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. 2023 Jun 16;16(6):888. doi: 10.3390/ph16060888

Phenothiazine- and Carbazole-Cyanochalcones as Dual Inhibitors of Tubulin Polymerization and Human Farnesyltransferase

Andreea Zubaș 1, Alina Ghinet 1,2,3,*, Amaury Farce 4, Joëlle Dubois 5, Elena Bîcu 1,*
Editor: Carlos Alberto Manssour Fraga
PMCID: PMC10305003  PMID: 37375835

Abstract

In the search for innovative approaches to cancer chemotherapy, a chemical library of 49 cyanochalcones, 1a-r, 2a-o, and 3a-p, was designed as dual inhibitors of human farnesyltransferase (FTIs) and tubulin polymerization (MTIs) (FTIs/MTIs), two important biological targets in oncology. This approach is innovative since the same molecule would be able to interfere with two different mitotic events of the cancer cells and prevent these cells from developing an emergency route and becoming resistant to anticancer agents. Compounds were synthesized by the Claisen–Schmidt condensation of aldehydes with N-3-oxo-propanenitriles under classical magnetic stirring and under sonication. Newly synthesized compounds were screened for their potential to inhibit human farnesyltransferase, tubulin polymerization, and cancer cell growth in vitro. This study allowed for the identification of 22 FTIs and 8 dual FTIs/MTIs inhibitors. The most effective molecule was carbazole-cyanochalcone 3a, bearing a 4-dimethylaminophenyl group (IC50 (h-FTase) = 0.12 µM; IC50 (tubulin) = 0.24 µM) with better antitubulin activity than the known inhibitors that were previously reported, phenstatin and (-)-desoxypodophyllotoxin. The docking of the dual inhibitors was realized in both the active site of FTase and in the colchicine binding site of tubulin. Such compounds with a dual inhibitory profile are excellent clinical candidates for the treatment of human cancers and offer new research perspectives in the search for new anti-cancer drugs.

Keywords: tubulin, inhibitor, dual, farnesyltransferase, cancer, cyanochalcone, sonication

1. Introduction

The majority of new cancer cases detected worldwide each year are treated with anti-cancer drugs. Unfortunately, many cancer mutations become resistant to these drugs. An alternative consists of using cocktails of drugs acting on various biological targets of interest in oncology to overcome resistant cells. This type of approach can give good results, but it often leads to a large increase in side effects. Advances in the field of cell biology have allowed for the identification of new targets for the treatment of cancer, opening up new therapeutic perspectives. Another alternative to avoid the use of multiple anticancer drugs to stop cancer cell growth proliferation is to use a single compound acting on two different biological targets [1,2]. Our approach concerns the inhibition of two aspects of cell division occurring at two very different times in the life of a cancer cell: one involving farnesyltransferase and the other involving tubulin. Designed compounds target farnesyltransferase, a zinc metalloenzyme, and also inhibit tubulin polymerization. Tubulin is involved in cell proliferation due to its ability to polymerize and form microtubules, key components of the cytoskeleton. This protein is the target of a large panel of small molecules that interfere with the dynamics of its polymerization or depolymerization. Most of them bind to the laulimalide, maytansine, taxane/epothilone, vinca alkaloid, and colchicine sites [3]. By interacting with tubulin and microtubules, the tubulin polymerization inhibitors block cells in mitosis; this results in their accumulation in the G2/M phase of the cancer cell cycle. For the design of our potential dual inhibitors, two known strong inhibitors of tubulin polymerization, combretastatin A-4 (CA-4) (I, Figure 1) and phenstatin (II, Figure 1), were considered as reference molecules. Most of the modifications previously described in the structure of CA-4 and phenstatin involved either the ethylenic or carbonyl bridge or the methoxyphenol B ring. However, the 3,4,5-trimethoxyphenyl group (ring A) has long been kept intact, as it was considered essential for cytotoxic activity as well as the inhibition of tubulin polymerization. Our group previously described that a completely different A ring consisting of a phenothiazine unit can successfully replace the 3,4,5-trimethoxyphenyl of phenstatin and provide effective tubulin polymerization (e.g., compounds III and IV, Figure 1) [4,5].

The other target of molecules from this study was human protein farnesyltransferase (FTase). FTase is a heterodimeric metalloenzyme that belongs to the protein prenyl transferase family and is composed of two subunits: α (48 kDa) and β (45 kDa). Farnesylation is a post-translational modification occurring in several cell signaling proteins such as small GTPases, including the oncogenic Ras proteins that play a fundamental role in cancer cell growth and division [6]. FTase catalyzes the transfer of a farnesyl group (C15) from farnesyl pyrophosphate or farnesyl diphosphate (FPP) to the free thiol group of a cysteine residue embedded in the C-terminal CaaX motif of proteins where C is a cysteine, a is an aliphatic amino acid, and X is a serine, a methionine, an alanine, or a glutamine [7]. Preventing the farnesylation process may constitute an approach in the treatment of cancers, and, therefore, farnesyltransferase inhibitors (FTIs) were developed for anticancer therapy, and diverse compounds with druglike properties are available [7,8,9,10,11]. The use of farnesyltransferase inhibitors was disappointing in clinical trials for cancer treatment. Indeed, even if FTase is completely inhibited, a bypass is always possible for the cancerous cell. This alternative path involves a protein very similar to FTase, which is geranylgeranyltransferase I (GGTase-I) [12]. However, proving the effectiveness of dual compounds FTIs/MTIs, which are inhibitors of FTase (FTIs) and of tubulin polymerization (MTIs), may lead to an innovative approach for the design of new anti-cancer compounds.

Several associations between FTIs and MTIs were described in the literature. The association of lonafarnib (SCH66336, compound V, Figure 1) with paclitaxel resulted in an enhanced cytotoxic effect in ovarian cancer cells in vitro and in vivo [13]. The same association of lonafarnib/paclitaxel (Taxol) or lonafarnib/docetaxel (Taxotere) is synergistic in vivo in NCI-460 lung cancer cells, and lonafarnib could also be used by patients who develop resistance to taxanes. Another FTI (FTI-277, compound VI, Figure 1) displayed synergistic effect with paclitaxel or docetaxel in cells resistant to paclitaxel [14].

Figure 1.

Figure 1

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Previously described potent inhibitors of tubulin polymerization (CA-4 (I), phenstatin (II), phenothiazines (III,IV)) [4,5] and of human farnesyltransferase (Lonafarnib (SCH66336) (V) [13] and FTI-277 (VI) [14]) investigated as anticancer compounds.

Based on all these previous findings presented above, a new series of anticancer agents, dual inhibitors of farnesyltransferase and tubulin polymerization FTIs/MTIs, were developed in this study (compounds 1a-r, 2a-o, and 3a-p, Figure 2). These compounds are not prodrugs or a combination of two known specific inhibitors, rather they are original structures rationally designed from previous studies and the literature data. These compounds, 1a-r, 2a-o, and 3a-p (Figure 2), share a common bridge between the A and B rings, which is a cyanochalcone group. Another particularity of these target molecules is phenothiazine (compounds 1a-r), 2-methylthiophenothiazine (compounds 2a-o), and carbazole (compounds 3a-p) as the A ring. The literature analysis allowed for the identification of some similar phenothiazine-cyanochalcones that are nitric oxide (NO) inhibitors, preventing diseases mediated by lipid peroxidation (compound VII, Figure 2) [15], or display antibacterial activity against the Gram-positive bacteria Bacillus subtilis (compounds VIII and IX, Figure 2) or the Gram-negative bacteria Escherichia coli (compound VIII, Figure 2) [16]. To the best of our knowledge, there is no phenothiazine- or carbazole-cyanochalcone described for its anticancer properties to date. Only one similar cyanoacetamide that integrated phenothiazine (compound XII, Figure 2) displayed a modest antitumor activity in vitro, inhibiting the growth of SW1990 (86.7%) and AsPC1 (74.68%) pancreatic tumor cells at 100 µM concentration [17]. On the contrary, chalcones with a phenothiazine (compounds X and XI, Figure 2) or carbazole (compound XIII, Figure 2) or azacarbazole ring (compounds XIV and XV, Figure 2) were previously reported for their antitumor activity. Phenothiazine-chalcone X inhibited human FTase with an excellent IC50 value of 9 nM [10], while phenothiazine derivative XI was active against Hep-G2 cells [18]. Carbazole chalcone XIII was more effective in inhibiting HL-60 leukemia cells with a submicromolar range (IC50 (HL-60) = 0.22 µM, Figure 2). Azacarbazole-chalcone XIV inhibited the growth of MCF-7 breast cancer cell lines in the low micromolar range [19], while azacarbazole-chalcone XV displayed similar IC50 value in melanoma B-16 cells (Figure 2) [20]. However, increasing interest was shown in the recent literature, reporting the development of phenothiazine hybrids with potential medicinal interest and other properties [21,22,23].

Figure 2.

Figure 2

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Structure of phenothiazine/(aza)carbazole-(cyano)chalcone derivatives with biological activities (VII–XV) and of target dual FTIs/MTIs compounds 1a-r, 2a-o, and 3a-p.

2. Results and Discussion

2.1. Synthetic Strategy

The cyanochalcones (1a-r, 2a-o, and 3a-p) of this study were prepared by the Claisen–Schmidt condensation of the corresponding N-3-oxo-propanenitriles 4a, 4b, and 5 and (hetero)aryl aldehydes 8–36 (Scheme 1). N-3-oxo-propanenitriles 4a, 4b, and 5 were obtained by treating phenothiazine 6a, 2-methylthiophenothiazine 6b, and carbazole 7 with the mixed anhydride of acetic acid and cyanoacetic acid obtained in situ from an equimolar mixture of cyanoacetic acid and acetic anhydride (Scheme 1). The resulting N-3-oxo-propanenitriles 4a and 5 were previously described, and their physicochemical characterization corresponded to that reported in the literature [24,25]. Next, the key condensation reaction was conducted under classical magnetic stirring, and, based on the effective results obtained previously for the Claisen–Schmidt condensation of (hetero)aryl ketones with (hetero)aryl aldehydes [11], also under sonication of the mixture instead of classical magnetic stirring. In the classical magnetic stirring procedure (procedure A, Scheme 1), piperidine and glacial acetic acid were used as catalysts and ethanol or acetonitrile as solvents. The reaction media were stirred under reflux for the phenothiazine derivatives and at rt for the carbazole derivatives. The sonication method (procedure B, Scheme 1) used LiOH as a base and ethanol as a solvent. Both procedures, applied to the corresponding N-3-oxo-propanenitriles 4a, 4b, and 5 and aldehydes 8–36, allowed for the obtainment of a large panel of 49 cyanochalcones in medium to high yields (40–87%) (see Table 1 and Chart 1 and Chart 2). All compounds were obtained as E-isomers. No trace of Z-isomers was detected in this series. In order to conduct the greenest and least energy-consuming synthetic method possible for obtaining cyanochalcones, the synthesis of the same compound was carried out following the two procedures. This resulted in comparable yields, but the reaction time was significantly reduced from hours to minutes by sonication. As an example, the phenothiazine derivative 1m was obtained in 74% yield under sonication and in 67% yield under magnetic stirring, but after only 2 min (procedure B) against 24 h (procedure A) (Chart 1). Consequently, a major part of 2-methylthiophenothiazine-cyanochalcones 2a, 2c-f, 2h-j, and 2o was further synthesized under sonication in less than 90 s (Table 1). Under the standard conditions of procedure A, the synthesis of these latter compounds would have required refluxing ethanol or acetonitrile for 24 h. Carbazole-cyanochacones 3a-p were synthesized using procedure A, since they could be easily obtained at room temperature (Scheme 1 and Chart 2).

Scheme 1.

Scheme 1

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Reagents and conditions: (i) 1.0 equiv 6a, 6b, or 7, 2.0 equiv cyanoacetic acid, 2.0 equiv Ac2O, 50 to 100 °C, 1 h; procedure A (classical magnetic stirring): (ii) 1.0 equiv 4a, 4b, or 5, 1.2 equiv (hetero)aromatic aldehyde, piperidine (drops), glacial acetic acid (drops), EtOH or ACN, reflux, 3–24 h (phenothiazine derivatives); rt, 12–48 h (carbazole derivatives); procedure B (ultrasound-mediated experiments): (iii) 0.7 equiv LiOH, EtOH, t °C, 45–120 s.

Table 1.

Conditions of Claisen–Schimdt ultrasound-mediated reaction of phenothiazin-10-yl-chalcone analogues 1f, 1m, 2a, 2c-f, 2h-j, and 2o.

Entry Compound No. EtOH (mL) Quantity of Reagent (mmol) Quantity
of Aldehyde
(mmol)		 LiOH
(equiv.)		 Amplitude Duration (s) Ti–Tf
(°C)		 Energy (J) Yield (%)
1 1f 25 0.94 1.13 0.7 0.3 120 20–50 539 75
2 1m 30 1.87 2.26 0.7 0.3 120 19–52 575 74
3 2a 30 1.28 1.41 0.7 0.3 45 19–35 125 68
4 2c 30 1.28 1.60 0.7 0.3 60 18–41 169 78
5 2d 30 1.28 1.44 0.7 0.3 60 19–41 158 61
6 2e 30 1.28 1.53 0.7 0.3 60 19–45 160 74
7 2f 30 1.28 1.41 0.7 0.3 60 18–45 149 67
8 2h 25 1.08 1.08 0.7 0.3 90 20–59 282 67
9 2i 25 0.80 0.82 0.7 0.3 90 20–59 343 72
10 2j 30 1.28 1.43 0.7 0.3 60 19–44 169 77
11 2o 30 1.28 1.41 0.7 0.3 60 19–44 166 61
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Ti = initial medium temperature; Tf = final medium temperature.

Chart 1.

Chart 1

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Compounds with phenothiazine units synthesized in this study: 1a-r and 2a-o (for compound 1m, regular writing corresponds to yield obtained by classical magnetic stirring (procedure A), and italic bold writing corresponds to ultrasound-mediated experiment (procedure B)).

Chart 2.

Chart 2

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Compounds with carbazole unit 3a-p synthesized in this study (procedure A).

