Azide derivatized anticancer agents of Vitamin K3: X-ray structural, DSC, resonance spectral and API studies

Journal of Molecular Structure 1006 (2011) 288–296

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Azide derivatized anticancer agents of Vitamin K3: X-ray structural, DSC, resonance spectral and API studies Kirti Badave a, Yogesh Patil a, Rajesh Gonnade b, Darbha Srinivas c, Rajan Dasgupta d, Ayesha Khan d, Sandhya Rane a,⇑ a

Department of Chemistry, University of Pune, Pune 411 007, India Center for Material Characterization, National Chemical Laboratory, Pune 411 008, India Catalysis Division, National Chemical Laboratory, Pune 411 008, India d Institute of Bioinformatics & Biotechnology, University of Pune, Pune 411 007, India b c

a r t i c l e

i n f o

Article history: Received 15 July 2011 Received in revised form 6 September 2011 Accepted 8 September 2011 Available online 14 September 2011 Keywords: Electronic isomers RAHB (resonance assisted H-bonding) API (Active Pharmaceutical Ingredients) Vitamin K3 Anticancer agents

a b s t r a c t Compound 1 [1-imino (acetyl hydrazino)–Vitamin K3], displays valence tautomerically related electronic isomers as Form I and Form II. Form I exhibits 2D packing fragment with 1D ribbon chains of N–H  O hydrogen bonds and shows EPR silent features. While Form II is EPR active and exhibits biradical nature with double quantum transitions at g = 2.0040. 1H NMR of compound 2, [1-imino (hydrazino carboxylate)–Vitamin K3] and Form II exhibit p delocalization via resonance assisted H-bonding [RAHB] effect compared to Form I. Molecular interactions in Form I and II are visualized by DSC. The electronic structures of compounds 1 and 2 have been correlated to their API values by measuring anticancer activities, mitochondrial potentials and DNA shearing patterns. Form II and compound 2 indicate mitochondria mediated apoptosis (75% cell death) while Form I causes 35% cell death. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Menadione (Vitamin K3) is a synthetic derivative of the naturally occurring Vitamins K1 and K2. In vitro, menadione is effective against a wide variety of tumor cells at concentrations that are clinically achievable IC50 values usually ranging from 10 to 50 lM [1,2]. Like other quinonoid compounds menadione, is cytotoxic via electrophilic arylation or redox cycling [3]. In case of menadione, the oxidative stress generated appears to be dosedependent and capable to induce both apoptosis and necrosis, depending on the doses used [4]. Verrax et al. have shown that menadione redox-cycling is greatly increased by the addition of ascorbate; a fact that explains the synergistic antitumor activity of the ascorbate/menadione combination. While neither menadione nor ascorbate alone exhibit cytotoxicity at the doses which they were used in their in vitro and in vivo studies [5]. The mode of cell death by ascorbate/menadione was necrotic-like as confirmed by annexin-V/propidium iodide labeled cells and light microscopy examination [6]. Necrosis is a form of traumatic cell death that results from acute cellular injury, in which the cellular debris can cause damage to the normal cell. Thus apoptosis, the

⇑ Corresponding author. Tel./fax: +91 20 25691728. E-mail address: [email protected] (S. Rane). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.09.022

process of programmed cell death (PCD) is a preferred mode of cell death. We have recently evaluated menadione derivatives for their acid phosphatase (APase) enzyme inhibitory activity to suggest its probable antioncogenic candidature [5], wherein the iminofunctionalized C-1 position of menadione oxime exhibited slightly higher activity than the parent menadione. Instead of simple imine functions such as oximes or Schiff’s bases, hydrazides exhibit carcinostatic activity against several types of tumors, which may be due to their ability of peptide like hydrogen bonding with greater stability as suggested by Cabezas and Satterthwait [6]. Azide derivatives of therapeutically relevant compounds are useful as prodrugs. In addition they are often better able to penetrate the blood–brain barrier than the corresponding drugs [7]. Hence to enhance and exploit antioncogenic potency of Vitamin K3 [8], we have coupled C-1 position of menadione with methyl and methoxy-derivative of acetyl hydrazine to perform iminofunctionalized azide derivatives as compounds 1 and 2 respectively, as hybrid drugs [9,10]. Compound 1 exhibits characteristic redox isomers as Form I and Form II. In pharmaceutical solids as drug compounds, rigid molecules are rather rare and most of the active pharmaceutical ingredients (API) formed by molecules with either one or number of rotatable bonds and molecular units with conformational flexibility [11] are preferred. Such API value deals with exceptional valence tautomers expressed by resonance assisted

