COMPOUNDS AS FLUORESCENCE PROBES

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from Indian Patent Application number 201841031942 filed on August 27, 2018, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to diphenylamino-methylene malononitrile based compounds, a process for preparing thereof and their use as photodynamic therapy agents in biological, biochemical and industrial applications such as in photodynamic therapeutics, diagnostics and as fluorescence probes for cell imaging applications.

BACKGROUND

Tubulin is a protein that polymerize in to dynamic microtubules in eukaryotic cells. Tubulin involves in various functions such as cell integrity, cell division, cell signalling, intracellular vesicles and organelle transport and locomotion. The minute changes of tubulin in microtubules is associated with different cancers. Thus, the control of these trace changes in tubulins has become new target of cancer therapy research. Therefore, the visualization of microtubules is critical to understand their intracellular function. Several imaging techniques have been developed to visualize and study specific organelles in living cell, such as electron microscopy, silver staining and fluorescence imaging. Among these, fluorescence probes with confocal microscopy present an exciting opportunity for the selective imaging of tubulin, microtubules and relevant events in living cells which has potential application in the cancer therapy research. However, the existing probes generally have shortcomings such as difficult to modify, possess high cytotoxicity, photo-instability, low sensitivity, poor selectivity and so on.

Therefore, there is a need in the art to provide new fluorescent probes, which can overcome one or more of the above-mentioned shortcomings, for detecting tubulin, microtubules and the like in living cells. SUMMARY

The present disclosure thus discloses a compound of formula (I) and/or formula (II):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein,

ring A is aryl, heteroaryl or is absent; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and -CORi;

ring B is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi;

Ri is hydroxy or alkoxy; and

D is a donor.

The present disclosure also discloses a pharmaceutical composition comprising a compound of formula (I) or formula (II).

In another aspect, the present disclosure discloses preparation of compounds of formula (I) or formula (II).

In yet another aspect, the present disclosure discloses a method of detecting the tubulin in a subject using the compound of formula (I) or (II).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates effect of compounds of formula (I) and formula (II) on viability of

HeLa cells;

FIG. 2 illustrates co-localization of Example 1 in Hela cells;

FIG. 3 illustrates co-localization of Example 2 in Hela cells;

FIG. 4 illustrates co-localization of Example 6 in Hela cells; and

FIG. 5 illustrates co -localization of Example 3, Example 4 and Example 5 in Hela cells DETAILED DESCRIPTION

The present disclosure discloses a compound of formula (I) or formula (II). The compound is useful as fluorescent probe for detecting the presence or absence of tubulin in a subject. These probes are easy to modify, possess low cytotoxicity, high photo stability, high sensitivity and site specific thereby good option for monitoring dynamic changes of tubulins in living biological samples. The probes of the present disclosure possess superior properties, such as accessibility to living cells, specificity for tubulin, and super-resolution imaging, compared to existing probes.

Each embodiment is provided by way of explanation of the disclosure and not by way of limitation of the disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the compounds, compositions and methods described herein without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment can be applied to another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure includes such modifications and variations and their equivalents. Other objects, features and aspects of the present disclosure are disclosed in or are obvious from, the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not to be construed as limiting the broader aspects of the present disclosure.

In an embodiment, the present disclosure discloses a compound of formula (I) or (II):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein,

ring A is aryl, heteroaryl or is absent; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and -CORi; ring B is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi;

Ri is hydroxy or alkoxy; and

D is a donor.

In certain embodiments, ring A, in formula (I), is monocyclic aryl. In further embodiments, ring A is phenyl.

In certain embodiments, ring A, in formula (I), is absent and ring B is phenyl.

In certain embodiments, ring D, in formula (

r or ^r ; wherein each of R ₂ , R3 and R ₄ is independently hydrogen or alkyl In certain embodiments, the compound of formula (I) is a compound of formula (III):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, ring A is aryl, heteroaryl or is absent; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and -CORi;

ring B is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi; and

Ri is hydroxy or alkoxy.

In certain embodiments, ring A, in formula (III), is monocyclic aryl. In further embodiments, ring A is phenyl. In certain embodiments, ring A, in formula (III), is monocyclic heteroaryl. In further embodiments, ring A is thiophenyl.

