Research ArticleExperimental Studies

Quantitative Structure–Cytotoxicity Relationship of Azulene Amide Derivatives

KANA IMANARI, MASASHI HASHIMOTO, HIDETSUGU WAKABAYASHI, NORIYUKI OKUDAIRA, KENJIRO BANDOW, JUNKO NAGAI, MINEKO TOMOMURA, AKITO TOMOMURA, YOSHIHIRO UESAWA and HIROSHI SAKAGAMI
Anticancer Research July 2019, 39 (7) 3507-3518; DOI: https://doi.org/10.21873/anticanres.13497

Abstract

Background/Aim: Very few studies of anticancer activity of azulene amides led us to investigate the cytotoxicity of 21 N-alkylazulene-1-carboxamides introduced either with 3-methyl [1-7], 7-isopropyl-3-methyl [8-14] or 2-methoxy group [15-21]. Materials and Methods: Tumor-specificity (TS) was calculated by the ratio of mean 50% cytotoxic concentration (CC50) against three normal human oral mesenchymal cells to that against four human oral squamous cell carcinoma (OSCC) cell lines. Potency-selectivity expression (PSE) was calculated by dividing TS value by CC50 value against OSCC cell lines. Apoptosis-inducing activity was evaluated by caspase-3 activation and appearance of subG1 cell population. Results: [8-14] showed higher TS and PSE values, than [1-7] and [15-21]. The most active compound [8-14] induced apoptosis in C9-22 OSCC cells at 4-times higher CC50. Quantitative structure-activity relationship analysis of [1-14] demonstrated that their tumor-specificity was correlated with chemical descriptors that explain the molecular shape and hydrophobicity. Conclusion: 7-Isopropyl-3-methyl-N-propylazulene-1-carboxamide [8] can be a potential candidate of lead compound for manufacturing new anticancer drug.

Azulene, an isomer of naphthalene, is well known for its beneficial antioxidant effects. Azulene gargle has been used to reduce the incidence of undesirable general anesthesia-induced postoperative sore (1). Sodium azulene sulfonate, a water-soluble derivative of azulene, inhibited the capsaicin-induced pharyngitis in rats, possibly by its antioxidative effect (2). Administration of 6-isopropyl-3-[4-(4-chlorophenylsulfonylamino)butyl]azulene-1-sulfonic acid sodium salt (KT2-962), a thromboxane A receptor antagonist, significantly reduced the myocardial ischemia/reperfusion injury in a dog, possibly by its direct hydroxyl radical scavenging activity (3). Azulenic retinobenzoic acid derivatives effectively suppressed the carcinogen-induced neoplastic transformation of mouse fibroblast C3H/10T1/2 cells (4). Guaiazulene, a lipophilic azulene derivative which is abundant in nature, protected rats from paracetamol-induced hepatocytotoxicity by its antioxidant activity (inhibition of lipid peroxidation and radical scavenging action) (5). Guaiazulene also inhibited CYP1A2 that participates in the formation of toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) and the metabolic activation of several toxic and carcinogenic compounds (6). On the other hand, there is a limited number of studies that have investigated the cytotoxicity of guaiazulene against human malignant (7-9) and non-malignant cells (9, 10).

We recently reported that among ten azulene-related compounds, N-propylguaiazulenecarboxamide showed the highest tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) vs. three normal human oral mesenchymal cells (gingival fibroblast, HGF; periodontal ligament fibroblast, HPLF; pulp cell, HPC) (10). Furthermore, quantitative structure-activity relationship (QSAR) analysis demonstrated the tight correlation between their tumor-specificity and hydrophobicity and molecular shape (11). PubMed search revealed only one study that has investigated the anticancer activity of azulene amides (10).

In order to obtain more tumor-selective guaiazulene derivatives, we have synthesized a total of 21 N-alkylazulene-1-carboxamide where 3-methyl, 7-isopropyl-3-methyl or 2-methoxy groups were introduced (Figure 1) and investigated their anticancer activity using four human oral squamous cell carcinoma (OSCC) cell lines and three normal human oral mesenchymal cells. We also investigated the effect of these compounds on apoptosis induction, since many anticancer drugs have been reported to induce apoptosis in clinical cancer tissues (12).

Figure 1.
Figure 1.

Structure of three groups of azulene amide derivatives used in this study.

Materials and Methods

Materials. The following chemicals were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) from GIBCO BRL (Grand Island, NY, USA); fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin-HCl (DXR) from Sigma-Aldrich Inc., (St. Louis, MO, USA); dimethyl sulfoxide (DMSO), actinomycin D (Act. D) from Wako Pure Chem. Ind., (Osaka, Japan); culture plastic dishes and 96-well plates from Techno Plastic Products AG, (Trasadingen, Switzerland). Protease and phosphatase inhibitors were purchased from Roche Diagnostics (Tokyo, Japan).

