Tumor suppressive activities of solvatochromic 3,3′-azadimethylene dinaphthospiropyran in colon cancer model

Abstract

Spiropyrans have been extensively investigated because of their thermo- and photochromic characteristics, but their biotherapeutic properties have not been explored much. We report anti-proliferative properties of a novel 3,3′-azadimethylene dinaphthospiropyran 11. Dibenzospiropyrans and dinaphthospiropyrans were synthesized by a simple and expedient method using acid-catalyzed aldol condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde, respectively, with cyclic ketones. Together with structural elucidation by 2D NMR and X-ray crystallography studies, we provide a putative mechanism for their formation. Compound 11 showed solvatochromism and exhibited altered spectral characteristics depending on the pH. In acidic conditions, 11 remains in open form, whereas upon alkalinization it reverts back to closed form. Based on the in vitro anti-proliferative activity in H441, HCT-116, MiaPaCa-2, and Panc-1 cancer cell lines, 11 was submitted to further investigation. It reduced HCT116 colonosphere formation and demonstrated induction of caspase cascade, suggesting apoptosis. In vitro proliferation assays also suggested that HCl and trifluoroacetate salts of 11 are more effective. Treatment of mice carrying HCT-116 xenografts with 11 (5 μg/day, intraperitoneal for 3 weeks) suppressed tumor growth by 62%. Overall, the results reveal a new series of structurally complex, but relatively easy to synthesize molecules of which compound 11 represents a lead for anticancer development.

1 |. INTRODUCTION

Reversible photochromism, thermochromism, electrochromism, and solvatochromism are exhibited by organic molecules belonging to azobenzene, spiropyran, spirooxazime, and naphthopyran classes. As a molecular class, spiropyrans are characterized by two orthogonal π-networks connected by a tetrahedral carbon center (Lukyanov & Lukyanov, 2005). They are characterized by a reversible molecular switching between two or more stable states in response to external stimuli (Fischer & Hirshberg, 1952). The stimuli could be a ultraviolet (UV) and visible light irradiation, or a change in acidity or basicity of the medium (Raymo & Giordani, 2001). They may exist as the colorless closed or spiro form (SP), the highly conjugated colored open merocyanine form (MC), and the protonated open form (Giordani & Raymo, 2003). The mechanisms for the thermal and photochemical transformations in the ring-opening and closure pathways have been extensively examined. In the traditional spiropyrans, two planar cycles of the SP isomer are attached by a common sp³ hybridized “spiro” carbon atom; photoexcitation breaks the carbon–oxygen bond and allows rotation across the C-C bond resulting in the MC form (Murugan, Chakrabarti, & Agren, 2011). In addition to the significant difference in the absorption spectra of the two isomers, several other molecular and bulk properties, like refractive index, dielectric constant, and redox potential, are also different in the two forms (Murugan et al., 2011).

Researchers have investigated spiropyran type of compounds for use in optics, but reports on the potential biological applications of spiropyran derivatives are very few. Their ability to serve as a molecular switch in biosensing and optical imaging is beginning to get investigated (Movia, Prina-Mello, Volkov, & Giordani, 2010; Wagner et al., 2011; Yan, Marriott, Petchprayoon, & Marriott, 2011). For instance, Shiraishi, et al. recently reported a sensitive coumarin–spiropyran conjugate as a cyanide-selective fluorescence-based chemosensor (Shiraishi, Sumiya, & Hirai, 2011). In another interesting application, a photochromic spiropyran has been reported as a photo-switchable regulator of l-DOPA release from spiropyran-capped gold nanoparticles (Ipe, Mahima, & Thomas, 2003). The ability of reversible transition between the SP and MC forms is crucial to the optic and biosensing applications of spiropyrans.

Thermodynamic stability and the existence of a preferred form, SP or MC, are affected by the nature of aromatic rings, substituents, solvents, etc. (Minkin, 2004; Roxburgh & Sammes, 1995; Roxburgh, Sammes, & Abdullah, 2009). While most common spiropyrans respond to external thermal and photochemical stimuli, modulation of SP-MC systems through acid–base equilibrium could have interesting consequences in biological systems (Cheng, Lai, Chiang, & Chiu, 2006; Wojtyk et al., 2007). In this article, we report a new series of pH-sensitive spiropyrans synthesized by an efficient and single-step method. We also made an attempt to explain the pH-dependent SP-MC transformations of this new spiropyran series. A selected compound 3,3′-azadimethylene dinaphthospiropyran (11) was evaluated in vivo in a mouse model. This compound was chosen as representative of the entire spiropyran series based on its in vitro anti-proliferative activity in cancer cells as well as its behavior illustrative of the series. While the use of spiropyrans as switchable drug release mechanism in nanoparticles has been reported (Tong, Hemmati, Langer, & Kohane, 2012; Zhu et al., 2010), this article appears to be the first in reporting spiropyrans with anticancer activity both in vitro and in vivo. The results provide a lead from which more potent and switchable anticancer molecules could be designed.

2 |. EXPERIMENTAL

All the chemicals were obtained from common vendors and were used without further purification. ¹H NMR spectra and ¹³C NMR spectra (DMSO-d6 and CDCl₃) were recorded at 300 MHz and 75 MHz on Mercury-VX 300 (Varian Inc.). The spectra were referenced to the residual protonated solvents. The chemical shifts and coupling constants were reported in δ parts per million (ppm) and hertz (Hz), respectively. For running HSQC and HMBC, the NMR data were collected on a Varian VNMRS-400 MHz NMR using an Auto X-indirect detection probe. The proton spectrum was collected with 1 scan and a 90 degree pulse width. The carbon chemical shifts were found indirectly using the HSQC and gradient HMBC experiments. The pulse widths were used as supplied by Varian in the VNMRJ 2.2D software using 32 transients and 128 increments. The proton–proton coupling relationships were found using the gradient-COSY experiment with 16 transients and 128 increments. Mass spectra were recorded by Finnigon Mat LCQ mass spectrometer. The melting points of the compounds were recorded on an Electrothermal Mel-Temp melting point apparatus (Thermo Scientific). The reported melting points (°C) are uncorrected.