2.2. Biological Evaluation

Synthesized compounds were further evaluated in vitro on the two biological targets: farnesyltransferase and tubulin. The results of these biological evaluations are presented in Table 2. Potential inhibitors were first screened at a high concentration (100 µM), and only compounds that generally inhibited more than 60% of the proteins were selected for IC50 calculation. Dimethylsulfoxide (DMSO) was used as a negative reference, while phenstatin (II) and (-)-desoxypodophyllotoxin were positive references for the tubulin polymerization assay, and FTI-276 was the positive reference for the evaluation on human FTase (Table 2). FTI-277 (VI, Figure 1) is a prodrug of FTI-276, the latter being more affine to FTase than parent FTI-277. Interestingly, a large part of the tested cyano-chalcones inhibited FTase and presented a moderate to potent effect (IC50 values ranging from tens of micromoles (e.g., phenothiazine 1q: IC50 (h-FTase) = 44.85 µM) on submicromolar (e.g., carbazole 3a: IC50 (h-FTase) = 0.12 µM) concentrations (Table 2). In the tubulin polymerization assay, fewer compounds were found to be tubulin polymerization compounds, but two of these inhibitors significantly outperformed the potencies of the positive references phenstatin (II) and (-)-desoxypodophyllotoxin (e.g., compare phenothiazine 1l: IC50 (tubulin) = 0.71 µM or carbazole 3a: IC50 (tubulin) = 0.24 µM with the positive reference phenstatin (II), IC50 (tubulin) = 3.43 µM, and with (-)-desoxypodophyllotoxin: IC50 (tubulin) = 1.76 µM, Table 2). Moreover, four carbazole-cyanochalcones 3b, 3i, 3j, and 3l displayed similar inhibitory activity to that of the reference inhibitors (Table 2). Considering both biological evaluations, it can be concluded that several compounds inhibited the two biological targets of interest and may be considered as dual FTIs/MTIs. This is the case for one phenothiazine-cyanochalcone, 1l, and seven carbazole-cyanochalcones, 3a, 3b, 3d, 3e, 3i, 3j, and 3l. The carbazole-cyanochalcone 3a was the most potent inhibitor discovered in this study, inhibiting both targets and presenting submicromolar IC50 (0.12 µM for h-FTase and 0.24 µM for tubulin polymerization, respectively). The corresponding phenothiazine 1c was not active (Table 2). Now, looking at the chemical structures of phenothiazine 1l and carbazole 3j, they have the same classical B-ring as CA-4 (I, Figure 1) and phenstatin (II, Figure 1). This ring seems important to the biological activity against tubulin, especially in the phenothiazine series. Its replacement by other substituents in the phenothiazine cyanochalcones (1a-k, 1m-r) abolished the inhibitory effect (Table 2). On the contrary, in the series of carbazoles, the replacement of the classical 3′-hydroxy-4′-methoxyphenyl B ring was tolerated. The 4-dimethylaminophenyl group in compound 3a had the best modulation in the current study. Moreover, the reverse substitution of the classical B ring (3′-methoxy-4′-hydroxyphenyl in compound 3l instead of 3′-hydroxy-4′-methoxyphenyl in compound 3j) conserved antitubulin activity. The 3′-fluoro-4′-methoxyphenyl substitution in compound 3i was also tolerated, while the substitution of the 3′-fluoro by a 3′-chloro in compound 3h resulted in the loss of the biological activity (Table 2). The suppression of the 4′-methoxy group in carbazole 3d dramatically decreased the antitubulin potential (compare carbazole 3d (IC50 (tubulin) = 69.8 µM) with 3j (IC50 (tubulin) = 2.92 µM), Table 2). The 4′-nitro substitution in carbazole 3e conserved an inhibitory potential (IC50 (tubulin) = 10.45 µM), Table 2) but was significantly reduced compared to that in carbazole 3a.

Table 2.

Inhibitory activities of studied molecules on human farnesyltransferase and tubulin polymerization in vitro.

Entry Compound % FTase a,b IC50 (µM) b R2 c % TPI d IC50 (µM) b R2 c
1 1a 0 n.d. e - n.d. - -
2 1b 42 - - 14 - -
3 1c 65 n.d. - n.d. - -
4 1d 0 n.d. - n.d. - -
5 1e 48 n.d. - n.d. - -
6 1g 76 7.27 0.9335 5 - -
7 1h 67 18.00 0.9346 21 - -
8 1i 68 12.78 0.9868 0 - -
9 1j 94 9.80 0.9908 14 - -
10 1k 50 - - 0 - -
11 1l 85 8.29 0.9811 100 0.71 0.9957
12 1m 68 n.d. - n.d. - -
13 1n 87 1.02 0.9483
14 1o 96 0.41 0.7235 22 - -
15 1p 89 3.21 0.9617 11 - -
16 1q 65 44.85 0.9145 n.d. - -
17 1r 84 3.32 0.9865 23 - -
18 2h 72 30.51 0.9818 n.d. - -
19 2i 58 n.d. - n.d. - -
20 3a 88 0.12 0.8096 100 0.24 0.8939
21 3b 85 42.90 0.8920 99 3.00 0.9510
22 3c 98 6.50 0.9920 6 - -
23 3d 87 23.86 0.9962 63 69.80 0.8829
24 3e 100 3.14 0.9102 86 10.45 0.8343
25 3f 98 11.48 0.9793 25 - -
26 3g 100 4.32 0.9640 16 - -
27 3h 97 6.45 0.9845 10 - -
28 3i 97 18.46 0.9886 64 2.59 0.9351
29 3j 89 11.15 0.9140 100 2.92 0.8226
30 3k 89 15.71 0.9035 2 - -
31 3l 91 11.36 0.9503 100 1.63 0.9135
32 3m 99 4.30 0.9414 3 - -
33 3n 92 23.24 0.9988 27 - -
34 3o - f n.d. - 41 - -
35 3p 49 - - 26 - -
36 Phenstatin II 40 - - 99 3.43 0.9378
37 (-)-Desoxypodophyllotoxin - - - 100 1.76 0.9740
38 FTI-276 100 7 0.8369 - - -
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a Inhibition of human farnesyltransferase at 100 µM concentration. b IC50 values are indicated as the mean of two independent experiments. c Regression factor. d Inhibition of tubulin polymerization at a 100 µM concentration. e Not determined. f Data not reliable. Intrinsic fluorescence at the test wavelength.

To better visualize the distribution of the cyanochalcones from this study into selective human FTase inhibitors or dual inhibitors, their clustering was realized using POWER BI Desktop software version 2.117.984.0.(Figure 3). Eight dual FTIs/MTIs (cluster in pink, Figure 3) and twenty-two inhibitors of human farnesyltransferase (cluster in green, Figure 3) were found. The dual inhibitors were generally carbazole cyanochalcones (3a, 3b, 3d, 3e, 3i, 3j, and 3l), except for phenothiazine 1l, which also displayed dual inhibitory potential. The phenothiazine cyanochalcones displayed an FTIs profile.

Figure 3.

Figure 3

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Clustering of the studied molecules in farnesyltransferase inhibitors (FTase) (green) and dual inhibitors of farnesyltransferase and tubulin polymerization FTIs/MTIs (pink) in vitro.

2-Methylthiophenothiazine-cyanochalcones 2a-o were submitted to the National Cancer Institute (NCI) and were further selected for biological evaluation based on their panel of 60 cancer cell lines in vitro. Thus, 2-methylthiophenothiazines 2a-o were tested at a single dose of 10 µM in the full NCI-60 panel. The one-dose data are reported as an average of the growth inhibition percentage of the treated cells (Table 3 and Figure S1 in the Supplementary Materials section). Only three compounds, 2k, 2l, and 2o, from the series were active. Cyanochalcone 2k, bearing an indole unit, was the most active among the tested compounds, inhibited the growth of the majority of the tested cancer cells by more than 50% (Table 3), and even reached an inhibition greater than 90% in HL-60(TB) leukemia, NCI-H522 non-small cell lung cancer, and SF-539 and SNB-75 CNS cancer cell lines. The two other 2-methylthiophenothiazines, 2l and 2o, were significantly less active. Compound 2l, bearing a 5-methoxyindole unit, displayed moderate activity in CCRF-CEM leukemia (49% inhibition), KM12 colon cancer (49% inhibition), PC-3 prostate cancer (60% inhibition), T-47D breast cancer (57% inhibition), and CAKI-1 renal cancer (49% inhibition) cells (Table 3). Finally, compound 2o, obtained from trans-cinnamaldehyde, was the least active and only inhibited the growth of MCF7 (59% inhibition) and MDA-MB-468 (60% inhibition) breast cancer cells (Table 3).

Table 3.

Results of the in vitro human cancer cell growth inhibition for selected compounds 2k, 2l, and 2o.

Compound 2k 2l 2o 2k 2l 2o
Cell Type Cell Line GI% a,b (10 µM) Cell Type Cell Line GI% a,b (10 µM)
Leukemia CCRF-CEM 80 49 46 CNS Cancer SF-295 85 20 0
HL-60(TB) 93 19 46 SF-539 93 0 0
K-562 84 43 42 SNB-19 59 0 20
MOLT-4 70 40 41 SNB-75 91 0 35
RPMI-8226 79 0 36 U251 58 0 24
SR 66 0 0 Melanoma M14 71 36 15
Non-Small Cell Lung Cancer A549/ATCC 53 34 23 MDA-MB-435 88 20 35
EKVX 65 29 25 SK-MEL-2 64 0 0
HOP-92 56 0 0 UACC-62 58 0 38
NCI-H226 50 35 0 Ovarian Cancer IGROV1 60 0 19
NCI-H460 55 0 20 OVCAR-3 63 16 34
NCI-H522 95 39 27 Renal Cancer 786-0 50 13 17
Colon Cancer COLO 205 59 0 0 A498 89 0 28
HCT-116 76 40 30 ACHN 60 23 0
HCT-15 73 45 43 CAKI-1 61 49 40
HT29 88 42 0 RXF 393 61 27 32
KM12 76 49 15 SN12C 66 20 23
SW-620 65 15 27
Prostate Cancer PC-3 71 60 45
Breast Cancer MCF7 77 25 59
HS 578T 62 20 0
T-47D 70 57 43
MDA-MB-468 88 32 60
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a Data obtained from NCI’s in vitro 60-cell one-dose screen at 10 µM concentration. b GI% is the percentage of growth inhibition of tumor cells.

2.3. Molecular Docking of Dual FTIs/MTIs Inhibitors

The docking study was next realized on the dual FTIs/MTIs inhibitors identified in this study in the active site of FTase and in the colchicine binding site of tubulin to understand their binding mode. All the data obtained for the docking of the eight dual inhibitors are available in the Supplementary Materials section (Figure S2). The structure of the human FTase was obtained from its complexed X-ray crystal structure in the RCSB Protein Data Bank (1LD7) with FPP and the inhibitor molecule described by Bell et al. [26]. The flexible docking of FTIs into the enzyme active site was performed using GOLD 5.1 [27]. The binding site was defined by a 10 Å sphere around the cocrystallized ligand of 1LD7, and 30 poses were generated for each compound using GoldScore as the scoring function. The solutions were selected by checking the superimposition of the poses, keeping the most representative of the largest clusters. The protocol used for the docking of the selected molecules in the tubulin binding site (colchicine site) was realized as previously reported [4].

2.3.1. Docking on FTase

All the investigated compounds fit well in the pocket (Figure 4 and Figure S2). The largest number, consisting of compounds 1l, 3b, 3e, 3i, 3j, and 3l, has their tricyclic group toward the entry of the pocket, and all form interactions with the zinc ion. Tyr 601 is involved in a hydrogen bond with 1l, 3j, and 3l (Figure 4a,d and Figure S2).

Figure 4.

Figure 4

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Docking of the most active dual FTIs/MTIs inhibitors in the active site of FTase: (a) compound 1l; (b) compound 3a; (c) compound 3d; (d) compound 3l.

The tricyclic part of compounds 3a (Figure 4b) and 3d (Figure 4c) is superimposed, though more toward Trp 407 than the other molecules. Moreover, only 3d can interact with zinc, as the fluorobenzene moiety of 3a is also oriented toward Trp 407, stabilizing the compound by a distant and not optimal double stacking with it.

2.3.2. Docking in the Tubulin Binding Site (Colchicine Site)

The reference, phenstatin II, binds to the backbone of Ala 732, while mostly being in a hydrophobic region (Figure 5f). Compounds 3b and 3a (Figure 5c) and the 40% highest score of compound 1l (Figure 5a) all form a cluster closer to the entry of the binding site, where they can form a hydrogen bond with Asn 682. Compounds 3d, 3e (Figure 5d), and 3i all superimpose well in a conformation deeper in the pocket, with the cyano able to form a hydrogen bond with the Ser 168 of α tubuline.

Figure 5.

Figure 5

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Docking of the most active dual FTIs/MTIs inhibitors in the tubulin binding site (colchicine site): (a) compound 1l—40% of the solutions; (b) compound 1l—60% of the solutions; (c) compound 3a; (d) compound 3e; (e) compound 3l; (f) phenstatin.

A third cluster is formed by compounds 3j and 3l (Figure 5e) and the 60% lowest score of compound 1l (Figure 5b), with a better occupation of the deepest part of the pocket than phenstatin but fully lacking any hydrogen bond and counting on their hydrophobic fitting to stay in the binding site.

2.4. Conclusions

In this study, a chemical collection of 49 cyanochalcones, 1a-r, 2a-o, and 3a-p, decorated with phenothiazine, 2-methylthiophenothiazine, and carbazole rings was designed and synthesized by the Claisen–Schmidt condensation of the corresponding N-3-oxo-propanenitriles and aldehydes. The synthetic procedure was realized either under classical magnetic stirring and heating or under sonication of the medium. The ultrasound-assisted condensation allowed for a reduction in the reaction time from hours to minutes, especially for the synthesis of phenothiazine cyanochalcones. The therapeutic strategy described in this report was used to obtain dual FTIs/MTIs inhibitors. This approach is innovative, since the same molecule would be able to interfere with two different mitotic events of cancer cells and prevent these cells from developing an emergency route and becoming resistant to anticancer agents. Synthesized compounds were evaluated in vitro on human farnesyltransferase, on tubulin polymerization, and on the NCI-60 cancer cell lines panel. Phenothiazine derivatives proved to be inhibitors of human FTase, while carbazole derivatives displayed dual inhibition of FTase and tubulin polymerization. Of interest, phenothiazine cyanochalcone 1l and carbazole cyanochalcone 3a displayed better antitubulin activity than that of the known inhibitors previously reported: phenstatin II and (-)-desoxypodophyllotoxin. Carbazole derivatives were more active than the phenothiazine analogues. This study allowed for the identification of 22 FTIs and 8 dual FTIs/MTIs inhibitors. The most effective molecule was carbazole-cyanochalcone 3a bearing a 4-dimethylaminophenyl group (IC50 (h-FTase) = 0.12 µM; IC50 (tubulin) = 0.24 µM). The docking of the dual inhibitors was realized both in the active site of FTase and in the colchicine binding site of tubulin and allowed for the visualization of their binding modes. The biological evaluation of these promising dual inhibitors in several cancer cell lines and the evaluation of their pharmacokinetic parameters will be realized in due course. Such compounds with a dual inhibitory profile are excellent clinical candidates for the treatment of human cancers and offer new research perspectives in the search for new anti-cancer drugs.

