Tricyclic Pyrrole-Based Compounds as Zika Virus Inhibitors

TL;DR

Researchers developed new compounds that effectively inhibit the Zika virus by reducing its protein production.

Contribution

The synthesis and evaluation of tricyclic pyrrole-based compounds as potent Zika virus inhibitors is presented.

Findings

Compound 2g showed the best antiviral activity against ZIKV with an EC50 of 0.4 μM and a high Selectivity Index of 501.

Three compounds (2g, 2h, and 2j) reduced ZIKV yield by impairing viral protein production.

The compounds were tested against both ZIKV and SARS-CoV for antiviral activity.

Abstract

A small library of 23 pyrrole-based tricyclic derivatives bearing bulky amine moieties was synthesized, and all were evaluated for their antiviral activities against ZIKV and SARS-CoV. Three compounds, derivatives 2g, 2h and 2j, elicited interesting activity against ZIKV: compound 2g, containing a bornylamine residue, showed the best activity against Huh-7 cells with EC50 and CC50 values of 0.4 μM and 230.5 μM, respectively, and a Selectivity Index (SI) of 501. All three compounds reduce ZIKV yield primarily by impairing viral protein.

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Full text

1. Introduction

Viral infections [1] include common viral diseases, such as respiratory infections (flu, colds), but also dangerous viral infections that emerge each year, such as SARS, the Middle East respiratory syndrome (MERS), or Ebola, which can spread epidemically worldwide. The first two are caused by coronaviruses and the third by Ebolavirus. Their treatment primarily involves the use of antiviral agents reducing the incidences of some of them, vaccination playing a pivotal preventive measure. Unfortunately, some viruses can reduce the effectiveness of vaccines through mutations, which currently represent one of the most significant problems of antiviral therapy [2] and, therefore, there is a continuous search for new antiviral drugs [3,4].

Nitrogen heterocycles are scaffolds widely used in medicinal chemistry to identify drug candidates due to their chemical–physical properties that characterize the pharmacokinetics and pharmacodynamics of the drug [5]. Among them, the therapeutic potential [6] of pyrrole-containing compounds is widely reported in the literature as well as their use to treat infectious diseases [7]. These nitrogen-containing heterocyclic templates are expressed both in natural products [8], like heme and cobalamin (vitamin B12), and in clinically approved drugs [9], like atorvastatin, a potent competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-CoenzimeA (HMG-CoA) reductase, and finafloxacin, a fluoroquinolone antibiotic indicated in the treatment of acute otitis externa.

Recent research has demonstrated that several novel pyrrole-based compounds, as pyrrolo[2,3-d]pyrimidines, exhibit significant inhibitory activity against several viruses [10], and several studies have demonstrated that pyrrole-containing compounds show a broad spectrum of antiviral activities, and resulting amide derivatives are particularly interesting (Figure 1) [11].

As part of a research project aimed at investigating the biological activity of compounds with a 1,4-dihydroindeno[1,2-b]pyrrol-3-yl-substituted core, recently we have identified a series of derivatives endowed with interesting tubulin polymerization inhibition (Figure 2) [12]. Therefore, to further investigate the potential of 1,4-dihydroindenopyrrole, we modulate the tricyclic system by N-benzylation of pyrrole and by introduction of bulky substituents on 3 position, as several examples of antiviral compounds bearing bicyclic monoterpenes are reported in the literature [13,14]. These structural modifications led us to obtain 1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamides and 3-carbohydrazides (Figure 2).

To evaluate the potential antiviral activity of these novel compounds, we characterized them against two distinct viruses that have caused global concern, like Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes and causes fever/rash with risks during pregnancy that may cause microcephaly, and SARS-CoV-2, that spreads through the respiratory tract causing COVID-19, most commonly manifesting acute respiratory syndrome.

2. Results

We describe the synthesis and anti ZIKV activity of two novel series of tricyclic pyrrole-based carbohydrazides and carboxamides 1a–k and 2a–l, (Table 1) which differ by the presence of a methyl group on 6-position of the tricyclic pyrrole.

2.1. Chemistry

Carbohydrazides 1,2a–d and carboxamides 1e–k and 2e–l were synthesized by seven synthetic steps starting from bromo-ketone 3 or 4 (Scheme 1). Its alkylation with ethyl cyanoacetate and K_2_CO_3_ in acetone gave the corresponding cyano-ketoester (5 or 6), which served as precursor for the synthesis of the key intermediate tricyclic pyrrole 3-carboxilate (9 or 10) in the designed synthetic route.

The cyclization of cyano-ketoesters 5 or 6 furnished derivatives 7 and 8 which were dehalogenated in the presence of ammonium formate and 10% Pd/C in EtOH to yield the key intermediates 9 and 10 (Scheme 1). Their N-benzylation with 1-(chloromethyl)-4-methylbenzene in the presence of 60% NaH in mineral oil gave the corresponding p-methylbenzyl derivates (11 or 12), which were hydrolysed to acid 13 or 14. Then, acid 13 was reacted with SOCl_2_ to give the corresponding acyl chlorides (not isolated), whereas 14 was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt), followed by reaction with the appropriate amine, furnished derivatives 1a–k and 2a–l, respectively.

While the literature reporting the synthesis of pyrrole–polycyclic systems bearing substituents in both positions α and β of the pyrrole ring is extensive, analogues with the β-carboxy group as single substituent are less described. The 5-exo-trig cyclization process [15] necessary for the preparation of the 1,4-dihydroindeno[1,2-b]pyrrole-3-carboxylic system (7 and 8) precursor of 9 or 10, proposes a single-stage mechanism, starting from cyano-ketoester (5 or 6), whose ethereal solution was insufflated with HCl. This process allows the isolation of ethyl 2-chloro-6-methyl-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxylate (6 or 7) via imidoyl chloride (5′ or 6′), which, by iminium ion cyclization (7′ or 8′) followed by dehydration and deprotonation, gave the tricyclic ester (Figure 3).

2.2. Biology

2.2.1. Antiviral Activity Against ZIKV and SARS-CoV-2

New compounds were screened for their potential antiviral activity against ZIKV^Br^ on Huh-7 cells (preliminary results are reported in Table S1 in Supplementary Materials). Each compound was tested in duplicate at concentrations from 180 μM to 0.003 μM. Treatment was applied immediately after infection, and at 24–48 h post-infection, supernatants were collected to assess viral yield for ZIKV determined as PFU/mL. The same treatments were applied to uninfected cells to evaluate the cytotoxicity of each compound, comparing them with reference sofosbuvir (SOF).

Compounds 2g, 2h and 2j showed promising antiviral effects and were further characterized to determine their IC_50_, CC_50_, and selectivity index (SI) values.

Concerning the antiviral activity against ZIKV, compound 2g showed the best values with a SI of 501 (EC_50_ = 0.4 μM; CC_50_ = 230.5 μM) (Figure 4 and Table 2). Derivative 2h had a SI of 232.5 (EC_50_ = 0.9 μM; CC_50_ = 211.6 μM), and compound 2j showed a good effect against ZIKV on Huh-7 cells, with a SI of 246.4 (EC_50_ = 0.6 μM; CC_50_ = 160.2 μM). In comparison, SOF exhibited an SI of 187.8 (EC_50_ = 1.5 μM; CC_50_ = 281.7 μM).

Compounds 2g and 2j were selected to test their activity against SARS-CoV-2^Mi^ on Vero-TMPRSS, applying the same treatments described above for ZIKV, and using as reference compound EIDD-1931. Neither of them showed activity against this virus. Therefore, we determined the CC_50_ values for 2g and 2j, 220.7 and 432.4 μM, respectively, whereas the control EIDD-1931 had a CC_50_ of 28.1 μM.

2.2.2. Determination of Viral Protein Synthesis During Treatment with 2g, 2h, 2j

To determine whether the reduction in ZIKV yield by 2g, 2h and 2j was due to inhibition of viral protein synthesis or viral release, Huh-7 were infected with ZIKV^Br^ and treated with 2g, 2h, 2j or SOF at 1×, 5×, and 10× their EC_50_. Viral protein levels in cell lysates were analyzed by immunoblotting (Figure 5). Compounds 2g, 2h and 2j downregulated viral protein expression in Huh-7 cells but did not fully suppress it; similarly, SOF strongly reduced protein synthesis at 7.5 μM (5 × EC_50_) (Figure 5). These findings indicate that 2g, 2h and 2j reduced viral protein synthesis.

