Inhibiting Melanoma Cells via Induction of Late-onset of Apoptosis and Autophagy

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Novel 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazides selectively inhibit melanoma cells via induction of late-onset of apoptosis and autophagy

Abstract

As earlier it was proven that thiadiazole and piperidin-4-one nucleus showed considerable anticancer activities and therefore, we assumed that the hybrid molecules of these scaffold may further enhance the  anti-tumour effects. To exploit the potential antitumour activities of compounds bearing piperidin-4-one nucleus, thiadiazole and azomethine group (–NH–N=CH–), a set of 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbo-hydrazide 5 (a-g)  hydrazones were synthesized. 4-Methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide 5a was identified as the most promising candidate that selectively inhibited the growth of melanoma cell lines carrying NRAS and BRAF mutations while sparing healthy HECK293 cells. However, parent compound 3a and 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide 4 showed toxicity towards a healthy control CHO-K1 and HECK293 cell lines with IC50 values ranging from at 1 to 15 uM. To explore structure-activity relationships, compounds 5a-g were compared their ability to inhibit the growth of WM-266-4, M14, A549, BT474 and HECK293 cells. Although compound 5a failed to influence A549, BT474 and HECK293 cell growth, it suppressed the growth of melanoma cells WM-266-4 (IC50 = 0.56uM) and M14 (IC50 = 8.57uM). None of individual compounds (5b-g) from this series exhibited good activity or selectivity towards any cells. Furthermore, 5a caused cell cycle arrest at G2/M phase, induced apoptosis and autophagy, and led to cell death by increasing the proportion of sub-G1 cells. Further studies on target identification and its mechanism of action (drug-resistant) may assist the development of novel piperidin-4-one skeleton bearing thiadiazole-inspired drugs to treat human cancer carrying NRAS and BRAF mutations.

Introduction

Today cancer is one of the leading cause of death worldwide. These startling statistics indicate that the most of the cancer drugs/drug candidates are toxic towards noncancerous cells and they develop resistance to many of the therapies. These features have relegated many of the current clinical products unusable [1]. To reduce nonspecific toxicity, overcome drug resistance and improve the overall efficacy of treatment, natural products and their derivatives have been found to be an excellent starting point for drug discovery and arguably the single most important source of therapeutic agents and of structural diversity [2-4]. Various natural phytochemical such as curcumin, resveratrol, and quercetin sensitize tumors to the various chemotherapeutic drugs with nonspecific toxicity towards normal cells [5]. A large number of natural phytochemicals have been demonstrated to exhibit anticancer activities by snooping with multiple signaling pathways aberrant in cancer [2,5,6]. However, in addition to the diverse biological functions, chemical structure diversity and biodiversity of natural products, it is essential to develop new technologies for screening of natural products in discovering new drugs. Pharmacological approach for early-stage drug discovery is a relatively slow and expensive mechanism. Because of these, most of lead molecules are abandoned  before reaching the clinical trials phases [7]. Therefore, we hypothesized that effort directed toward to assembly of different biological structural scaffolds in the same molecule could be a valid shortcut to demonstrate the new paradigm for accelerated drug discovery and the development of new anticancer agents.

The piperidin-4-one nucleus, thiadiazole and azomethine group (–NH–N=CH–) are present in several biologically active natural products and commercial drugs [8-10]. The piperidin-4-one nucleus exhibits a wide spectrum of biological activities ranging from antibacterial to anticancer [8]). Modification of position 3 of the piperidin-4-one nucleus as well as a substitution of certain functional groups in the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety would result in compounds with potent biological activities [11]. In particular, an increasing the electron-withdrawing properties of substituents on aromatic ring has an advantageous effect on cytotoxicity. The dimethyl groups at C3 atoms in piperidone may increases its hydrophobic binding affinity towards various proteins [11]. Therefore, many researchers have focused on modifying the piperidin-4-one pharmacophore to achieve better biological activities. Hydrazones contain an azomethine that constitutes an important class of compounds for new drug development. Azomethine group (–NH–N=CH–) connected with carbonyl group is responsible for their different biological effects and makes possible the synthesis of different heterocyclic scaffold [10,12]. The recent literature review has proved that the thiadiazole scaffold is having a broad spectrum of pharmacological activities including antimicrobial, anti-inflammatory, anticancer, and anti-oxidant activities [9].

The above findings inspired us to combine hydrazide with thiadiazoles ring  into the piperidin-4-one pharmacophore by condensation would result in potent biologically active compounds with low toxicity because of the blockage of –NH group in hydrazides (10). The compounsd 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide 5 (a-g) has been synthesized by using a reaction of piperidin-4-one with 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide in the presence of acetic acid in methanol. The chemical structures are established by using IR, 1H-NMR, 13C-NMR and elemental analysis.

Given the propensity of single-target-based compounds to cause resistance, a potential of phenotypic screening to discover compounds that favorably interact with multiple targets (i.e., polypharmacology), thus avoiding or diminishing the chances for resistance, represents an additional benefit as compared to the target-based screening [1,13]. The above considerations prompted us to screen our newly synthesized compounds to discover potentially first-in-class selective inhibitors of various cancer cells such as A549 nonsmall cell lung cancer, BT474,  melanoma cells, M14 with NRAS mutation and WM-266-4 cells with  BRAF (V600E) mutation and a healthy control HECK293 cell line. 