2.1 |. Chemistry

2.1.1 |. General synthesis of oxaspiro compounds

Hydrochloric acid gas (generated in situ) was bubbled into a solution of cyclic ketone (1 eq) in glacial acetic acid for approximately 15 min. Aromatic hydroxyl aldehyde (2 eq) was added to the solution, and the reaction mixture was left at room temperature for 24–48 hr. The crystals formed were filtered on a Buchner funnel, suspended in methylene chloride, and washed with 20% ammonia solution. The organic phase was separated and dried to obtain the corresponding spiro-compounds.

2.1.2 |. 3,3′-Azadimethylene dibenzospiropyran (3)

From (0.50 g, 3.27 mmol) of 4-piperidone hydrochloride monohydrate and (0.69 ml, 6.53 mmol) of salicylaldehyde, the title compound 3 was obtained as red crystalline solid (0.83 g, 89% yield). ¹H NMR (400 MHz, DMSO-d6): δ 9.93 (br, NH), 7.47 (dd, 2H, Ar-H, J = 7.6, 1.6 Hz), 7.29 (s, 2H, 2C=CH), 7.29 (td, 2H, Ar-H, J = 7.6, 1.0 Hz), 7.11 (td, 2H, Ar-H, J = 7.6, 1.0 Hz), 6.87 (dd, 2H, Ar-H, J = 7.6, 1.0 Hz), 4.11 (dt, 2H, CH₂, J = 13.5, 3.3 Hz), 3.71 (dt, 2H, CH₂, J = 13.5, 7.0 Hz). ¹³C NMR (100 MHz, DMSO-d6): δ 149.5, 130.6, 128.6, 127.4, 127.4, 122.4, 120.7, 120.1, 116.3, 40.9. ESI mass calculated for C₁₉H₁₆NO₂ (M + H)⁺ 290.12 found 290.00.

The HSQC and HMBC data of compound 3 are included in Table 1, whereas it’s COSY and NOESY data are shown in Table 2.

TABLE 1.

HSQC and HMBC data of compound 3

13C chemical shift	Multiplicity	One-bond C–H coupling (HSQC)	Long-range C–H coupling (HMBC)
40.9	CH2	3.71, 4.11	7.29
92.9	C	—	7.29, 4.11
116.3	CH	6.87	7.11,
120.1	C	—	7.29, 7.11, 6.87
120.7	C	—	3.71
122.4	CH	7.11	6.87, 7.47
127.4	CH	7.47	7.29
128.6	CH	7.29	7.47, 3.71
130.6	CH	7.29	7.47
149.5	C	—	7.47, 7.29, 6.87

TABLE 2.

COSY and NOESY data of compound 3

1H Chemical Shift	Multiplicity	Integration	Coupling Constant, J (Hertz)	gCOSY (1H−1H coupling)	NOESY (1H−1H through space)
3.71	dt	2	13.5, 7.0	4.11, 7.29 9.93
4.11	dt	2	13.5, 3.3	3.71, 9.93
6.87	dd	2	7.6, 1.0	7.29, 7.11
7.11	td	2	7.6, 7.6, 1.0	7.47, 7.29, 6.87
7.29	td	2	7.6, 7.6, 1.6	7.47, 7.11, 6.87
7.29	s	2	—	3.71	4.11
7.47	dd	2	7.6, 1.6	7.29, 7.11
9.93	broad	2	—	3.71, 4.11

2.1.3 |. 3,3′-(N-tosyl)azadimethylene dibenzospiropyran (4)

Pyridine (1 ml) and tosyl chloride (73 mg, 0.36 mmol) were added to a solution of 3,3′-azadimethylene dibenzospiropyran (3, 100 mg, 0.34 mmol) in anhydrous methylene chloride (3 ml). The reaction mixture was stirred at room temperature for 16 hr. After completion of the reaction, the mixture was diluted with methylene chloride and washed with water. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to obtain yellow solid. The crude compound was purified through silica column using 50% ethyl acetate in hexanes. The fractions containing the desired compound were collected and dried to get the tosylate derivative 4 as white solid (130 mg, 81% yield).¹H NMR (300 MHz, DMSO-d6): δ 7.69 (d, 2H, Ar-H, J = 8.0 Hz), 7.24–7.14 (m, 6H, Ar-H), 7.01 (dd, 2H, Ar-H, J = 7.6 Hz), 6.75 (d, 2H, Ar-H, J = 8.0 Hz), 6.65 (s, 2H, C=CH), 4.32 (d, 2H, CH₂, J = 13.4 Hz), 3.99 (dd, 2H, CH₂, J = 13.4, 1.8 Hz), 2.28 (s, 3H, CH₃). ¹³C NMR (75 MHz, DMSO-d6): δ 150.06, 143.84, 134.74, 130.20, 129.88, 129.70, 127.40, 127.09, 126.14, 123.67, 122.36, 120.58, 116.89, 46.39, 21.43. ESI mass calculated for C₂₆H₂₁NNaO₄S (M + Na)⁺ 466.11 found 466.01.

2.1.4 |. 3-(2, β-Thiodimethylene) [2-(2-hydroxy)-1-benzylvinyl)]benzopyrilium chloride (5)

4-Tetrahydrothiophenone (0.50 g, 4.30 mmol) and salicylaldehyde (0.90 ml, 8.60 mmol) were allowed to react in presence of dry hydrochloric acid gas. The solid obtained was filtered on Buchner funnel to obtain title compound 5 as red crystalline solid. The solid was washed with ether (1.20 g, 91% yield). The crystal structure of compound 5 was determined by X-ray diffraction. ¹H NMR (300 MHz, DMSO-d6): δ 8.27 (br s, OH), 7.34 (d, 2H, Ar-H, J = 7.5 Hz), 7.22 (t, 2H, Ar-H, J = 7.5 Hz), 7.10–7.00 (m, 4H, Ar-H, C=CH), 6.81 (d, 2H, Ar-H, J = 8.1 Hz), 3.61 (d, 2H, CH₂, J = 12.0 Hz), 3.46 (d, 2H, CH₂, J = 12.0 Hz).

The HSQC and HMBC data of compound 5 are included in Table S1, whereas it’s COSY and NOESY data are shown in Table S2. The hydrogen bonds present in the open form of compound 5 are described in Table S4. The hydrogen bond between the oxygen atom of open ring with hydrogen of HCl could be noted in Figure S1.