After testing compounds 2h, 2g and 2j on SARS-CoV-2 and observing that it was not possible to calculate an IC_50_ based on viral yield because the compounds were not active against this virus, we decided to test them again, evaluating their effect on viral protein production. A Western blot analysis was performed to detect the viral nucleocapsid protein. In agreement with previous results, the levels of viral protein were comparable to those observed in the infected, untreated control, confirming that the compounds had no antiviral activity. In contrast, treatment with the reference compound EIDD-1931 resulted in an almost complete inhibition of viral protein synthesis, which was statistically significant (Figure 6).

3. Conclusions

We synthesized 23 novel 1,4-dihydroindeno[1,2-b]pyrrole-based derivatives (1a–k, 2a–l) and evaluated their antiviral and cytotoxic activity against ZIKV and SARS-CoV-2.

In the series 1a–k the introduction of different amines on the carboxylic function do not confer antiviral activity against the selected viruses. On the contrary, the introduction of a methyl group on C-6 position of the tricyclic system, as in series 2a–l, is decisive for their activity against ZIKV. Three compounds with the best SI and the lowest IC_50_ values, 2g, 2h and 2j, were selected to further evaluate their activity against ZIKV. Two, 2g and 2j, were used to assess the anti-SARS-CoV-2 activity. From these preliminary assays, we can assume that only compounds of series 2a–l, bearing a methyl group on the C-6 position of the tricyclic system, showed antiviral activity against ZIKV, whereas SARS-CoV-2 was not affected. These preliminary data suggest that the methyl group on the phenyl ring of the tricyclic system, missing in series 1a–k, plays a role in downregulation of viral protein expression in Huh-7 cells. Moreover, it was confirmed that bulky amines as bornyl (2g), isopinocampheyl (2h) and 1-adamantylbornyl (2j) in the 3-carboxy-position of the pyrrole-based structure are preferred for increased activity of such compounds. Particularly, derivative 2g was identified as the most active of all the pyrrole-based compounds and the immunoblotting analyses showed a not fully suppression of viral protein expression in Huh-7 cells, suggesting that the reduction in ZIKV yield is due primarily to an impairing of viral protein synthesis.

Further studies are in progress to assess the impact of other structural modifications, with the aim of obtaining new information on this template and deepening the structure-activity relationships, aiming at the development of potential novel antiviral agents.

4. Experimental Section

4.1. Chemistry

4.1.1. General Methods

The melting point range of compounds was measured with a Köpfler hot stage microscopy and is uncorrected.

Thin Layer Cromatography (TLC) was performed on Poligram^®^ SIL N-HR/HV_254_ silica plates (0.2 mm). Compounds were purified using flash chromatography (FC), either automatically on a Biotage^®^ Flash-master system using pre-packed Biotage^®^ SNAP silica gel cartridges or manually with Merck^®^ Kieselgel 60 (0.040–0.063 mm) silica gel.

^1^H and ^13^C NMR spectra were acquired at room temperature using a Bruker AVANCE III Nanobay 400 MHz or a Varian Unity-200 MHz spectrophotometer. Tetramethylsilane (TMS) was used as internal standard. Spectra were acquired using as deuterated solvents dimethyl sulfoxide (DMSO-d_6_) or chloroform (CDCl_3_). Chemical shifts (δ) and coupling constants (J, expressed in Hz) were reported and multiplicities are indicated as s (singlet), br s (broad singlet), d (doublet), dd (double doublet), t (triplet) and m (multiplet). (Figures S1–S23 in Supplementary Materials).

Structures were confirmed by spectroscopic data.

IR spectra of compounds were recorded as KBr tablet using a Jasco FT/IR460 plus spectrophotometer and absorbance was indicated as wave number (ν, cm^−1^).

Reactions with air- or moisture-sensitive compounds were conducted under a nitrogen or argon atmosphere.

Agilent 1100 LC/MSD system, consisting of a single quadrupole detector (SQD) mass spectrometer (MS) equipped with an electrospray ionization (ESI) interface and a photodiode array (PDA) detector, range 120–550 nm, was used to perform LC/MS analyses applying ESI in positive mode. Mobile phases: (A) MeOH in H_2_O (8:2). Analyses were performed at a flow rate of 0.9 mL/min, temperature 350 °C. The purity of final compounds was verified through elemental analysis (C, H, and N) using a PerkinElmer 240 B elemental analyser. All synthesized final compounds were found to have a purity exceeding 95%.

Reagents and solvents were purchased from Merck^®^, Alfa Aesar^®^, and Acros Organics^®^, and used without further purification.

Ethyl 1,4-dihydroindeno[1,2-b]pyrrole-3-carboxylate (9) and ethyl 6-methyl-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxylate (10) were prepared as reported in the literature [16] or by analogy, respectively.

4.1.2. General Synthesis of Esters 11 and 12

To a solution of ester 9 or 10 (3.47 mmol) in DMF (11,56 mL) NaH 60% in mineral oil (4,16 mmol) was added under Argon and stirred for 30 min. Then, a solution of 4-methyl-benzyl chloride (3.47 mmol) in THF (3.10 mL) was added to the suspension and the whole stirred at room temperature for 5h. The mixture was taken up with H_2_O and extracted with CHCl_3_, dried (Na_2_SO_4_) and concentrated, to give a crude solid which was purified by FC furnishing the desired ester.

4.1.3. Ethyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pirrole-3-carboxilate (11)

A general procedure was used to prepare the title compound from ester 9. Subsequent FC purification (petroleum benzine/EtOAc: 8/2) furnished 11 as a white solid (750 mg, 64.99%), mp = 106–108 °C. IR (cm^−1^): 1702 (C=O). ^1^H NMR (400 MHz, CDCl_3_) δ: 1.37 (t, 3H, J = 7.0 Hz), 2.31 (s, 3H), 3.69 (s, 2H), 4.31 (q, 2H, J 6.8 Hz), 5.31 (s, 2H), 7.02–7.38 (m, 7H), 7.35 (s, 1H), 7.46 (d, 1H, J = 6.8 Hz). ^13^C NMR (100 MHz, CDCl_3_) δ: 14.15 (CH_3_), 21.33 (CH_3_), 22.54 (CH_2_), 51.57 (CH_2_), 56.12 (CH_2_), 114.28 (C), 118.63 (C), 122.70 (CH), 124.62 (CH), 126.21 (CH), 127.25 (CH × 2), 128.63 (CH), 128.94 (CH × 2), 130.01 (CH), 134.36 (C), 135.42 (C), 137.60 (C), 139.41 (C), 140.85 (C), 165.90 (C=O). Anal. calcd for C_22_H_21_NO: C, 83.78; H, 6.71; N, 4.44. Found: C, 82.37; H, 6.60; N, 4.36.

4.1.4. Ethyl-6-methyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pirrole-3-carboxilate (12)

General procedure was used to prepare title compound from ester 10. Subsequent FC purification (petroleum benzine/EtOAc: 9/1) furnished 12 as a yellow solid (680 mg, 57.05%), mp = 127–129 °C. IR (cm^−1^): 1706 (C=O). ^1^H NMR (400 MHz, CDCl_3_) δ: 1.37 (t, 3H, J = 6.8 Hz), 2.31 (s, 3H), 2.36 (s, 3H), 3.66 (s, 2H), 4.30 (q, 2H, J = 6.6 Hz), 5.31 (s, 2H), 6.96–7.10 (m, 5H), 7.26 (d, 1H, J_m_ = 1.4 Hz), 7.31 (d, 2H, J = 6.6 Hz). ^13^C NMR (100 MHz, CDCl_3_) δ: 14.21 (CH_3_), 21.33 (CH_3_), 21.65 (CH_3_), 22.30 (CH_2_), 51.46 (CH_2_), 56.24 (CH_2_), 114.10 (C), 118.62 (C), 122.75 (CH), 124.54 (CH), 126.53 (CH), 127.41 (CH × 2), 128.75 (CH × 2), 131.40 (CH), 134.44 (C), 134.62 (C), 135.35 (C), 137.31 (C), 138.34 (C), 141.02 (C), 165.71 (C=O). Anal. calcd for C_23_H_23_NO: C, 83.85; H, 7.04; N, 4.25. Found: C, 82.39; H, 7.07; N, 4.21.

4.1.5. General Synthesis of Carboxylic Acids 13 and 14

To a suspension of ester 11 or 12 (1.25 mmol) in EtOH (5.30 mL), a solution of KOH (2.61 mmol) in EtOH (4.23 mL) and H_2_O (4 drops) was added: the mixture was refluxed overnight. The solution was poured onto ice and acidified with 1N HCl and the resulting precipitate was filtered under vacuum and dissolved in KHCO_3_ aq. The basic solution was acidified with 1N HCl to precipitate the desired compound which was washed (H_2_O) and air dried to give the corresponding carboxylic acid 13 or 14.