Results and discussion

Chemistry.In order to bring the piperidin-4-one nucleus, thiadiazole and azomethine group (–NH–N=CH–) scaffolds in the same molecule, we have synthesized the 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbo-hydrazide 5 (a-g) as described inScheme 1. First, commercially available appropriate ketones, aldehydes and ammonium acetate in a 1:2:1 ratio were converted to the 3-alkyl-2r,6c-diaryl piperidin-4-one 3 (a-l) by condensation according to the method described by Noller and Baliah [14]. Then, 3-alkyl-2r,6c-diaryl piperidin-4-one 3 (a-l) (1mmol), and 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide (1.5mmol) in the solvent mixture of chloroform and methanol (1:1 v/v) wasrefluxed for about 3-4 hours in the presence of   acetic acid (2 ml) as a catalyst to provide the target compounds 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-car-bohydrazide 5 (a-g). The structure of the target compounds  5 (a-g) was further unambiguously determined by the detailed investigation of  IR, 1H NMR and C13 NMR spectral data with CHN analysis (detailed parameters are included in the Supporting Information).

Antiproliferative Activities and the SARs. As earlier it was proven that thiadiazole ring [9] and piperidin-4-one nucleus [8] show considerable anticancer activities and therefore, we assumed that the hybrid molecules of these scaffold may further enhance the  anti-tumour effects. Therefore, in the present study, we first report the synthesis of 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbo-hydrazide 5a and compared the effect of compound 3a, 4 and 5a for toxicity towards a healthy control CHO-K1 and HECK293 cell lines. To confirm the selective nature of 5a and to estimate the potency, dose−response experiments were performed using 10-point 3-fold serial dilutions. As a result of this screen, compounds 3a and 4  exhibited pronounced inhibitory activity against CHO-K1 and HECK293 cell line viability with IC50 values ranging from at 1 to 15 uM, whereas compound 5a did not significantly inhibit growth of these healthy cell line (Figure 1A and 1B).  Since compound 5a showed no toxicity to noncancerous cells, which is  the most common liabilities of cancer drugs, we further evaluated the antiproliferative effects of compound 5a against melanoma cells such as M14 and WM-266-4. In the present study, 5a exerted cytotoxic effects on M14 and WM-266-4 in a dose- and time-dependent manner (figure 1C and D). Compound 5a exhibited approximately 100-fold selectivity for WM-266-4 cells over normal healthy cells and more than 20-fold selectivity for M14 cells over HECK293 cells (Figure 1C and 1D, IC50 = 0.52,  2.88, and >100μM for WM-266-4, M14, and HECK293 cells, respectively). The differences in IC50 values may be attributed to the duration of exposure and the differential sensitivities of cell lines to the cytotoxic effects of compound 5a.

In search for a chemical modification that would increase the activity of the parent scaffolds, compounds 5a with different substituents at the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety (X and Y) were prepared and evaluated for their antiproliferative activities against WM-266-4, M14, and HECK293 cells. None of individual compounds (5b-g) from this series exhibited good activity or selectivity toward M14 (Table 1). However, Compounds 5a deprived of any substitutes at the para position of the phenyl groups at the C-2 and C-6 positions of the piperidine ring exhibited dose-dependent inhibition of viability of WM-266-4 and M14 cell lines. 5a was the most efficient against the WM-266-4 ((IC50 = 0.56uM) than M14 (8.57uM). Compounds (5b and 5c) containing electron donating functional groups (–CH3;IC50=2.83uM , –OCH3; IC50=8.76uM) exhibited moderate cytotoxic effects against WM-266-4 cells compared to the electron withdrawing functional groups (–F,–Cl and -Br) present on the aryl rings attached to piperidones of compouds 5d-g. Further, we were interested to see compounds 5a-g could also inhibit other cancer cell line. Therefore, we tested compound 5a-g against the A549, BT474 and HECK293. Interestingly, compounds 5a-g were not selective against A549 (lung cancer), BT474 (A human breast tumor cell line) and HECK293 cell lines inhibition (Table 1).

Thus, compounds 5a that lack any substitutes at the aryl groups accounts for the enhanced inhibitory effects against melanoma cells. This may be due to the presence of azomethine–NHNvCH– groups as well as thiadiazole scaffold. Thiadiazole is a prevalent and important five-membered heterocyclic system containing two nitrogen atoms and a sulfur atom [9]. The electron-withdrawing effect of the nitrogen atoms and a sulfur atom with lone electron pairs in the ring provide an electron deficient nature, obvious aromaticity, and pretty high thermotic stability [9,15]. A substitution on the C3′ or C5′ position in the ring are highly activated and ready to react. The nitrogen atoms tend to nucleophilic attack, while the carbon atoms can susceptible to both nucleophilic substitutions and electrophilic attacks [9]. The compounds with thiadiazole ring display anticancer properties via a mechanism of action comprising that the electron-donating ability of two nitrogen atoms built a favorable H bonds with the amino hydrogen of aminoacids in proteins or to chelate certain metal ions [9]. Several studies have reported that thiadiazoles and its isomers showed broad-spectrum anticancer activities against human cancers and targeted molecular involved in proliferation, survival, and metastasis [9,16-20].