Synthesis of alkylaminogroups. 3-methyl-N-propylazulene-1-carboxamide [1], 3-methyl-N-butylazulene-1-carboxamide [2], 3-methyl-N-pentylazulene-1-carboxamide [3], 3-methyl-N-(2-hydroxyethyl)azulene-1-carboxamide [4], 3-methyl-N-(3-hydroxypropyl) azulene-1-carboxamide [5], 3-methyl-N-(2-methoxyethyl)azulene-1-carboxamide [6], 3-methyl-N-(3-methoxypropyl)azulene-1-carboxamide [7], 7-isopropyl-3-methyl-N-propylazulene-1-carboxamide [8], 7-isopropyl-3-methyl-N-butylazulene-1-carboxamide [9], 7-isopropyl-3-methyl-N-pentylazulene-1-carboxamide [10], 7-isopropyl-3-methyl-N-(2-hydroxyethyl)azulene-1-carboxamide [11], 7-isopropyl-3-methyl-N-(3-hydroxypropyl)azulene-1-carboxamide [12], 7-isopropyl-3-methyl-N-(2-methoxyethyl)azulene-1-carboxamide [13], 7-isopropyl-3-methyl-N-(3-methoxypropyl)azulene-1-carboxamide [14], 2-methoxy-N-propylazulene-1-carboxamide [15], 2-methoxy-N-butylazulene-1-carboxamide [16], 2-methoxy-N-pentylazulene-1-carboxamide [17], 2-methoxy-N-(2-hydroxyethyl)azulene-1-carboxamide [18], 2-methoxy-N-(3-hydroxypropyl)azulene-1-carboxamide [19], 2-methoxy-N-(2-methoxyethyl)azulene-1-carboxamide [20], 2-methoxy-N-(3-methoxypropyl)azulene-1-carboxamide [21] (structures shown in Figure 1) were synthesized, according to previous reports (13-17). All compounds were dissolved in DMSO at 40 mM and stored at −20°C before use.

Cell culture. Human normal oral cells (HGF, HPLF, HPC) cells (18) at 12~20 population doubling level (PDL) and OSCC cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) (Riken Cell Bank, Tsukuba, Japan) were cultured at 37°C in DMEM supplemented with 10% heat-inactivated FBS and antibiotics as described previously (11).

Assay for cytotoxic activity. Cells were inoculated at 2×103 cells/0.1 ml in a 96-microwell plate. After 48 h, the medium was replaced with 0.1 ml of fresh medium containing different concentrations of test compounds. Control cells were treated with the same amounts of DMSO present in each diluent solution. Cells were incubated for 48 h and the relative viable cell number was then determined by the MTT method, as described previously (11). The CC50 was determined from the dose–response curve of triplicate samples.

Calculation of tumor-specificity index (TS). TS was calculated as the ratio of mean CC50 (HGF+HPLF+HPC) to mean CC50 (Ca9-22+HSC-2+HSC-3+HSC-4), using seven cell lines [(D/B) in Table I] (19), and as the ratio of CC50 (HGF) to CC50 (Ca9-22) [(C/A) in Table I], using two cell lines derived from the gingival tissue (20). Normal keratinocytes, which are highly sensitive to many anticancer drugs (21), were not used in this study.

View this table:
Table I.

Cytotoxicity of 21 azulene amide derivatives and doxorubicin against human oral squamous cell carcinoma cell lines and human oral normal cells.

Calculation of potency-selectivity expression (PSE). PSE was calculated by dividing TS value by CC50 against tumor cells (19) [(D/B2) ×100 and (C/A2) ×100] (Table I).

Western blot analysis. The cells were washed, lysed and their protein extracts subjected to western blot (WB) analysis, as described previously (11). The blots were probed with the primary antibody [antibodies against cleaved caspase-3 (Cell Signaling Technology Inc., Beverly, MD, USA), poly ADP-ribose polymerase (PARP) (Cell Signaling Technology Inc.) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Trevigen, Gaithersburg, MD, USA), followed by incubation with a horseradish peroxidase-conjugated secondary antibody [α-rabbit IgG (DAKO, Tokyo Japan)]. The immune complexes were visualized using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific). WB results were documented and quantified using ImageQuant LAS 500 (GE Healthcare, Tokyo, Japan) (22).