4.1.6. 1-(4-Methylbenzyl)-1,4-dihydroindeno[1,2-b]pirrole-3-carboxilic acid (13)

Title compound was prepared from ester 11 following the general procedure, to yield 13 as a white solid (380 mg, 99.73%), mp = 232–234 °C. IR (cm^−1^): 1670 (C=O). ^1^H NMR (400 MHz, CDCl_3_) δ: 2.32 (s, 3H), 3.72 (s, 2H), 5.35 (s, 2H), 7.05–7.30 (m, 6H), 7.42 (s, 1H), 7.62 (d, 2H, J = 8.0 Hz). ^13^C NMR (100 MHz, CDCl_3_) δ: 21.32 (CH_3_), 22.51(CH_2_), 51.40 (CH_2_), 56.22 (CH_2_), 114.21 (C), 118.67 (C), 122.82 (CH), 124.53 (CH), 126.15 (CH), 127.21 (CH × 2), 128.50 (CH), 128.92 (CH × 2), 130.10 (CH), 134.33 (C), 135.42 (C), 137.53 (C), 139.34 (C), 140.81 (C), 166.25 (C=O). Anal. calcd for C_20_H_17_NO_2_: C, 79.19; H, 5.65; N, 4.62. Found: C, 78.12; H, 5.57; N, 4.56.

4.1.7. 6-Methyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pirrole-3-carboxilic acid (14)

Title compound was prepared from ester 12 following the general procedure, to yield 14 as a white solid (300 mg, 75.75%), mp = 230–232 °C. IR (cm^−1^): 1670 (C=O). ^1^H NMR (400 MHz, CDCl_3_) δ: 2.31 (s, 3H), 2.36 (s, 3H), 3.69 (s, 2H), 5.32 (s, 2H), 6.97–7.12 (m, 5H), 7.28 (d, 2H, J = 8.2 Hz), 7.39 (s, 1H). ^13^C NMR (100 MHz, CDCl_3_) δ: 21.33 (CH_3_), 21.72 (CH_3_), 22.35 (CH_2_), 51.40 (CH_2_), 56.22 (CH_2_), 114.10 (C), 118.67 (C), 122.72 (CH), 124.55 (CH), 126.53 (CH), 127.42 (CH × 2), 128.74 (CH × 2), 131.41 (CH), 134.42 (C), 134.74 (C), 135.32 (C), 137.21 (C), 138.23 (C), 141.02 (C), 166.31 (C=O). Anal. calcd for C_21_H_19_NO_2_: C, 79.47; H, 6.03; N, 4.41. Found: C, 78.42; H, 5.95; N, 4.35.

4.1.8. General Synthesis of Carbohydrazides 1a–d, 2a–d and Carboxamides 1e–k, 2e–l

Method A. A solution of acid 13 (0.49 mmol) and SOCl_2_ (1.47 mmol) in toluene (4 mL) was refluxed for 3 h. After evaporation of SOCl_2_ in excess, the residue was dissolved in CH_2_Cl_2_ (5.5 mL) and the solution was dropwise added of the appropriate hydrazine or amine (0.637 mmol) and TEA (0.64 mmol for 1a,c–g,i–k, or 1.27 mmol for 1b,h), cooling with an ice bath. The resulting solution was stirred at room temperature for 3 h, and then washed (saturated NaCl_aq_), dried (Na_2_SO_4_) and concentrated, to give a crude product which was purified by FC to yield compounds 1a–k.

Method B. To a suspension of acid 14 (0.315 mmol) in CH_2_Cl_2_ (2.12 mL) HOBt (0.378 mmol) and EDC (0.378 mmol) were added and the whole stirred at room temperature for 1.5 h. The appropriate hydrazine or amine (0.63 mmol) and TEA (0.756 mmol only for 2a,k) were added and the resulting solution was stirred at room temperature for another 2 h. Then it was washed (saturated NaCl_aq_), dried (Na_2_SO_4_) and concentrated, to give a crude product which was purified by FC to give derivatives 2a–l.

4.1.9. 1-(4-Methylbenzyl)-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-b]pirrole-3-carbohydrazide (1a)

Compound 1a was prepared following general procedure I, Method A, by a reaction of acid 13 and 1-aminopiperidine. After FC purification (petroleum ether/EtOAc 3/7), carbohydrazide 1a was isolated as a beige solid (28 mg, 14.81%). M.p.: 152.5–154.6 °C; IR (nujol) ν: 1629 (C=O), 3154 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.32–1.44 (m, 2H), 1.47–1.71 (m, 4H), 2.25 (s, 3H), 2.79–3.03 (m, 4H), 3.63 (s, 2H), 5.42 (s, 2H), 7.05 (t, 2H, J = 7.6 Hz), 7.13–7.19 (m, 4H, NH exch. with D_2_O), 7.32–7.34 (m, 1H), 7.40–7.46 (m, 2H), 7.53–7.56 (m, 1H); ^13^C NMR (100 MHz, DMSO) δ: 21.07 (CH_3_), 23.50 (CH_2_), 25.74 (CH_2_ × 2), 31.34 (CH_2_), 51.51 (CH_2_ × 2), 55.99 (CH_2_), 114.80 (C), 117.13 (CH), 119.54 (CH), 123.73 (CH), 124.93 (CH), 125.75 (CH), 126.76 (CH), 127.16 (CH), 127.73 (CH), 128.28 (C), 134.87 (C), 135.26 (C), 137.21 (C), 137.94 (C), 146.94 (C), 162.18 (C=O). MS (ESI): C_25_H_27_N_3_O requires m/z 385.22, found 386.22 [M + H]^+^. Anal. calcd for C_25_H_27_N_3_O: C, 77.89; H, 7.06; N, 10.90. Found: C, 76.58; H, 7.03; N, 10.71.

4.1.10. 1-(4-Methylbenzyl)-N-(pyrrolidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (1b)

Compound 1b was prepared following general procedure I, Method A, by a reaction of acid 13 and 1-aminopyrrolidine. After FC purification (petroleum ether/EtOAc 3/7), carbohydrazide 1b was isolated as a beige solid (39 mg, 21.97%). M.p.: 120–124 °C; IR (nujol) ν: 1629 (C=O), 3226 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.86–1.99 (m, 4H), 2.24 (s, 3H), 3.05–3.11 (m, 4H), 3.61 (s, 2H), 5.46 (s, 2H), 7.09 (t, 1H, J = 7.6 Hz), 7.13–7.20 (m, 6H, NH exch. with D_2_O), 7.36 (d, 1H, J = 7.2 Hz), 7.47 (d, 1H, J = 7.2 Hz), 7.74 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 20.61 (CH_3_), 22.17 (CH_2_ × 2), 30.89 (CH_2_), 51.03 (CH_2_), 51.17 (CH_2_), 56.12 (CH_2_), 111.78 (C), 116.96 (CH), 123.65 (CH), 125.39 (CH), 126.14 (CH), 126.42 (CH), 126.73 (CH × 2), 128.86 (C), 129.27 (CH × 2), 134.04 (C), 134.50 (C), 136.84 (C), 138.10 (C), 146.36 (C), 162.69 (C=O). MS (ESI): C_24_H_25_N_3_O requires m/z 371.20, found 372.20 [M + H]^+^. Anal. calcd for C_24_H_25_N_3_O: C, 77.60; H, 6.78; N, 11.31. Found: C, 77.37; H, 6.76; N, 11.28.

4.1.11. 1-(4-Methylbenzyl)-N-(homopiperidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (1c)

Compound 1c was prepared following general procedure I, Method A, by a reaction of acid 13 and 1-aminohomopiperidine. After FC purification (petroleum ether/EtOAc 3/7), carbohydrazide 1c was isolated as a beige solid (73 mg, 3.73%). M.p.: 169–173 °C; IR (nujol) ν: 1635 (C=O), 3226 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.58–1.70 (m, 8H), 2.25 (s, 3H), 2.97–3.06 (m, 4H), 3.61 (s, 2H), 5.40 (s, 2H), 7.05 (t, 1H, J = 7.2 Hz), 7.12–7.16 (m, 5H), 7.32 (d, 1H, J = 7.2 Hz), 7.44 (d, 1H, J = 7.2 Hz), 7.49 (s, 1H), 8.90 (br s, 1H, NH exch. with D_2_O); ^13^C NMR (100 MHz, DMSO) δ: 21.09 (CH_3_), 26.98 (CH_2_), 27.10 (CH_2_), 31.32 (CH_2_), 51.49 (CH_2_), 57.89 (CH_2_), 59.52 (CH_2_), 60.70 (CH_2_), 72.72 (CH_2_), 114.98 (C), 117.11 (CH), 123.69 (CH), 125.76 (CH), 126.76 (CH), 127.16 (CH), 127.50 (CH × 2), 129.04 (C), 129.70 (CH × 2), 134.89 (C), 135.31 (C), 137.18 (C), 137.84 (C), 146.95 (C), 162.45 (C=O). MS (ESI): C_26_H_29_N_3_O requires m/z 399.23, found 400.23 [M + H]^+^. Anal. calcd for C_26_H_29_N_3_O: C, 78.16; H, 7.32; N, 10.52. Found: C, 78.39; H, 7.34; N, 10.55.