3. Materials and Methods for Synthesis and Characterizations

Starting materials were commercially available and were used without further purification (suppliers: Carlo Erba Reagents S.A.S., Val de Reuil, France, Thermo Fisher Scientific Inc., Illkirch-Graffenstaden, France, and Sigma-Aldrich Co., Saint-Quentin-Fallavier, France). Ultrasound-mediated reactions were realized using Q700S apparatus (QSonica, LLC, Newton, MA, USA) and CL-334 model probe. Melting points were measured on a MPA 100 OptiMelt® apparatus (Stanford Research Systems, Sunnyvale, CA, USA) and a KRÜSS Optronic KSP1N apparatus (A.KRÜSS Optronic GmbH, Hamburg, Germany) and were uncorrected. Nuclear magnetic resonance (NMR) spectra were acquired at 500 MHz for 1H NMR and at 125 MHz for 13C NMR on a Bruker Avance III spectrometer (Bruker, Mannheim, Germany) and at 400 MHz for 1H NMR and at 100 MHz for 13C NMR on a Varian 400-MR spectrometer (Varian, Les Ulis, France) with tetramethylsilane (TMS) as internal standard, at room temperature (RT). All spectra were realized using deuterated solvents (CDCl3 99.8%D + 0.03% TMS V/V or DMSO-d6 99.8%D + 0.03% TMS V/V), purchased from Eurisotop, Saint-Aubin, France. The calibration was realized using TMS pic as the 0.00 ppm value in the registered spectra. Chemical shifts (δ) were expressed in ppm relative to TMS. Splitting patterns were designed: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; td, triplet of doublets; q, quadruplet; quint, quintuplet; m, multiplet; sym m, symmetric multiplet; br s, broaden singlet; br t, broaden triplet. Coupling constants (J) were reported in hertz (Hz). Thin-layer chromatography (TLC) was realized on Macherey Nagel silica gel plates with fluorescent indicator and were visualized under a UV lamp at 254 nm and 365 nm. Column chromatography was performed with a CombiFlash Rf Companion (Teledyne-Isco System, Serlabo Technologies, Entraigues sur la sorgues, France) using RediSep packed columns. IR spectra were recorded on a FT-IR Bruker Tensor 27 Spectrometer (Bruker, MA, USA) or a Cary 630 FT-IR Spectrometer (Agilent Technologies, Les Ulis, France). Elemental analyses (C, H, N, S) of new compounds were determined on a Thermo Electron apparatus by “Pôle Chimie Moléculaire-Welience”, Faculté de Sciences Mirande, Université de Bourgogne, Dijon, France.

3.1. General Procedure for the Synthesis of N-Cyanoacetyl-phenothiazines 4a and 4b and N-Cyanoacetyl-carbazole 5

A mixture of cyanoacetic acid (2 equiv.) and acetic anhydride (2 equiv.) was stirred at 50–80 °C. After complete solubilization, phenothiazine derivative 6a or 6b or carbazole 7 (1 equiv.) was added, and the mixture was stirred at 100 °C for 1 h. The precipitate formed was filtered and purified by recrystallization from ethanol to provide pure product 4a, 4b, or 5.

The physicochemical characterization of compounds 4a and 5 corresponded to that previously described in the literature [24,25].

3-(2-(Methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile (4b)

The general procedure was used with cyanoacetic acid (6.93 g, 81.6 mmol), acetic anhydride (8.34 g, 81.6 mmol), and 2-(methylthio)-10H-phenothiazine (10.00 g, 40.8 mmol) to obtain pure compound 4b (10.58 g, 33.8 mmol, 83% yield) as a mint-green solid; mp 127–128 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.51 (s, 3H, SCH3), 3.60 (s, 2H, CH2), 7.17 (dd, J = 7.5, 2.0 Hz, 1H, ArH), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.38 (dt, J = 7.5, 1.5 Hz, 2H, ArH), 7.42 (br s, 1H, ArH), 7.46–7.56 (m, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 61.0 (SCH3), 26.3 (CH2), 113.7 (CN), 123–133 (7CH+C), 137.4 (2C), 138.2 (2C), 161.1 (C=O); IR ν (cm−1): 2218, 1668, 1446, 1363, 1316, 1257, 1126, 990, 762, 730. Anal. Calcd for C16H12N2OS2: C, 61.51; H, 3.87; N, 8.97. Found: C, 61.37; H, 3.64; N, 8.81%.

3.2. General Procedure for the Synthesis of Chalcone Analogues (1a-e, 1g-r, 2b, 2g, 2k-n, and 3a-p) by Claisen–Schmidt Condensation under Classical Magnetic Stirring)—Procedure A

To a solution of phenothiazine derivative 4a or 4b or carbazole derivative 5 (1.0 equiv.) and an aromatic/heteroaromatic aldehyde (1.2 equiv.) in ethanol or acetonitrile, piperidine (3–4 drops) and glacial acetic acid (1–3 drops) were added dropwise, and the resulting solution was stirred at reflux for 3–24 h. The reaction was monitored by TLC (EtOAc:Cyclohexane) until complete consumption of starting substrate 4a, 4b, or 5. The formed precipitate was filtered, washed with ethanol, and purified by recrystallization from ethanol or by flash column chromatography (silica gel 60 (0.063–0.200 mm, 60 Å), mobile phase: gradient cyclohexane/EtOAc 100/0 to 0/100) to obtain pure cyanochalcone (1a-e, 1g-r, 2b, 2g, 2k-n, and 3a-p).

3.2.1. (E)-2-(10H-Phenothiazine-10-carbonyl)-3-phenylacrylonitrile (1a)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.67 g, 2.51 mmol), benzaldehyde 8 (0.32 g, 3.02 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 10 mL acetonitrile to obtain pure 1a (0.68 g, 1.92 mmol, 76% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 7.23–7.36 (m, 4H, ArH), 7.39–7.52 (m, 5H, ArH), 7.62 (d, J = 7.6 Hz, 2H, ArH), 7.81 (d, J = 7.6 Hz, 2H, ArH), 8.01 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 107.3 (C), 114.1 (CN), 126.3 (2CH), 127.2 (2CH), 127.4 (2CH), 128.1 (2CH), 129.1 (2CH), 130.4 (2CH), 132.0 (C), 132.5 (CH), 132.8 (2C), 138.2 (2C), 153.8 (=CH), 162.0 (C=O). IR ν (cm−1): 2207, 1661, 1590, 1327, 1182, 807, 759. Anal. Calcd for C22H14N2OS: C, 74.55; H, 3.98; N, 7.90. Found: C, 74.69; H, 4.02; N, 8.11%.

3.2.2. (E)-2-(10H-Phenothiazine-10-carbonyl)-3-(p-tolyl)acrylonitrile (1b)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 4-methylbenzaldehyde 9 (0.26 g, 2.16 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 1b (0.45 g, 1.22 mmol, 65% yield) as a yellowish solid; mp 221–223 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.40 (s, 3H, CH3), 7.22–7.35 (m, 6H, ArH), 7.49 (dd, J = 8.0, 1.0 Hz, 2H, ArH), 7.61 (d, J = 8.0 Hz, 2H, ArH), 7.73 (d, J = 8.0 Hz, 2H, ArH), 8.00 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 21.9 (CH3), 106.0 (C), 114.6 (CN), 126.4 (2CH), 127.4 (2CH), 127.5 (2CH), 128.2 (2CH), 129.6 (C), 130.0 (2CH), 130.7 (2CH), 132.9 (2C), 138.5 (2C), 143.9 (C), 154.1 (=CH), 162.4 (C=O); IR ν (cm−1): 2207, 1660, 1588, 1460, 1326, 1262, 1181, 1125, 1032, 951, 807, 766, 665, 603. Anal. Calcd for C23H16N2OS: C, 74.98; H, 4.38; N, 7.60. Found: C, 75.31; H, 4.65; N, 7.91%.

3.2.3. (E)-3-(4-(Dimethylamino)phenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1c)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.25 g, 0.94 mmol), 4-(dimethylamino)benzaldehyde 10 (0.17 g, 1.13 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL ethanol to obtain pure 1c (0.31 g, 0.78 mmol, 85% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.07 (s, 6H, 2CH3), 6.65 (d, J = 9.0 Hz, 2H, ArH), 7.22–7.28 (m, 2H, ArH), 7.30 (td, J = 7.5, 1.5 Hz, 2H, ArH), 7.48 (dd, J = 7.5, 1.5 Hz, 2H, ArH), 7.65 (dd, J = 7.5, 1.5 Hz, 2H, ArH), 7.81 (d, J = 9.0 Hz, 2H, ArH), 7.97 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 40.3 (2CH3), 98.6 (C), 111.6 (2CH), 116.2 (CN), 120.2 (C), 126.5 (2CH), 127.1 (2CH), 127.3 (2CH), 128.1 (2CH), 132.9 (2C), 133.5 (2CH), 139.1 (2C), 153.3 (C), 154.6 (=CH), 163.7 (C=O); IR ν (cm−1): 2202, 1665, 1613, 1571, 1531, 1460, 1384, 1319, 1263, 1181, 1132, 1063, 1029, 946, 889, 810, 753, 664. Anal. Calcd for C24H19N3OS: C, 72.52; H, 4.82; N, 10.57. Found: C, 72.77; H, 4.93; N, 10.68%.

3.2.4. (E)-3-(4-Methoxyphenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1d)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 4-methoxylbenzaldehyde 11 (0.31 g, 2.25 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 10 mL ethanol to obtain pure 1d (0.57 g, 1.48 mmol, 79% yield) as a yellow solid; mp 230–231 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.78 (s, 3H, OCH3), 6.87 (d, J = 9.0 Hz, 2H, ArH), 7.18–7.30 (m, 4H, ArH), 7.42 (dd, J = 7.5, 2.0 Hz, 2H, ArH), 7.55 (dd, J = 7.5, 2.0 Hz, 2H, ArH), 7.75 (d, J = 9.0 Hz, 2H, ArH), 7.88 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 55.0 (OCH3), 102.7 (C), 114.0 (2CH), 114.1 (C), 114.2 (CN), 124.2 (C), 125.7 (2CH), 125.8 (C), 126.6 (2CH), 126.7 (2CH), 127.4 (2CH), 131.9 (C), 132.2 (2CH), 137.7 (2C), 153.0 (=CH), 162.6 (C=O); IR ν (cm−1): 2213, 2156, 1677, 1592, 1511, 1459, 1427, 1320, 1259, 1180, 1121, 1060, 1019, 926, 891, 828, 757, 726, 663. Anal. Calcd for C23H16N2O2S: C, 71.85; H, 4.19; N, 7.29. Found: C, 72.05; H, 4.30; N, 7.61%.

3.2.5. (E)-3-(4-Bromophenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1e)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.25 g, 0.94 mmol), 4-bromobenzaldehyde 12 (0.21 g, 1.13 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 10 mL ethanol to obtain pure 1e (0.29 g, 0.67 mmol, 71% yield) as a yellow solid; mp 233–235 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.28–7.37 (m, 4H, ArH), 7.51 (dd, J = 7.5, 1.5 Hz, 2H, ArH), 7.55–7.63 (m, 4H, ArH), 7.68 (d, J = 7.5 Hz, 2H, ArH), 7.96 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 108.0 (C), 114.1 (CN), 126.4 (2CH), 127.4 (2CH), 127.5 (C), 127.7 (2CH), 128.3 (2CH), 131.0 (C), 131.8 (2CH), 132.6 (2CH), 132.9 (2C), 138.2 (2C), 152.6 (=CH), 161.8 (C=O); IR ν (cm−1): 2218, 1679, 1584, 1489, 1460, 1407, 1323, 1262, 1188, 1076, 1009, 816, 756. Anal. Calcd for C22H13BrN2OS: C, 60.98; H, 3.02; N, 6.46. Found: C, 61.19; H, 3.34; N, 6.62%.

3.2.6. (E)-3-(2,5-Dimethoxyphenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1g)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.30 g, 1.13 mmol), 2,5-dimethoxybenzaldehyde 19 (0.23 g, 1.38 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 15 mL ethanol to obtain pure 1g (0.35 g, 0.84 mmol, 75% yield) as a yellow solid; mp 184–186 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.74 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.87 (d, J = 9.0 Hz, 1H, ArH), 7.03 (dd, J = 9.0, 2.0 Hz, 1H, ArH), 7.27–7.36 (m, 4H, ArH), 7.61–7.67 (m, 3H, ArH), 7.90 (dd, J = 7.5, 2.0 Hz, 2H, ArH), 8.50 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 55.9 (OCH3), 56.3 (OCH3), 106.4 (C), 112.1 (CH), 112.5 (CH), 114.9 (CN), 121.5 (C), 121.5 (CH), 126.5 (2CH), 127.3 (2CH), 127.4 (2CH), 128.1 (2CH), 132.9 (C), 138.5 (2C), 148.6 (=CH), 153.4 (C), 153.5 (2C), 162.5 (C=O); IR ν (cm−1): 2201, 1666, 1575, 1495, 1457, 1358, 1306, 1228, 1161, 1041, 945, 847, 812, 159, 701, 666. Anal. Calcd for C24H18N2O3S: C, 69.55; H, 4.38; N, 6.76. Found: C, 69.90; H, 4.56; N, 7.02%.

3.2.7. (E)-3-(3-Chloro-4-methoxyphenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1h)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 3-chloro-4-methoxybenzaldehyde 20 (0.38 g, 2.25 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 1h (0.56 g, 1.35 mmol, 73% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.96 (s, 3H, OCH3), 6.93–6.99 (m, 1H, ArH), 7.27–7.38 (m, 4H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.61 (d, J = 7.5 Hz, 2H, ArH), 7.81–7.86 (m, 2H, ArH), 7.92 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.6 (OCH3), 105.5 (C), 112.2 (CH), 114.5 (CN), 123.5 (C), 125.7 (C), 126.4 (2CH), 127.4 (2CH), 127.5 (2CH), 128.3 (2CH), 130.8 (CH), 132.7 (CH), 132.9 (2C), 138.4 (2C), 152.3 (=CH), 158.5 (C), 162.2 (C=O); IR ν (cm−1): 2209, 2159, 1674, 1587, 1498, 1459, 1326, 1260, 1186, 1058, 1013, 915, 812, 757, 727, 690. Anal. Calcd for C23H15ClN2O2S: C, 65.95; H, 3.61; N, 6.69. Found: C, 66.23; H, 3.83; N, 6.92%.

