically significant.