4.1.12. 1-(4-Methylbenzyl)-N-(morpholin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (1d)

Compound 1d was prepared following general procedure I, Method A, by a reaction of acid 13 and 1-aminomorpholine. After FC purification (chloroform/acetone 8/2), carbohydrazide 1d was isolated as a beige solid (44 mg, 23.28%). M.p.: 198–205 °C; IR (nujol) ν: 1629 (C=O), 3193 (NH); ^1^H NMR (400 MHz, DMSO) δ: 2.25 (s, 3H), 2.90–3.00 (m, 4H), 3.55–3.70 (m, 6H), 5.43 (s, 2H), 7.05 (t, 1H, J = 8.0 Hz), 7.13–7.21 (m, 5H,), 7.33 (d, 1H, J = 8.0 Hz), 7.41–7.53 (m, 2H), 9.00 (br s, 1H, NH exch. with D_2_O); ^13^C NMR (100 MHz, DMSO) δ: 21.09 (CH_3_), 51.51 (CH_2_), 54.57 (CH_2_), 54.73 (CH_2_), 55.16 (CH_2_), 66.19 (CH_2_), 66.33 (CH_2_), 112.13 (C), 117.22 (CH), 125.79 (CH × 2), 126.70 (CH), 126.80 (CH), 127.53 (CH), 128.00 (C), 129.72 (CH × 2), 129.76 (CH), 134.79 (C), 135.20 (C), 136.62 (C), 137.23 (C), 146.91 (C), 162.18 (C=O). MS (ESI): C_24_H_25_N_3_O requires m/z 387.19, found 388.19 [M + H]^+^. Anal. calcd for C_24_H_25_N_3_O_2_: C, 74.39; H, 6.50; N, 10.84. Found: C, 74.20; H, 6.48; N, 10.80.

4.1.13. 1-(4-Methylbenzyl)-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1e)

Compound 1e was prepared following general procedure I, Method A, by a reaction of acid 13 and piperidine. After FC purification (petroleum ether/EtOAc 7/3), carbohydrazide 1e was isolated as a beige solid (83 mg, 46.41%). M.p.: 102–104 °C; IR (nujol) ν: 1606 (C=O); ^1^H NMR (400 MHz, DMSO) δ: 1.53–1.54 (m, 4H), 1.62–1.63 (m, 2H), 2.24 (s, 3H), 3.51 (s, 2H), 3.59 (t, 4H, J = 5.2 Hz), 5.42 (s, 2H), 7.04 (t, 1H, J = 7.6 Hz), 7.10–7.18 (m, 5H), 7.31 (d, 1H, J = 7.6 Hz), 7.33 (s, 1H), 7.42 (d, 1H, J = 7.6 Hz); ^13^C NMR (100 MHz, DMSO) δ: 21.08 (CH_3_), 24.71 (CH_2_ × 2), 26.41 (CH_2_), 31.56 (CH_2_ × 2), 51.24 (CH_2_ × 2), 115.08 (C), 117.16 (CH), 123.71 (CH), 125.72 (CH), 126.83 (CH), 127.09 (CH × 2), 128.67 (C), 128.82 (CH), 129.68 (CH × 2), 134.83 (C), 135.43 (C), 137.11 (C), 137.19 (C), 146.59 (C), 165.21 (C=O). MS (ESI): C_25_H_26_N_2_O requires m/z 370.20, found 371.20 [M + H]^+^. Anal. calcd for C_25_H_26_N_2_O: C, 81.05; H, 7.07; N, 7.56. Found: C, 79.81; H, 7.05; N, 7.54.

4.1.14. N-Cyclohexyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1f)

Compound 1f was prepared following general procedure I, Method A, by a reaction of acid 13 and cyclohexylamine. After FC purification (petroleum ether/EtOAc 8/2), carboxamide 1f was isolated as a beige solid (69 mg, 36.17%). M.p.: 189–190 °C; IR (nujol) ν: 1617 (C=O), 3272 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.11–1.38 (m, 5H), 1.58–1.86 (m, 5H), 2.25 (s, 3H), 3.67 (s, 2H), 3.70–3.73 (m, 1H), 5.41 (s, 2H), 6.67 (d, 1H, J = 7.6 Hz, NH exch. with D_2_O), 7.04 (t, 1H, J = 7.6 Hz), 7.10–7.19 (m, 5H), 7.30–7.33 (m, 1H), 7.43 (d, 1H, J = 7.6 Hz), 7.54 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 21.09 (CH_3_), 25.41 (CH_2_ × 2), 25.79 (CH_2_), 31.29 (CH_2_), 33.17 (CH_2_ × 2), 47.92 (CH), 51.49 (CH_2_), 116.32 (C), 117.09 (CH), 123.65 (CH), 125.77 (CH), 126.76 (CH), 127.14 (CH × 2), 128.93 (CH), 129.13 (C), 129.70 (CH × 2), 134.95 (C), 135.39 (C), 137.16 (C), 137.82 (C), 146.97 (C), 163.22 (C=O). MS (ESI): C_26_H_28_N_2_O requires m/z 384.22, found 385.22 [M + H]^+^. Anal. calcd for C_26_H_28_N_2_O: C, 81.21; H, 7.34; N, 7.29. Found: C, 81.40; H, 7.32; N, 7.27.

4.1.15. N-Bornyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1g)

Compound 1g was prepared following general procedure I, Method A, by a reaction of acid 13 and bornylamine. After FC purification (petroleum ether/EtOAc 8/2), carboxamide 1g was isolated as a beige solid (72 mg, 32.56%). M.p.: 151.7–153 °C; IR (nujol) ν: 1617 (C=O), 3222 (NH); ^1^H NMR (400 MHz, DMSO) δ: 0.76 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 1.08 (dd, 1H, J = 12 Hz, J = 4 Hz), 1.27 (t, 1H, J = 8.00 Hz), 1.38 (t, 1H, J = 8.00 Hz), 1.63–1.70 (m, 3H), 1.79 (t, 1H, J = 4 Hz), 2.17 (t, 1H, J = 12 Hz), 2.24 (s, 3H), 3.71 (d, 1H, J = 21.6 Hz), 3.55 (d, 1 H, J = 21.6 Hz), 4.34 (br s, 1H, NH exch. with D_2_O) 5.42 (s, 2H), 7.05 (t, 1H, J = 7.2 Hz), 7.11–7.19 (m, 5H), 7.31 (d, 1H, J = 7.6 Hz), 7.46 (d, 1H, J = 7.2 Hz), 7.65 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 14.41 (CH_3_), 18.95 (CH_3_), 20.28 (CH_3_), 21.08 (CH_3_), 28.15 (CH_2_), 28.27 (CH_2_), 31.27 (CH_2_), 35.79 (CH_2_), 44. 93 (CH), 48.23 (C), 49.86 (C), 51.50 (CH_2_), 53.04 (CH), 116.33 (C), 117.09 (CH), 123.68 (CH), 125.79 (CH), 126.78 (CH), 127.04 (CH × 2), 127.95 (CH), 129.15 (C), 129.69 (CH × 2), 134.96 (C), 135.46 (C), 137.14 (C), 137.82 (C), 146.92 (C), 164.34 (C=O). MS (ESI): C_30_H_34_N_2_O requires m/z 438.27, found 439.27 [M + H]^+^. Anal. calcd for C_30_H_34_N_2_O: C, 82.15; H, 7.81; N, 6.39. Found: C, 81.99; H, 7.79; N, 6.38.