2.1.5 |. 3,3′-Thiodimethylene dibenzospiropyran (6)

The styrylpyrilium salt 5 was suspended in ether, and 20% aqueous ammonium hydroxide solution was added. The organic phase was washed with water, separated, dried over anhydrous sodium sulfate, and concentrated to obtain 6. The solid was recrystallized from chloroform to obtain white crystals. The single crystal X-ray analysis of this compound was performed (Figure S2), and the data are included in Table S4. The bond lengths and angles are reported in Table S5. ¹H NMR (300 MHz, DMSO-d6): δ 7.28 (d, 2H, Ar-H, J = 7.5 Hz), 7.67 (t, 2H, Ar-H, J = 7.5 Hz), 7.05–6.90 (m, 4H, 2Ar-H, 2C=CH), 6.77 (d, 2H, Ar-H, J = 7.8 Hz), 3.51 (dd, 4H, CH₂, J = 12.0 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 149.74, 130.14, 128.08, 127.39, 124.07, 122.76, 121.05, 116.69, 95.63, 26.77. FT IR (cm⁻¹): 1,485, 1,459, 1,229, 957.4, 762.4, 756.8. ESI mass calculated for C₁₉H₁₅O₂S (M + H)⁺ 307.08 found 307.00.

2.1.6 |. 3,3′-Oxydimethylene dibenzospiropyran (7)

From (0.40 g, 0.40 mmol) of 4-tetrahydropyranone and (0.84 ml, 0.80 mmol) of salicylaldehyde, we obtained the title compound 7 as crystalline solid (0.89 g, 73% yield). ¹H NMR (300 MHz, DMSO-d6): δ 7.37 (d, 2H, Ar-H, J = 7.0 Hz), 7.21 (t, 2H, Ar-H, J = 7.3 Hz), 7.10–7.00 (m, 4H, 2Ar-H, 2C=CH), 6.78 (d, 2H, Ar-H, J = 7.9 Hz), 4.40 (d, 2H, CH₂, J = 11.9 Hz), 4.29 (d, 2H, CH₂, J = 11.9 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 150.25, 130.55, 127.81, 127.21, 125.44, 122.90, 121.53, 116.93, 93.29, 63.59. ESI mass calculated for C₁₉H₁₅O₃ (M + H)⁺ 291.10 found 291.10.

2.1.7 |. 3,3′-Dimethylene dibenzospiropyran (8)

From cyclopentanone (1.0 g, 11.89 mmol) and salicylaldehyde (2.5 ml, 23.74 mmol), we obtained the title compound 8 as crystalline solid (2.91 g, 89% yield). ¹H NMR (300 MHz, DMSO-d6): δ 7.37 (dd, 2H, Ar-H, J = 7.3, 1.8 Hz), 7.17 (td, 2H, Ar-H, J = 7.6, 1.8 Hz), 7.05 (td, 2H, Ar-H, J = 7.6, 1.2 Hz), 6.91 (s, 2H, C=CH), 6.76 (d, 2H, Ar-H, J = 7.9 Hz), 2.82–7.72 (m, 2H, CH₂), 2.65–2.56 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d6): δ 147.80, 132.07, 130.37, 129.75, 128.96, 127.64, 122.43, 118.10, 115.09, 55.38. ESI mass calculated for C₁₉H₁₅O₂ (M + H)⁺ 275.12 found 275.00.

2.1.8 |. 3,3′-Trimethylene dibenzospiropyran (9)

From cyclohexanone (1 g, 10.4 mmol) and salicylaldehyde (2.2 ml, 20.4 mmol), we obtained the title compound 9 as crystalline solid (2.54 g, 85% yield). ¹H NMR (300 MHz, DMSO-d6): δ 7.19 (dd, 2H, Ar-H, J = 7.3,1.8 Hz), 7.07 (td, 2H, Ar-H, J = 6.2, 1.5 Hz), 6.94 (td, 2H, Ar-H, J = 7.6, 1.2 Hz), 6.71 (s, 2H, C=CH), 6.69–6.65 (m, 2H, Ar-H), 2.60–2.44 (m, 2H, CH₂), 2.41–2.26 (m, 2H, CH₂), 1.80–1.68 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d6): δ 150.00, 136.59, 131.90, 129.01, 126.51, 122.18, 116.54, 95.85, 27.10, 26.31. ESI mass calculated for C₂₀H₁₇O₂ (M + H)⁺ 289.12 found 289.10.

2.1.9 |. 3,3′-Tetramethylene dibenzospiropyran (10)

From of cycloheptanone (1 g, 8.91 mmol) and salicylaldehyde (1.90 ml, 18.4 mmol), we obtained the title compound 10 as crystalline solid (2.09 g, 78% yield). ¹H NMR (300 MHz, DMSO-d6): δ 7.18 (dd, 2H, Ar-H, J = 7.6,1.6 Hz), 7.09 (td, 2H, Ar-H, J = 7.6, 1.6 Hz), 6.93 (td, 2H, Ar-H, J = 7.3, 0.9 Hz), 6.71 (s, 2H, C=CH), 6.68 (d, 2H, Ar-H, J = 0.9 Hz), 2.60–2.44 (m, 2H, CH₂), 2.24 (t, 2H, CH₂, J = 11.7 Hz), 2.08–1.95 (m, 2H, CH₂), 1.45 (t, 2H, CH₂, J = 10.8 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 149.17, 135.32, 129.02, 126.37, 123.50, 121.98, 120.98, 116.09, 113.80, 33.42, 32.22. ESI mass calculated for C₂₁H₁₉O₂ (M + H)⁺ 303.14 found 303.20.

2.1.10 |. 3,3′-Azadimethylene dinaphthospiropyran (11)

From 4-piperidone hydrochloride monohydrate (0.50 g, 3.27 mmol) and 2-hydroxy-1-naphthaldehyde (1.12 g, 6.50 mmol), we obtained the title compound 11 as crystalline solid (0.63 g, 49% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.32 (d, 2H, Ar-H, J = 7.9 Hz), 8.00–7.80 (m, 4H, Ar-H), 7.70–7.60 (m, 2H, Ar-H), 7.54–7.42 (m, 4H, Ar-H, 2C=CH), 7.20–7.00 (m, 2H, 2Ar-H), 3.90 (d, 2H, CH₂, J = 14.2 Hz), 3.77 (dd, 2H, CH₂, J = 14.2 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 147.78, 130.31, 129.79, 128.97, 127.63, 126.81, 125.33, 124.87, 122.46, 122.89, 118.12, 115.15, 114.10, 55.39, 44.84. ESI mass calculated for C₂₇H₂₀NO₂ (M + H)⁺ 390.15 found 390.10.