K. Badave et al. / Journal of Molecular Structure 1006 (2011) 288–296

H-bonding (RAHB) effect in 1 and it is compared with compound 2 for substituent effect on hydrazides in the present investigations. The importance of RAHB in Vitamin K3 family members was established in our laboratory [5]. To gain insight of such RAHB in azide derivatized menadione towards stability, the anticancer activity against HeLa, HL-60 and CAPAN-1 cell lines via hybrid drug uptake is reported in the current paper. The DNA laddering studies and mitochondrial membrane potential studies were performed to confirm apoptotic mode of cell death. 2. Experimental 2.1. Measurements 2.1.1. C, H, N, S analytical data C, H, N, S analytical data were done on FLASH EA 1112 series (Thermo Electron Corporation). 2.1.2. 1H NMR spectra 1 H NMR spectra were recorded on Varian Mercury YH 300 NMR instrument at 298 K. Chemical shift reported as ppm relative to TMS as internal standard. 2.1.3. Single crystal X-ray crystallography Single crystal X-ray diffraction studies were conducted on selected good quality single crystals of Form I of 1 using Leica polarizing microscope and mounted on glass fibers with epoxy cement. The X-ray data were collected on a Brüker-AXS Smart Apex CCD diffractometer at 297(2) K with graphite-monochromatized (Mo Ka = 0.71073 Å) radiation. The X-ray generator was operated at 50 kV and 30 mA. The intensity measurements were monitored by the SMART program (version 5.63, Brüker AXS Inc., Madison, Wisconsin, USA, 1997). Data reduction was performed with the SAINT software (version 6.45, Brüker AXS Inc., Madison, Wisconsin, USA, 2003). All the data were corrected for Lorentz and polarization effects. A semi-empirical absorption correction based on symmetry equivalent reflections was applied by using the SADABS program, G.M. Sheldrick, SADABS (version 2.10, Brüker AXS Inc., Madison, Wisconsin, USA, 2003). Lattice parameters were determined from the least squares analysis of all reflections. The structure was solved by direct method and refined by full matrix least squares, based on F2, using the SHELXTL software package (G.M. Sheldrick, SHELXTL 6.14, Program for the structure solution and refinement, Brüker AXS Inc., Madison, Wisconsin, USA, 2000). All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms in all structures were included in the refinement as per the riding model option in SHELXL (Bruker, 2003). Hydrogen atoms in all the structures were located from a difference Fourier map, their positional coordinates and isotropic thermal parameters were refined. Molecular and packing diagrams were generated using ORTEP-32 and Mercury-2.3. Geometrical calculations were performed using SHELXTL (Brüker, 2003) and PLATON. 2.1.4. DSC analysis The thermal behavior of tautomers of 1 (Form I, Form II) and 2 were investigated by measuring enthalpy change on DSC-60, Shimadzu Scientific Instrument at Department of Chemistry, University of Pune, Pune 411 007. Indium (99.99%) DHfusion = 97 cal/g, M.P. 156.4° were used to prepare calibration curve and to calibrate temperature axis. Crystals (wt. 1 mg) were placed on an aluminum pan (5 mm diameter) and were analyzed from low temperature (120 °C) using an empty pan as the reference. The heating rate was 5 °C/min and nitrogen gas was used for purging.

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2.1.5. EPR measurements The X band EPR spectra of Form II of 1 were recorded on a Brüker EMX EPR spectrometer at National Chemical Laboratory, Pune 411 008, operating at X-band 9.42 GHz frequency and 100 kHz field modulation. 2.2. Materials Synthetic starting material, reagents and solvents were of analytical grade. Menadione (3-methyl-1,4-naphthoquinone), acetyl hydrazide, methyl hydrazino carboxylate were obtained from Sigma–Aldrich. Solvents used in synthesis and crystallization are of extra pure HPLC grade purchased from RANCHEM (India). The progress of the reaction was monitored by thin layer chromatography with F254 silica-gel precoated sheets (Merck) using hexane/acetone 60/40 as eluent; UV light was used for detection. 2.3. Synthesis 2.3.1. Synthesis of 1 (Form I and Form II) A mixture of acetyl hydrazine (0.74 g, 0.01 mol) and menadione (1.72 g, 0.01 mol) was dissolved in mixture of methanol (30 ml) and acetic acid (5.12 ml, 2 mol). The solution was refluxed for 24 h and then mixture was allowed to cool, yielded yellow crystalline compound (Form I). It was collected by filtration, rinsed thoroughly with methanol, dried. The clear filtrate yielded deep red brown micro crystals of Form II after 15 days, which were collected by filtration, washed with methanol and dried under vacuum. Form I: Yellow crystals, Yield: (1.16 g) 50.86%, Anal. Calc.: C, 68.41; H, 5.30; N, 12.27. Found: C, 68.38; H, 5.10; N, 12.73. 1H NMR (300 MHz, CDCl3) [C2–H2 at 7.5 d(s), C5–H5 at 8.3 d(d); C8–H8 at 8.2 d(d); C6–H6 at 7.65 d(t); C7–H7 at 7.6 d(t)]. Form II: Red brown crystals, Yield: (0.320 g) 14.03%, Anal. Calc.: C, 68.41; H, 5.30; N, 12.27. Found: C, 67.75; H, 4.98; N, 12.44. 1H NMR (300 MHz, CDCl3) [C2–H2 at 7.5 d(s), C5–H5 at 8.3 d(d); C8–H8 at 8.2 d(d); C6–H6 at 7.65 d(t); C7–H7 at 7.6 d(t)]. Retention factor (Rf) of menadione, Form I of 1 and Form II of 1 are 0.825, 0.675, 0.600 respectively using solvent hexane:acetone: 3:2 ratio. 2.3.2. Synthesis of 2 A mixture of menadione (1.72 g, 0.01 mol) and methyl hydrazino carboxylate (0.90 g, 0.01 mol) was dissolved in mixture of methanol (30 ml) and acetic acid (5.12 ml, 2 mol). The solution was refluxed for 5 h and then mixture was allowed to cool, yielded yellow crystalline compound. It was collected by filtration, rinsed thoroughly with methanol, dried. Yellow crystalline powder of 2: Yield: (1.85 g) 75.81%, Anal. Calc.: C, 63.99; H, 4.95; N, 11.45. Found: C, 63.55; H, 4.80; N, 11.65. 1H NMR data in d6-DMSO: aromatic protons: C5–H5 at 8.237 d(d); C8–H8 at 8.06 d(d); C6–H6 at 7.7 d(t); C7–H7 at 7.59 d(t); quinonoidal ring protons C2–H2 at 8.1 d(s); methyl proton 2.082 d(s); hydrazino fragment protons-methoxy at 3.8 d(s); amide N2–H2 at 11.59 d(s). Retention factor (Rf) of menadione and compound 2 are 0.825, 0.725 respectively using solvent hexane:acetone: 3:2 ratio. 2.4. Biological activity 2.4.1. Anticancer activity The cells were trypsinized using TPVG solution. 1 ml of 1  105 cells/ml of medium and dilutions of concentration 5, 10, 15, 20 and 25 lM was added in 96 well plates and kept in the CO2 incubator for 24, 28, 72 and 96 h. The compounds were dissolved in 20% DMSO to obtain a solution of 1 mM concentration each. These