Contrary to reports of a positive correlation between the cytotoxicity and the electron withdrawing functional groups (–F,–Cl and -Br) present on the aryl rings attached to piperidones [11,21], we found modarate cytotxic effects in synthetic compound 5b and 5c despite its electron donating functional groups (–CH3, –OCH3) present on the aryl rings attached to piperidones. Although compounds 5b-g consist of azomethine–NHNvCH– groups and thiadiazole scaffold, we speculated that substitution of certain functional groups in the para position of the phenyl ring attached to C-2 and C-6 carbons of the piperidine moiety (compounds 5b-g) was not well tolerated due to steric  hindrance with the putative target(s). In addition, very low bioavailability and absorption rate could also affects the biological effects of the compound 5b-g, compared to 5a.  However, multiple mechanisms regulating the cytotoxic effects of the hybrid molecules need to be further investigated

Knowledge of the mechanism of cell death caused by a lead compound can help predict potential compound liabilities and allow prioritization of compounds. For example, compounds that cause primary necrosis usually do not make good drug candidates because of their general toxicity, whereas cell-cycle inhibitors have proven to be very selective and well-tolerated in melanoma clinical trials [22]. Our lead compounds were discovered as a result of a phenotypic assay; therefore, to exclude the possibility of necrosis as a mechanism of death, we performed a time-course study using the MTT viability assay. Primary necrosis is characterized by the rapid loss of cell viability, which can be detected as early as 3 h after compound addition. We determined the effect of lead compound application on the viability of WM-266-4 and M14 cells at 4, 24, 48, and 72 h. The test were screened in 10-point, 1:3 serial dilution dose −response format starting at 300 μM. Compound 5a did not exhibited signs of cell viability loss at any concentration at the 4 h time point and only slight loss of viability at the 24 h time point. All compounds reached their full potency at 48 h (Figure 1C and D). These data suggested that lead compound 5a is unlikely to cause primary necrosis in WM-266-4 and M14 cells. Selective inhibitory of 5da against melanoma cells in vitro were comparable to the first-line chemotherapeutic agent Dabrafinib, and this compound was chosen to further investigate the mechanism of its cytotoxic effects.

5da Induces cell cycle arrest in WM-266-4 Cells. To study the potential mechanistic pathways responsible for cell proliferation inhibition by 5a, we tested the change of cell cycle in WM-266-4 cancer cell lines. As shown in Figure 2 A,  5a treatment (doses from 5, 25, 50 and 100 μM, 24 h) clearly increased the cell proportion of G2/M phase, while the cell proportions in G0/S phase were markedly reduced (Figure 2).  Collectively, these results documented the antiproliferation potency of 5a in WM-266-4 cancer cell lines were potentially due to the G2/M phase arrest-induced growth inhibition.

5a Induces Cell apoptosis and autophagy. To gain further insight into the mechanisms of tumor suppression activity of 5a, we wanted to confirm first whether the G2/M phase cells induced by 5a were due to apoptosis or autophagy. The 5a -treated WM-266-4 cells were therefore investigated with PI and FITC-annexin V staining for apoptosis identification. We performed flow cytometry-based annexin V assays (Fig. 3) to detect populations of viable, necrotic, early and late apoptotic cells at 4, 24 and 48 h treatment of compound 5a (100uM). At 4 hrs apoptosis was detected only in staurosporine-treated cells (Fig. 3A and B). At 24 h and 48 h, 23% and 45% of 5a-treated cells, respectively, were undergoing late apoptosis (Fig. 3A and B).  Early apoptotic and necrotic cells were less than  8% of total population sugge sting that 5a causes late apoptosis. Staining of compound 5a treated WM-266-4 cells with  PI and FITC-annexin V, as depicted in Figure 3C, displayed obvious cell apoptosis in WM-266-4 cells in a dose-dependent manner. At the 50 and 100 uM concentration unstained cells treated with compound 5a exhibited traits of apoptosis (Fig. 3C, top panel; note smaller cell size, rounded shape, membrane blebbing, and apoptotic bodies). However, the apoptosis intensified leading to the extensive cell death as evidence by increased anexin V and PI staining at 50 and 100uM concentration of compound 5a treatment (Fig. 3A).

To further investigate the mechanism of cell death caused by our lead compounds, we stained WM-266-4 cells treated with 5a at 25, 50 and 100 uM for autophagy detection at 24 h  after addition of the compounds using autophagosome stain. Interestingly, cells in the presence of 5a stained positive for autophagy as lower concentration at 25uM (Fig. 4A), where apoptotic stain was not observed. At 24 h, the intensity of autophagy staining increased in cells treated with increasing concentration of 5a (Fig. 4A). Quantitation of the representative image of cells showed that 60% of cells treated with compound 5a at 100uM were undergoing autophagy, whereas untreated control cells showed no signs of autophagy (Fig. 3B). These results collectively indicated that 5a-induced late-onset apoptosis and autophagy in WM-266-4. However, the mechanism of apoptosis and autophagy induction  are warrentd to study in future.

Our results provide evidence that compound 5a caused cell cycle arrest at G2/M phase, induced apoptosis and autophagy, and led to cell death by increasing the proportion of sub-G1 cells.  Thus, compound 5a exhibited potency towards melanoma cell lines with specific molecular backgroung comparable to Dabrafinib (Table 1). Dabrafinib is a first-in-class, specific small-molecule inhibitor of V600EBRAF and has been approved by the U.S. Food and Drug Administration for the treatment of late-stage (metastatic) or unresectable melanoma in patients whose tumors express  V600EBRAF [23]. This suggests that 5a may potentially act via inhibition of the MAPK pathway, which is constitutively activated in melanomas carrying V600EBRAF and NRAS mutations [1,24].