We prepared trifluoroacetate and hydrochloride salts by adding acids into the methanolic solution of 11. The salts were precipitated by adding the solution dropwise into water. The precipitates were washed with water (thrice ×10 ml) and dried under vacuum to obtain respective salts.

2.1.11 |. 3,3′-Thiodimethylene dinaphthospiropyran (12)

From 4-tetrahydrothiophenone (0.50 g, 4.30 mmol) and 2-hydroxy-1-naphthaldehyde (1.48 g, 8.60 mmol), we obtained the title compound 12 as crystalline solid (1.13 g, 58% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.16 (d, 2H, Ar-H, J = 8.2), 7.80 (d, 2H, Ar-H, J = 7.9), 7.70 (d, 2H, Ar-H, J = 9.1 Hz), 7.63 (s, 2H, C=CH), 7.58 (td, 2H, Ar-H, J = 7.0, 1.2 Hz), 7.43 (td, 2H, Ar-H, J = 7.9, 0.9 Hz), 7.02 (d, 2H, Ar-H, J = 8.8 Hz), 3.88 (d, 2H, CH₂, J = 12.2 Hz), 3.64 (d, 2H, CH₂, J = 12.2 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 130.08, 129.67, 129.59, 128.64, 126.99, 125.98, 124.27, 123.85, 122.13, 121.42, 119.99, 117.96, 28.16. ESI mass calculated for C₂₇H₁₉O₂S (M + H)⁺ 407.11 found 407.10.

2.1.12 |. 3,3′-Oxadimethylene dinaphthospiropyran (13)

From 4-tetrahydropyranone (0.50 g, 5.0 mmol) and 2-hydroxy-1-naphthaldehyde (1.72 g, 0.01 mmol), we obtained the title compound 13 as amorphous solid (1.07 g, 53% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.27 (d, 2H, Ar-H, J = 8.5 Hz), 7.89 (s, 2H, C=CH), 7.85 (d, 2H, Ar-H, J = 8.8 Hz), 7.80 (d, 2H, Ar-H, J = 8.8 Hz), 7.61 (t, 2H, Ar-H, J = 7.6 Hz), 7.48 (t, 2H, Ar-H, J = 7.6 Hz), 7.01 (d, 2H, Ar-H, J = 8.8 Hz), 4.61 (d, 2H, CH₂, J = 12.3 Hz), 4.50 (d, 2H, CH₂, J = 12.3 Hz). ¹³C NMR (75 MHz, DMSO-d6): δ 130.91, 129.92, 129.78, 127.94, 125.77, 125.51, 124.95, 123.70, 122.45, 121.72, 118.11, 114.79. 64.13. ESI mass calculated for C₂₇H₁₉O₃ (M + H)⁺ 391.13 found 391.10.

2.1.13 |. 3,3′-Dimethylene dinaphthospiropyran (14)

From cyclopentanone (1 g, 11.89 mmol) and 2-hydroxy-1-naphthaldehyde (4.09 g, 23.77 mmol), we obtained the title compound 14 as amorphous solid (1.68 g, 38% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.23 (d, 2H, Ar-H, J = 8.2 Hz), 7.88 (d, 2H, Ar-H, J = 7.6 Hz), 7.76 (d, 2H, Ar-H, J = 8.8 Hz), 7.65 (s, 2H, C=CH), 7.60 (td, 2H, Ar-H, J = 7.0, 1.5 Hz), 7.46 (td, 2H, Ar-H, J = 7.0, 1.2 Hz), 7.00 (d, 2H, Ar-H, J = 8.8 Hz), 3.02–2.86 (m, 2H, CH₂), 2.84–2.70 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d6): δ 135.02, 134.38, 130.02, 129.79, 128.86, 127.51, 124.82, 123.02, 118.19, 117.87, 116.62, 99.07, 33.13. ESI mass calculated for C₂₇H₁₉O₂ (M + H)⁺ 375.14 found 375.10.

2.1.14 |. 3,3′-Trimethylene dinaphthospiropyran (15)

From cyclohexanone (1 g, 10.40 mmol) and 2-hydroxy-1-naphthaldehyde (3.58 g, 20.80 mmol), we obtained the title compound 15 as amorphous solid (1.69 g, 42% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.28 (d, 2H, Ar-H, J = 8.2 Hz), 7.85 (d, 2H, Ar-H, J = 7.9 Hz), 7.72 (d, 2H, Ar-H, J = 9.1 Hz), 7.64 (s, 2H, C=CH), 7.58 (td, 2H, Ar-H, J = 6.7, 1.2 Hz), 7.42 (td, 2H, Ar-H, J = 6.7, 0.9 Hz), 6.98 (d, 2H, Ar-H, J = 8.8 Hz), 2.80–2.66 (m, 2H, CH₂), 2.58–2.42 (m, 2H, CH₂), 1.84–1.78 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d6): δ 130.42, 130.39, 129.52, 129.05, 128.46, 126.69, 124.03, 123.78, 122.19, 121.66, 119.10, 118.08, 117.95, 26.91. ESI mass calculated for C₂₈H₂₁O₂ (M + H)⁺ 389.15 found 389.20.

2.1.15 |. 3,3′-Tetramethylene dinaphthospiropyran (16)

From cycloheptanone (1 g, 8.90 mmol) and 2-hydroxy-1-naphthaldehyde (3.07 g, 23.8 mmol), we obtained the title compound 16 as crystalline solid (1.78 g, 48% yield). ¹H NMR (300 MHz, DMSO-d6): δ 8.16 (d, 2H, Ar-H, J = 7.3 Hz), 7.77 (d, 2H, Ar-H, J = 7.9 Hz), 7.67–7.62 (m, 2H, Ar-H), 7.58–7.52 (m, 2H, Ar-H), 7.46 (s, 2H, C=CH), 7.42–7.34 (m, 2H, Ar-H), 6.98 (d, 2H, Ar-H, J = 7.6 Hz), 2.82–2.72 (m, 2H, CH₂), 2.64–2.52 (m, 2H, CH₂), 2.20–2.12 (m, 2H, CH₂), 1.70–1.64 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d6): δ 134.09, 130.41, 129.22, 128.59, 126.64, 125.33, 123.88, 121.36, 119.47, 118.52, 117.99, 113.43, 34.18, 32.40. ESI mass calculated for C₂₉H₂₃O₂ (M + H)⁺ 403.17 found 403.15.