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samples were then filter sterilized using a 0.22 lm filter using syringe filter. All experiments were carried out in Laminar flow hoods, Laminar Flow Ultraclean Air Unit, Microfilt, India. The cells were visualized using an Inverted Microscope, Olympus. The number of viable cells remaining after appropriate treatment was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) assay [12]. After 24, 48, 72 h, and 96 h MTT was added to each well at a final volume of 0.5 mg/ml and the microplates were incubated at 37 °C for 3 h. After the supernatant was removed, the formazan salt resulting from the reduction of MTT was solubilized in dimethyl sulfoxide (DMSO, Sigma Chemical Co.) and the absorbance was read at 570 nm using an automatic plate reader (Thermo Corporation). The cell viability was extrapolated from the absorbance values and expressed as percent survival. Fluorescence Microscopic studies were carried out on a Carl Zeiss Fluorescence Microscopes. 2.4.2. DNA Ladder assay 1.0 ml of 1  105 cells/ml of the cell culture was taken and centrifuged at 10,000 rpm for 15 min. To the supernatant 100 ll lysis buffer (without SDS) was added and kept at room temperature for 5 min and mixed on a cyclomixer (vortex). Lysis buffer (300 ll with SDS) was added and gently mixed by inversion. This was followed by the addition of 66 ll of 5 M NaCl. The mixture was centrifuged at 10,000 rpm for 30 min at room temperature and the supernatant transferred to a new micro-centrifuge tube. Equal volumes of chloroform was added and mixed gently till it turned milky. The mixture was treated with RNase for 5 min. Equal volume of chloroform was added and mixed gently till it turned milky. Ice-cold isopropanol was added and incubated for 1 h on ice. The mixture was centrifuged at 10,000 rpm for 15 min and the supernatant was decanted. The DNA pellet was air dried to remove traces of isopropanol and re-suspended in about 50 ll of Tris EDTA buffer. The DNA isolated was run on a gel and absorbance at 260/280 nm was noted to assess the purity of extracted DNA. The DNA obtained was 69 ng/ml. 2.4.3. Mitochondrial membrane potential measurements Mitochondria from rat heart were isolated as described previously [13]. Incubations were conducted at either 28 °C or 37 °C in a medium composed of 135 mM KCl, 20 mM MOPS, 5 mM K2HPO4 and 5 mM MgCl2 at pH 7.00. Substrates were added as indicated in the figure legends. Incubations also contained R123 at concentrations indicated in the figure legends. Isolated mitochondria were kept at 4 °C and used within 4 h after isolation. Rhodamine 123 dissolved in methanol was used directly. The methanol concentrations in all incubations of mitochondria were kept at 0.5% (v/v). Fluorescent measurements of mitochondria and extracts were made using a Spectra Max5 (Molecular Probes) Multiscanner using a 90° excitation and emission optical path and a thermostat cuvette holder. The excitation and emission slits were 0.5 mm, to yield a bandpass of 2.1 nm.

3.2. Electronic isomer in Form I of 1 with X-ray crystal structure such as ketohydrazone Recently, we have established valence tautomers or electronic isomers in derivatized naphthoquinones [5,14,15] with respect to their polymorphic behavior and chemical nature. A single crystal X-ray structure is solved to identify such redox form of 1 as thermodynamic yellow crystalline stable Form I at high temperature and metastable kinetic brick red fine crystalline Form II obtained on cooling the filtrate of Form I. Fig. 1 shows a typical ORTEP view of Form I of 1.  space group with two indeForm I crystallized in triclinic P 1 pendent molecules in the asymmetric unit which are stacked one over the other via off-centered p  p interactions (Fig. 2). Both molecules in the asymmetric unit form their respective centrosymmetric dimeric assembly across the inversion center linked via conventional N–H  O hydrogen bond engaging N–H and carbonyl oxygen attached to the azide moiety (H2 N  O2 = 2.05 Å, N2  O2 = 2.896(4) Å, \N2–H2 N  O2 = 169°; H20 N  O20 = 2.09 Å, N20   O20 = 2.931(4) Å, \N20 –H20 N  O20 = 167°) (Fig. 3). Additionally, the same carbonyl oxygen O2 accepts H atom from C2 of the naphthoquinone group to form short C–H  O contact, thereby supporting the dimeric synthon (H2  O2 = 2.33 Å, C2  O2 = 3.245(5) Å, \N2–H2  O2 = 168°; H20   O20 = 2.34 Å, C20   O20 = 3.265(5) Å, \C20 -H20   O20 = 171°). Thus, carbonyl

Fig. 1. ORTEP view of Form I of compound 1 with atom numbering (ellipsoids are drawn with 50% probability level).