3.2.8. (E)-3-(3-Fluoro-4-methoxyphenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1i)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 3-fluoro-4-methoxybenzaldehyde 21 (0.35 g, 2.25 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 1i (0.52 g, 1.29 mmol, 69% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.95 (s, 3H, OCH3), 6.99 (t, J = 8.0 Hz, 1H, ArH), 7.27–7.36 (m, 4H, ArH), 7.50 (d, J = 7.5 Hz, 2H, ArH), 7.61 (d, J = 7.5 Hz, 3H, ArH), 7.67 (d, J = 12.0 Hz, 1H, ArH), 7.94 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.5 (OCH3), 105.5 (C), 113.2 (d, J = 2.5 Hz, CH), 114.4 (CN), 117.6 (d, J = 18.75 Hz, CH), 125.3 (d, J = 7.5 Hz, C), 126.4 (2CH), 127.4 (2CH), 127.5 (2CH), 128.3 (2CH), 128.7 (d, J = 2.5 Hz, CH), 133.0 (2C), 138.4 (2C), 151.3 (d, J = 56.25 Hz, C), 152.4 (d, J = 180.0 Hz, C-F), 152.5 (d, J = 2.5 Hz, =CH), 162.2 (C=O); IR ν (cm−1): 2212, 2026, 1676, 1599, 1573, 1515, 1480, 1441, 1330, 1288, 1260, 1238, 1200, 1141, 1016, 973, 924, 871, 819, 761, 629. Anal. Calcd for C23H15FN2O2S: C, 68.64; H, 3.76; N, 6.96. Found: C, 68.90; H, 3.89; N, 7.13%.

3.2.9. (E)-3-(4-Methoxy-3-nitrophenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1j)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 4-methoxy-3-nitrobenzaldehyde 25 (0.39 g, 2.16 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 1j (0.56 g, 1.31 mmol, 70% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 4.03 (s, 3H, OCH3), 7.15 (d, J = 8.5 Hz, 1H, ArH), 7.28–7.37 (m, 4H, ArH), 7.51 (dd, J = 8.0, 2.0 Hz, 2H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 7.96 (s, 1H, =CH), 8.17–8.19 (m, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 57.1 (OCH3), 107.7 (C), 114.0 (CN), 114.2 (CH), 124.7 (C), 126.4 (2CH), 127.5 (2CH), 127.7 (2CH), 128.3 (2CH), 128.6 (CH), 133.0 (2C), 135.1 (CH), 138.2 (2C), 139.8 (C), 150.8 (=CH), 155.6 (C), 161.5 (C=O); IR ν (cm−1): 2207, 2160, 1666, 1595, 1532, 1460, 1330, 1283, 1220, 1184, 1082, 1003, 926, 865, 828, 761, 728, 666, 606. Anal. Calcd for C23H15N3O4S: C, 64.33; H, 3.52; N, 9.78. Found: C, 64.50; H, 3.72; N, 9.93%.

3.2.10. (E)-3-(2,6-Dichlorophenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1k)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 2,6-dichlorobenzaldehyde 26 (0.39 g, 2.22 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 1k (0.56 g, 1.31 mmol, 70% yield) as a yellow solid; mp 237–239 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.27–7.32 (m, 3H, ArH), 7.33–7.39 (m, 4H, ArH), 7.49 (d, J = 8.0 Hz, 2H, ArH), 7.65–7.72 (m, 2H, ArH), 8.06 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 112.2 (C), 118.1 (CN), 126.5 (2CH), 127.6 (2CH), 127.8 (2CH), 128.2 (2CH), 128.5 (2CH), 130.9 (2C), 131.5 (CH), 132.7 (C), 134.3 (2C), 137.9 (2C), 150.0 (=CH), 160.3 (C=O); IR ν (cm−1): 2159, 2032, 1663, 1618, 1578, 1479, 1459, 1430, 1343, 1264, 1186, 1031, 957, 821, 783, 768, 748, 726, 681. Anal. Calcd for C22H12ClN2OS: C, 62.42; H, 2.86; N, 6.62. Found: C, 62.78; H, 3.09; N, 6.83%.

3.2.11. (E)-3-(3-Hydroxy-4-methoxyphenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1l)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.30 g, 1.13 mmol), 3-hydroxy-4-methoxybenzaldehyde 23 (0.21 g, 1.38 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 10 mL ethanol to obtain pure 1l (0.25 g, 0.62 mmol, 55% yield) as a yellow solid; mp 218–220 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 3.89 (s, 3H, OCH3), 6.15 (br s, 1H, OH), 6.96 (d, J = 8.0 Hz, 1H, ArH), 7.21–7.38 (m, 5H, ArH), 7.49 (d, J = 7.6 Hz, 2H, ArH), 7.63 (d, J = 7.6 Hz, 2H, ArH), 7.68 (d, J = 1.2 Hz, 1H, ArH), 7.99 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 56.1 (OCH3), 103.2 (C), 110.7 (CH), 114.8 (CH), 115.1 (CN), 124.9 (C), 126.3 (2CH), 127.2 (2CH), 127.3 (2CH), 128.0 (2CH), 128.1 (CH), 132.8 (2C), 138.5 (2C), 146.7 (C), 150.2 (C), 154.3 (=CH), 162.5 (C=O). IR ν (cm−1): 3341, 2207, 1666, 1572, 1459, 1261, 757. Anal. Calcd for C23H16N2O3S: C, 68.98; H, 4.03; N, 7.00. Found: C, 68.79; H, 3.94; N, 6.87%.

3.2.12. (E)-3-(1H-Indol-3-yl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1n)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 1H-indole-3-carbaldehyde 30 (0.33 g, 2.27 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 10 mL ethanol to obtain pure 1n (0.63 g, 1.60 mmol, 85% yield) as a yellow solid; mp > 250 (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 7.27–7.35 (m, 6H, ArH), 7.44 (td, J = 8.8, 4.0 Hz, 1H, ArH), 7.50 (d, J = 7.6 Hz, 2H, ArH), 7.68 (d, J = 7.6 Hz, 2H, ArH), 7.80 (td, J = 8.8, 4.0 Hz, 1H, ArH), 8.44 (d, J = 3.2 Hz, 1H, ArH), 8.56 (s, 1H, =CH), 8.98 (br s, 1H, NH); 13C NMR (CDCl3, 100 MHz) δ ppm: 98.8 (C), 111.5 (C), 112.1 (CH), 116.7 (CN), 118.3 (CH), 122.5 (CH), 124.1 (CH), 126.5 (2CH), 127.1 (2CH), 127.2 (2CH), 127.4 (C), 128.0 (2CH), 129.7 (CH), 132.8 (2C), 135.5 (C), 138.8 (2C), 146.7 (=CH), 162.9 (C=O). IR ν (cm−1): 3293, 2216, 1651, 1561, 1459, 1329, 1291, 1227, 734. Anal. Calcd for C24H15N3OS: C, 73.26; H, 3.84; N, 10.68. Found: C, 73.38; H, 3.93; N, 10.82%.

3.2.13. (E)-3-(5-Methoxy-1H-indol-3-yl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1o)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.30 g, 1.13 mmol), 5-methoxy-1H-indole-3-carbaldehyde 31 (0.24 g, 1.37 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 1o (0.33 g, 0.78 mmol, 61% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.91 (s, 3H, OCH3), 6.96 (dd, J = 8.5, 2.5 Hz, 1H, ArH), 7.22 (d, J = 2.5 Hz, 1H, ArH), 7.27–7.34 (m, 5H, ArH), 7.50 (dd, J = 7.5, 1.5 Hz, 2H, ArH), 7.68 (dd, J = 7.5, 1.5 Hz, 2H, ArH), 8.38 (d, J = 3.0 Hz, 1H, ArH), 8.54 (s, 1H, =CH), 8.89 (br s, 1H, NH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.0 (OCH3), 98.4 (C), 100.1 (CH), 111.7 (C), 113.0 (CH), 114.7 (CH), 116.8 (CN), 126.6 (2CH), 127.3 (2CH), 127.4 (2CH), 128.2 (2CH), 128.4 (C), 130.1 (CH), 130.4 (C), 133.0 (2C), 139.0 (2C), 146.8 (=CH), 156.4 (C), 163.2 (C=O); IR ν (cm−1): 3290, 2200, 1658, 1568, 1480, 1319, 1260, 1216, 1141, 1059, 1027, 927, 867, 803, 767, 729, 662, 632. Anal. Calcd for C25H17N3O2S: C, 70.90; H, 4.05; N, 9.92. Found: C, 71.29; H, 4.41; N, 9.62%.

3.2.14. (E)-3-(5-Methoxy-1-methyl-1H-indol-3-yl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1p)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.30 g, 1.13 mmol), 5-methoxy-1-methyl-1H-indole-3-carbaldehyde 32 (0.26 g, 1.37 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 20 mL acetonitrile to obtain pure 1p (0.35 g, 0.80 mmol, 63% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.80 (s, 3H, NCH3), 3.91 (s, 3H, OCH3), 6.97 (dd, J = 8.5, 2.5 Hz, 1H, ArH), 7.22 (d, J = 2.5 Hz, 1H, ArH), 7.22–7.28 (m, 3H, ArH), 7.33 (td, J = 7.5, 1.0 Hz, 2H, ArH), 7.49 (dd, J = 7.5, 1.0 Hz, 2H, ArH), 7.68 (d, J = 7.5 Hz, 2H, ArH), 8.26 (s, 1H, ArH), 8.51 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 34.2 (CH3), 56.0 (OCH3), 96.7 (C), 100.2 (CH), 110.2 (C), 111.4 (CH), 114.2 (CH), 117.2 (CN), 126.6 (2CH), 127.1 (2CH), 127.3 (2CH), 128.1 (2CH), 129.4 (C), 131.8 (C), 133.0 (2C), 134.2 (CH), 139.1 (2C), 146.4 (=CH), 156.5 (C), 163.4 (C=O); IR ν (cm−1): 2205, 1667, 1629, 1575, 1517, 1459, 1395,1355, 1313, 1259, 1221, 1114, 1040, 836, 798, 764, 729, 702, 655, 609. Anal. Calcd for C26H19N3O2S: C, 71.38; H, 4.38; N, 9.60. Found: C, 71.26; H, 4.88; N, 9.73%.

3.2.15. (E)-3-(1-Acetyl-1H-indol-3-yl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1q)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.30 g, 1.13 mmol), 1-acetyl-1H-indole-3-carbaldehyde 33 (0.25 g, 1.35 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 1q (0.35 g, 0.80 mmol, 71% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.65 (s, 3H, CH3), 7.28–7.38 (m, 4H, ArH), 7.39–7.49 (m, 2H, ArH), 7.52 (d, J = 7.6 Hz, 2H, ArH), 7.66 (d, J = 7.6 Hz, 2H, ArH), 7.72 (d, J = 7.6 Hz, 1H, ArH), 8.44 (s, 1H, ArH), 8.47 (d, J = 7.6 Hz, 1H, ArH), 8.57 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 23.8 (CH3), 104.7 (C), 115.3 (C), 115.6 (CN), 117.0 (CH), 118.0 (CH), 124.9 (CH), 126.4 (2CH), 126.7 (CH), 127.3 (2CH), 127.5 (2CH), 128.1 (2CH), 128.4 (CH),128.9 (C), 132.8 (2C), 135.3 (C), 138.3 (2C), 144.4 (=CH), 161.5 (C=O), 168.8 (C=O). Anal. Calcd for C26H17N3O2S: C, 71.71; H, 3.93; N, 9.65. Found: C, 72.02; H, 4.06; N, 9.88%.

3.2.16. (E)-3-(Anthracen-9-yl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1r)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), anthracene-9-carbaldehyde 34 (0.45 g, 2.18 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 1r (0.72 g, 1.59 mmol, 85% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.35 (td, J = 8.0, 1.0 Hz, 2H, ArH), 7.44 (td, J = 8.0, 1.0 Hz, 2H, ArH), 7.47–7.51 (m, 4H, ArH), 7.54 (dd, J = 7.5, 1.0 Hz, 2H, ArH), 7.63–7.70 (m, 2H, ArH), 7.82 (d, J = 7.5 Hz, 2H, ArH), 7.98–8.03 (m, 2H, ArH), 8.50 (s, 1H, =CH), 8.79 (s, 1H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 113.4 (C), 117.2 (CN), 124.7 (2CH), 125.5 (C), 125.7 (2CH), 126.5 (2CH), 127.1 (2CH), 127.7 (4CH), 128.4 (2CH), 129.1 (2C), 129.2 (2CH), 130.2 (CH), 131.1 (3C), 132.9 (C), 138.4 (2C), 152.9 (=CH), 161.7 (C=O); IR ν (cm−1): 2227, 2156, 1673, 1598, 1459, 1330, 1263, 1200, 1127, 1076, 1030, 939, 885, 842, 793, 760, 729, 666, 629. Anal. Calcd for C30H18N2OS: C, 79.27; H, 3.99; N, 6.16. Found: C, 79.11; H, 3.75; N, 6.02%.

3.2.17. (E)-3-(4-(Dimethylamino)phenyl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2b)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.28 g, 0.90 mmol), 4-(dimethylamino)benzaldehyde 10 (0.16 g, 1.08 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 5 mL acetonitrile to obtain pure 2b (0.28 g, 1.58 mmol, 70% yield) as an orange solid; mp 221–223 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.46 (s, 3H, SCH3), 3.08 (s, 6H, 2CH3), 6.65 (d, J = 9.0 Hz, 2H, ArH), 7.15 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.22–7.31 (m, 2H, ArH), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.47 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.56 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.64 (d, J = 2.0 Hz, 1H, ArH), 7.81 (d, J = 9.0 Hz, 2H, ArH), 7.98 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 16.5 (SCH3), 40.1 (2NCH3), 98.4 (C), 111.6 (2CH), 116.2 (C≡N), 120.1 (C), 124.7 (CH), 125.6 (CH), 126.4 (CH), 127.2 (CH), 127.3 (CH), 128.0 (CH), 128.2 (CH), 129.1 (C), 133.1 (C), 133.6 (2CH), 138.1 (C), 139.0 (C), 139.6 (C), 153.4 (C), 154.6 (=CH), 163.8 (C=O); IR ν (cm−1): 2200, 1664, 1571, 1526, 1313, 1182, 811, 751. Anal. Calcd for C25H21N3OS2: C, 67.69; H, 4.77; N, 9.47. Found: C, 67.90; H, 4.62; N, 9.33%.