4.1.16. N-(1S,2S,3S,5R)-(+)-Isopinocampheyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1h)

Compound 1h was prepared following general procedure I, Method A, by a reaction of acid 13 and (1S,2S,3S,5R)-(+)-isopinocampheyamine. After FC purification (petroleum ether/EtOAc 8/2), carboxamide 1h was isolated as a beige solid (52 mg, 23.72%). M.p.: 192–195 °C; IR (nujol) ν: 1619 (C=O), 3289 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.04 (s, 3H), 1.06 (s, 3H), 1.23 (s, 3H), 1.64–1.68 (m, 1H), 1.88–1.92 (m, 1H), 1.90–1.95 (m, 2H), 1.96–2.06 (m, 1H), 2.25 (s, 3H), 2.30–2.46 (m, 2H), 3.62 (d, 2H, J = 5.2 Hz), 4.26–4.38 (m, 1H), 5.42 (s, 2H), 7.06 (d, 1H, J = 6.4 Hz), 7.03–7.19 (m, 5H), 7.32 (d, 1H, J = 7.6 Hz), 7.45 (d, 1H, J = 7.6 Hz), 7.49 (d, 1H, J = 8.0 Hz, 1H, NH exch. with D_2_O), 7.57 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 20.61 (CH_3_), 20.64 (CH_3_), 23.17 (CH_3_), 27.96 (CH_3_), 30.84 (CH_2_), 33.90 (CH_2_), 36.41 (CH_2_), 38.31 (C), 41.21 (CH), 43.98 (CH), 46.56 (CH), 47.34 (CH), 51.04 (CH_2_), 115.83 (C), 116.61 (CH), 123.19 (CH), 125.31 (CH), 126.29 (CH), 126.64 (CH × 2), 128.44 (CH) 128.87 (C), 129.24 (CH × 2), 134.48 (C), 134.95 (C), 136.69 (C), 137.38 (C), 146.54 (C), 163.15 (C=O). MS (ESI): C_30_H_34_N_2_O requires m/z 438.27, found 439.27 [M + H]^+^. Anal. Calcd for C_30_H_34_N_2_O: C, 82.15; H, 7.81; N, 6.39. Found: C, 82.82; H, 7.78; N, 6.37.

4.1.17. 1-(4-Methylbenzyl)-N-myrtanyl-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1i)

Compound 1i was prepared following general procedure I, Method A, by a reaction of acid 13 and cis-myrtanylamine. After FC purification (petroleum ether/EtOAc 83/17), carboxamide 1i was isolated as a beige solid (49 mg, 22.32%). M.p.: 88–91 °C; IR (nujol) ν: 1623 (C=O), 3284 (NH); ^1^H NMR (200 MHz, CDCl_3_) δ: 0.80–0.97 (m, 2H), 1.09 (s, 3H), 1.22 (s, 3H), 1.82–2.15 (m, 5H), 2.31 (s, 3H), 2.33–2.40 (m, 2H), 3.40–3.50 (m, 2H), 3.64 (s, 2H), 5.32 (s, 2H), 5.68 (br s, 1H, NH exch. with D_2_O), 7.02–7.30 (m, 7H), 7.46 (d, 2H, J = 7.8 Hz). ^13^C NMR (50 MHz, CDCl_3_) δ: 19.93 (CH_3_), 21.08 (CH_3_), 23.26 (CH_3_), 26.05 (CH_2_), 28.02 (CH_2_), 30.88 (CH_2_), 33.30 (CH_2_), 38.75 (C), 41.38 (CH), 41.64 (CH), 43.97 (CH), 45.00 (CH_2_), 52.15 (CH_2_), 95.87 (C), 115.79 (C), 116.77 (CH), 123.62 (CH), 125.40 (CH), 126.33 (C), 126.66 (CH), 126.73 (CH × 2), 127.96 (CH), 129.60 (CH × 2), 133.55 (C), 134.86 (C), 137.69 (C), 147.19 (C), 164.63 (C=O). MS (ESI): C_30_H_34_N_2_O requires m/z 438.27, found 439.27 [M + H]^+^. Anal. calcd for C_30_H_34_N_2_O: C, 82.15; H, 7.81; N, 6.39. Found: C, 82.08; H, 7.78; N, 6.37.

4.1.18. 1-Adamantyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1j)

Compound 1j was prepared following general procedure I, Method A, by a reaction of acid 13 and 1-adamantanamine. After FC purification (petroleum ether/EtOAc 85/15), carboxamide 1j was isolated as a beige solid (83 mg, 39.43%). M.p.: 124–126 °C; IR (nujol) ν: 1631 (C=O), 3421 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.66 (s, 6H), 2.06 (s, 8H), 2.24 (s, 4H), 3.59 (s, 2H), 5.39 (s, 2H), 6.67 (br s, 1H, NH exch with D_2_O), 7.02 (t, 1H, J = 6.8 Hz), 7.09–7.18 (m, 5H), 7.31 (d, 1H, J = 7.6 Hz), 7.43 (d, 1H, J = 7.2 Hz), 7.55 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 21.08 (CH_3_), 29.21 (CH × 3), 31.21 (CH_2_), 36.60 (CH_2_ × 3), 41.77 (CH_2_ × 3), 51.35 (CH_2_), 51.47 (C), 117.08 (C), 117.13 (CH), 123.63 (CH), 125.75 (CH), 126.77 (CH), 127.09 (CH × 2), 128.05 (CH), 128.91 (C), 129.67 (CH × 2), 134.96 (C), 135.43 (C), 137.13 (C), 137.73 (C), 146.93 (C), 163.69 (C=O). MS (ESI): C_30_H_32_N_2_O requires m/z 436.25, found 437.25 [M + H]^+^. Anal. calcd for C_30_H_32_N_2_O: C, 82.53; H, 7.39; N, 6.42. Found: C, 82.37; H,7.37; N, 6.41.

4.1.19. 2-Adamantyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (1k)

Compound 1k was prepared following general procedure I, Method A, by a reaction of acid 13 and 2-adamantanamine. After FC purification (petroleum ether/EtOAc 75/25), carboxamide 1k was isolated as a beige solid (43 mg, 20.66%). M.p.: 81.9–82.3 °C; IR (nujol) ν: 1639 (C=O), 3276 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.55–2.16 (m, 14H), 2.24 (s, 3H), 3.66 (s, 2H), 4.04 (s, 1H,), 5.61 (s, 2H), 6.98 (d, 1H, J = 6.8 Hz, NH exch with D_2_O), 7.06 (t, 1H, J = 7.6 Hz), 7.04–7.18 (m, 5H), 7.32 (d, 1H, J = 7.2 Hz), 7.47 (d, 1H, J = 7.6 Hz), 7.68 (s, 1H); ^3^C NMR (100 MHz, DMSO) δ: 20.60 (CH_3_), 26.80 (CH), 30.67 (CH_2_), 31.28 (CH_2_ × 2), 31.44 (CH × 3), 36.87 (CH_2_ × 2), 37.19 (CH_2_), 51.01 (CH_2_), 52.93 (CH), 115.72 (C), 116.66 (CH), 123.23 (CH), 125.34 (CH), 126.33 (CH), 126.60 (CH × 2), 128.06 (CH), 128.39 (C), 129.20 (CH × 2), 134.47 (C), 134.94 (C), 136.66 (C), 137.36 (C), 146.34 (C), 163.17 (C=O). MS (ESI): C_30_H_32_N_2_O requires m/z 436.25, found 437.25 [M + H]^+^. Anal. calcd for C_30_H_32_N_2_O: C, 82.53; H, 7.39; N, 6.42. Found: C, 82.28; H, 7.37; N, 6.40.

4.1.20. 6-Methyl-1-(4-methylbenzyl)-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (2a)

Compound 2a was prepared following general procedure I, Method B, by a reaction of acid 14 and 1-aminopiperidine. After FC purification (petroleum ether/EtOAc 3/7), carbohydrazide 2a was isolated as a beige solid (20 mg, 16.80%). M.p.: 165–168 °C; IR (nujol) ν: 1614 (C=O); ^1^H NMR (400 MHz, DMSO) δ: 0.84–0.89 (m, 2H), 1.22–1.30 (m, 2H), 1.40–1.60 (m, 2H), 1.89–1.74 (m, 4H), 2.25 (s, 3H), 2.30 (s, 3H), 3.63 (s, 2H), 5.45 (s, 2H), 7.02 (t, 1H, J = 8.0 Hz), 7.06 (s, 1H), 7.11–7.16 (m, 4H,), 7.25 (d, 1H, J = 7.6 Hz), 7.35 (d, 1H, J = 6.8 Hz), 7.66–7.68 (m, 1H, NH exch. with D_2_O); ^13^C NMR (100 MHz, DMSO) δ: 20.61 (CH_3_), 20.94 (CH_3_), 22.36 (CH_2_), 22.90 (CH_2_), 28.32 (CH_2_), 29.76 (CH_2_), 30.75 (CH_2_), 51.19 (CH_2_), 56.18 (CH_2_), 114.80 (C), 116.74 (CH), 126.31 (CH), 126.77 (CH), 126.93 (CH), 128.21 (C), 128.81 (CH), 129.26 (CH × 2), 129.30 (CH), 131.42 (C), 134.45 (C × 2), 136.87 (C × 2), 146.67 (C), 162.11 (C=O). MS (ESI): C_26_H_29_N_3_O requires m/z 399.23, found 400.23 [M + H]^+^. Anal. calcd for C_26_H_29_N_3_O: C, 78.16; H, 7.32; N, 10.52. Found: C, 77.85; H, 7.29; N, 10.48.