5a could potentially be a better inhibitor of mutant V600EBRAF than the wild-type BRAF, which could explain the difference in potency toward WM-266-4 and M14 cells. Another possibility is that 5a could be acting on the HSP90 chaperone that has multiple client proteins in the MAPK pathway. Inhibition of HSP90 by small molecule (17-AAG) resulted in melanoma stabilization in patients carrying BRAF or NRAS mutation [1]. Fatkhutdinov et al [25] have demonstrated that inhibition of nucleotide metabolism through knockdown of RRM2 in combination with mutant BRAF inhibitor induced melanoma cell apoptosis and prolonged treatment response. Our compound 5a may exert it inhibitory effects against melanoma cells via targeting both RRM2 and mutant V600EBRAF. Further studies are requied to determine the identity of its target(s) and the possibility of utilizing this novel piperidin-4-one nucleus bearing thiadiazole and azomethine group (–NH–N=CH–) based skeleton for oncological drug discovery.

Experimental Section

Materials. All chemicals that were purchased were used without further purification. All the reported melting points that were measured in open capillaries were uncorrected.  FT-IR analysis was done by making a pellet of compound with KBr. Both One and Two dimensional NMR spectra were recorded in the NMR spectrometer. A sample was prepared with a 5mm diameter tube using DMSO-d6 solvent (10mg in 0.5 ml). 1H NMR and 13C NMR data were collected in 400.13 MHz and 100.62 MHz operating frequency, respectively. Chemical shifts (δ) were expressed in ppm with respect to TMS. Splitting patterns were designated as follows: s-singlet, d-doublet, t-triplet, q-quartet and m-multiplied. 

Chemistry

General procedure for the synthesis of 4-methyl-N’-(3-alkyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazides 5 (a-l)

The 3-alkyl-2r,6c-diaryl piperidin-4-one 3 (a-l), were prepared by the condensation of appropriate ketones, aldehydes and ammonium acetate in a 1:2:1 ratio, according to the method described by Noller and Baliah [14]. A reaction mixture containing 3-alkyl-2r,6c-diaryl piperidin-4-one 3 (a-l) (1mmol), 4-methyl-1,2,3-thiadiazole-5-carboxylic acid hydrazide (1.5mmol) was dissolved in the solvent mixture of chloroform and methanol (1:1 v/v) acetic acid (2 ml)  was added as a catalyst. The reaction mixture was refluxed for about 3-4 hours. After completion of the reaction, the crude product was formed, filtered and washed with a cold mixture of ethanol and water. The pure compounds 5 (a-l) were obtained by crystallization from distilled ethanol. Analytical data of compounds 5a-5l were shown in table-1.

4-Methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5a).

White solid, yield: 70%; mp: 189 ºC. IR (KBr, max cm-1): 1656 (C=N) 1492 (C=O), 3431 (N−H st pip), 3307 (N−H st amide), 3053, 2978, 2802 (C−H st).1H NMR (400 MHz, DMSO-d6) δ:  1.28 (d, 6H, 2CH3 at piperidin ring), 1.97 (s, 1H, NH at piperidin ring), 2.56 (d, 1H, C5-1Ha), 2.56 (s, 3H, methyl at thiadiazole ring), 3.19 (dd, 1H, C5-1He), 3.81 (d, 1H, C2-1Ha), 3.89 (d, 1H, C6-1Ha), 7.26–7.52 (m, 10H, Ar–H), 10.42 (s, 1H, N–H, amide NH). 13C (400 MHz, DMSO-d6) δ: 21.62 and  24.37 (2CH3 at piperidin ring), 14.91 (CH3 at thiadiazole), 33.49 (C-5), 44.51 (C-3), 61.51 (C-6), 70.52 (C-2), 126.76–129.17 (Ar–C), 135.11 (C-5 at thiadiazole), 139.77 and 143.04 (ipso carbons) and 163.47 (C-4), 162.05 (NHCO) and 164.59 (C-4 at thiadiazole).

4-Methyl-N’-(3,3-dimethyl-2r,6c-bis(4-methylyphenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5b).

White solid, yield: 65%, m.p: 150 ºC. IR (KBr, νmax cm−1):1656 (C=N) 1494 (C=O), 3419 (N−H st pip), 3300 (N−H st amide), 3055, 2978, 2924 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ: 1.26 (d, 6H, 2CH3 at piperidin ring), 2.17 (s, 1H, NH at piperidin ring), 2.17 (s, 6H, CH3 at phenyl ring), 2.36 (dd, 1H,C5-1Ha),  2.72 (s, 3H, CH3 at thiadiazole ring), 3.02 (d, 1H, C5-1He), 3.76 (d, 1H, C2-1Ha), 3.84 (d, 1H,C6-1Ha), 7.13–7.39 (m, 8H, Ar–H), 9.86 (s, 1H, N–H amide N–H). 13C (400 MHz, DMSO-d6) δ: 21.54 and 24.28  (2CH3 at piperidin ring), 15.13 (CH3 at thiadiazole ring), 30.99 (CH3 at phenyl group), 33.28 (C-5), 44.51 (C-3), 61.13 (C-6), 70.27 (C-2), 126.59 –129.47 (Ar–C), 135.18 (C-5 at thiadiazole ring),137.98–140.06 (ipso carbons), 163.25 (C-4), 161.52 (NHCO) and 164.60 (C-4 at thiadiazole ring).

4-Methyl-N’-(3,3-dimehyl-2r,6c-bis(4-methoxyphenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5c).