3.2.18. (E)-3-(4-(Dimethylamino)-2-methoxyphenyl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2g)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.30 g, 0.96 mmol), 2-methoxy-4-(dimethylamino)benzaldehyde 22 (0.21 g, 1.16 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 5 mL acetonitrile to obtain pure 2g (0.37 g, 0.78 mmol, 82% yield) as a yellow solid; mp 184–186 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.46 (s, 3H, SCH3), 3.08 (s, 6H, 2CH3), 3.88 (s, 3H, OCH3), 6.02 (d, J = 2.5 Hz, 1H, ArH), 6.27 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 7.13 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.21–7.30 (m, 3H, ArH), 7.33 (d, J = 7.5 Hz, 1H, ArH), 7.58 (d, J = 7.5 Hz, 1H, ArH), 7.67 (d, J = 2.0 Hz, 1H, ArH), 8.20 (d, J = 9.0 Hz, 1H, ArH), 8.53 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 16.5 (SCH3), 40.3 (2NCH3), 55.5 (OCH3), 93.2 (CH), 96.9 (C), 105.1 (CH), 110.2 (C), 116.9 (C≡N), 124.7 (CH), 125.4 (CH), 126.4 (CH),127.0 (CH), 127.3 (CH), 127.9 (CH), 128.1 (CH), 129.1 (C), 130.5 (CH), 133.0 (C), 138.0 (C), 139.3 (C), 139.8 (C), 148.2 (=CH), 155.3 (C), 161.5 (C), 164.4 (C=O); IR ν (cm−1): 2204, 1669, 1560, 1461, 1247, 1122, 809, 748, 667. Anal. Calcd for C26H23N3O2S2: C, 65.94; H, 4.89; N, 8.87. Found: C, 66.23; H, 5.04; N, 9.12%.

3.2.19. (E)-3-(1H-Indol-3-yl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2k)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.28 g, 0.90 mmol), indole-3-carboxaldehyde 30 (0.16 g, 1.08 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 6 mL ethanol to obtain pure 2k (0.25 g, 0.57 mmol, 64% yield) as a yellowish solid; mp 194–195 °C (EtOH); 1H NMR (DMSO-d6, 500 MHz) δ ppm: 2.42 (s, 3H, SCH3), 7.20–7.30 (m, 3H, ArH), 7.34 (td, J = 7.5, 1.5 Hz, 1H, ArH), 7.39 (td, J = 7.5, 1.5 Hz, 1H, ArH), 7.52 (d, J = 8.0 Hz, 1H, ArH), 7.56 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.60 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.73 (dd, J = 8.0, 1.5 Hz, 2H, ArH), 7.82 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 8.35 (s, 1H, ArH), 8.42 (s, 1H, =CH), 12.40 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 15.0 (SCH3), 96.5 (C), 110.1 (C), 112.9 (CH), 116.6 (C≡N), 118.2 (CH), 122.0 (CH), 123.5 (CH), 123.9 (CH), 124.8 (CH), 126.6 (CH), 126.9 (C), 127.3 (CH), 127.5 (CH), 127.7 (C), 128.0 (2CH), 130.9 (CH), 131.8 (C), 136.0 (C), 137.9 (C), 138.4 (C), 139.0 (C), 146.7 (=CH), 162.4 (C=O); IR ν (cm−1): 3331, 2214, 1650, 1562, 1329, 1140, 943, 735, 665. Anal. Calcd for C25H17N3OS2: C, 68.31; H, 3.90; N, 9.56. Found: C, 68.55; H, 3.73; N, 9.41%.

3.2.20. (E)-3-(5-Methoxy-1H-indol-3-yl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2l)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.30 g, 0.96 mmol), 5-methoxyindole-3-carboxaldehyde 31 (0.20 g, 1.14 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 8 mL acetonitrile to obtain pure 2l (0.32 g, 0.68 mmol, 70% yield) as a yellow solid; mp 218–220 °C (EtOH); 1H NMR (DMSO-d6, 500 MHz) δ ppm: 2.42 (s, 3H, SCH3), 3.84 (s, 3H, OCH3), 6.89 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.22 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.30–7.34 (m, 2H, ArH), 7.38 (t, J = 7.5 Hz, 1H, ArH), 7.44 (d, J = 8.5 Hz, 1H, ArH), 7.51 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.59 (d, J = 7.5 Hz, 1H, ArH), 7.70 (d, J = 7.5 Hz, 2H, ArH), 8.29 (s, 1H, ArH), 8.46 (s, 1H, =CH), 12.29 (s, 1H, NH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 15.0 (SCH3), 55.4 (OCH3), 95.6 (C), 100.1 (CH), 110.3 (C), 113.6 (CH), 113.7 (CH), 116.8 (C≡N), 123.8 (CH), 124.7 (CH), 126.5 (CH), 127.3 (CH), 127.5 (CH), 127.7 (C), 127.9 (C), 128.0 (2CH), 130.8 (C), 131.1 (CH), 131.8 (C), 137.9 (C), 138.5 (C), 139.1 (C), 147.1 (=CH), 155.6 (C), 162.6 (C=O); IR ν (cm−1): 3277, 2217, 1648, 1564, 1460, 1313, 1215, 1115, 929, 793, 739, 660. Anal. Calcd for C26H19N3O2S2: C, 66.50; H, 4.08; N, 8.95. Found: C, 66.72; H, 4.33; N, 8.87%.

3.2.21. (E)-3-(5-Methoxy-1-methyl-1H-indol-3-yl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2m)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.30 g, 0.96 mmol), 5-methoxy-1-methylindole-3-carboxaldehyde 32 (0.22 g, 1.16 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 5 mL acetonitrile to obtain pure 2m (0.36 g, 0.74 mmol, 79% yield) as a yellow solid; mp 235–237 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.47 (s, 3H, SCH3), 3.82 (s, 3H, NCH3), 3.91 (s, 3H, OCH3), 6.98 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.16 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.23 (d, J = 2.0 Hz, 1H, ArH), 7.25–7.33 (m, 3H, ArH), 7.37 (d, J = 8.5 Hz, 1H, ArH), 7.49 (dd, J = 7.5, 2.0 Hz, 1H, ArH), 7.59 (dd, J = 7.5, 2.0 Hz, 1H, ArH), 7.69 (d, J = 2.0 Hz, 1H, ArH), 8.28 (s, 1H, ArH), 8.53 (s, 1H, =CH); 13C NMR (125 MHz, CDCl3) δ ppm: 16.4 (SCH3), 34.3 (NCH3), 56.0 (OCH3), 96.5 (C), 100.1 (CH), 110.2 (C), 111.4 (CH), 114.3 (CH), 117.2 (C≡N), 124.7 (CH), 125.4 (CH), 126.5 (CH), 127.3 (CH), 127.4 (CH), 128.0 (CH), 128.2 (CH), 129.0 (C), 129.4 (C), 131.8 (C), 133.2 (C), 134.2 (CH), 138.2 (C), 139.1 (C), 139.6 (C), 146.5 (=CH), 156.5 (C), 163.4 (C=O); IR ν (cm−1): 3117, 2203, 1647, 1566, 1460, 1301, 1223, 1115, 1076, 804, 733, 642. Anal. Calcd for C27H21N3O2S2: C, 67.06; H, 4.38; N, 8.69. Found: C, 67.35; H, 4.24; N, 8.75%.

3.2.22. (E)-3-(2-Methoxynaphthalen-1-yl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2n)

General procedure A was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.30 g, 0.96 mmol), 2-methoxy-1-naphthaldehyde 35 (0.21 g, 1.16 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 6 mL acetonitrile to obtain pure 2n (0.28 g, 0.58 mmol, 61% yield) as a yellowish-green solid; mp 208–211 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.50 (s, 3H, SCH3), 3.96 (s, 3H, OCH3), 7.19 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.26–7.34 (m, 2H, ArH), 7.36–7.41 (m, 3H, ArH), 7.51 (t, J = 8.0 Hz, 2H, ArH), 7.58 (d, J = 8.5 Hz, 1H, ArH), 7.62 (s, 1H, ArH), 7.75 (d, J = 7.5 Hz, 1H, ArH), 7.79 (d, J = 8.5 Hz, 1H, ArH), 7.92 (d, J = 8.5 Hz, 1H, ArH), 8.51 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 16.0 (SCH3), 56.0 (OCH3), 113.0 (CH), 114.1 (C), 114.6 (C), 123.3 (CH), 124.2 (CH), 124.5 (CH), 125.5 (CH), 126.7 (CH), 127.3 (CH), 127.4 (CH), 128.0 (CH), 128.2 (2CH), 128.5 (2C), 128.8 (CH), 128.9 (C), 132.1 (C), 132.9 (C), 133.4 (CH), 138.4 (C), 138.6 (C), 139.1 (C), 149.9 (=CH), 156.0 (C), 162.3 (C=O); IR ν (cm−1): 2218, 1662, 1462, 1316, 1258, 1152, 1088, 939, 812, 745. Anal. Calcd for C28H20N2O2S2: C, 69.97; H, 4.19; N, 5.83. Found: C, 70.23; H, 4.35; N, 5.94%.

3.2.23. (E)-2-(9H-Carbazole-9-carbonyl)-3-(4-(dimethylamino)phenyl)acrylonitrile (3a)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 4-(dimethylamino)benzaldehyde 10 (0.31 g, 2.05 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3a (0.54 g, 1.48 mmol, 87% yield) as a white solid; mp 228–230 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.11 (s, 6H, 2CH3), 6.71 (d, J = 9.0 Hz, 2H, ArH), 7.36 (t, J = 7.5 Hz, 2H, ArH), 7.44 (t, J = 7.5 Hz, 2H, ArH), 7.91–8.04 (m, 7H, =CH+6ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 40.2 (2CH3), 98.3 (C), 111.8 (2CH), 115.4 (2CH), 117.7 (CN), 119.7 (C), 120.2 (2CH), 123.5 (2CH), 126.0 (2C), 127.0 (2CH), 134.5 (2CH), 138.7 (2C), 154.1 (C), 154.5 (=CH), 164.8 (C=O); IR ν (cm−1): 2210, 1651, 1608, 1556, 1515, 1435, 1383, 1296, 1169, 1070, 939, 889, 822, 760, 682, 617. Anal. Calcd for C24H19N3O: C, 78.88; H, 5.24; N, 11.50. Found: C, 78.66; H, 5.09; N, 11.37%.

3.2.24. (E)-2-(9H-Carbazole-9-carbonyl)-3-(4-methoxyphenyl)acrylonitrile (3b)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 4-methoxybenzaldehyde 11 (0.28 g, 2.04 mmol), piperidine (4 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3b (0.44 g, 1.25 mmol, 74% yield) as a yellow solid; mp 173–175 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.92 (s, 3H, OCH3), 7.04 (d, J = 7.0 Hz, 2H, ArH), 7.40 (td, J = 7.5, 1.0 Hz, 2H, ArH), 7.46 (td, J = 7.5, 1.0 Hz, 2H, ArH), 7.96 (d, J = 7.5 Hz, 2H, ArH), 7.97 (s, 1H, =CH), 8.01 (d, J = 7.5 Hz, 2H, ArH), 8.05 (d, J = 7.0 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 55.9 (OCH3), 104.0 (C), 115.1 (2CH), 115.5 (2CH), 116.2 (CN), 120.3 (2CH), 124.1 (2CH), 124.7 (C), 126.3 (2C), 127.3 (2CH), 133.8 (2CH), 138.5 (2C), 154.7 (=CH), 163.4 (C), 164.3 (C=O); IR ν (cm−1): 2208, 2168, 1670, 1577, 1512, 1442, 1311, 1271, 1217, 1177, 1070, 1025, 977, 893, 839, 751, 687, 617. Anal. Calcd for C23H16N2O2: C, 78.39; H, 4.58; N, 7.95. Found: C, 78.47; H, 4.68; N, 8.11%.

3.2.25. (E)-3-(4-Bromophenyl)-2-(9H-carbazole-9-carbonyl)acrylonitrile (3c)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 4-bromobenzaldehyde 12 (0.38 g, 2.04 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3c (0.52 g, 1.30 mmol, 76% yield) as a yellow solid; mp 193–194 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.42 (t, J = 7.5 Hz, 2H, ArH), 7.47 (t, J = 7.5 Hz, 2H, ArH), 7.68 (d, J = 7.5 Hz, 2H, ArH), 7.89 (d, J = 7.5 Hz, 2H, ArH), 7.92 (s, 1H, =CH), 7.95 (d, J = 7.5 Hz, 2H, ArH), 8.01 (d, J = 7.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 108.3 (C), 115.2 (CN), 115.6 (2CH), 120.4 (2CH), 124.5 (2CH), 126.5 (2C), 127.4 (2CH), 128.7 (C), 130.6 (C), 132.2 (2CH), 133.0 (2CH), 138.3 (2C), 153.3 (=CH), 162.3 (C=O); IR ν (cm−1): 2211, 1654, 1579, 1478, 1443, 1404, 1369, 1332, 1185, 1119, 1067, 1006, 953, 916, 822, 748, 692, 623. Anal. Calcd for C22H13BrN2O: C, 65.85; H, 3.27; N, 6.98. Found: C, 65.93; H, 3.31; N, 7.06%.

3.2.26. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3-hydroxyphenyl)acrylonitrile (3d)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 3-hydroxybenzaldehyde 15 (0.25 g, 2.05 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3d (0.28 g, 0.82 mmol, 48% yield) as a yellow solid; mp 202–204 °C (EtOH); 1H NMR (DMSO-d6, 500 MHz) δ ppm: 7.10 (s, 1H, ArH), 7.21–7.65 (m, 7H, ArH), 7.80–8.45 (m, 5H, =CH+4ArH), 10.06 (br s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 106.1 (C), 115.4 (2CH), 115.7 (CN), 116.3 (CH), 120.6 (2CH), 120.9 (CH), 122.2 (CH), 124.1 (2CH), 125.6 (2C), 127.3 (2CH), 130.6 (CH), 132.9 (C), 137.9 (2C), 155.8 (=CH), 157.9 (C), 162.7 (C=O); IR ν (cm−1): 3346, 2226, 1666, 1583, 1442, 1359, 1329, 1300, 1275, 1214, 1177, 984, 957, 867, 750, 683, 627. Anal. Calcd for C22H14N2O2: C, 78.09; H, 4.17; N, 8.28. Found: C, 78.40; H, 4.36; N, 8.51%.