3. Results and discussion 3.1. Synthesis and characterization Iminofunctionalized azide derivatives 1 and 2 were synthesized by coupling at C-1 position of Vitamin K3 in good yields. The redox isomeric electronic structures of 1 as Form I and Form II are characterized in solid state and in solution with the help of thermochemical DSC and resonance spectral (ESR and 1H NMR) studies. The active pharmaceutical ingredients (API) are identified from their anticancer activity.

Fig. 2. Two molecules in the asymmetric unit displaying aromatic p  p stacking interactions.

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oxygen O2 is involved in bifurcated H-bond formation N–H  O and C–H  O. The two centrosymmetric neighboring dimers made by each symmetry independent molecules are connected through C– H  O contact to form a 2D planar sheet diagonal to the ac-plane (Fig. 4). The other carbonyl oxygens O1 and O10 of the naphthoquinone group forms C–H  O contacts with the C6 and C60 but with varying strength, the former being the weaker and the latter being short and linear (H6  O10 = 2.54 Å, C6  O10 = 3.205(5) Å, \C6– H6  O10 = 128°; H60   O1 = 2.44 Å, C60   O1 = 3.345(6) Å, \C60 – H60   O1 = 165°). These 2D sheets are stacked along the c-axis; with the inter-planar spacing being 3.32–3.39 Å via off-centered aromatic p  p interactions (Fig. 5). The electronic or redox isomers in parent Vitamin K3 are identified from their magnetostructural studies [5]. Similarly, ketohydrazino isomer as imino naphthoquinone form is observed in Form I crystals of 1. Its p-iminonaphthoquinone segment exhibits C4–O1 distance equals to 1.218 Å and acetyl-hydrazino segment

291

shows C12–O2 distance equal to 1.223 Å which indicate that Form I is an oxidized keto-hydrazino isomer as its N1–N2 distance is 1.351 Å [16]. At the same time C2–C3 bond length (1.342) Å of quinonoidal ring is shorter than remaining average C–C bond lengths (1.464 Å) of the ring by 0.12 Å unit suggesting its quinone character [17–19]. Due to strong p–p stacking (3.32– 3.39 Å) interactions in it, analogues to naphthoquinone Form I of iodolawsone polymorph (3.408 Å) [20]. Thus resulted in thermodynamically stable crystal in diamagnetic form. 3.3. Differential scanning calorimetry The Form I and Form II are tautomers related to valence forms of 1 which are well differentiated from their differential scanning calorimetry (DSC) measurements. DSC curves of Form I and Form II of 1 and compound 2 are presented in Fig. 6. The DSC curve of Form I exhibits two broad endotherms at 29.0 °C and 164.1 °C with

Fig. 3. Ball and stick plot displaying dimeric association via N–H  O and C–H  O interactions.

Fig. 4. 2D planar sheet made by two symmetry independent molecules of Form I of 1.

Fig. 5. Stacking of 2D planar sheets via off-centered aromatic p  p stacking interactions viewed down the b-axis.

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(A)

6 5 4 ο

mW

3

Peak 284.94 C ο Onset 281.85 C ο End set 285.92 C Heat 240.32 J/g 54.79 kJ/mole

2 1 0

ο

ο

Peak 29.04 C ο Onset 26.72 C ο End set 72.31 C Heat -75.47 J/g -16.97 KJ/mole

-1 -2 -100

0

Peak 280.26 C ο Onset 273.81 C ο End set 281.18 C Heat -70.50 J/g -16.07 kJ/mole

ο

Peak 164.07 C ο Onset 162.73 C ο End set 246.02 C Heat -105.45 J/g -24.04 kJ/mole

100

200

300

400

Temp. oC

(B)

5 4

mW

3 2

ο

Peak 278.26 C ο Onset 275.36 C ο End set 279.70 C Heat 202.14 J/g 46.08 kJ/mole

ο

Peak -31.85 C ο Onset - 58.33 C ο End set -29.66 C Heat 223.70J/g 51.00 kJ/mole

1

ο

Peak 268.92 C ο Onset 255.05 C ο End set 272.96 C Heat 10.58 J/g 2.41 kJ/mole

0 -1 -100

0

100

200

300

400

Temp. oC

(C)

8 ο

Peak 253.93 C ο Onset 251.17 C ο End set 256.19 C Heat 538.38 J/g 131.36 KJ/mole

6

mW

ο

4 ο

2

0

Peak -6.86 C ο Onset -38.99 C ο End set 29.36 C Heat 941.21 J/g 299.65KJ/mole

-2 -200

-100

Peak 52.00 C ο Onset 42.65 C ο End set 72.34 C Heat -45.26 J/g -11.04KJ/mole

ο

ο

Peak 96.25 C ο Onset 91.30 C ο End set 120.52 C Heat -62.23 J/g -15.18 KJ/mole

0

100

200

Peak 321.74 C ο Onset 290.66 C ο End set 347.60 C Heat 76.60 J/g 18.69 KJ/mole

300

400

Temp. oC Fig. 6. DSC profiles of (A) Form I of 1, (B) Form II of 1 and (C) 2; with heating rate 5 °C/min.

enthalpies DH1 = 17.0 kJ/mole and DH2 = 24.0 kJ/mole, respectively. A sharp melting endothermic peak is observed at 280.3 °C (DH3 = 16.1 kJ/mole) and a sharp exothermic peak is seen at 284.9 °C with DfusH4 = 54.8 kJ/mole. In contrast, the Form II tautomer of 1, exhibits three exotherms in its DSC curve, wherein two (broad ones) are observed at 31.9 °C with DH1 = 51.0 kJ/mole and 269.0 °C with DH2 = 2.4 kJ/mole. The third sharp exotherm is observed at 278.3 °C (DfusH3 = 46.1 kJ/mole).