White solid, yield: 73%, m.p: 151 ºC. IR (KBr, νmax cm−1):1654 (C=N) 1498 (C=O), 3437 (N−H st pip), 3302 (N−H st amide), 3061, 2974, 2821 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  1.25 (d, 6H, 2CH3 at piperidine ring), 2.17 (s, 1H,NH at piperidin ring), 2.52 (t, 1H, C5-1Ha), 2.64 (s, 1H, CH3 at thiadiazole ring), 3.10 (dd 1H, C5-1He), 3.74 (d, 1H, C2-1Ha), 3.82 (dd,1H, C6-1Ha), 3.82 (s, 6H, OCH3), 6.86–7.42 (m, 8H, Ar–H), 10.26 (s, 1H, N–H amide N–H). 13C (400 MHz, DMSO-d6) δ: 21.52 and 24.32 (CH3 at piperidin ring),15.06 (CH3 at thiadiazole ring), 33.50 (C-5), 44.59 (C-3), 55.41 (OCH3), 60.87 (C-6), 69.92(C-2), 113.17–131.92 (Ar–C), 135.23 (C-5 at thiadiazole ring), 159.21 and 159.48 (ipso carbons) , 163.57 (C-4), 161.80 (NHCO), and 164.56  (C-4 at thiadiazole ring).

4-Methyl-N’-(3,3-dimethyl-2r,6c-bis(2-chlorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5d).

White solid, yield: 70%, m.p: 169 ºC. IR (KBr, νmax cm−1):1653 (C=N) 1506 (C=O), 3433 (N−H st pip), 3304 (N−H st amide), 3064, 2972, 2831 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  1.35 (d, 6H, 2CH3 at piperidin ring), 2.17 (s, 1H, NH at  piperidin ring), 2.37 (t, 1H, C5-1Ha), 2.77 (s, 3H, CH3 at thiadiazole ring), 3.20 (dd, 1H, C5-1He), 4.41 (d,1H, C2,-1Ha), 4.56 (dd, 1H, C6-1Ha), 7.26–7.91 (m, 8H, Ar–H), 9.78 (s, 1H, N–H, amide N–H). 13C (400 MHz, DMSO-d6) δ: 21.97 and 23.72 (2CH3 at piperidin ring), 15.13(CH3 at thiadiazole ring), 30.97 (C-5), 45.54 (C-3), 64.18 (C-6), 76.72 (C-2), 126.47–132.46 (Arc), 134.66 (C-5 at thiadiazole ring), 137.38 and 139.91 (ipso carbons), 161.54 (C-4), 161.54 (NHCO) and 164.59 (C-4 at thiadiazole ring).

4-Methyl-N’-(3,3-dimethyl-2r,6c-bis(4-chlorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5e).

White solid, yield: 70%, m.p: 169 ºC. IR (KBr, νmax cm−1):1656 (C=N) 1487 (C=O), 3439 (N−H st pip), 3309 (N−H st amide), 3068, 2976, 2854 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  1.12 (d, 6H, 2CH3 at piperidin ring), 2.09 (s, 1H, NH at  piperidin ring), 2.21 (t, 1H, C5-1Ha), 2.50 (s, 3H, CH3 at thiadiazole ring), 3.17 (dd, 1H, C5-1He), 3.74 (d,1H, C2,-1Ha), 3.82 (dd, 1H, C6-1Ha), 7.40–7.63 (m, 8H, Ar–H), 11.40 (s, 1H, N–H, amide N–H). 13C (400 MHz, DMSO-d6) δ: 20.89 and 24.09 (2CH3 at piperidin ring), 14.91 (CH3 at thiadiazole ring), 30.67 (C-5), 43.33 (C-3), 59.10 (C-6), 68.70 (C-2), 127.39 –131.84 (Arc), 135.85 (C-5 at thiadiazole ring), 139.10 and 142.86  (ipso carbons), 163.13(C-4), 160.72 (NHCO) and 164.45 (C-4 at thiadiazole ring).

4-Methyl-N’-(3,3-dimethyl-2r,6c-bis(4-fluorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5f).

White solid, yield: 78%, m.p: 168 ºC. IR (KBr, νmax cm1): 1658 (C=N) 1502 (C=O), 3439 (N−H st pip), 3302 (N−H st amide), 3068, 2976, 2848 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ: 1.12 (d, 6H, 2CH3 at piperidin ring), 2.21 (s, 1H, NH at piperidin ring), 2.50 (t, 1H, C5-1Ha), 2.95 (s, 3H, CH3 at thiadiazole ring), 3.36 (dd, 1H, C5-1He), 3.72 (d,1H, C2-1Ha), 3.80 (dd, 1H, C6-1Ha), 7.42–7.56 (m, 8H, Ar–H), 11.41 (s, 1H, N–H, amide N–H). 13C (400 MHz, DMSO-d6) δ: 20.88 and 24.08 (CH3 at piperidin ring), 14.91 (CH3 at  thiadiazole ring), 33.03 (C-5), 43.27 (C-3), 59.14 (C-6), 68.75 (C-2), 129.09 –131.25 (Arc), 135.85 (C-5 at thiadiazole ring), 139.50 and 143.26 (ipso carbons), 160.73 (C-4), 159.28 (NHCO) and 163.11 (C-4 at thiadiazole ring).

4-Methyl-N’-(3,3-dimethyl-2r,6c-bis(p-bromophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5g).