3.2.27. (E)-2-(9H-Carbazole-9-carbonyl)-3-(4-nitrophenyl)acrylonitrile (3e)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.30 g, 1.28 mmol), 4-nitrobenzaldehyde 16 (0.23 g, 1.52 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3e (0.31 g, 0.84 mmol, 66% yield) as an orange solid; mp 221–223 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.41–7.51 (m, 4H, ArH), 7.96 (d, J = 8.0 Hz, 2H, ArH), 8.01 (s, 1H, =CH), 8.03 (d, J = 8.0 Hz, 2H, ArH), 8.17 (d, J = 8.5 Hz, 2H, ArH), 8.38 (d, J = 8.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 112.0 (C), 114.5 (CN), 115.7 (2CH), 120.6 (2CH), 124.7 (2CH), 124.9 (2CH), 126.7 (2C), 127.6 (2CH), 131.4 (2CH), 137.2 (C), 138.1 (2C), 150.0 (C), 151.0 (=CH), 161.3 (C=O); IR ν (cm−1): 2210, 1688, 1589, 1519, 1437, 1329, 1301, 1215, 1182, 1108, 1034, 1009, 937, 849, 798, 745, 654, 615. Anal. Calcd for C22H13N3O3: C, 71.93; H, 3.57; N, 11.44. Found: C, 71.77; H, 3.42; N, 11.49%.

3.2.28. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3,4-dimethoxyphenyl)acrylonitrile (3f)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.71 mmol), 3,4-dimethoxybenzaldehyde 18 (0.34 g, 2.04 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3f (0.49 g, 1.28 mmol, 76% yield) as a yellow solid; mp 191–193 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.98 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 6.98 (d, J = 8.5 Hz, 1H, ArH), 7.41 (t, J = 7.5 Hz, 2H, ArH), 7.45–7.51 (m, 3H, ArH), 7.88 (d, J = 1.5 Hz, 1H, ArH), 7.95 (s, 1H, =CH), 7.97 (d, J = 8.0 Hz, 2H, ArH), 8.03 (d, J = 8.0 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.3 (OCH3), 56.4 (OCH3), 104.0 (C), 111.3 (CH), 111.7 (CH), 115.5 (2CH), 116.4 (CN), 120.3 (2CH), 124.2 (2CH), 125.0 (C), 126.3 (2C), 127.3 (2CH), 128.2 (CH), 138.5 (2C), 149.7 (C), 154.2 (C), 155.0 (=CH), 163.4 (C=O); IR ν (cm−1): 2849, 2208, 1672, 1586, 1510, 1441, 1357, 1327, 1274, 1217, 1164, 1071, 1017, 966, 853, 814, 779, 748, 690, 628. Anal. Calcd for C24H18N2O3: C, 75.38; H, 4.74; N, 7.33. Found: C, 75.80; H, 4.91; N, 7.56%.

3.2.29. (E)-2-(9H-Carbazole-9-carbonyl)-3-(2,5-dimethoxyphenyl)acrylonitrile (3g)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.36 g, 1.54 mmol), 2,5-dimethoxybenzaldehyde 19 (0.31 g, 1.87 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3g (0.35 g, 0.92 mmol, 60% yield) as a yellow solid; mp 156–158 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.81 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.93 (d, J = 9.0 Hz, 1H, ArH), 7.15 (dd, J = 9.0, 2.0 Hz, 1H, ArH), 7.41 (t, J = 7.5 Hz, 2H, ArH), 7.48 (t, J = 7.5 Hz, 2H, ArH), 7.96–8.05 (m, 5H, =CH+4ArH), 8.50 (s, 1H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.1 (OCH3), 56.3 (OCH3), 106.9 (C), 112.3 (CH), 112.9 (CH), 115.8 (2CH), 115.9 (CN), 120.3 (2CH), 121.1 (C), 122.7 (CH), 124.2 (2CH), 126.4 (2C), 127.3 (2CH), 138.5 (2C), 149.4 (=CH), 153.7 (C), 154.2 (C), 163.2 (C=O); IR ν (cm−1): 2214, 1671, 1496, 1444, 1361, 1333, 1302, 1259, 1232, 1186, 1075, 1039, 944, 823, 749, 616. Anal. Calcd for C24H18N2O3: C, 75.38; H, 4.74; N, 7.33. Found: C, 75.75; H, 4.88; N, 7.51%.

3.2.30. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3-chloro-4-methoxyphenyl)acrylonitrile (3h)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.30 g, 1.28 mmol), 3-chloro-4-methoxybenzaldehyde 20 (0.26 g, 1.52 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3h (0.42 g, 1.09 mmol, 84% yield) as a yellow solid; mp 196–198 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 4.00 (s, 3H, OCH3), 7.05 (d, J = 8.0 Hz, 1H, ArH), 7.40 (t, J = 7.5 Hz, 2H, ArH), 7.46 (t, J = 7.5 Hz, 2H, ArH), 7.87 (s, 1H, ArH), 7.94 (d, J = 8.0 Hz, 2H, ArH), 7.99–8.06 (m, 4H, =CH+3ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.7 (OCH3), 105.7 (C), 112.4 (CH), 115.5 (2CH), 115.6 (CN), 120.4 (2CH), 124.0 (C), 124.3 (2CH), 125.2 (C), 126.4 (2C), 127.3 (2CH), 131.6 (CH), 133.1 (CH), 138.4 (2C), 153.0 (=CH), 159.3 (C), 162.8 (C=O); IR ν (cm−1): 2205, 1665, 1570, 1502, 1443, 1319, 1273, 1183, 1067, 1017, 955, 882, 846, 809, 760, 722, 676, 618. Anal. Calcd for C23H15ClN2O2: C, 71.41; H, 3.91; N, 7.24. Found: C, 71.29; H, 3.78; N, 7.10%.

3.2.31. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3-fluoro-4-methoxyphenyl)acrylonitrile (3i)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.30 g, 1.28 mmol), 3-fluoro-4-methoxybenzaldehyde 21 (0.24 g, 1.56 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3i (0.27 g, 0.72 mmol, 56% yield) as a yellow solid; mp 214–216 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 4.00 (s, 3H, OCH3), 7.09 (t, J = 8.5 Hz, 1H, ArH), 7.41 (t, J = 7.5 Hz, 2H, ArH), 7.47 (t, J = 7.5 Hz, 2H, ArH), 7.79 (d, J = 8.5 Hz, 1H, ArH), 7.88–7.92 (m, 2H, =CH+ArH), 7.95 (d, J = 7.5 Hz, 2H, ArH), 8.02 (d, J = 7.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.6 (OCH3), 105.7 (C), 113.5 (d, J = 2.5 Hz, CH), 115.6 (2CH), 115.7 (CN), 118.0 (d, J = 18.75 Hz, CH), 120.4 (2CH), 124.3 (2CH), 124.8 (d, J = 6.25 Hz, C), 126.4 (2C), 127.3 (2CH), 129.5 (d, J = 3.75 Hz, CH), 138.4 (2C), 151.9 (d, J = 145 Hz, C-F), 152.9 (d, J = 92.5 Hz, C), 153.4 (d, J = 2.5 Hz, =CH), 163.9 (C=O); IR ν (cm−1): 2208, 1668, 1562, 1518, 1445, 1327, 1283, 1187, 1128, 1019, 961, 875, 808, 760, 685, 621. Anal. Calcd for C23H15FN2O2: C, 74.59; H, 4.08; N, 7.56. Found: C, 74.77; H, 4.32; N, 7.74%.

3.2.32. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3-hydroxy-4-methoxyphenyl)acrylonitrile (3j)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 3-hydroxy-4-methoxybenzaldehyde 23 (0.31 g, 2.04 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3j (0.51 g, 1.39 mmol, 81% yield) as a yellow solid; mp 211–213 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 4.01 (s, 3H, OCH3), 5.76 (br s, 1H, OH), 6.99 (d, J = 8.5 Hz, 1H, ArH), 7.41 (td, J = 7.5, 1.0 Hz, 2H, ArH), 7.47 (td, J = 7.5, 1.0 Hz, 2H, ArH), 7.62 (dd, J = 8.5, 2.0 Hz, 1H, ArH), 7.70 (d, J = 2.0 Hz, 1H, ArH), 7.91 (s, 1H, =CH), 7.96 (d, J = 7.5 Hz, 2H, ArH), 8.02 (d, J = 7.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.4 (OCH3), 104.8 (C), 111.0 (CH), 115.6 (2CH), 115.9 (CN), 116.4 (CH), 120.3 (2CH), 124.2 (2CH), 125.5 (C), 125.9 (CH), 126.4 (2C), 127.3 (2CH), 138.5 (2C), 146.3 (C), 151.4 (C), 154.8 (=CH), 163.4 (C=O); IR ν (cm−1): 3325, 2208, 1641, 1590, 1509, 1440, 1370, 1331, 1277, 1209, 1147, 1072, 1022, 980, 933, 872, 816, 779, 742, 714. Anal. Calcd for C23H16N2O3: C, 74.99; H, 4.38; N, 7.60. Found: C, 75.26; H, 4.49; N, 7.78%.

3.2.33. (E)-2-(9H-Carbazole-9-carbonyl)-3-(2,6-dichlorophenyl)acrylonitrile (3k)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 2,6-dichlorobenzaldehyde 26 (0.36 g, 2.05 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3k (0.48 g, 1.23 mmol, 72% yield) as a yellow solid; mp 173–175 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 7.10–7.60 (m, 8H, ArH), 7.90–8.25 (m, 4H, =CH+3ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 110.7 (CH), 113.3 (C), 115.9 (CH), 118.7 (CN), 119.6 (CH), 120.4 (2CH), 123.5 (C), 124.8 (CH), 125.9 (CH), 126.6 (C), 127.4 (CH), 128.8 (CH), 130.4 (C), 132.0 (2CH), 134.4 (C), 138.1 (2C), 139.6 (C), 150.8 (=CH), 160.4 (C=O); IR ν (cm−1): 2158, 1679, 1599, 1449, 1365, 1325, 1213, 1187, 1093, 995, 928, 859, 788, 721, 678. Anal. Calcd for C22H12Cl2N2O: C, 67.54; H, 3.09; N, 7.16. Found: C, 67.31; H, 3.02; N, 7.04%.

3.2.34. (E)-2-(9H-Carbazole-9-carbonyl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile (3l)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 4-hydroxy-3-methoxybenzaldehyde 24 (0.31 g, 2.04 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 15 mL acetonitrile to obtain pure 3l (0.48 g, 1.30 mmol, 77% yield) as a yellow solid; mp 212–214 °C (EtOH); 1H NMR (DMSO-d6, 500 MHz) δ ppm: 3.90 (s, 3H, OCH3), 7.16 (d, J = 8.5 Hz, 1H, ArH), 7.45 (t, J = 7.5 Hz, 2H, ArH), 7.48–7.55 (m, 3H, ArH), 7.71 (d, J = 2.0 Hz, 1H, ArH), 7.93 (d, J = 8.5 Hz, 2H, ArH), 8.16 (s, 1H, =CH), 8.24 (d, J = 7.5 Hz, 2H, ArH), 9.77 (br s, 1H, OH); 13C NMR (DMSO-d6, 125 MHz) δ ppm: 55.9 (OCH3), 102.0 (C), 112.2 (CH), 115.2 (2CH), 115.8 (CH), 116.3 (CN), 120.5 (2CH), 123.8 (2CH), 124.5 (C), 125.4 (2C), 126.4 (CH), 127.2 (2CH), 137.9 (2C), 147.0 (C), 153.2 (C), 155.9 (=CH), 163.4 (C=O); IR ν (cm−1): 3322, 2208, 1641, 1590, 1509, 1440, 1370, 1332, 1277, 1209, 1148, 1072, 1023, 980, 933, 872, 816, 779, 742, 714, 626. Anal. Calcd for C23H16N2O3: C, 74.99; H, 4.38; N, 7.60. Found: C, 75.27; H, 4.61; N, 7.82%.

3.2.35. (E)-2-(9H-Carbazole-9-carbonyl)-3-(4-methoxy-3-nitrophenyl)acrylonitrile (3m)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.30 g, 1.28 mmol), 3-nitro-4-methoxybenzaldehyde 25 (0.28 g, 1.54 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3m (0.35 g, 0.88 mmol, 68% yield) as a yellow solid; mp 233–235 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 4.10 (s, 3H, OCH3), 7.28 (d, J = 8.5 Hz, 1H, ArH), 7.43 (t, J = 7.5 Hz, 2H, ArH), 7.49 (t, J = 7.5 Hz, 2H, ArH), 7.93–7.96 (m, 3H, =CH+2ArH), 8.03 (d, J = 7.5 Hz, 2H, ArH), 8.38 (d, J = 2.0 Hz, 1H, ArH), 8.44 (dd, J = 8.5, 2.0 Hz, 1H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 57.3 (OCH3), 107.9 (C), 114.6 (CH), 115.2 (CN), 115.6 (2CH), 120.5 (2CH), 124.3 (C), 124.6 (2CH), 126.6 (2C), 127.5 (2CH), 129.1 (CH), 135.6 (CH), 138.3 (2C), 140.1 (C), 151.5 (=CH), 156.3 (C), 162.1 (C=O); IR ν (cm−1): 2208, 1665, 1617, 1578, 1534, 1501, 1444, 1357, 1330, 1294, 1167, 1090, 1011, 959, 896, 862, 819, 762, 665, 622. Anal. Calcd for C23H15N3O4: C, 69.52; H, 3.80; N, 10.57. Found: C, 69.73; H, 3.95; N, 10.89%.

3.2.36. (E)-2-(9H-Carbazole-9-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (3n)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.40 g, 1.70 mmol), 3,4,5-trimethoxybenzaldehyde 27 (0.40 g, 2.05 mmol), piperidine (3 drops), and glacial acetic acid (3 drops) in 15 mL acetonitrile to obtain pure 3n (0.28 g, 0.68 mmol, 40% yield) as a yellow solid; mp 178–180 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.93 (s, 6H, 2OCH3), 3.99 (s, 3H, OCH3), 7.34 (s, 2H, ArH), 7.42 (t, J = 7.5 Hz, 2H, ArH), 7.48 (t, J = 7.5 Hz, 2H, ArH), 7.91 (s, 1H, =CH), 7.97 (d, J = 7.5 Hz, 2H, ArH), 8.03 (d, J = 7.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.5 (2OCH3), 61.3 (OCH3), 105.9 (C), 108.7 (2CH), 115.6 (2CH), 116.0 (CN), 120.4 (2CH), 124.3 (2CH), 126.4 (2C), 126.9 (C), 127.3 (2CH), 138.4 (2C), 143.3 (C), 153.6 (2C), 154.8 (=CH), 163.0 (C=O); IR ν (cm−1): 2210, 1673, 1568, 1502, 1441, 1326, 1297, 1222, 1160, 1129, 1074, 1034, 995, 938, 867, 829, 754, 643, 615. Anal. Calcd for C25H20N2O4: C, 72.80; H, 4.89; N, 6.79. Found: C, 73.11; H, 5.03; N, 6.98%.