The single sharp exotherm with heat of fusion (DfusH4 = 54.8 kJ/ mole) in Form I corresponds to breaking of one dimensional N– H  O hydrogen bonded ribbon chains analogous to aprepitant molecules [21]. A similar sharp exotherm in Form II requires less heat of fusion (DfusH3 = 46.1 kJ/mole) when N  H–O bond becomes stabilized with less energy (DH3). This deals with general criterion that H-bond energies steeply decrease when the bond is resonantly stabilized [22] which may be the situation in Form II. At the same time such interaction along N–H  O chain is reported in 2,6-dimethyl-N-/-formamide [23] with enthalpy change of DH = 44 kJ/mole. The endothermic enthalpy change in Form I (DH3 = 16.1 kJ/ mole) starts at 273.8 °C with Tc = 280.3 °C probably gives rise to phase transition in redox isomers of 1 such as Form I to Form II. Such temperature induced magnetic phase transition with characteristic enthalpy change in derivatized organic naphthoquinone compound [20] and their complexes [24] have been reported by our group. Hence, the phase transition in ordered Form I to disordered Form II is entropy driven with DSt = 0.029 kJ/mole. A broad exotherm (DH1 = 51.0 kJ/mole) at low temperature (31.9 °C) in thermal curve of Form II which is absent in Form I may correspond to stabilization of kinetic crystal of Form II with additional N–H  O linkage within molecular chains. Form II is obtained on cooling of Form I solution similar to the polymorphic forms of local anesthetic drug oxybuprocaine [11], this exotherm may lead to phase transformation process of Form I to Form II. An exotherm in Form II with small enthalpy change (DH2 = 2.41 kJ/mole) prior to melting point is indicative of required proton transfer (PT) pathway as seen in RAHB model in ketohydrazone  azoenol tautomers with N  H  O transition state where energy barrier is 2.34 kJ/mole quantized from DFT data [22]. This is in concurrence with the ‘‘PET’’ mechanism in spin carrying naphthoquinones via H-bonding resulting in compound Form II of 1 as an intrinsic radical such as iminonaphthosemiquinone (INSQ) [14] compared to diamagnetic iminonaphthoquinone (INQ) isomer Form I. A broad endotherm with enthalpy change of DH2 = 24.0 kJ/ mole at 164 °C in the DSC curve of Form I is absent in Form II. This may correspond to p  p interaction analogues to 2,6-dichloro-N-/ formamide [23] and it suggests absence of such interaction in Form II of 1. X-ray structural observations are in support of strong p  p stacking in Form I. Form I tautomer of 1 compared to Form II possesses additional thermodynamic stabilization of crystal by the cooperativity between stacking, hydrogen bonding and electrostatic interactions. Its endotherms can be interpreted as suggestive of stronger stacking interactions compared to Form II similar to intercalating coordination compounds [25]. The DSC curve presented in Fig. 6 for methoxy substituted compound 2 is very similar to paramagnetic Form II of compound 1. The DSC curve of 2 exhibits two broad and one sharp exotherms together with two very small broad endotherms analogous to Form II tautomer of 1. A broad exotherm of 2 is observed at 321.7 °C with DH3 = 18.6 kJ/mole. This energy is very close to radical stabilization energy via H-bondings in case of methoxy substituents in antioxidants [26] or aroxyl radicals [27]. It indicates that DH3 in methoxy substituted compound 2 may be the radical stabilization energy. One broad and one sharp exotherms at 6.9 °C (DH1 = 299.7 kJ/ mole) and 253.9 °C (DH2 = 131.4 kJ/mole) respectively with large enthalpy changes similar to tautomers of 1 may be due to stabilizing interaction via N–H  O chains in 2. The most stabilizing geometries in substituted N-/-formamides contribute to about 11.0 kJ/ mole energy towards the lattice stability, similarly in 2, a broad endotherm at 52 °C with enthalpy change DH4 = 11.0 kJ/mole is probably due to lattice stability. This energy is close to stabilization

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(a)

(b)

20000

3000 g = 2.0038

2000

Intensity

Intensity

10000

0

-10000

1000

g = 2.0043

0 -1000

-20000 2000

2500

3000

3500

4000

4500

-2000 3000

3200

3400

3600

3800

4000

Field (G)

Field (G)

Fig. 7. Solid state X-band EPR spectra of Form-II of 1 at 298 K: (a) fine brown colored powder and (b) crystalline form.

Table 1 Theoretically generated molecular parameters.

HeLa

S. no.

Compound

miLogP (calculated)

Log P (observed)

TPSA

nrotb

1 2 3 4

Menadione Form I (1) Form II (1) 2

2.044 1.876 1.972 2.376

1.928 2.054 2.025 2.613

34.142 58.533 41.674 67.767

0 1 1 2

% Cell Viablility

100 80 60 40 20 0 5

3.4. Resonance spectral studies The degree of p-delocalization of ketohydrazino fragment in 1 and 2 depends on chemical substitution enhancing RAHB mechanism which is supported by infra resonance spectral studies.