2.2 |. Optical properties

UV-visible spectra were acquired using Shimadzu UV160A spectrophotometer; the fluorescence spectra were recorded at 90° detection angle on Shimadzu 5000U-DR15 spectro-fluorophotometer equipped with a Xenon lamp excitation source (λ_abs = 305 nm or 425 nm). The spectra were obtained with (PDNA) = 50 μM in acetonitrile, acetonitrile with 2.5% trifluoroacetic acid, and acetonitrile with 2.5% triethylamine.

2.3 |. Crystal structures

X-ray crystal structure analyses were performed with a CCD Bruker APEX diffractometer (Bruker-AXS, 1998) after crystallizing in triclinic P‾1 space system. The structures were determined by direct method using SHELXL (Sheldrick, 2007) and refined on F² by full-matrix least squares with the SHELXL (Sheldrick, 2008). Colorless plate-shaped (4), red prism-shaped, (5) and colorless prism-shaped crystals (6) of dimensions 0.26 × 0.14 × 0.03, 0.32 × 0.10 × 0.03, and 0.38 × 0.31 × 0.16 mm, respectively, were selected for structural analysis. The intensity data for these compounds were collected using a diffractometer with a Bruker APEX CCD area detector and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) (Bruker-AXS, 1998). The cell parameters were determined from a non-linear least squares fit of 9,662 peaks in the range 2.34 < θ < 28.30°, 2.28 < θ < 28.31°, and 4.29 < θ < 60.62°. A total of 44,544, 12,295, and 4,146 data were collected in the range 0.76 < θ < 28.31°, 2.28 < θ < 28.32°, and 4.44 < θ < 53.41° using ω oscillation frames. The data were merged to form a set of 21,160 independent data with R(int) = 0.0413 and a coverage of 99.5% for 4, 4,357 independent data with R(int) = 0.0325 and a coverage of 100.0% for 5, and 21,160 independent data with R(int) = 0.0977 and a coverage of 98.2% for 6.

2.4 |. Biological studies

2.4.1 |. In-house cell proliferation assay

Human lung adenocarcinoma cell line NCI-H441 (ATCC Number: HTB-174) was obtained from the American Type Culture Collection. H441 cells were maintained at 37°C with 5% CO₂ in McCoy’s 5A Medium (Invitrogen) supplemented with 5% heat-inactivated Fetal Bovine Serum (FBS) and Gentamicin (GIBCO Laboratories). Pancreatic cancer Panc-1 and MiaPaCa-2 cells and colon cancer HCT-116 were acquired from the laboratory of Dr. Shrikant Anant (Biomedical Research Center, University of Oklahoma Health Sciences Center). The pancreatic cancer cells were maintained in RPMI 1640 in 10% heat-inactivated FBS. Antibiotics, penicillin at 100 U and streptomycin at 100 μg/ml of medium, were added to the cell culture medium.

To evaluate the cytotoxicity, cancer cells were seeded in a 96-well flat-bottom tissue culture plates at a density of 5 × 10³ cells per well. The cells were allowed to adhere and grow overnight. The test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the cells at various concentrations in medium supplemented with 5% FBS. The DMSO concentration was maintained at 0.1% per well. Equivalent volume of DMSO without any drugs was added to the control wells. The cells were allowed to remain in the treatment medium for 24, 48, and 72 hr. The total number of viable cells was estimated by hexosaminidase assay. Briefly, the medium was removed and hexosaminidase substrate solution in citrate buffer pH 5 (7.5 mM), p-nitrophenol-N-acetyl-beta-D-glucosaminidase (Calbiochem) was added at 60 μl per well. The plates were incubated at 37°C in 100% humidity for 30 min, before stopping the reaction by adding 90 μl of 50 mM glycine containing 5 mM of EDTA (pH 10.4); absorbance was measured at 405 nm.

2.4.2 |. Cell cycle analyses

HCT116 cells were treated with compound 11 for 48 hr and subsequently trypsinized and suspended in phosphate buffered saline (PBS). Single-cell suspensions were fixed using 70% ethanol for 2 hr, before permeabilizing the cells with 1 mg/ml propidium iodide (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), and 2 mg DNase-free RNase (Sigma-Aldrich) at room temperature. Flow cytometry was performed using a FACSCalibur analyzer (Becton Dickinson), capturing 50,000 events for each sample. Results were analyzed with ModFit LT TM software (Verity Software House).

2.4.3 |. Colonosphere formation assay

In order to examine the ability of compound 11 to inhibit colonosphere formation of colon cancer cell lines, HCT-116 cells were harvested and diluted as single-cell suspensions. Approximately 2,500 cells were added into each well of 24-well in ultralow-binding plate, with or without compound 11. The cells were maintained in serum-free Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco) supplemented with B27 (Life Technologies), 20 ng/ml epidermal growth factor (Sigma), 10 ng/ml fibroblast growth factor (Sigma), and antibiotic–antimycotic agent in 24-well ultralow attachment plates (Corning Inc). Fresh aliquots of the growth factors were added to the culture medium every other day. The cells were cultured for 10 days, and the effect of compound 11 on colonospheres was observed.

2.4.4 |. Immunoblotting

Antibodies against poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-7, and caspase-9, were purchased from Cell Signaling. Anti–β-actin antibody was obtained from Aldrich-Sigma. After treatment with compound 11, HCT-116 cells were incubated on ice for 30 min in 0.5 ml of ice-cold lysate buffer consisting of 10% NP-40, 5 M NaCl, 1 M HEPES, 0.1 M ethylene glycol tetraacetic acid, 0.5 M ethylene diamine tetraacetic acid, 0.1 M phenylmethylsulfonyl fluoride, 0.2 sodium orthovanadate, 1 M NaF, 2 μg/ml aprotinin, and 2 μg/ml leupeptin. The protein was extracted by homogenization using a dounce homogenizer and centrifugation at 19,060 xg at 4ºC for 10 min. The proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electro-transferred on to nitrocellulose membranes, blotted with respective primary antibodies, followed by HRP-conjugated secondary antibody. The final detection was performed by enhanced chemiluminescence reagent Pierce SuperSignal West Femto reagent (Thermo Scientific).