White solid, yield:65%, m.p: 177ºC. IR (KBr, νmax cm−1):1653 (C=N) 1498 (C=O), 3441 (N−H st pip), 3290 (N−H st amide), 3061, 2980, 2926 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  1.25 (d, 6H, 2CH3 at piperidin ring), 1.91 (s, 1H, NH at piperidin ring), 2.50 (t, 1H, C5-1Ha), 2.71 (s, 3H, CH3 at thiadiazole ring), 3.06 (dd, 1H, C5-1He), 3.79 (d,1H, C2-1Ha), 3.87 (d, 1H, C6-1Ha), 7.02 –7.50 (m, 8H, Ar–H), 9.98 (s, 1H, N–H amide N–H). 13C (400 MHz, DMSO-d6) δ: 21.44 and 24.21 (CH3 at piperidin ring), 15.09 (CH3 at thiadiazole ring), 33.32 (C-5), 44.37 (C-3), 60.68 (C-6), 69.71 (C-2), 114.72 –130.58 (arc), 135.09 (C-5 at thiadiazole ring), 135.26 and 138.65 (ipso carbons), 163.78 (C-4), 161.33 (NHCO) and 164.53 (C-4 at thiadiazole ring).

4-Methyl-N’-(3-isopropyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5h).

White solid, yield: 70%, mp: 189 ºC. IR (KBr, νmax cm−1):1651 (C=N) 1496 (C=O), 3439 (N−H st pip), 3300 (N−H st amide), 3055, 2958, 2881 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  0.84, 1.13 (t, 6H, 2CH3 at piperidin ring), 2.27 (m, 1H, CHat piperidin ring), 1.85 (s, 1H, NH at piperidin ring), 2.86 (d, 1H, C5-1Ha), 2.57 (s, 3H, methyl at thiadiazole ring), 2.74 (t, 1H, C3-1Ha), 2.86 (dd, 1H, C5-1He), 4.18 (d, 1H, C2-1Ha), 4.23 (d, 1H, C6-1Ha), 7.26–7.47 (m, 10H, Ar–H), 10.64 (s, 1H, N–H, amide NH).13C (400 MHz, DMSO-d6) δ: 21.16 and 21.34 (2CH3 at piperidin ring), 15.08 (CH3 at thiadiazole), 31.50 (CH at piperidin ring), 36.79 (C-5), 56.27 (C-3), 58.15 (C-6), 63.86 (C-2), 126.59–128.87 (Ar–C), 135.16 (C-5 at thiadiazole), 143.55 and 144.96 (ipso carbons), 158.11 (C-4), 162.56 (NHCO) and 164.21 (C-4 at thiadiazole).

4-Methyl-N’-(3-isopropyl-2r,6c-bis(2-chlorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5i).

White solid, yield: 70%, m.p: 169 ºC. IR (KBr, νmax cm−1):1647 (C=N) 1483 (C=O), 3444 (N−H st pip), 3296 (N−H st amide), 3062, 2980, 2872 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  0.84, 1.12 (t, 6H, 2CH3 at piperidin ring), 2.25 (m, 1H, CHat piperidin ring), 1.79 (s, 1H, NH at piperidin ring), 2.87 (d, 1H, C5-1Ha), 2.56 (s, 3H, methyl at thiadiazole ring), 2.65 (t, 1H, C3-1Ha), 2.87 (dd, 1H, C5-1He), 4.17 (d, 1H, C2-1Ha), 4.22 (d, 1H, C6-1Ha), 7.26–7.41 (m, 8H, Ar–H), 10.92 (s, 1H, N–H, amide NH).13C (400 MHz, DMSO-d6) δ: 21.14 and 21.34 (2CH3 at piperidin ring), 15.10 (CH3 at thiadiazole), 31.39 (CH at piperidin ring), 36.62 (C-5), 57.32 (C-3), 56.31 (C-6), 62.84 (C-2), 127.87–133.93 (Ar–C), 135.09 (C-5 at thiadiazole), 141.94 and 143.28 (ipso carbons), 157.30 (C-4), 162.68 (NHCO) and 164.06 (C-4 at thiadiazole).

4-Methyl-N’-(3-isopropyl-2r,6c-bis(4-chlorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5j).

White solid, yield: 70%, m.p: 169 ºC. IR (KBr, νmax cm−1):1651 (C=N) 1500 (C=O), 3435 (N−H st pip), 3302 (N−H st amide), 3062, 2968, 2875 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  0.84, 1.12 (t, 6H, 2CH3 at piperidin ring), 2.25 (m, 1H, CHat piperidin ring), 1.81 (s, 1H, NH at piperidin ring), 2.83 (d, 1H, C5-1Ha), 2.61 (s, 3H, methyl at thiadiazole ring), 2.65 (t, 1H, C3-1Ha), 2.83 (dd, 1H, C5-1He), 4.17 (d, 1H, C2-1Ha), 4.22 (d, 1H, C6-1Ha), 7.27–7.40 (m, 8H, Ar–H), 10.69 (s, 1H, N–H, amide NH).13C (400 MHz, DMSO-d6) δ: 21.11 and 21.33 (2CH3 at piperidin ring), 15.15 (CH3 at thiadiazole), 31.33 (CH at piperidin ring), 36.53 (C-5), 57.33 (C-3), 56.27 (C-6), 62.87 (C-2), 127.95–133.96 (Ar–C), 135.08 (C-5 at thiadiazole), 141.89 and 143.22 (ipso carbons), 157.11 (C-4), 162.48 (NHCO) and 164.13 (C-4 at thiadiazole).

4-Methyl-N’-(3-isopropyl-2r,6c-bis(4-fluorophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5k).