50

100

200

Conc. (μ μ M)

(A) HeLa cell lines

% Cell Viability

100

CAPAN-1

80 60 40 20 0 5

20

50

100

200

Conc. (μM)

(B) CAPAN-1 cell lines HL-60

100

% Cell Viability

by edge to face C–H  p interaction as seen in methyl substituted formamides [23]. In DSC curve of 2 a small endotherm prior to melting exotherm, with DH5 = 15.2 kJ/mole at 96.3 °C, may govern the phase change analogous to spin carrying 3-iodo-2-hydroxy-1,4-naphthoquinone [20]. Lastly, if we compare the energy changes in terms of total enthalpy change for exothermic N–H  O interactions in Form I and Form II of 1 and 2 compounds, it exhibits increasing order. Comparison between non-resonant static and stable Form I (DEHB = 54.8 kJ/mole) with resonant Form II (DEHB = 51.0 + 46.1 = 97.1 kJ/ mole) of compound 1, there is 1.8 times increase in energy due to contribution of resonance analogues to tautomeric b-aminone and b-enaminone [28] forms. In the case of compound 2 (DEHB = 299.7 + 131.4 = 431.1 kJ/mole) which is 7.8 times greater than non-resonant Form I and 4.4 times higher than resonant Form II of compound 1. This indicates that in compound 2, e donating methoxy substituents shift the RAHB more towards iminoenol form compared to ketohydrazino Form I similar as azonaphthol tautomers [22,28] which is shown in Scheme-I [see Supplementary material].

20

80 60 40 20 0 5

20

50

100

200

Conc. (μM) 3.4.1. 1H NMR spectra Form I and Form II tautomers of 1 exhibit in aprotic CDCl3 similar spectra for aromatic protons in the region 7.5–8.3 ppm. [C5–H5 at 8.3 d(d); C8–H8 at 8.2 d(d); C6–H6 at 7.65 d(t); C7–H7 at 7.6 ppm d(t)] [29]. The methyl protons of menadione fragments and acetyl hydrazine fragments are observed at 2.3 d(s) and 2.5 d(s), respectively. The quinonoidal ring proton (C2–H2) of Form II, shows upfield shift (7.3 d) compared to Form I (7.5 d). Similarly, the D2O exchangeable amide –NH proton in Form II

(C) HL-60 cell lines Fig. 8. Effect of the menadione , Form I of 1 , Form II of 1 and 2 on cell survival. Cells were incubated for 24 h at different concentrations of ranging from 5 to 200 lM. Results represent the mean values from at least three experiments.  p < 0.05 as compared with control values (untreated conditions).

exhibits upfield shift (9.46 d) [30] compared to Form I (10 d). Like b-enaminonic system [26] remarkable r delocalizations via

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1

2

3

4

Fig. 9. 0.8% Agarose gel showing the cleavage of genomic DNA by Form I and II and compound 2. [DNA] = 300 ng, [1 and 2] = 300 ng. Lane 1, DNA control; Lane 2, DNA from cells treated with Form I; Lane 3, DNA from cells treated with Form II; Lane 4, DNA from cells treated with 2.

conjugation [31] and geometric changes in Form I compared to Form II resulted in more downfield shift of amide-NH protons. While spin delocalization in Form II via p-conjugation exhibits anti-spin polarization effect on amide –NH proton and results in upfield shift due to RAHB mechanism. Such contact shifts are well known in paramagnetic compounds [32]. 1 H NMR spectra of tautomer Form I of 1 in polar d6-DMSO resulted in similar chemical shifts as in CDCl3 but Form II performs mixture of Form I and Form II with more downfield chemical shifts of amide N2–H2 protons at 11.8 d and 11.6 d respectively. This suggests that in polar solvent, the compound undergoes r delocalizations [32] via RAHB. Similar effect of RAHB is seen on the 1H NMR spectrum of compound 2 also [1H nmr data in d6DMSO: aromatic protons: C5–H5 at 8.2 d(d); C8–H8 at 8.1 d(d); C6–H6 at 7.7 d(t); C7–H7 at 7.6 ppm d(t); quinonoidal ring protons C2–H2 at 8.1 ppm d(s); methyl protons of menadione fragment 2.1 ppm d(s); methoxy protons of hydrazino fragment at 3.8 ppm d(s); amide N2–H2 at 11.6 ppm d(s)]. Finally we can conclude that in non-polar solvent Form II of 1 exhibiting upfield shifts of quinonoidal and amide protons imply microscopic hydrophobicity making nearly electrostatic and Hbonds more effective like DNA-intercalators [25]. Further this may lead to its API activity as anticancer agent in Form II compared to Form I as seen in infra biological anticancer activities. At the same time, e donor methoxy substituent in hydrazino fragment of compound 2, activates p-conjugation pathway of quinonoidal ring with upfield shift of methyl-menadione ring protons compared to Form II of 1. And it relates to its highest biological anticancer activity via higher permeability and due to highest mitochondrial membrane potential compared to Form I and Form II of compound 1 as seen in infra biological studies.