3.2.37. (E)-2-(9H-Carbazole-9-carbonyl)-3-(5-methoxy-1H-indol-3-yl)acrylonitrile (3o)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.25 g, 1.07 mmol), 5-methoxy-1H-indole-3-carbaldehyde 31 (0.22 g, 1.26 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3o (0.29 g, 0.74 mmol, 69% yield) as a yellow solid; mp > 250 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.85 (s, 3H, OCH3), 6.98 (d, J = 8.5 Hz, 1H, ArH), 7.16 (s, 1H, ArH), 7.33–7.51 (m, 5H, ArH), 7.98 (d, J = 7.0 Hz, 2H, ArH), 8.04 (d, J = 7.0 Hz, 2H, ArH), 8.58 (s, 1H, =CH), 8.72 (s, 1H, ArH), 9.21 (br s, 1H, NH); 13C NMR (CDCl3, 125 MHz) δ ppm: 56.0 (OCH3), 98.8 (C), 100.3 (CH), 112.0 (C), 113.3 (CH), 115.0 (CH), 115.4 (2CH), 116.0 (CN), 120.3 (2CH), 123.8 (2CH), 126.1 (2C), 127.1 (2CH), 128.6 (C), 130.6 (C), 131.5 (CH), 138.7 (2C), 147.9 (=CH), 156.8 (C), 164.1 (C=O); IR ν (cm−1): 3412, 2917, 2206, 2019, 1978, 1665, 1571, 1481, 1441, 1360, 1299, 1245, 1212, 1137, 1056, 939, 880, 827, 800, 768, 679, 618. Anal. Calcd for C25H17N3O2: C, 76.71; H, 4.38; N, 10.74. Found: C, 76.96; H, 4.59; N, 10.92%.

3.2.38. (E)-2-(9H-Carbazole-9-carbonyl)-3-(5-methoxy-1-methyl-1H-indol-3-yl)acrylonitrile (3p)

General procedure A was used with 3-(9H-carbazol-9-yl)-3-oxopropanenitrile 5 (0.25 g, 1.07 mmol), 5-methoxy-1-methyl-1H-indole-3-carbaldehyde 32 (0.24 g, 1.27 mmol), piperidine (3 drops), and glacial acetic acid (1 drop) in 10 mL acetonitrile to obtain pure 3p (0.37 g, 0.92 mmol, 85% yield) as a yellow solid; mp 227–229 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 3.85 (s, 3H, NCH3), 3.92 (s, 3H, OCH3), 6.98–7.03 (m, 1H, ArH), 7.14–7.18 (m, 1H, ArH), 7.31 (t, J = 8.0 Hz, 1H, ArH), 7.39 (t, J = 8.0 Hz, 2H, ArH), 7.46 (t, J = 8.0 Hz, 2H, ArH), 7.96 (d, J = 8.0 Hz, 2H, ArH), 8.03 (dd, J = 8.0 Hz, 2H, ArH), 8.55 (s, 1H, =CH), 8.59 (s, 1H, ArH); 13C NMR (CDCl3, 125 MHz) δ ppm: 34.5 (CH3), 56.0 (OCH3), 97.0 (C), 100.5 (CH), 110.6 (C), 111.7 (CH), 114.5 (CH), 115.3 (2CH), 118.6 (CN), 120.2 (2CH), 123.5 (2CH), 126.0 (2C), 127.0 (2CH), 129.6 (C), 132.1 (C), 135.5 (CH), 138.8 (2C), 147.5 (=CH), 157.0 (C), 164.4 (C=O). IR ν (cm−1): 2198, 1658, 1621, 1568, 1519, 1476, 1442, 1360, 1296, 1233, 1195, 1133, 1070, 1044, 935, 862, 851, 743, 683, 641. Anal. Calcd for C26H19N3O2: C, 77.02; H, 4.72; N, 10.36. Found: C, 76.89; H, 4.56; N, 10.22%.

3.3. General Procedure for the Ultrasound-Mediated Synthesis of Chalcone Analogues (1f, 1m, 2a, 2c-f, 2h-j, and 2o) by Claisen–Schmidt Condensation—Procedure B

In a beaker, to a mixture of N-3-oxo-propanenitrile 4a or 4b (1 equiv.), aldehyde (1–1.2 equiv.) and lithium hydroxide (0.7 equiv.) in ethanol, at room temperature, an ultrasonic agitation was applied for 45 to 120 s (amplitude = 0.3). After cooling to rt for 1 to 5 h, a precipitate was formed, filtered, and purified by recrystallization from ethanol to obtain target cyanochalcone 1f, 1m, 2a, 2c-f, 2h-j, or 2o as a pure solid.

3.3.1. (E)-3-(2,4-Dichlorophenyl)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (1f)

General procedure B was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.25 g, 0.94 mmol), 2,4-dichlorobenzaldehyde 17 (0.20 g, 1.13 mmol), and lithium hydroxide (0.02 g, 0.66 mmol) in 25 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 120 s (amplitude = 0.3; ti = 20 °C; tf = 50 °C; E = 539 J). After 3 h, a precipitate was formed, filtered, and purified by recrystallization from ethanol to obtain pure compound 1f (0.30 g, 0.71 mmol, 75% yield) as a yellow solid; mp 176–178 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 7.27–7.39 (m, 5H, ArH), 7.48–7.52 (m, 3H, ArH), 7.62 (d, J = 8.0 Hz, 2H, ArH), 7.92 (d, J = 8.0 Hz, 1H, ArH), 8.20 (s, 1H, CH=); 13C NMR (CDCl3, 100 MHz) δ ppm: 110.7 (C), 113.5 (CN), 126.3 (2CH), 127.4 (2CH), 127.6 (2CH), 127.8 (CH), 128.2 (2CH), 128.9 (C), 130.1 (C), 130.1 (CH), 132.7 (2C), 136.3 (C), 137.9 (CH), 138.6 (2C), 148.1 (=CH), 161.2 (C=O); IR ν (cm−1): 2205, 1660, 1579, 1479, 1460, 1325, 1291, 1263, 1239, 1196, 1155, 1100, 1029, 960, 927, 865, 825, 753, 728, 653. Anal. Calcd for C22H12Cl2N2OS: C, 62.42; H, 2.86; N, 6.62. Found: C, 62.37; H, 2.94; N, 6.80%.

3.3.2. (E)-2-(10H-Phenothiazine-10-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (1m)

General procedure A was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 3,4,5-trimethoxybenzaldehyde 27 (0.44 g, 2.26 mmol), piperidine (4 drops), and glacial acetic acid (3 drops) in 15 mL ethanol to obtain pure 1m (0.55 g, 1.24 mmol, 74% yield) as a yellow solid.

General procedure B was used with 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile 4a (0.50 g, 1.87 mmol), 3,4,5-trimethoxybenzaldehyde 27 (0.44 g, 2.26 mmol), and lithium hydroxide (0.03 g, 1.25 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 120 s (amplitude = 0.3; ti = 19 °C; tf = 52 °C; E = 575 J). After 1 h, a precipitate was formed, filtered, and purified by recrystallization from ethanol to obtain pure compound 1m (0.61 g, 1.38 mmol, 74% yield) as a yellow solid; mp 192–194 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 3.86 (s, 6H, 2OCH3), 3.92 (s, 3H, OCH3), 7.14 (s, 2H, ArH), 7.22–7.38 (m, 4H, ArH), 7.50 (d, J = 7.6 Hz, 2H, ArH), 7.62 (d, J = 7.6 Hz, 2H, ArH), 7.99 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 56.3 (2OCH3), 61.1 (OCH3), 105.5 (C), 108.0 (2CH), 114.6 (CN), 126.3 (2CH), 127.2 (C), 127.3 (2CH), 127.4 (2CH), 128.1 (2CH), 132.8 (2C), 138.3 (2C), 142.2 (C), 153.2 (2C), 154.1 (=CH), 162.1 (C=O); IR ν (cm−1): 2218, 1662, 1462, 1316, 1258, 1152, 812, 745. Anal. Calcd for C25H20N2O4S: C, 67.55; H, 4.54; N, 6.30. Found: C, 67.41; H, 4.36; N, 6.68%.

3.3.3. (E)-2-(2-(Methylthio)-10H-phenothiazine-10-carbonyl)-3-(p-tolyl)acrylonitrile (2a)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), 4-methylbenzaldehyde 9 (0.17 g, 1.41 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 45 s (amplitude = 0.3; ti = 19 °C; tf = 35 °C; E = 125 J). After cooling to rt for 1 h, the formed precipitate was filtered and washed with water and ethanol to obtain pure 2a (0.35 g, 0.87 mmol, 68% yield) as a green solid; mp 129–131 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.40 (s, 3H, CH3), 2.45 (s, 3H, SCH3), 7.16 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.22–7.34 (m, 4H, ArH), 7.36 (d, J = 8.0 Hz, 1H, ArH), 7.48 (dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.53 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.59 (d, J = 1.6 Hz, 1H, ArH), 7.73 (d, J = 8.0 Hz, 2H, ArH), 8.00 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 16.2 (SCH3), 21.8 (CH3), 105.7 (C), 114.4 (C≡N), 124.4 (CH), 125.7 (CH), 126.2 (CH), 127.3 (CH), 127.4 (CH), 127.9 (CH), 128.1 (CH), 128.9 (C), 129.4 (C), 129.9 (2CH), 130.5 (2CH), 132.9 (C), 138.2 (C), 138.3 (C), 138.8 (C), 143.8 (C), 154.0 (=CH), 162.2 (C=O); IR ν (cm−1): 2212, 1662, 1587, 1460, 1400, 1329, 1262, 1183, 1105, 1032, 949, 868, 806, 748,665. Anal. Calcd for C24H18N2OS2: C, 69.54; H, 4.38; N, 6.76. Found: C, 69.71; H, 4.57; N, 6.95%.

3.3.4. (E)-4-(2-Cyano-3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxoprop-1-en-1-yl)benzonitrile (2c)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), 4-cyanobenzaldehyde 13 (0.21 g, 1.60 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 18 °C; tf = 41 °C; E = 169 J). After cooling to rt for 2 h, the formed precipitate was filtered, washed with water and ethanol, and then purified by recrystallization from ethanol to give pure 2c (0.43 g, 0.1 mmol, 78% yield) as a yellow solid; mp 207–209 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.47 (s, 3H, SCH3), 7.19 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.29–7.36 (m, 2H, ArH), 7.40 (d, J = 8.4 Hz, 1H, ArH), 7.48–7.57 (m, 3H, ArH), 7.73 (dd, J = 8.0, 2.0 Hz, 2H, ArH), 7.87 (d, J = 8.0 Hz, 2H, ArH), 8.01 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz,) δ ppm: 16.1 (SCH3), 111.0 (C), 113.3 (C≡N), 115.4 (C≡N), 117.8 (C), 124.2 (CH), 125.7 (CH), 126.1 (CH), 127.4 (CH), 127.8 (CH), 128.1 (CH), 128.3 (CH), 128.7 (C), 130.3 (2CH), 132.8 (2CH), 133.0 (C), 135.8 (C), 137.7 (C), 138.2 (C), 138.6 (C), 151.0 (=CH), 160.9 (C=O); IR ν (cm−1): 2227, 1670, 1459, 1329, 1194, 1108, 840, 798, 746, 664. Anal. Calcd for C24H15N3OS2: C, 67.74; H, 3.55; N, 9.87. Found: C, 68.11; H, 3.68; N, 10.03%.

3.3.5. (E)-2-(2-(Methylthio)-10H-phenothiazine-10-carbonyl)-3-(4-(trifluoromethyl)phenyl)acrylonitrile (2d)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), 4-(trifluoromethyl)benzaldehyde 14 (0.25 g, 1.44 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 19 °C; tf = 41 °C; E = 158 J). A precipitate formed immediately after and was filtered and washed with water and ethanol to afford pure 2d (0.13 g, 0.28 mmol, 22% yield) as a green-yellow solid. After 3h at rt, the filtrate precipitated, and the precipitate was then filtered and purified by recrystallization from ethanol to give additional mass of pure 2d (total 0.37 g, 0.79 mmol, 61% yield) as a green-yellow solid; mp 167–169 °C (EtOH); 1H NMR (CDCl3, 400 MHz,) δ ppm: 2.47 (s, 3H, SCH3), 7.18 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.28–7.36 (m, 2H, ArH), 7.39 (d, J = 8.4 Hz, 1H, ArH), 7.48–7.58 (m, 3H, ArH), 7.70 (d, J = 8.4 Hz, 2H, ArH), 7.89 (d, J = 8.4 Hz, 2H, ArH), 8.03 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 16.1 (SCH3), 110.1 (C), 113.5 (C≡N), 122.0 (C), 124.3 (CH), 124.7 (C), 125.7 (CH), 126.0 (CH), 126.1 (CH), 126.2 (CH), 127.4 (CH), 127.8 (CH), 128.1 (CH), 128.3 (CH), 128.8 (C), 130.3 (2CH), 133.4 (t, J = 39.5 Hz, CF3), 135.1 (C), 137.8 (C), 138.4 (C), 138.5 (C), 151.7 (=CH), 161.2 (C=O); IR ν (cm−1): 2365, 1671, 1458, 1317, 1160, 1109, 1067, 929, 806, 748, 642. Anal. Calcd for C24H15F3N2OS2: C, 61.53; H, 3.23; N, 5.98. Found: C, 61.27; H, 3.08; N, 5.76%.

3.3.6. (E)-3-(3-Fluoro-4-methoxyphenyl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2e)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), 3-fluoro-4-trimethoxybenzaldehyde 21 (0.24 g, 1.53 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 19 °C; tf = 45 °C; E = 160 J). A precipitate formed immediately after and was filtered and washed with water and ethanol to afford the pure compound 2e (0.42 g, 0.90 mmol, 74% yield) as a yellow solid; mp 178–180 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.46 (s, 3H, SCH3), 3.95 (s, 3H, OCH3), 7.00 (t, J = 8.0 Hz, 1H, ArH), 7.17 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.26–7.35 (m, 2H, ArH), 7.37 (d, J = 8.0 Hz, 1H, ArH), 7.47–7.54 (m, 2H, ArH), 7.57–7.63 (m, 2H, ArH), 7.66 (dd, J = 12.0, 2.0 Hz, 1H, ArH), 7.94 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 16.2 (SCH3), 56.3 (OCH3), 105.2 (C), 113.1 (d, J = 1.5 Hz, CH), 114.2 (CN), 117.4 (d, J = 19.7 Hz, CH), 124.4 (CH), 125.1 (d, J = 7.6 Hz, C), 125.6 (CH), 126.2 (CH), 127.3 (CH), 127.5 (CH), 128.0 (CH), 128.2 (CH), 128.5 (d, J = 3.0 Hz, CH), 128.9 (C), 133.0 (C), 138.1 (C), 138.3 (C), 138.7 (C), 151.5 (d, J = 10.7 Hz, C), 151.9 (d, J = 247.5 Hz, CF3), 152.4 (d, J = 2.2 Hz, =CH), 162.0 (C=O); IR ν (cm−1): 2213, 1674, 1598, 1514, 1320, 1288, 1256, 1140, 1018, 816, 752. Anal. Calcd for C24H17FN2O2S2: C, 64.27; H, 3.82; N, 6.25. Found: C, 64.38; H, 3.90; N, 6.44%.