Cell cycle analysis. Cells (approximately 106 cells) were harvested, fixed with paraformaldehyde (Wako) in PBS(-), treated with ribonuclease (RNase) A (Sigma-Aldrich Inc.), stained propidium iodide (PI) (Wako) in the presence of 0.01% NonidetP-40 (Nakalai Tesque Inc., Kyoto, Japan), filtered through Falcon® cell strainers (Corning, NY, USA) and then were subjected to cell sorting (SH800 Series, SONY Imaging Products and Solutions Inc., Atsugi, Kanagawa, Japan), as described previously (19). Cell cycle analysis was performed with Cell Sorter Software version 2.1.2. (SONY Imaging Products and Solution Inc.) (19).

Calculation of chemical descriptors. pCC50 (i.e., the −log CC50) was used for the comparison of the cytotoxicity between the compounds, since the CC50 values had a distribution pattern close to a logarithmic normal distribution. The mean pCC50 values for normal cells and tumor cell lines were defined as N and T, respectively (23). The 3D-structure of each chemical structure was drawn by Marvin Sketch ver 16 (ChemAxon, Budapest, Hungary, http://www.chemaxon.com), and optimized by CORINA Classic (Molecular Networks GmbH, Nürnberg, Germany) with partial charge calculations (amber-10: EHT) in Molecular Operating Environment (MOE) version 2018.0101 (Chemical Computing Group Inc., Quebec, Canada) and Dragon (Dragon 7 version 7.0.2, Kode srl., Pisa, Italy).

Figure 2.
Figure 2.

Cytotoxicity of 3-methyl-N-pentylazulene-1-carboxamide [3], 7-isopropyl-3-methyl-N-propylazulene-1-carboxamide [8], 7-isopropyl-3-methyl-N-butylazulene-1-carboxamide [9] and 2-methoxy-N-pentylazulene-1-carboxamide [17] against human malignant and non-malignant cells. Human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) and human oral normal cells (HGF, HPLF, HPC) were treated for 48 h with the indicated concentrations of [3], [8], [9], or [17] and viable cell number was determined by the MTT method. Each value represents mean±S.D. of triplicate assays.

Statistical analysis. Each experimental value was expressed as the mean±standard deviation (SD) of triplicate or quadruplicate measurements. The correlation between chemical descriptors and cytotoxicity or tumor specificity was investigated using simple regression analyses by JMP Pro version 14.0.0 (SAS Institute Inc., Cary, NC, USA). The significance level was set at p<0.05.

Results

Cytotoxicity. Twenty one azulene amides used in this study were classified into three groups, 3-methyl-N-alkylazulene-1-carboxamides [1-7] (Group A), 7-Isopropyl-3-methyl-N-alkylazulene-1-carboxamides [8-14] (Group B) and 2-methoxy-N-alkylazulene-1-carboxamides [15-21] (Group C) (Figure 1). We compared their cytotoxic activity against four human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) and three normal oral cells (HGF, HPLF, HPC), by comparing the 50% cytotoxic concentration (CC50) determined from the dose-response curve (example shown in Figure 2) (Table I).

Generally, group B compounds showed higher cytotoxicity against OSCC cell lines (mean CC50=107 μM) than group C (246 μM) and group A compounds (280 μM). Among 21 compounds, [8] showed the highest cytotoxicity (CC50=42 μM), followed by [3] (63 μM) and [17] (67 μM). From the dose-response curve, [3, 8, 17] and [9] showed cytotoxic and cytostatic growth inhibition against four OSCC cell lines, respectively (Figure 2). On the other hand, group A, B and C compounds showed comparable cytotoxicity against normal oral cells (mean CC50=313, 301 and 318 μM, respectively).

Tumor-specificity (TS). TS was determined by dividing the mean CC50 value towards the three normal cells by the mean CC50 value towards the four OSCC cell lines (D/B, Table I) or by dividing the CC50 value against HGF cells by the CC50 value against Ca9-22 cells, two cells derived from the gingival tissue (C/ A, Table I). Among Group A, [3] showed the highest tumor-specificity (TS=3.9 in D/B; 3.6 in C/A). Among Group B, [8] showed the highest tumor-specificity (TS= 7.1 in D/B; 10.1 in C/A), followed by [9] (TS=3.7, in D/B; 11.1 in C/A). Among Group C, [17] showed the highest tumor-specificity (TS=2.3 in D/B, 2.9 in C/A) (Table I).

Figure 3.
Figure 3.

Assessment of caspase-3 activation by [3], [8], or [17]. (A) Comparison between three compounds. (B) Dose-response and (C) time course of apoptosis induction by [8]. Ca9-22 cells were incubated for 24 h with the indicated concentrations of [3], [8], or [17] or 1 μM actinomycin D (Act D) as positive control and subjected to western blot analysis.