2.4.5 |. HCT-116 tumor xenograft in mice

The animal experiments were performed according to the NIH Animal Use and Care Guidelines and were approved by the Institutional Animal Care Committee of the University of Oklahoma Health Sciences Center. Approximately 5-week-old male athymic nude (NCr-nu) mice were purchased from the Jackson Laboratory; they were maintained with water and standard mouse chow ad libidum in pathogen-free conditions. The mice were injected with 1 × 10⁶ HCT-116 cells in the left and right flank and allowed to form xenograft tumors. Compound 11 (200 μg/kg body weight) in 5% NaHCO₃ buffer was intraperitoneally administered daily for 15 days.

The tumor size was measured on weekly basis. At the end of the treatment schedule, the animals were sacrificed, and the tumors were removed and weighed.

2.5 |. Statistical analysis

All values are expressed as the mean ± SEM. The data were analyzed using a paired two-tailed t test. A p value of <.05 was considered statistically significant.

3 |. RESULTS AND DISCUSSION

Despite being reported over half a century ago in 1952 (Fischer & Hirshberg, 1952), therapeutic applications of spiropyrans have been relatively less intensely investigated. In this article, we describe our somewhat serendipitous discovery of a series of spiropyrans that are optically responsive to changes in pH. We obtained these compounds by aldol condensation of salicylaldehyde or 2-hydroxy-1-naphthaldehyde with 4-piperidone, 4-tetrahydrothiophenone, 4-tetrahydropyranone, or cyclopentanones, respectively (Scheme 1). The resultant 3,3′-azadimethylene dibenzospiropyran (3) upon tosylation afforded 3,3′-(N-tosyl)azadimethylene dibenzospiropyran (4). The product of these reactions was unexpected because we expected 3,5-bis(2-hydroxybenzylidene)-4-piperidone (1) and its N-tosyl derivative (2), based on our previous work (Lagisetty, Powell, & Awasthi, 2009; Lagisetty, Vilekar, Sahoo, Anant, & Awasthi, 2010). However, instead of six N-tosyl methyl protons expected in compound 2, the ¹H NMR spectrum revealed only three protons corresponding to N-tosyl methyl protons at δ 2.40 ppm. The ESI mass of the intermediate and final product showed molecular ion peaks at 466.10 and 290.12, respectively. Both NMR and mass spectroscopy led us to hypothesize and propose 3,3′-azadimethylene dibenzospiropyran tosylate as the structure of compound 4 consistent with these observations. We further investigated the crystal structure of 4 by X-ray diffraction (Figure 1). The analysis of X-ray data suggested that the skeleton of this compound consisted of a central piperidone ring connected to benzopyran rings via the spiro-C2 atom. The piperidone ring was in twist-boat conformation, and the tosylate group was present at the piperidine N-atom. The bond lengths at C(3)-C(4) and C(3′)-C(4′) were 1.334(7) and 1.332(7) Å, respectively, suggestive of the presence of double bonds at these positions. The benzene rings, with the exception of the oxygen atoms, appeared to be nearly coplanar (Figure 1).

SCHEME 1.

The scheme showing the synthesis of 3,3′-azadimethylene dibenzospiropyran 3 and its tosylate derivative 4

FIGURE 1.

(a) Crystal structure of (a) 3,3′-azadimethylene dibenzospiropyran tosylate, and (b) a perspective view of the crystal packing of 3,3′-azadimethylene dibenzospiropyran tosylate

The C_spiro-O bond length was determined to be 1.44 Å which is considerably longer than a normal C_Sp3-O bond (1.41 Å). The angle around the carbon atoms C(3)–C(2)–C(3′) was 111.3(4); the ring strain appears to be considerably less because of the presence of cyclohexenone structure. The crystal data and the selected bond distances and angles of compound 4 are listed in Table 3. The complete set of structural parameters in CIF format is available as an Appendix S1 from the Cambridge Crystallographic Data Centre (CCDC 843989).

TABLE 3.

Crystal data and selected bond lengths (Å), bond angles (°), and torsion angles (°) in compound 4

Parameter	Particulars	Bond	Length
Empirical formula	C26H21NO4S	O(1)–C(2)	1.435(6)
Formula Mass	443.5	C(2)–O(1′)	1.427(6)
system	Triclinic	C(2)–C(3)	1.514(7)
Space group	P‾1	C(2)–C(3′)	1.500(7)
Unit cell dimensions		C(3)–C(4)	1.332(7)
a (Å)	11.3703(18)	C(3′)–C(4′)	1.334(7)
b (Å)	14.065(2) Å	C(3)–C(11)	1.508(7)
c (Å)	27.439(4)	O(1)–C(2)–C(3)	111.1(4)
Volume (Å3)	4,297.3(11)	O(1)–C(2)–C(3′)	107.1(4)
Z, Z′	8, 4	O(1′)–C(2)–C(3′)	113.0(4)
Density (calculated)	1.371 Mg/m3	C(3)–C(2)–C(3′)	111.3(4)
Temperature	100(2) K	C(9)–O(1)–C(2)	115.8(4)
F(000)	394	O(1′)–C(2)–C(3)–C(11)	89.3 (5)
Absorption coefficient	0.185 mm−1	O(1)–C(2)– C(3′)–C(11′)	96.8 (4)
Max. and min. transmission	0.995 and 0.948	O(1)–C(2)–C(3)–C(4)	30.7(6)
θ range for data collection	0.76 to 28.31°	O(1′)–C(2)–C(3)–C(4)	88.1(5)
Reflections collected	44,544	C(9)–O(1)–C(2)–C(3)	43.2(5)
Data/restraints/parameters	21,160/0/1,158	C(9)–O(1)–C(2)–C(3′)	164.3(4)
wR(F2 all data)	wR2 = 0.2762	O(1)–C(2)–C(3′)–C(4′)	85.5(5)
R(F obsd data)	R1 = 0.1000	C(3)–C(2)–C(3′)–C(4′)	152.5(5)
Goodness of fit on F2	1.057	O(1′)–C(2)–C(3′)–C(4′)	32.8(6)
Observed data [I > 2s(I)]	3,737	C(3′)–C(2)–O(1′)–C(9′)	42.2(6)
Largest diff. peak and hole(e/Å3)	0.554 and −0.561	C(7′)–C(8′)–C(9′)–O(1′) C(2)–O(1′)–C(9′)–C(8′) C(2)–O(1′)–C(9′)–C(10′)	177.0(5) 157.5(4) 28.8(6)