4.1.21. 6-Methyl-1-(4-methylbenzyl)-N-(pyrrolidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (2b)

Compound 2b was prepared following general procedure I, Method B, by a reaction of acid 14 and 1-aminopyrrolidine. After FC purification (petroleum ether/EtOAc 3/7), carbohydrazide 2b was isolated as a white solid (30 mg, 25.34%). M.p.: 102–105 °C; IR (nujol) ν: 1460 (C=O); ^1^H NMR (200 MHz, CDCl_3_) δ: 1.80–2.10 (m, 4H), 2.30 (s, 3H), 2.36 (s, 3H), 2.98–3.20 (m, 4H), 3.64 (s, 2H), 5.30 (s, 2H), 6.86–7.38 (m, 8H), 7.62 (br s, 1H, NH exch. with D_2_O). MS (ESI): C_26_H_29_N_3_O requires m/z 385.22, found 386.22 [M + H]^+^. Anal. calcd for C_25_H_27_N_3_O: C, 77.89; H, 7.06; N, 10.90. Found: C, 77.58; H, 7.03; N, 10.86.

4.1.22. 6-Methyl-1-(4-Methylbenzyl)-N-(homopiperidin-1-yl)-1,4-dihydroindeno[1,2-b]pirrole-3-carbohydrazide (2c)

Compound 2c was prepared following general procedure I, Method B, by a reaction of acid 14 and 1-aminohomopiperidine. After FC purification (petroleum ether/EtOAc 1/1), carbohydrazide 1c was isolated as a white solid (66 mg, 16.15%). M.p.: 118–120 °C; IR (nujol) ν: 1637 (C=O), 2360 (NH); ^1^H NMR (200 MHz, CDCl_3_) δ: 1.50–1,90 (m, 8H), 2.31 (s, 3H), 2.36 (s, 3H), 3.12–3.39 (m, 4H), 3.62 (s, 2H), 5.30 (s, 2H), 6.92–7.10 (m, 8H), 7.62 (br s, 1H, NH exch. with D_2_O). MS (ESI): C_27_H_31_N_3_O requires m/z 431.25, found 432.25 [M + H]^+^. Anal. calcd for C_27_H_31_N_3_O: C, 78.42; H, 7.56; N, 10.16. Found: C, 78.11; H, 7.53; N, 10.12.

4.1.23. 1-(4-Methylbenzyl)-N-(morpholin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carbohydrazide (2d)

Compound 2d was prepared following general procedure I, Method B, by a reaction of acid 14 and 1-aminomorpholine. After FC purification (chloroform/acetone 9/1), carbohydrazide 2d was isolated as a white solid (26 mg, 20.39%). M.p.: 226–228 °C; IR (nujol) ν: 1631 (C=O), 3197 (NH); ^1^H NMR (200 MHz, CDCl_3_) δ: 2.31 (s, 3H), 2.36 (s, 3H), 2.83–3.17 (m, 4H), 3.65 (s, 2H), 3.70–4.05 (m, 4H), 5.31 (s, 2H), 6.22–6.57 (m, 1H, NH exch. with D_2_O), 6.20–7.20 (m, 8H); ^13^C NMR (50 MHz, CDCl_3_) δ: 21.09 (CH_3_), 21.38 (CH_3_), 31.35 (CH_2_), 52.18 (CH_2_), 56.54 (CH_2_ × 2), 66.50 (CH_2_ × 2), 116.41 (CH), 118.08 (C), 125.76 (CH), 126.42 (CH × 2), 126.89 (CH), 127.00 (CH), 127.20 (C), 129.62 (CH × 2), 132.09 (C), 133.11 (C), 133.43 (C), 133.62 (C), 137.80 (C), 146.95 (C). MS (ESI): C_25_H_27_N_3_O_2_ requires m/z 401.21, found 402.21 [M + H]^+^. Anal. calcd for C_25_H_27_N_3_O_2_: C, 74.79; H, 6.78; N, 10.47. Found: C, 74.49; H, 6.75; N, 10.43.

4.1.24. 6-Methyl-1-(4-Methylbenzyl)-N-(piperidin-1-yl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2e)

Compound 2e was prepared following general procedure I, Method B, by a reaction of acid 14 and piperidine. After FC purification (petroleum ether/EtOAc 1/1), carbohydrazide 2e was isolated as a white solid (93 mg, 24.72%). M.p.: 125–130 °C; IR (nujol) ν: 1614 (C=O), 3430 (NH); ^1^H NMR (200 MHz, CDCl_3_) δ: 1.43–1.83 (m, 6H), 2.30 (s, 3H), 2.34 (s, 3H), 3.55 (s, 2H), 3.62–3.71 (m, 4H), 5.29 (s, 2H), 6.90–7.18 (m, 8H); ^13^C NMR (50 MHz, CDCl_3_) δ: 21.07 (CH_3_), 21.35 (CH_3_), 24.78 (CH_2_ × 2), 26.29 (CH_2_), 31.51 (CH_2_ × 2), 51.91 (CH_2_ × 2), 115.36 (C), 116.25 (CH), 126.30 (CH), 126.71 (CH × 2), 126.90 (CH), 127.18 (CH),127.56 (C), 129.52 (CH × 2), 132.25 (C), 133.11 (C), 133.89 (C), 137.50 (C), 137.78 (C), 147.01 (C), 166.37 (C=O). MS (ESI): C_25_H_26_N_2_O requires m/z 384.22, found 385.22 [M + H]^+^. Anal. calcd for C_26_H_28_N_2_O: C, 81.21; H, 7.34; N, 7.29. Found: C, 80.88; H, 7.31; N, 7.26.

4.1.25. 6-Methyl-N-Cyclohexyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2f)

Compound 2f was prepared following general procedure I, Method B, by a reaction of acid 14 and cyclohexylamine. After FC purification (petroleum ether/EtOAc 6/4), carboxamide 1f was isolated as a white solid (104 mg, 86.66%). M.p.: 200–202 °C; IR (nujol) ν: 1627 (C=O), 3274 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.14–1.28 (m, 6H), 1.58–1.82 (m, 4H), 2.24 (s, 3H), 2.28 (s, 3H), 3.56 (s, 2H), 3.70–3.72 (m, 1H), 5.37 (s, 2H), 6.97 (d, 1H, J = 8.0 Hz), 7.11 (q, 4H, J = 7.6 Hz), 7.20 (d, 1H, J = 7.6 Hz), 7.26 (s, 1H), 7.29 (d, 1H, J = 8.0 Hz, NH exch. with D_2_O), 7.49 (s, 1H,); ^13^C NMR (100 MHz, DMSO) δ: 20.60 (CH_3_), 20.92 (CH_3_), 24.94 (CH_2_ × 2), 25.32 (CH_2_), 30.70 (CH_2_), 32.69 (CH_2_ × 2), 47.44 (CH), 51.00 (CH_2_), 115.86 (C), 116.28 (CH), 126.16 (CH), 126.68 (CH × 2), 126.71 (CH), 126.73 (CH), 127.89 (C), 129.18 (CH × 2), 131.96 (C), 132.28 (C), 134.96 (C), 136.65 (C), 137.44 (C), 146.86 (C), 162.80 (C=O). MS (ESI): C_27_H_30_N_2_O requires m/z 398.24, found 399.24 [M + H]^+^. Anal. calcd for C_27_H_30_N_2_O: C, 81.37; H, 7.59; N, 7.03. Found: C, 81.21; H, 7.57; N, 7.02.