PSE value. Next, the potency-selectivity expression (PSE) value that reflects both the cytotoxicity against OSCC and tumor-specificity was determined. [8] showed much higher PSE value [16.9 in (D/B2) ×100; 28.5 in (C/A2) ×100)], followed by [3] (PSE=6.1; 4.7), [9] (PSE=3.8; 30.8) and [17] (PSE=3.5; 5.6).

Apoptosis induction. Western blot analysis (Figure 3A) demonstrated that among [3], [8], and [17] compounds, [8] induced the cleavage of PARP, one of the substrates of caspase-3, most potently, followed by [17] and [3], while [17] induced cleavage of caspase-3 (producing active form) most potently, followed by [8] and [3]. Dose-response study (Figure 3B) showed that cleavage of PARP by [8] was detected above 80 μM, while activation of capsase-3 was detected above 160 μM, suggesting the induction of apoptosis (24). Time course study (Figure 3C) shows that cleavage of PARP was observed at early stage (1, 2 and 4 h after treatment with 80 μM [8]), and declined thereafter (at 24 h).

Light microscopical observation (upper column, Figure 4) revealed that cells were damaged and began to detach by treatment with higher concentrations (160 or 200 μM) of all three compounds [3, 8, 17]. Both [17] and actinomycin D (positive control) produced apoptotic cells (upper column, Figure 4). Cell-cycle analysis demonstrated that [17] increased the subG1 cell population to an extent similar with that of actinomycin D, while [8] was slightly less potent in apoptosis induction (lower panel, Figure 4).

Figure 4.
Figure 4.

Production of subG1 population by [3], [8], or [17]. Ca9-22 cells were incubated for 24 or 48 h with the indicated concentrations of [3], [8], [17] or 1 μM actinomycin D (Act D) as a positive control and subjected to cell sorter analysis.

Figure 5.
Figure 5.

Determination of coefficient between chemical descriptors and cytotoxicity of 14 Group A and B compounds against tumor cells (defined as T). The mean (pCC50 i.e., the −log CC50) values for tumor cell lines were defined as T.

Computational analysis. Since 3-methyl-N-alkylazulene-1-carboxamides [1-7] (Group A), 7-Isopropyl-3-methyl-N-alkylazulene-1-carboxamides [8-14] (Group B) showed a higher tumor-specificity than 2-methoxy-N-alkylazulene-1-carboxamides [15-21] (Group C), QSAR analysis was performed with Group A and B compounds [1-14]. The number of descriptors calculated from MOE and dragon was 344 and 5,255, respectively. As a result of excluding duplicate descriptors, the number of descriptors reduced to 301 and 2,765, respectively. Among a total of 3,066 descriptors, the top six descriptors that showed the highest correlation coefficient (r2) to T, N and T-N are shown in Table II.

Figure 6.
Figure 6.

Determination of coefficient between chemical descriptors and cytotoxicity of 14 Group A and B compounds against normal cells (defined as N). The mean (pCC50 i.e., the −log CC50) values for normal cells were defined as N.

Cytotoxicity against human OSCC cell lines was correlated with descriptors SpMin1_Bh(m) (topological shape) (r2=0.827, p<0.0001), BCUT_SLOGP_3 (topological shape) (r2=0.796, p<0.0001), MATS8m (topological shape and size) (r2=0.741, p=0.0001), H% (percentage of H atoms) (r2=0.734, p=0.0001), Eig04_EA(ri) (topological shape and energy) (r2=0.726, p=0.0001), AMW (average molecular weight) (r2=0.725, p=0.0001) (Figure 5).

Figure 7.
Figure 7.

Determination of coefficient between chemical descriptors and tumor specificity of 14 Group A and B compounds (defined as T–N).

Cytotoxicity against human normal oral mesenchymal cells was correlated with descriptors Depressant-50 (Drug-like indices) (p=0.0025), Neoplastic-50 (Drug-like indices) (p=0.0025), BCUT_SLOGP_2 (topological shape) (r2=0.540, p=0.0027), Eig05_AEA(dm) (topological shape and dipole moment) (r2=0.514, p=0.0039), IC2 (topological shape) (r2=0.424, p=0.0117), F10[C-O] (topological shape) (r2=0.403, p=0.0147) (Figure 6).

View this table:
Table II.

Top six chemical descriptors that correlate with cytotoxicity to tumor cells, normal cells and tumor-specificity (having the highest r2 values).