3.1 |. Mechanism for the formation of 3,3′-azadimethylene dibenzospiropyran (4)

The mechanistic explanation for the unexpected formation of 4 is provided in Figure 2. The initial acid-catalyzed aldol condensation of salicylaldehyde and 4-piperidone produces the anticipated 3,5-bis(2-hydroxybenzylidene)-4-piperidone (1) as a labile intermediate, which in presence of acid undergoes further transformations. The initial attack of H⁺ on carbonyl oxygen results in a carbocation at the carbonyl carbon which rearranges with the benzylidene double bond through conjugation. Since this carbon is a planar sp² carbon, concomitant isomerization of cis- (the protons cis- to the piperidone methylene protons) to the trans-isomer occurs (the double bond protons are trans- to the piperidone methylene protons). The carbonyl carbon again forms an acid-catalyzed carbocation to eventually generate a styryl pyrilium salt. Similar styryl pyrilium salts of 2-hydroxy-1-naphthaldehyde by condensation with dibenzylketone (Dickinson, Heilbron, & O’Brien, 1928) and salicylaldehyde have been reported in the literature (Dickinson & Heilbron, 1927; Heilbronn, Heslop, & Irving, 1933). The proposed mechanism was also supported by the crystal structure of [3-(2,β-thiodimethylene)[2-(2-hydroxy)-1-benzylvinyl)]benzo pyrilium chloride] (5) which was obtained by the condensation of 4-tetrahydrothiopyranone and salicylaldehyde. The treatment of styryl pyrilium salt 5 with a base produced a cyclized product 6. The structural determinations of 5 and 6 in relation to those of 4 are described in supplemental material.

FIGURE 2.

A putative mechanism for the formation of spiro-derivatives

3.2 |. General synthesis of spirodibenzopyrans and spirodinaphthopyrans

The condensation of salicylaldehyde with 6-membered cyclic ketones containing various heteroatoms, such as oxygen or sulfur (i.e., 4-tetrahydropyranone and 4-tetrahydrothiopyranone), resulted in the corresponding spirodibenzopyrans in good yields. Similarly, condensation of cycloalkanones with salicylaldehyde resulted in their corresponding spirodibenzopyrans in quantitative yields (Scheme 2a). All the compounds were characterized by NMR and mass spectroscopy. Our attempts to establish the structures of open form of spiro-compounds by NMR met with failure, because in solution phase (DMSO-d6 or acetone-d6), the open form kept getting converted into the closed form. This phenomenon was observed even at low temperature.

SCHEME 2.

The general schemes for the synthesis of (a) spirodibenzopyrans and (b) spirodinaphthopyrans

We extended the observed chemistry to perform a reaction between 4-piperidone and 2-hydoxy-1-naphthaldehyde. This reaction resulted in 3,3′-azadimethylene dinaphthospiropyran (11). Similar condensations of 4-tetrahydrothiophenone, 4-tetrahydropyranone, cyclopentanone, cyclohexanone, and cycloheptanone with 2-hydoxy-1-naphthaldehyde produced the respective spirodinaphthopyrans (12–16) (Scheme 2b). The complete list of reactants and resultant spiro-derivatives and their characteristics are summarized in Table 4. As could be observed, the yields of spirodinaphthopyrans are lower than the corresponding spirodibenzopyrans (entries (i) vs. (vii), (iii) vs. (x), and (iv) vs. (xi)), perhaps because of enhanced steric congestion.

TABLE 4.

Precursors, structures, yield, and melting point (M.P.) of spiro-compounds

Entry	Reactants		Producta
			Structure	Comp No.	Yieldb (%)	M.P. (°C)
(i)				3	89	198–200
(ii)				6	91	178–179
(iii)				7	3	124–126
(iv)				8	89	200–202
(v)				9	85	118–120
(vi)				10	78	206–208
(vii)				11	49	223–225
(viii)				12	58	265–266
(ix)				13	53	213–215
(xi)				14	38	233–235
(xii)				15	42	155–160
(xiii)

^aAll compounds were characterized by spectral data.

^bYields refer to the isolated pure compounds

3.3 |. Optical properties of spirodinaphthopyrans

We focused our attention on spirodinaphthopyrans because of its novelty and the exciting observations from in vitro anti-proliferative studies that were conducted in a simultaneous fashion (see results below). Of the various compounds, we selected 11 to study the optical characteristics. The absorption spectra of solution of compound 11 in acetonitrile are shown in Figure 3. In neutral and alkalinized solution, compound 11 showed the absorption maxima (λ_abs) at 305 nm. In acidified solution, however, an additional peak at 425 nm appeared. This suggests that a new molecular species was generated by acidification, characterized by more effective delocalization of π electrons in open form. The fluorescence spectra (Figure 4) of compound 11 showed two emission bands with maxima (λem) at 387 and 521 nm; upon alkalization, <di> almost disappeared. On the other hand, acidification resulted in reduced <di> and increased <di> intensity, creating an isosbestic point at 415 nm (Figure 4a). This indicates that in solution compound 11 exists in equilibrium between two species, which can be shifted forward and backward by adding acid or base (Figure 4c).

FIGURE 3.

(a) UV-visible absorbance spectra of compound 11 dissolved in acetonitrile. The compound solution was alkalinized with triethylamine or acidified with trifluoroacetic acid. The acidified solution acquired additional absorbance maxima at 425 and 560 nm as magnified in the inset. (b) A visual of the optical changes corresponding to the change in pH

FIGURE 4.