4.1.26. 6-Methyl-N-Bornyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2g)

Compound 2g was prepared following general procedure I, Method B, by a reaction of acid 14 and bornylamine. After FC purification (petroleum ether/EtOAc 7/3), carboxamide 2g was isolated as a white solid (117 mg, 83.80%). M.p.: 165–167 °C; IR (nujol) ν: 1619 (C=O), 3313 (NH); ^1^H NMR (400 MHz, DMSO) δ: 0.76 (s, 3H), 0.86 (s, 3H), 0.94 (s, 3H), 1.07 (dd, 1H, J = 12.4 Hz, J = 4.8 Hz), 1.27 (t, 1H, J = 12 Hz), 1.40 (t, 1H, J = 8.8 H), 1.63–1.70 (m, 2H), 1.78 (t, 1H, J = 8.4 Hz), 2.16 (t, 1H, J = 12 Hz), 2.24 (s, 3H), 2.29 (s, 3H), 3.50 (d, 1 H, J = 21.6 Hz), 3.66 (d, 1 H, J = 21.6 Hz), 4.29–4.39 (m, 1H), 5.39 (s, 2H), 6.97 (d, 1H, J = 7.6 Hz), 7.09–7.20 (m, 6H), 7.27 (s, 1H, NH exch. with D_2_O), 7.60 (s, 1H,); ^13^C NMR (100 MHz, DMSO) δ: 14.41 (CH_3_), 18.95 (CH_3_), 20.28 (CH_3_), 21.08 (CH_3_), 21.40 (CH_3_), 28.15 (CH_2_), 28.26 (CH_2_), 31.16 (CH_2_), 35.78 (CH_2_), 44.93 (CH), 48.22 (C), 49.86 (C), 51.49 (CH_2_), 53.03 (CH), 116.35 (C), 116.77 (CH), 126.77 (CH), 127.06 (CH × 2), 127.23 (CH), 127.33 (CH), 128.45 (C), 129.66 (CH × 2), 132.44 (C), 132.78 (C), 135.51 (C), 137.10 (C), 137.92 (C), 147.29 (C), 164.38 (C=O). MS (ESI): C_31_H_36_N_2_O requires m/z 452.28, found 453.28 [M + H]^+^. Anal. calcd for C_31_H_36_N_2_O: C, 82.26; H, 8.02; N, 6.19. Found: C, 82.10; H, 8.00; N, 6.18.

4.1.27. 6-Methyl-N-(1S,2S,3S,5R)-(+)-Isopinocampheyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2h)

Compound 2h was prepared following general procedure I, Method B, by a reaction of acid 14 and (1S,2S,3S,5R)-(+)-isopinocampheyamine. After FC purification (petroleum ether/EtOAc 6/4), carboxamide 2h was isolated as a white solid (92 mg, 65.50%). M.p.: 225–228 °C; IR (nujol) ν: 1617 (C=O), 3278 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.03 (s, 3H), 1,06 (s, 3H), 1.22 (s, 3H), 1.62–1.67 (m, 1H), 1.79 (t, 1H, J = 4.8 Hz), 1.90–1.98 (m, 2H), 2.00–2.02 (m, 1H), 2.24 (s, 3H), 2.29 (s, 3H), 2.45–2.31 (m, 2H), 3.58 (d, 2 H, J = 4.8 Hz), 4.20–4.34 (m, 1H,) 5.38 (s, 2H), 6.97 (d, 1H, J = 7.6 Hz), 7.12 (q, 4H, J = 8.0 Hz,), 7.20 (d, 1H, J = 7.6 Hz), 7.27 (s, 1H), 7.49 (d, 1H, J = 8.4 Hz, NH exch. with D_2_O), 7.52 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 20.61 (CH_3_), 20.63 (CH_3_), 20.93 (CH_3_), 23.17 (CH_3_), 27.96 (CH_3_), 30.73 (CH_2_), 33.89 (CH_2_), 36.39 (CH_2_), 38.31 (C), 41.20 (CH), 43.97 (CH), 46.54 (CH), 47.34 (CH), 51.02 (CH_2_), 115.84 (C), 116.28 (CH), 126.19 (CH), 126.50 (CH), 126.66 (CH × 2), 126.73 (CH), 128.14 (C), 129.20 (CH × 2), 131.95 (C), 132.30 (C), 134.99 (C), 136.66 (C), 137.49 (C), 146.91 (C), 163.20 (C=O). MS (ESI): C_31_H_36_N_2_O requires m/z 452.28, found 453.28 [M + H]^+^. Anal. calcd for C_31_H_36_N_2_O: C, 82.26; H, 7.02; N, 6.19. Found: C, 82.01; H, 8.00; N, 6.17.

4.1.28. 6-Methyl-1-(4-Methylbenzyl)-N-myrtanyl-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2i)

Compound 2i was prepared following general procedure I, Method B, by a reaction of acid 14 and cis-myrtanylamine. After FC purification (petroleum ether/EtOAc 7/3), carboxamide 2i was isolated as a white solid (119 mg, 85.38%). M.p.: 88–91 °C; IR (nujol) ν: 1623 (C=O), 3268 (NH); ^1^H NMR (400 MHz, DMSO) δ: 0.81–0.87 (m, 3H), 1.06 (s, 3H), 1.18 (s, 3H), 1.49–1.59 (m, 1H), 1.79–1.97 (m, 4H), 2.24 (s, 3H), 2.28 (s, 3H), 2.30–2.36 (m, 1H), 3.23 (t, 2H, J = 6.0 Hz), 3.57 (s, 2H), 5.37 (s, 2H), 6.97 (d, 1H, J = 7.6 Hz), 7.11 (q, 4H, J = 8.4 Hz), 7.20 (d, 1H, J = 7.6 Hz), 7.26 (s, 1H), 7.44 (s, 1H), 7.52 (t, 1H, J = 6 Hz, NH exch. with D_2_O). ^13^C NMR (100 MHz, DMSO) δ: 19.64 (CH_2_), 21.08 (CH_3_), 21.39 (CH_3_), 23.38 (CH_3_), 26.18 (CH_2_), 28.31 (CH_3_), 31.17 (CH_2_), 33.30 (CH_2_), 38.75 (C), 41.32 (CH), 41.41 (CH), 43.63 (CH), 44.51 (CH_2_), 51.45 (CH_2_), 116.30 (C), 116.78 (CH), 126.64 (CH), 127.17 (CH × 2), 127.20 (CH), 127.23 (CH), 128.03 (C), 129.67 (CH × 2), 132.41 (C), 132.80 (C), 135.40 (C), 137.13 (C), 137.98 (C), 147.28 (C), 164.12 (C=O). MS (ESI): C_31_H_36_N_2_O requires m/z 452.28, found 453.28 [M + H]^+^. Anal. calcd for C_31_H_36_N_2_O: C, 82.26; H, 8.02; N, 6.19. Found: C, 82.10; H, 8.00; N, 6.18.

4.1.29. 6-Methyl-1-Adamantyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2j)

Compound 2j was prepared following general procedure I, Method B, by a reaction of acid 14 and 1-adamantanamine. After FC purification (petroleum ether/EtOAc 7/3), carboxamide 2j was isolated as a white solid (114 mg, 81.76%). M.p.: 158–162 °C; IR (nujol) ν: 1770 (C=O), 3336 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.65 (s, 6H), 2.06 (s, 9H), 2.27 (s, 3H), 2.32 (s, 3H), 3.55 (s, 2H), 5.36 (s, 2H), 6.65 (s, 1H, NH exch with D_2_O), 6.97 (d, 1H, J = 7.6 Hz), 7.05–7.10 (m, 4H), 7.19 (d, 1H, J = 7.6 Hz), 7.25 (s, 1H), 7.50 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 20.60 (CH_3_), 20.91 (CH_3_), 28.91 (CH × 3), 30.62 (CH_2_), 36.12 (CH_2_ × 2), 41.29 (CH_2_ × 3), 50.84 (CH_2_), 50.97 (C), 116.28 (CH), 116.67 (C), 126.16 (CH), 126.63 (CH × 2), 126.74 (CH), 126.96 (CH), 127.69 (C), 129.16 (CH × 2), 131.96 (C), 132.27 (C), 135.00 (C), 136.62 (C), 137.36 (C), 146.81 (C), 163.28 (C=O). MS (ESI): C_31_H_34_N_2_O requires m/z 450.27, found 451.27 [M + H]^+^. Anal. calcd for C_31_H_34_N_2_O C, 82.63; H, 7.61; N, 6.22. Found: C, 82.30; H, 7.58; N, 6.20.