Menadione

Form I of 1

3.4.2. EPR spectra Among the two tautomers of 1, Form I is EPR inactive while kinetic Form II is EPR active. The room temperature EPR spectrum of Form II in fine red brown colored powder (a) and in crystalline (b) forms is presented in Fig. 7. Analogous to the iminofunctionalized Vitamin K3 [14], EPR of Form II in fine powder state exhibits typical bi-and monoradical nature due to hydrogen bondings [5]. The biradical interaction gives rise to inhomogeneously broaden signal with narrow central line type EPR for micro crystals of Form II [Fig. 7b]. Here broad line is superposition of the transitions in ms states viz. |1> ? 10 > & |0 > ? |+1> while narrow central line is due to double quanta absorption leading to |1 > ? l + 1> transition at moderate microwave power [33]. Such intrinsic radical formation in Form II may be effective via H-bondings compared to Form I and may act as API in 1, demonstrated in infra anticancer activity studies. 3.5. Active pharmaceutical ingredient (API) studies The active pharmaceutical ingredients (API) in pharmaceutical solids either relates with rotatable bonds with conformational flexibility [11] and drug permeability. In supra part we have projected ‘‘molecular interactions’’ of hybrid drug molecules 1 and 2 with respect to their chemical valence tautomeric structures and their activation by RAHB effect. Such hybrid drug molecules are further investigated for their API studies. 3.5.1. Theoretical molecular parameters The total molecular polarizable surface area (TPSA) is the sum of fragment contributions of O- and N-centered polar fragments. It describes characterization of drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood–brain barrier penetration. The above parameters were calculated using the Molinspiration software (Table 1). It is observed that all the four ligands obey the Lipinsky rule of 5. The number of rotatable bonds (–nrotb) is a measure of molecular flexibility. It has been shown to be a very good descriptor of oral bioavailability of drugs [34,35]. Thus, comparing the rotatable bonds, flexibility and Log P values, all the four ligands have potential for drug likeliness. Since theoretically the compounds exhibited potency we tried to analyze the same using experimental protocols such as anticancer activity. 3.5.2. Anticancer activity The cytotoxicity of the compounds was tested on three cell lines, viz. the adherent cell lines (A) HeLa, (B) CAPAN1 and a non-adherent leukemic cell line, (C) HL-60. The percentage cell viability was evaluated by the MTT assay [36]. A significant decrease in cell viability was observed for all the compounds in a time and dose dependant manner (Fig. 8) after 24 h of treatment. The microscopic examination of the plates was done to observe the cell death qualitatively (refer Supplementary data).

Form II of 1

2

Fig. 10. Morphological studies by Fluorescence microscopy. HeLa cells were incubated at 37 °C in the presence of menadione, Form I of 1, Form II of 1, 2 (25 lM each) and photographed after 24 h. Small white arrows indicate some cells presenting large blisters of empty cytoplasm and a relocation of organelles around the nucleus.

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Preliminary microscopic studies of the cell suggested apoptotic mode of cell death. Compound 2 exhibited the highest toxicity while among the two forms of 1 viz. Form II showed activity similar to Compound 2. The activity of menadione was comparable with an earlier report [37]. 3.5.3. Effect of compounds on DNA Gel electrophoresis was carried out for detecting DNA fragmentation to confirm the mode of cell death either necrosis or apoptosis. The genomic DNA was extracted after treating the HeLa cell lines with the ligands Form I and Form II of compound 1 and compound 2. Fig. 9 describes the cleaving pattern observed by Agarose gel electrophoresis (0.8–1%) to visualize the DNA. A shearing pattern was observed instead of ladder formation, which is usually obtained for planar compounds [38]. Similar DNA patterns were also observed by us for HL-60 as well as CAPAN1 cell lines. 3.5.4. Fluorescence microscopic images Since the compounds exhibited intrinsic fluorescence the drug uptake was monitored using fluorescence microscopy. The drug uptake could be most likely due to the adherence of the compounds to the plasma membrane by electrostatic attraction before its transport across the membrane facilitated by difference in concentration gradient or some other unknown mechanism. The emission spectra of the compounds show that there is greater excitation at 430 nm, which is a typical minimum excitation wavelength for single-photon confocal microscopy experiments [39]. After 24 h of treatment the cells were washed with PBS and fixed onto a glass slide. Menadione, Form II of 1 and 2 showed that the compounds (25 lM) were easily taken up by the HeLa cells. The mode of cell death for menadione is found to be ‘‘autochizis’’ [40]. In case of Form I, Form II of 1 and compound 2, apoptotic cell deaths were observed by the inverted microscopic images. These results were confirmed with DNA laddering studies [41]. However for Form I it is probable that the compound just adhered to the cell surface and brought about some morphological changes leading to cell death. However, no drug uptake was observed in this case as compared to menadione, Form II of 1 and 2. Thus the activity mechanism for Form I of 1 is different and needs to be explored. Although the chem-informatics data indicated drug likeliness for all the four ligands, while the MTT data observed was different. This difference in cell death may be due to the differential drug uptake as observed by the fluorescence microscopic images (see Fig. 10). 3.5.5. Mitochondrial membrane potential measurements Since the DNA data indicate apoptotic mode of cell death, it was essential to confirm whether the cell death was mediated through the mitochondrial membrane. As Form I of 1 did not show drug uptake, this effect needed to be confirmed using mitochondrial membrane potential measurements [42]. In coupled mitochondria, the excitation and emission wavelengths of peak intensities of Rhodamine 123 were 510 and 534 nm, respectively. Upon deenergizing with dinitrophenol, these wavelengths shifted to 500 and 526 nm. The maximum difference in the excitation spectra between the coupled and uncoupled state with Rhodamine 123 occurred at 497 and 520 nm. The calibration curve in Fig. 11 was obtained using 0.33 mM Rhodamine 123 and 0.25 mg/ml mitochondria. This curve was then used to estimate Dw for the samples treated with the mitochondria. Dw was calculated (Table 2) from the fluorescence ratio using the equation of Dw = 171.9 (573/546 ratio) + 26.2. Since Form I was unable to penetrate the cells and interact with the DNA, it also showed negligible membrane potential. However, it still shows apoptotic cell death. It may be that the small amount of H2O2 generated is leading to apoptotic cell death. Further inves-

Fig. 11. Calibration curve.