White solid, yield: 78%, m.p: 168 ºC. IR (KBr, νmax cm−1): 1635 (C=N) 1556 (C=O), 3417 (N−H st pip), 3334 (N−H st amide), 3061, 2966, 2870 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  0.85, 1.12 (t, 6H, 2CH3 at piperidin ring), 2.25 (m, 1H, CHat piperidin ring), 2.17 (s, 1H, NH at piperidin ring), 2.81 (d, 1H, C5-1Ha), 2.64 (s, 3H, methyl at thiadiazole ring), 2.64 (t, 1H, C3-1Ha), 2.81 (dd, 1H, C5-1He), 4.16 (d, 1H, C2-1Ha), 4.22 (d, 1H, C6-1Ha), 7.26–7.41 (m, 8H, Ar–H), 10.54 (s, 1H, N–H, amide NH).13C (400 MHz, DMSO-d6) δ: 21.09 and 21.32 (2CH3 at piperidin ring), 15.18 (CH3 at thiadiazole), 30.97 (CH at piperidin ring), 36.47 (C-5), 56.25 (C-3), 57.33 (C-6), 62.87 (C-2), 127.94–133.95 (Ar–C), 135.08 (C-5 at thiadiazole), 141.86 and 143.18 (ipso carbons), 157.02 (C-4), 162.38 (NHCO) and 164.16 (C-4 at thiadiazole).

4-Methyl-N’-(3-isopropyl-2r,6c-bis(4-bromophenyl)piperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide (5l).

White solid, yield: 73%, m.p: 151ºC. IR (KBr, νmax cm−1):1643 (C=N) 1562 (C=O), 3412 (N−H st pip), 3273 (N−H st amide), 3062, 2962, 2870 (C−H st). 1H NMR (400 MHz, DMSO-d6) δ:  0.84, 1.12 (t, 6H, 2CH3 at piperidin ring), 2.26 (m, 1H, CHat piperidin ring), 2.04 (s, 1H, NH at piperidin ring), 2.94 (d, 1H, C5-1Ha), 2.83 (s, 3H, methyl at thiadiazole ring), 2.67 (t, 1H, C3-1Ha), 2.94 (dd, 1H, C5-1He), 4.16 (d, 1H, C2-1Ha), 4.21 (d, 1H, C6-1Ha), 6.99–7.45 (m, 8H, Ar–H), 9.97 (s, 1H, N–H, amide NH). 13C (400 MHz, DMSO-d6) δ: 21.00 and 21.28 (2CH3 at piperidin ring), 15.32 (CH3 at thiadiazole), 31.11 (CH at piperidin ring), 36.49 (C-5), 56.29 (C-3), 57.40 (C-6), 62.96 (C-2), 115.46–129.23 (Ar–C), 135.28 (C-5 at thiadiazole), 141.48 and 143.20 (ipso carbons), 157.18 (C-4), 162.07 (NHCO) and 164.24 (C-4 at thiadiazole).

Cell culture

Cells were grown at 37°C in a humidified 5% CO2-95% air atmosphere. All tissue culture media and serum were purchased from Gibco (Invitrogen). CHO-K1 and and A549 were maintained in F12.  WM-266-4 and HECK293 cells were maintained in EMEM.  BT474 cells were cultured in Hybri-Care Medium. All media were EMEM supplemented with 10% FCS and  1% penicillin/streptomycin.

Cell viability assays by CellTiter Glo reagent.

Briefly, cells were plated in 384-well plates in 8 μL of media. Test  compounds and dabrafenib (pharmacological assay control) were prepared as 10-point, 1:3 serial dilutions starting at 300 μM, then added to the cells (4 μL per well) using the Biomek NXP. Plates were incubated for 4, 24, 48, and 72 h at 37°C, 5% CO2 and 95% relative humidity. After incubation, 4 μL of CellTiter-G lo® (Promega cat#: G7570) were added to each well, and incubated for 15 min at room temperature. Luminescence was recorded using a Biotek Synergy H4 multimode microplate reader. Viability was expressed as a percentage relative to wells containing media only (0%) and wells containing cells treated with DMSO only (100%). Three parameters were calculated on a per-plate basis: (a) the signal-to-background ratio (S/B); (b) the coefficient for variation [CV; CV = (standard deviation/mean) x 100)] for all compound test wells; and (c) the Z’-factor (18). The IC50 value of the pharmacological control (dabrafenib, LC Labora-tories # G-4408) was also calculated to ascertain the assay robustness.

Annexin V flow cytometry assay. WM266-4 cells were seeded at 1,000,000 cells/flask in EMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin in T25 flasks (Nunc cat# 75008384) and allowed to adhere overnight. 24 hours after plating, the cells were treated with compound 5a (100μM) and staurosporine (1μM) for 4, 24 and 48 hrs. After 4, 24 and 48 hrs of compound exposure the adherent and floating cells were combined and stained using the TACS Annexin V-FITC Apoptosis detection kit (Trevigen Inc, Gaithersburg, MD, USA) using manufacturer’s protocol. Viable, necrotic, early and late apoptotic cells were counted using Accuri flow cytometer as per the manufacturer instructions.