4.1.30. 6-Methyl-2-Adamantyl-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2k)

Compound 2k was prepared following general procedure I, Method B, by a reaction of acid 14 and 2-adamantanamine. After FC purification (petroleum ether/EtOAc 6/4), carboxamide 2k was isolated as a white solid (79 mg, 56.52%). M.p.: 178–180 °C; IR (nujol) ν: 1644 (C=O), 3440 (NH); ^1^H NMR (400 MHz, DMSO) δ: 1.55 (d, 2H, J = 12.8 Hz), 1.73 (s, 1H), 1.78–1.84 (m, 5H), 1.94 (s, 1H), 2.05 (d, 2H, J = 12.4 Hz), 2.24 (s, 3H), 2.29 (s, 3H), 2.50–2.40 (m, 2H), 3.60 (s, 2H), 4.01–4.03 (m, 1H), 5.38 (s, 2H), 6.97 (t, 2H, J = 7.2 Hz), 7.12 (q, 4H, J = 8.4 Hz), 7.19 (d, 1H, J = 7.6 Hz), 7.28 (s, 1H), 7.44 (s, 1H, NH exch with D_2_O), 7.63 (s, 1H); ^3^C NMR (100 MHz, DMSO) δ: 21.08 (CH_3_), 21.41 (CH_3_), 27.25 (CH), 27.28 (CH), 31.04 (CH_2_), 31.75 (CH_2_ × 2), 31.93 (CH × 2), 37.35 (CH_2_ × 2), 37.66 (CH_2_), 51.47 (CH_2_), 53.39 (CH), 116.21 (C), 116.81 (CH), 126.70 (CH), 127.10 (CH × 2), 127.26 (CH), 126.94 (CH), 127.96 (C), 129.64 (CH × 2), 132.43 (C), 132.82 (C), 135.46 (C), 137.11 (C), 137.94 (C), 147.18 (C), 163.69 (C=O). MS (ESI): C_31_H_34_N_2_O requires m/z 450.27, found 451.27 [M + H]^+^. Anal. calcd for C_31_H_34_N_2_O: C, 82.63; H, 7.61; N, 6.22. Found: C, 82.47; H, 7.60; N, 6.21.

4.1.31. 6-Methyl-N-((1S,2R,5R)-menthyl)-1-(4-methylbenzyl)-1,4-dihydroindeno[1,2-b]pyrrole-3-carboxamide (2l)

Compound 2l was prepared following general procedure I, Method B, by a reaction of acid 14 and (1S,2R,5R)-menthyl-amine. After FC purification (petroleum ether/EtOAc 8/2), carboxamide 2l was isolated as a white solid (99 mg, 70.43%). M.p.: 179–181 °C; IR (nujol) ν: 1640 (C=O), 3430 (NH); ^1^H NMR (400 MHz, DMSO) δ: 0.74 (d, 3H, J = 6.8 Hz), 0.86–0.89 (m, 6H), 0.90–1.10 (m, 1H), 1.23–1.43 (m, 4H), 1.61–1.71 (m, 2H), 1.77–1.80 (m, 1H), 2.24 (s, 3H), 2.29 (s, 3H), 3.56 (q, 2H, J = 21.4 Hz), 3.70–3.78 (m, 1H), 5.37 (s, 2H), 6.97 (d, 2H, J = 7.6 Hz), 7.12 (q, 4H, J = 8.4 Hz), 7.19–7.23 (m, 2H), 7.26 (s, 1H, NH exch with D_2_O), 7.47 (s, 1H); ^13^C NMR (100 MHz, DMSO) δ: 16.04 (CH_3_), 20.61 (CH_3_), 20.92 (CH_3_), 21.08 (CH_3_), 22.24 (CH_3_), 23.52 (CH_2_), 26.12 (CH), 30.74 (CH_2_), 31.70 (CH), 34.33 (CH_2_), 42.66 (CH_2_), 46.42 (CH), 48.75 (CH), 50.99 (CH_2_), 115.97 (C), 116.28 (CH), 126.17 (CH), 126.47 (CH), 126.51 (CH), 126.72 (CH × 2), 127.84 (C), 129.20 (CH × 2), 131.95 (C), 132.29 (C), 134.95 (C), 136.66 (C), 137.47 (C), 146.88 (C), 162.92 (C=O). MS (ESI): C_31_H_38_N_2_O requires m/z 454.30, found 455.30 [M + H]^+^. Anal. calcd for C_31_H_38_N_2_O: C, 81.89; H, 8.42; N, 6.16. Found: C, 81.73; H, 8.40; N, 6.15.

4.2. Biology

4.2.1. Cell Culture

Huh-7 [17], and Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA), 1 mM glutamine, 1 mM sodium pyruvate, 7% fetal bovine serum (FBS, Gibco). Vero E6 TMPRSS2 (Vero TMPRSS) [18] were cultured like VeroE6 cells, with 1mg/mL of G418 (Merck) added once a month. All cells were cultured without antibiotics and checked for Mycoplasma as described [19].

4.2.2. Compounds Preparation and Storage

All compounds were stored at 10mM in DMSO at room temperature for 1 month. For longer storage, aliquots were kept at −20 °C.

4.2.3. Viruses

ZIKV, strain Brazil/2016/INMI1 (ZIKV^Br^) (“Lazzaro Spallanzani”—National Institute for Infectious Diseases, Rome, Italy) was propagated on Huh-7 cells. SARS-CoV-2, strain VR PV10734 (SARS-CoV2-Mi, GISAID EPI_ISL_2544194) (Università San Raffaele, Milan, Italy) was propagated on Vero-TMPRSS cells. SARS-CoV-2 and all related experiments were conducted in a biosafety level 3 (BSL-3) laboratory available at AOUP, University Hospital of Pisa.

4.2.4. Determination of Compound Cytotoxicity and Cytotoxic Concentration 50 (CC50)

Cytotoxic effects on uninfected cells were evaluated using the Orangu WST-8 assay (Cell Guidance Systems, Cambridge, UK) and the crystal violet assay [20]. Cells were seeded in 96-well plates and treated with three-fold serial dilutions of the compounds (from 0.25 to 180 μM). Huh-7 and Vero TMPRSS cells were seeded in preparation for antiviral assays. The former were seeded at 1 × 10^4^ cells/well and incubated for 48 h, whereas Vero TMPRSS cells were seeded at 1.2 × 10^4^ cells/well and incubated for 24 h to match the specific conditions, in terms of timing and culture medium, required for their antiviral assays. After the incubation, the medium was replaced with a 10% WST-8 solution in DMEM and incubated for 1 h at 37 °C. Then, optical density was measured at 450 nm with a Varioskan^®^ LUX multimode microplate reader (ThermoFisher Scientific^®^, Milan, Italy). Subsequently, cells were fixed with 4% buffered formalin solution (Merck^®^, Darmstadt, Germany), stained with 1% crystal violet (Merck^®^, Darmstadt, Germany), and the dye was solubilized in 30% acetic acid for 30 min under shaking. Optical density was then measured at 595 nm to confirm experimental outcomes. The percentage of viability at each concentration was calculated using the formula:

$$[eqn]$$

where OD_compound_ and OD_untreated_ are the OD of the cells treated with compound or DMSO, respectively.

The 50% cytotoxic concentration (CC_50_) value was calculated using nonlinear regression analysis on GraphPad Prism 7 (San Diego, CA, USA).

4.2.5. Determination of Viral Yield Reduction and Effective Concentration 50 (EC50)

Viral yield reduction assays for ZIKV and SARS-CoV-2 were performed as previously described [21]. Briefly, 1 × 10^4^ Huh-7 cells/well and 1.2 × 10^4^ Vero TMPRSS cells/well were infected with ZIKV (MOI 1) or SARS-CoV-2 (MOI 0.05) in a primary 96-well plate for 2 h at 37 °C, 5% CO_2_. Following inoculum removal, cells were treated with serial dilutions of the compounds (from 0.5 to 50 μM for initial screening; from 0.12 to 180 μM for EC_50_ determination of the most promising compounds) in DMEM with 2% FBS. Supernatants were collected after 48 h (Huh-7) or 24 h (VERO TMPRSS), diluted three-fold, and added onto VERO E6 or VERO TMPRSS monolayers, respectively. After 2 h at 37 °C, the inoculum was replaced with 0.75% carboxymethyl cellulose (Merck^®^, Darmstadt, Germany) in DMEM 2% FBS. After 48–72 h, cells were then fixed with 4% buffered formalin solution (Merck^®^, Darmstadt, Germany) and stained with 1% crystal violet (Merck^®^, Darmstadt, Germany). Titers and percentage of inhibition were calculated as follows:

$$[eqn]$$

where n is the dilution where plaques were counted:

$$[eqn]$$

where Titer-compound and Titer-untreated control are the titers (PFU/mL) obtained on cells treated with compound or DMSO, respectively.

The EC_50_ value was determined by nonlinear regression analysis on GraphPad Prism (San Diego, CA, USA). Selectivity index (SI) was obtained by dividing CC_50_ by EC_50_ for each drug/virus/cell line. Compounds with SI values ≥ 10 were active in vitro.

4.2.6. Western Blot Analysis

Immunoblotting was performed as described [22]. Briefly, cells were washed in phosphate-buffered saline (PBS) and lysed using RIPA lysis buffer added with protease and phosphatase inhibitors (ThermoFisher Scientific, Milano, Italy). Lysates were obtained in loading buffer, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% PAGE gels, and transferred onto nitrocellulose (Millipore). The antibodies used (Table 3) were diluted in PBS, 5% Skim milk, 0.1% Tween 20.