Fluorescence spectra of compound 11 dissolved in acetonitrile and acidified or alkalinized with trifluoroacetic acid or triethylamine, and excited at (a) 305 nm and (b) 425 nm. The fluorescence emission maxima for neutral, acidic, and alkaline solutions were 518.4, 521.6, and 491.21 nm, respectively. (c) Delocalization of π electrons alters the spectral characteristics compound 11

3.4 |. Compound 11 suppresses cancer cell proliferation in vitro in a timedependent manner

Since the primary objective of this exercise was to synthesize novel anticancer chalcone derivatives based on our previous work (Lagisetty et al., 2009, 2010), we hypothesized that these spiro-compounds will also have anti-proliferative activity in cancer cells. The synthesized spiro-compounds were evaluated in lung adenocarcinoma (H441), colon cancer (HCT-116), and pancreatic cancer cells (MiaPaCa-2 and Panc-1). The total number of cells after 24, 48, and 72 hr of treatment was estimated by the MTT and/or hexosaminidase assay (Landegren, 1984). The comprehensive results from these experiments are provided in supplementary data (Figure S2), based on which we chose compound 11 for further investigations. In Figure 5a, we show the anti-proliferative activity of compound 11 in various cell lines carried out in our laboratory. To further substantiate the efficacy of open versus closed form, we prepared hydrochloride and trifuoracetate salts of 11. As shown in Figure 5b, acid salts appear to be more effective in suppressing the proliferation of A549 and Pacn-1 cells. Whether the salt forms actually remain in open forms or revert back to closed forms under culture conditions is not known at present.

FIGURE 5.

(a) Anti-proliferative activity of compound 11 in lung adenocarcinoma H441, colon cancer HCT-116, and pancreatic cancer MiaPaCa-2 and Panc-1 cells. (b) A light micrograph of H441 cells treated with compound 11 shows massive loss of adhered cell population. (c) HCl and trifluoroacetate salts of 11 demonstrate better anti-proliferative effect than compound 11 itself

3.5 |. Compound 11 causes apoptotic cell death and inhibits colonosphere formation

Treatment of HCT-116 cells with compound 11 decreased the number of cells in G2/M phase, followed by a corresponding increase in the number of cells in subG0 phase (Figure 6a). There was no evidence of cell cycle arrest by compound 11 in HCT-116 cells. This suggests that compound 11 induces apoptotic cell death in HCT-116 cells that are in the G2/M phase of the cell cycle. The cell population in sub-G0 phase increased in a dose-dependent manner, which is an indication of apoptotic cell death. That compound 11 induces apoptosis in HCT-116 cells was confirmed by induction of typical apoptotic biomarkers (Figure 6b); there was dose-dependent cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3 and −7. The cleavage of caspase 9 is also suggestive of induction of intrinsic apoptotic pathway. Caspase-9 is an initiator caspase capable of cleaving procaspase-3 and procaspase-7, which in turn cleave several cellular targets, including PARP. Cleavage of PARP inactivates it, prevents it from participating in DNA repair mechanisms, and forces cells toward programmed cell death.

FIGURE 6.

(a) Cell cycle profiles of HCT-116 cells untreated and treated with compound 11 (10, 20, and 30 μM) for 48 hr. The flow cytometric determination of DNA content was performed by using propidium iodide staining. Compound 11 treatments significantly decreased the cells in the G2M phase of cell cycle, while increasing the number of dying cells as is evident from subsequent increase of signal in subG0 phase in 48 hr. (b) Compound 11 induces apoptosis in HCT116 cells. (c) A representative photomicrograph showing formation of HCT-116 colonospheres with and without compound 11 (30 μM)

At this point, it is not clear what molecular and biochemical pathway(s) is responsible for the induction of apoptotic cell death by compound 11.

As a prelude to in vivo testing, we further confirmed anti-proliferative activity of compound 11 by colonosphere formation assay. Colon cancer is the second leading cause of cancer-related deaths in United States. There is nearly 50% of recurrence of colon cancer in patients treated with conventional chemotherapeutics. This is due to the chemotherapy resistant cancer stem cells (CSCs). To assess whether compound 11 is efficient in inhibiting CSCs, we performed an assay for the formation of colonospheres that are taken as an in vitro surrogate for tumor formation. The functional CSCs have the ability to form colonosphere in ultralow attachment plates. The results revealed that compound 11-treated wells had no or very small spheres as compared to the spherical colonies in control wells (Figure 6c). Clonogenic assay has a proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs (Fiebig, Maier, & Burger, 2004).

3.6 |. Compound 11 inhibits tumor growth in a xenograft model in mice

To evaluate the effect of compound 11 on tumor growth in vivo, we examined the change in tumor volume of xenograft human colon HCT-116 cancer cells in nude mice. The xenograft tumors were allowed to develop and grow to a size of 500 mm³, following which, compound 11 was intraperitoneally administered daily (5 μg/day) until euthanasia in the third week. Compound 11 remarkably inhibited the growth of the tumor xenografts (Figure 7). The tumor volumes significantly decreased to 62% by treatment as compared to the control (p < .05). Whereas the control tumors continued to grow during the treatment period reaching a size of 2,000 mm³, tumors in the treated animals did not show any growth. The excised tumors from the treated mice weighed from 400 to 700 mg, whereas those from the control group weighed more than 2,220 mg (Figure 7b). Although we did not perform an elaborate histological examination, gross observation of organs upon necropsy did not provide any indication of apparent toxic effects of prolonged administration of compound 11.

FIGURE 7.

(a) Compound 11 suppresses tumor growth in a xenograft tumor of HCT-116 cells in mice. (b) Tumor weight upon necropsy shows a significant reduction after treatment with compound 11

Overall, we report a novel class of anticancer spiro-compounds that could be easily synthesized in high yields from inexpensive and commercially available materials. Several of the synthesized compounds appeared to suppress proliferation of cancer cells in vitro. Compound 11 was found to inhibit tumor growth in a xenograft model in mice. It thus represents an interesting lead from which more potent anticancer compounds could be derived in future. To our knowledge, this unique synthetic pathway for spiro-compounds and their anticancer activity has not been reported thus far. However, their efficacy and inadvertent toxicities, other than killing of cancer cells, compared to the other known and investigational anticancer drugs need to be carefully assessed. The potential toxicities of these complex compounds could be related to their metabolic fate dictated by solvatochromic behavior exhibited by spiropyrans. The utility of SP-MC transformation in anticancer efficacy is not clear yet and could only be speculated at best. Perhaps, the mechanistic target and signaling pathway responsible for the anticancer activity will be able to shed some light on these open questions about the toxicity, biocompatibility, and metabolism of reported compounds.