Cell staining for autophagy. WM266-4 cells were seeded at 10,000 cells/well in 0.1 mL of E-MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin in 96 well plates (Greiner BioOne CellStar cat# 655180) and allowed to adhere overnight. After overnight incubation 100 μM compound 5a were added and incubated for various length of time. Cells were rinsed with warm PBS and stained with CYTO-ID®1.0 autophagy reagent (Enzo ENZ-51031-0050) and counterstained with DAPI. Cells were imaged using Cytation 5 imager (Biotek Inc, Winooski, VT) using GFP and DAPI filter sets. For % autophagic cells calculations cell counts were conducted using DAPI-stained nuclei and Object Sum Area value was obtained from at least 1,000 cells/well using DAPI channels. To obtain number of cells undergoing autophagy the cell count was conducted using GFP channel. Both GFP and DAPI cell counts were conducted using optimized algorithm in automatic mode. 8 replicate wells were used. % autophagic cells was calculated using Equation 1:

% autophagic cells=100%∗(number of cells,GFP channel/number of cells,DAPI channel)

To calculate staining intensity, the Object Sum Area value from GFP channel was obtained and divided by number of cells using DAPI channel. Both GFP and DAPI cell counts were conducted using optimized algorithm in automatic mode. 3 replicate wells were used.

Cell cycle arrest assay. 3 x 106 cells seeded in 5 mL of E-MEM medium supplemented with 10% FBS  and 1% penicillin/streptomycin in 10 cm plates. After 24 h, 100 μM compound 5a wase added and incubated for various length of time. After incubation,  the cells were harvested in 15 mL tubes, 2 x 106 cells were fixed with 70% ice cold ethanol and stained using cell cycle reagent (Life Technologies # F10797). The cell cycle analysis was performed using Accuri flow cytometer (Biorad).

Western blotting for LC3

1 x 106 of WM266-4 cells were seeded in 3 mL of E-MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin in 6 cm plates. After 24 h, the cells were treated with autophagy control (rapamycin (10 μM) and chloroquine (5 μM)) and compound 5a at 100µM. After treatment, the cells were trypsinized and collected in 15 mL tubes followed by lysis, SDS-PAGE, and western blot analysis of LC3 using polyclonal LC3 A/B anti-body (Cell Signaling Cat# 4108, RP: 1:1000; 2% BSA) and actin using monoclonal β-actin antibody (Sigma-Aldrich A5441). After washing with TBST, the membranes were treated with chemilumiscent horseradish peroxidase detection reagent (Thermo Scientific, Cat# 32209) and exposed to autoradiography film (Denville Scientific, In c., Metuchen, NJ, USA, cat# E3018). ImageJ software (NIH, Bethesda, MD) was used to quantify the intensity of proteins bands. The protein bands were normalized against loading controls (β-actin) and expressed as a fold of an untreated control.

Statistics

Data are expressed as mean ± standard deviation (SD); n=3. The IC50 for in vitro cell viability assay was calculated using linear regression analysis. Analysis of multiple groups was performed by analysis of variance (ANOVA). One-way ANOVA was used followed by Dunnett post hoc test. **** – p < 0.0001, *** – p < 0.001, ** – p <0.01, * – p < 0.05, ns – not significant. 

ASSOCIATED CONTENT

Supporting Information

Spectral data (1H NMR, C13NMR, H−H COSY, NOESY,HSQC, and HMBC) for compound 5a-g.

Notes

The authors declare no competing financial interest

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Table1.   Analytical data of compounds 5a–5g

compounds Molecular formula Molecular weight Elemental analysis found (Calculated) (%)
C H N  
5a C23H25N5OS 419.54 65.23(65.84) 6.00(6.01) 16.21(16.69)
5b C25H29N5OS 447.60 67.02 (67.08) 6.49 (6.53) 15.57(15.65)
5c C25H29N5O3S 479.59 62.59 ( 62.61) 6.05 (6.09) 14.57 (14.60)
5d C23H23Cl2N5OS 488.43 56.55 (56.56) 4.70 (4.75) 14.32 (14.34)
5e C23H23Cl2N5OS 488.43 56.50 (56.56) 4.74 (4.75) 14.31 (14.34)
5f C23H23F2N5OS 455.52 60.59 (60.64) 5.00 (5.09) 15.40 (15.37)
5g C23H23Br2N5OS 577.33  45.80 (47.85) 4.00 (4.02) 12.10 (12.13)

* The observed microanalysis values for C, H and N were within ± 0.4 % of the theoretical values.

Table 2. IC50 of 4-methyl-N’-(3,3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-car-bohydrazide 5a-g against multiple cancer cell Lines.

Cells 151 152 153 154 155 156 157 Dabrafenib Trametinib
Heck293 318.8 523 156.7 191.5 149 294.7 >300 >100 >100
WM-266-4 0.54 2.83 8.76 38.14 10.3 >300 >300 0.40 0.23
M14 8.57 >300 >300 >300 >300 >300 >300 4.21 2.80
A549 >300 >300 >300 >300 >300 >300 >300
BT474 >300 >300 >300 >300 >300 >300 >300

Each IC50 value was calculated from three independent experiments performed in triplicate.

Scheme 1. Schematic representation of the synthesis of 4-methyl-N’-(3-dimethyl-2r,6c-diarylpiperidin-4-ylidene)-1,2,3-thiadiazole-5-carbohydrazide 5 (a-g).  Reagents and conditions: a) CH3CO2NH4, EtOH, warm. b) MeOH: CHCl3, AcOH, reflux.

Compound R X Y
3a 5a H H
3b 5b p-CH3 H
3c 5c p-OCH3 H
3d 5d H o-Cl
3e 5e p-Cl H
3f 5f p-F H
3g 5g p-Br H

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