Table 2 Mitochondrial membrane potentials (mV) for samples. S. no.

Sample

Potential (mV)

1 2 3 4

Menadione Form I of 1 Form II of 1 2

142 ± 0.033 Undetectable 137 ± 0.087 159 ± 0.023

tigations are being carried out to reveal a suitable mechanism of cell death for all the four ligands.

4. Conclusion Azide derivatized Vitamin K3, compound 1, is tautomerized in two forms viz. Form I (INQ) and II (INSQ), wherein Form I has a stronger stacking effect while Form II possesses a stronger intermolecular RAHB effect on N  H  O bonding due to which different thermochemical and spectral behaviors were observed. To elucidate the effect of substituents on azide derivatized segment of Vitamin K3, a methoxy group was selected in compound 2. The anticancer activity is correlated with RAHB effect assisted by N  H  O bondings and their mitochondrial potentials by fluorescence microscopic studies. Accordingly, the anticancer activity at 50 lM follows the order such as Form I (35%) < menadione (50%) < Form II (75%) < compound 2 (81%). The mitochondrial potential which deals with penetration ability follows the order Form I  Form II < menadione < compound 2. As Form I performs least penetration which is reflected in its anticancer activity. By the theoretical studies, compound 2 showed greatest drug likeliness due to highest log P value and two rotational bonds compared to others. All compounds exhibited apoptotic mode of cell death observed by DNA shearing pattern. However if we compare activity of azide derivatized Form II and compound 2 with parent menadione both have the advantage of greater stability and potential for peptide like H-bonding at binding site [6,43] due to flexible RAHB effect compared to naphthoquinone type isomeric compound viz. menadione. Thus we conclude intermediate electronic isomers [44] Form II of 1 and compound 2 have better API value compared to parent menadione.

Supplementary material Crystallographic data of Form I in 1 has been deposited with the Cambridge Crystallographic Data Centre and may be obtained on request by quoting the deposition number CCDC-812940 from the CCDC, 12 Union Road, Cambridge CB21EZ, UK (Fax: +44 1223 336 033: E-mail address: [email protected]). Figures of quantitative microscopic examination of plates are shown for HeLa

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and CAPAN-1 cell lines. Scheme-I for probable tautomeric forms is deposited. Acknowledgements Professor A.N. Latey, Academic Co-ordinator, National Institute of Virology, Pune is thanked for the technical assistance. K.B. is thankful to UGC for financial assistance as JRF under Meritorious fellowship [F.4-1/2006(BSR)/5-82/2007(BSR)]. A.K. acknowledges Department of Science and Technology (DST), New Delhi, India, for providing a DST fast track fellowship under the scheme [SR/ FTP/CS-96/2007]. References [1] E.C. Cranenburg, L.J. Schurgers, C. Vermer, Thronb. Haemost. 98 (2007) 120. [2] D.W. Lanrson, S.M. Plaza, Altern. Med. Rev. 8 (2003) 303. [3] T. Akiyoshi, S. Matzno, M. Sakai, N. Okamura, K. Matsuyama, Cancer Chemother. Pharmacol. 65 (2009) 143. [4] (a) J. Verrax, M. Delvaux, N. Beghein, H. Taper, B. Gallez, P. Buc Calderon, Free Radical Res. 39 (2005) 649; (b) J. Verrax, J. Cadrobbi, C. Marques, H. Taper, Y. Habraken, J. Piette, P. Buc Calderon, Apoptosis 9 (2004) 223; (c) N. Sata, H. Klonowski-Stumpe, B. Han, D. Haussinger, C. Niederau, Free Radical Biol. Med. 23 (1997) 844. [5] S. Rane, K. Ahmed, S. Gawali, S. Zaware, D. Shriniwas, R. Gonnande, M. Bhadbhade, J. Mol. Struct. 892 (2008) 74. [6] E. Cabezas, A.C. Satterthwait, J. Am. Chem. Soc. 121 (1999) 3862. [7] A.A. Farooqui, W-Y. Ong, L.A. Horrocks, Pharmacol. Rev. 58 (2006) 591. [8] S. Matzno, Y. Yamaguchi, T. Akiyoshi, T. Nakabayashi, K. Matsuyama, Biol. Pharm. Bull. 31 (2008) 1270. [9] N. Hulsman, J.P. Medema, C. Bos, A. Jongejan, R. Leurs, M.J. Smit, I.J.P. de Esch, D. Richel, M. Wijtmans, J. Med. Chem. 50 (2007) 2424. [10] R. Morphy, C. Kay, Z. Rankovic, Drug Discov. Today 9 (2004) 641. [11] U.J. Griesser, R.K.R. Jetti, M.F. Haddow, T. Brehmer, D.C. Apperley, A. King, R.K. Harris, Cryst. Growth Des. 8 (2008) 44. [12] K. Subbaramaiah, N. Telang, J.Y. Ramonetti, R. Araki, B. Devito, B.B. Weksler, A.J. Dannenberg, Cancer Res. 56 (1996) 4424. [13] R.C. Scaduto, Eur. J. Biochem. 223 (1994) 751. [14] A.V. Todkary, R. Dalvi, S. Salunke-Gawali, J. Linares, F. Varret, J. Marrot, J.V. Yakhmi, M. Bhadbhade, D. Srinivas, S.P. Gejji, S.Y. Rane, Spectrochim. Acta A 63 (2006) 130.

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