3.3.7. (E)-3-(Benzo[d][1,3]dioxol-5-yl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2f)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), 1,3-benzodioxole-5-carboxaldehyde 28 (0.21 g, 1.41 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 18 °C; tf = 45 °C; E = 149 J). A precipitate formed immediately after and was filtered and washed with water and ethanol to afford the pure compound 2f (0.38 g, 0.85 mmol, 67% yield) as a yellow-green solid; mp 192–194 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.46 (s, 3H, SCH3), 6.05 (s, 2H, CH2), 6.86 (d, J = 8.4 Hz, 1H, ArH), 7.16 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.26–7.38 (m, 4H, ArH), 7.47–7.55 (m, 3H, ArH), 7.59 (d, J = 2.0 Hz, 1H, ArH), 7.93 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 16.2 (SCH3), 102.1 (CH2), 104.0 (C), 108.6 (CH), 108.8 (CH), 114.5 (C≡N), 124.4 (CH), 125.6 (CH), 126.2 (CH), 126.4 (C), 127.3 (CH), 127.4 (CH), 127.9 (CH), 128.1 (CH), 128.6 (CH), 128.9 (C), 133.0 (C), 138.3 (2C), 138.8 (C), 148.5 (C), 151.7 (C), 153.6 (=CH), 162.3 (C=O); IR ν (cm−1): 2214, 1671, 1584, 1445, 1310, 1256, 1036, 920, 810, 753, 625. Anal. Calcd for C24H16N2O3S2: C, 64.85; H, 3.63; N, 6.30. Found: C, 64.72; H, 3.55; N, 6.12%.

3.3.8. (E)-3-(2,4-Dichlorophenyl)-2-(2-(methylthio)-10H-phenothiazine-10-carbonyl)acrylonitrile (2h)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.34 g, 1.09 mmol), 2,4-dichlorobenzaldehyde 17 (0.19 g, 1.09 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 25 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 90 s (amplitude = 0.3; ti = 20 °C; tf = 59 °C; E = 282 J). After 3 h, a precipitate formed and was filtered and purified by recrystallization from ethanol to obtain the pure compound 2h (0.34 g, 0.72 mmol, 67% yield) as a yellow solid; mp 176–178 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.47 (s, 3H, SCH3), 7.16 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.27–7.36 (m, 3H, ArH), 7.38 (d, J = 7.5 Hz, 1H, ArH), 7.45–7.51 (m, 2H, ArH), 7.55 (s, 2H, ArH), 7.90 (d, J = 8.5 Hz, 1H, ArH), 8.20 (s, 1H, CH=); 13C NMR (CDCl3, 125 MHz) δ ppm: 16.2 (SCH3), 110.9 (C), 113.6 (C≡N), 124.2 (CH), 125.8 (CH), 126.3 (CH), 127.5 (CH), 127.8 (CH), 128.0 (CH), 128.2 (CH), 128.3 (CH), 128.8 (C), 129.0 (C), 130.2 (2CH), 133.0 (C), 136.3 (C), 137.9 (C), 138.5 (C), 138.7 (2C), 148.3 (=CH), 161.3 (C=O); IR ν (cm−1): 2214, 1670, 1456, 1396, 1327, 1262, 1241, 1192, 1103, 1047, 972, 828, 793, 744, 728, 695. Anal. Calcd for C23H14Cl2N2OS2: C, 58.85; H, 3.01; N, 5.97. Found: C, 59.14; H, 3.26; N, 6.25%.

3.3.9. (E)-2-(2-(Methylthio)-10H-phenothiazine-10-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (2i)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.25 g, 0.80 mmol), 3,4,5-trimethoxybenzaldehyde 27 (0.16 g, 0.82 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 25 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 90 s (amplitude = 0.3; ti = 20 °C; tf = 59 °C; E = 343 J). After 3 h, the formed precipitate was filtered and purified by recrystallization from ethanol to obtain the pure compound 2i (0.28 g, 0.57 mmol, 72% yield) as a yellow solid; mp 198–199 °C (EtOH); 1H NMR (CDCl3, 500 MHz) δ ppm: 2.44 (s, 3H, SCH3), 3.76 (s, 3H, OCH3), 3.78 (s, 6H, 2OCH3), 7.25 (s, 2H, ArH), 7.31–7.78 (m, 7H, ArH), 8.12 (s, 1H, =CH); 13C NMR (CDCl3, 125 MHz) δ ppm: 15.0 (SCH3), 56.0 (2OCH3), 60.3 (OCH3), 105.1 (C), 107.8 (2CH), 114.5 (C≡N), 123.9 (CH), 125.2 (CH), 126.6 (CH), 127.2 (C), 127.5 (CH), 127.7 (CH), 127.8 (C), 128.0 (CH), 128.1 (CH), 131.8 (C), 137.6 (C), 138.1 (C), 138.3 (C), 141.3 (C), 152.9 (2C), 154.3 (=CH), 161.2 (C=O); IR ν (cm−1): 2208, 1672, 1573, 1504, 1461, 1421, 1394, 1308, 1292, 1258, 1241, 1186, 1161, 1133, 1110, 991, 937, 928, 863, 808, 754, 635. Anal. Calcd for C26H22N2O4S2: C, 63.65; H, 4.52; N, 5.71. Found: C, 63.79; H, 4.68; N, 5.94%.

3.3.10. (E)-2-(2-(Methylthio)-10H-phenothiazine-10-carbonyl)-3-(thiophen-2-yl)acrylonitrile (2j)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), thiophene-2-carboxaldehyde 29 (0.16 g, 1.43 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 19 °C; tf = 44 °C; E = 169 J). The formed precipitate 3 h after reaction was filtered, washed with water, and purified by recrystallization from ethanol to give the pure compound 2j (0.40 g, 0.98 mmol, 77% yield) as a yellow solid; mp 160–162 °C (EtOH); 1H NMR (CDCl3, 400 MHz) δ ppm: 2.47 (s, 3H, SCH3), 7.16–7.20 (m, 2H, ArH), 7.27–7.34 (m, 2H, ArH), 7.37 (d, J = 8.4 Hz, 1H, ArH), 7.47–7.55 (m, 2H, ArH), 7.60 (d, J = 2.0 Hz, 1H, ArH), 7.68–7.71 (m, 1H, ArH), 7.74 (dd, J = 3.6, 2.0 Hz, 1H, ArH), 8.28 (s, 1H, =CH); 13C NMR (CDCl3, 100 MHz) δ ppm: 16.3 (SCH3), 103.0 (C), 114.3 (C≡N), 124.5 (CH), 125.8 (CH), 126.2 (CH), 127.3 (CH), 127.5 (CH), 127.9 (CH), 128.2 (CH), 128.4 (CH), 128.9 (C), 133.0 (C), 134.1 (CH), 136.1 (CH), 136.4 (C), 138.2 (C), 138.3 (C), 138.8 (C), 146.7 (=CH), 161.7 (C=O); IR ν (cm−1): 2216, 1672, 1593, 1458, 1301, 1192, 1109, 943, 817, 755, 719. Anal. Calcd for C21H14N2OS3: C, 62.04; H, 3.47; N, 6.89. Found: C, 62.35; H, 3.58; N, 7.06%.

3.3.11. (E)-2-(2-(Methylthio)-10H-phenothiazine-10-carbonyl)-5-phenylpenta-2,4-dienenitrile (2o)

General procedure B was used with 3-(2-(methylthio)-10H-phenothiazin-10-yl)-3-oxopropanenitrile 4b (0.40 g, 1.28 mmol), cinnamaldehyde 36 (0.19 g, 1.41 mmol), and lithium hydroxide (0.02 g, 0.84 mmol) in 30 mL ethanol, at room temperature, and an ultrasonic agitation was applied for 60 s (amplitude = 0.3; ti = 19 °C; tf = 44 °C; E = 166 J). The precipitate formed immediately after was filtered and washed with water and ethanol to afford the pure compound 2o (0.22 g, 0.52 mmol, 40% yield) as an orange solid. After 3h at rt, the filtrate precipitated, and the precipitate was then filtered and purified by recrystallization from ethanol to give additional mass of pure 2o (total 0.33 g, 0.77 mmol, 61% yield) as an orange solid; mp 161–163 °C (EtOH); 1H NMR (400 MHz, CDCl3) δ ppm: 2.48 (d, J = 1.2 Hz, 3H, SCH3), 7.15–7.20 (m, 3H, =CH+2ArH), 7.26–7.42 (m, 6H, ArH), 7.47–7.57 (m, 4H, =CH+3ArH), 7.58 (d, J = 2.0 Hz, 1H, ArH), 7.94 (dd, J = 6.4, 4.8 Hz, 1H, =CH); 13C NMR (100 MHz, CDCl3) δ ppm: 16.2 (SCH3), 107.9 (C), 113.1 (CN), 123.3 (CH), 124.4 (CH), 125.6 (CH), 126.3 (CH), 127.3 (CH), 127.5 (CH), 127.9 (CH), 128.1 (CH), 128.4 (2CH), 128.8 (C), 129.1 (2CH), 130.9 (CH), 133.0 (C), 134.8 (C), 138.1 (C), 138.3 (C), 138.7 (C), 147.4 (=CH), 155.3 (=CH), 161.3 (C=O); IR ν (cm−1): 2212, 1674, 1562, 1443, 1298, 1167, 980, 819, 745, 687. Anal. Calcd for C25H18N2OS2: C, 70.39; H, 4.25; N, 6.57. Found: C, 70.48; H, 4.47; N, 6.81%.

3.4. Human FTase Assay

Assays were realized in 96-well plates, prepared with a Biomek NKMC and a Biomek 3000 from Beckman Coulter, and read on a Wallac Victor fluorimeter from PerkineElmer [28]. Per well, 20 μL of farnesyl pyrophosphate (10 μM) was added to 180 μL of a solution containing 2 μL of varied concentrations of potential inhibitors (dissolved in DMSO) and 178 μL of a solution composed of 10 μL of partially purified recombinant human FTase (5 mg/mL) and 1.0 mL of Dansyl-GCVLS peptide (in the following buffer: 5.6 mM DTT, 5.6 mM MgCl2, 12 μM ZnCl2 and 0.2% (w/v) octyl-s-D-glucopyranoside, 52 mM Tris/HCl, pH 7.5). Fluorescence was recorded for 15 min (0.7 s per well, 20 repeats) at 30 °C with an excitation filter of 340 nm and an emission filter of 486 nm. Each measurement was reproduced twice (two independent experiments on different 96-well plates) in duplicate. The kinetic experiments were realized under the same conditions, either with FPP as varied substrate with a constant concentration of Dns-GCVLS of 2.5 μM or with Dns-GCVLS as varied substrate with a constant concentration of FPP of 10 μM. Nonlinear regressions were performed with Excel software.

3.5. Tubulin Polymerization Assay

Sheep brain tubulin was purified according to the method of Shelanski [29] by two cycles of assembly–disassembly and then dissolved in the assembly buffer containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM of GTP (pH 6.6) to give a tubulin concentration of about 2–3 mg/mL. Tubulin assembly was monitored by fluorescence according to reported procedure [30] using DAPI as fluorescent molecule. Assays were realized on 96-well plates prepared with Biomek NKMC and Biomek 3000 from Beckman coulter (Villepinte, France) and read at 37 °C on Wallac Victor fluorimeter from Perkin–Elmer (Villebon-sur-Yvette, France). The IC50 value of each compound was determined as tubulin polymerization inhibition by 50% compared to the rate in the absence of compound. The IC50 values for all compounds were compared to the IC50 values of phenstatin and (-)-desoxypodophyllotoxin and were measured the same day under the same conditions.

3.6. Cell Proliferation Assay

Compounds 2k, 2l, and 2o were tested on a panel of 60 human cancer cell lines at the National Cancer Institute, Germantown, MD [31]. The cytotoxicity studies were conducted using a 48h exposure protocol using the sulforhodamine B assay [32,33].

Acknowledgments

The authors acknowledge the Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI), Romania, for financial support of this work. The authors also acknowledge the National Cancer Institute (NCI) for the biological evaluation of compounds on their 60-cell panel. The testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (http://dtp.cancer.gov, accessed on 20 April 2023). The authors also thank the CERNESIM Center within the Interdisciplinary Research Institute at “Alexandru Ioan Cuza” University of Iasi, Romania, for the infrastructure used in recording NMR experiments.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ph16060888/s1. Copies of 1H and 13C NMR spectra and IR spectra for all synthesized cyanochalcones 1a-r, 2a-o, and 3a-p; copies of two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) correlations for compounds 1g, 2l, and 3k; copies of one-dose full graphs obtained on NCI-60 cancer cell lines panel for compounds 2k, 2l, and 2o (Figure S1); and molecular docking poses for all the dual inhibitors FTIs/MTIs identified in this study (Figure S2) are provided in this section.

Click here for additional data file. (17.2MB, zip)

Author Contributions

A.G. and E.B.: conceptualization, resources, supervision, data curation, project administration, validation. A.G.: biological evaluation of tubulin polymerization and human farnesyltransferase, writing—original draft, writing—review and editing. J.D.: resources, supervision of biological evaluation. A.Z.: data curation, formal analysis, investigation, methodology. A.F.: docking of compounds in tubulin and FTase binding sites. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors. Supplementary materials include copies of NMR spectra of all synthesized compounds.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research was funded by the Executive Unit for Financing Higher Education, Research, Development and In-novation (UEFISCDI), Bucharest, Romania, grant number PN-III-P4-ID-PCE-2020-0818, acronym: REPAIR. The APC was also funded by UEFISCDI.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Click here for additional data file. (17.2MB, zip)

Data Availability Statement

The data presented in this study are available on request from the corresponding authors. Supplementary materials include copies of NMR spectra of all synthesized compounds.

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