Tumor specificity was correlated with descriptors vsurf_D6 (3D shape and size) (r2=0.815, p<0.0001), P_VSA_ppp_L (topological shape and lipophilicity) (r2=0.813, p<0.0001), E_str (3D shape and energy) (r2=0.792, p<0.0001), PEOE_VSA_NEG (topological shape and partial charge) (r2=0.790, p<0.0001), Q_VSA_NEG (topological shape and partial charge) (r2=0.790, p<0.0001), Mor27u (3D shape and size) (r2=0.789, p<0.0001) (Figure 7).

Discussion

The present study demonstrated that 7-isopropyl-3-methyl-N-alkylazulene-1-carboxamides [8-14] (Group B) showed slightly higher OSCC-specific cytotoxicity than 3-methyl-N-alkylazulene-1-carboxamides [1-7] (Group A) and 2-methoxy-N-alkylazulene-1-carboxamides [15-21] (Group C). Among them, 7-isopropyl-3-methyl-N-propylazulene-1-carboxamide [8] showed the highest tumor-specificity towards OSCC over normal oral cells, based on TS and PSE values. [8] induced apoptosis markers such as caspase-3 activation (24) and production of subG1 population (25) only at 160 μM (Figure 3), four times the CC50 (42 μM) (Table I). On the other hand, [17], that showed lower tumor-specificity than [8] (TS=2.3 vs. 7.1 (D/B), 2.9 vs. 10.1 (C/A); PSE=3.5 vs. 16.9 in (D/B2)×100, 5.6 vs. 28.5 in C/A2)×100) (Table I), activated caspase-3 and produced a higher subG1 population (Figure 4). These data suggest that induction of tumor-specific cytotoxicity (anti-tumor activity) by [8] may not be mediated via apoptosis induction. There are many types of cell death, such as intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition (26). Further study is required to investigate the possibility of the involvement of necrotic cell death, such as pyroptosis and necroptosis, in which the activation of caspase-1, IL-1β and IL-18IL-pathways is involved (27).

QSAR analysis with 14 Group A and B compounds demonstrated that their tumor-specificity was correlated with molecular shape (descripted by vsurf_D6, P_VSA_ppp_L, E_str, PEOE_VSA_NEG, Q_VSA_NEG, Mor27u) and lipophilicity (descripted by P_VSA_ppp_L) (Table II). We also reported previously that tumor-specificity of ten N-alkylguaiazulenecarboxamides was correlated with molecular shape (descripted by vsurf_ID1, vsurf_ID5, vsurf_ID4, vsurf_CW4, vsurf_ID3, vsurf_CW3) and hydrophobicity (descripted by vsurf_ID1, vsurf_ID5, vsurf_ID4, vsurf_ID3) (11). These data suggest that the antitumor-potential of guaiazulenes can be estimated by their molecular shape and hydrophobicity. By closer inspection, [9] (100~400 μM) and [17] (13~100 μM) were found to stimulate the growth of normal oral cells. The biological significance of this hormetic growth stimulation (28) remains to be investigated.

[8] and [9] have similar chemical structure to guaiazulene (1,4-dimethyl-7-isopropylazulene), all of them having isopropyl group in seven-membered ring and methyl group at the different positions. As far as we know only two studies have been published on the anticancer activity against OSCC (8, 29). More tumor-specific derivatives of [8] as a lead compound are being synthesized to examine their anti-cancer effects.

Acknowledgements

This work was partially supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (16K11519).

Footnotes

  • Author's Contributions

    Hiroshi Sakagami designed the study and wrote the draft of the manuscript Masashi Hashimoto and Hidetsugu Wakabayashi synthesized all compounds. Hidetsugu Wakabayashi supervised the overall study and revised the manuscript. Kana Imanari and Noriyuki Okudaira performed the western blot analysis. Kenjiro Bandow, Mineko Tomomura and Akito Tomomura performed the cell cycle analysis. Junko Nagai and Yoshihiro Uesawa performed the QSAR analysis.

  • Conflicts of Interest

    The Authors confirm that there are no known conflicts of interest associated with this publication and there was no significant financial support for this work that could have influenced its outcome.

  • Received March 18, 2019.
  • Revision received April 12, 2019.
  • Accepted April 15, 2019.

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Quantitative Structure–Cytotoxicity Relationship of Azulene Amide Derivatives
KANA IMANARI, MASASHI HASHIMOTO, HIDETSUGU WAKABAYASHI, NORIYUKI OKUDAIRA, KENJIRO BANDOW, JUNKO NAGAI, MINEKO TOMOMURA, AKITO TOMOMURA, YOSHIHIRO UESAWA, HIROSHI SAKAGAMI
Anticancer Research Jul 2019, 39 (7) 3507-3518; DOI: 10.21873/anticanres.13497
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