In certain embodiments, in formula (III), ring A is absent and ring B is phenyl.

In certain embodiments, the compound of formula (III) is a compound of formula (IIIA):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein,

ring A is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi; and

Ri is hydroxy or alkoxy.

In certain embodiments, ring A, in formula (IIIA), is monocyclic aryl. In further embodiments, ring A is phenyl.

In certain embodiments, ring A, in formula (IIIA), is monocyclic heteroaryl. In further embodiments, ring A is thiophenyl.

In certain embodiments, the compound of formula (II) is a compound of formula (IV):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein, ring A is aryl, heteroaryl or is absent; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and -CORi;

ring B is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi; and

Ri is hydroxy or alkoxy.

In certain embodiments, ring A, in formula (IV) is monocyclic aryl. In further embodiments, ring A is phenyl.

In certain embodiments, ring A, in formula (IV) is monocyclic heteroaryl. In further embodiments, ring A is thiophenyl.

In certain embodiments, in formula (III), ring A is absent and ring B is phenyl.

In certain embodiments, the compound of formula (IV) is a compound of formula

(IV A):

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein,

ring A is aryl or heteroaryl; wherein said aryl and heteroaryl are optionally substituted with one or more substituents independently selected from alkyl, haloalkyl, cyano, nitro and - CORi; and

Ri is hydroxy or alkoxy.

In certain embodiments, ring A, in formula (IVA) is monocyclic aryl. In further embodiments, ring A is phenyl.

In certain embodiments, ring A, in formula (IVA) is monocyclic heteroaryl. In further embodiments, ring A is thiophenyl. In certain embodiments, the compounds of formula (I), formula (III) and formula (IIIA) are selected from:

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In certain embodiments, the compounds of formula (II), formula (IV) and formula (IVA) are selected from:

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a compound as disclosed herein.

In certain embodiment, the compounds of the formula (I) and (II) may be useful in photodynamic therapy as fluorescent probe for the diagnosis of tubulin and its associated diseases like cancer.

In certain embodiments, the present disclosure provides compounds of formula (I) and/or formula (II) for use both in vitro and in vivo photodynamic therapeutic treatment.

In certain embodiments, compounds of the formula (I) and/or formula (II) can be used as cellular components in fixed or live cell imaging applications.

In certain embodiments, the compound of formula (I) and/or formula (II) are tubulin specific fluorescent compounds. The compounds have low cytotoxicity, specifically binds to tubulins and emitting green fluorescence. Therefore, the compounds are useful for fluorescence imaging, observing and detecting trace changes of tubulin in microtubules during cancer therapy.

In certain embodiments, the present disclosure provides a method of detecting tubulin in a sample, wherein the method comprises contacting the compound of formula (I) or (II) with the sample and detecting the fluorescence generated due to the reaction of the compound with the sample. In certain embodiments, this method is further characterized by the fact that the fluorescence detection is visualized using fluorescent imaging means. In certain embodiments, the amount of the compound of formula (I) or (II) (fluorescent probe) is not particularly restricted and the amount of compound can be selected as appropriate by one skilled in the art.

In certain embodiments, the sample is selected from a group comprising cells, biological fluids and chemical mixture.

In certain embodiments, the disclosure provides a kit for detecting tubulin in a sample, wherein the kit comprises a compound of formula (I) or formula (II). The kit may also comprise instructions for carrying out a method of detecting the tubulin in the sample. This kit may also include other reagents and the like as needed. For example, dissolution auxiliaries, buffers, isotonifying agents, pH adjusters and other such additives, and the amounts compounded can be selected as appropriate by one skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, "optionally substituted alkyl" refers to the alkyl may be substituted as well as the event or circumstance where the alkyl is not substituted.

The term "compounds of the present disclosure " comprises compounds of formula (I) or formula (II) or a pharmaceutically acceptable salt or a stereoisomer thereof.

As used herein, the term "alkyl" refers to a straight chain or branched saturated hydrocarbon group containing no unsaturation. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, Ci _-6 alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec -butyl, tert-butyl, l-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, l-hexyl, 2-hexyl, 3-hexyl, l-heptyl, 2- heptyl, 3-heptyl, 4-heptyl, l-octyl, 2-octyl, 3-octyl and 4-octyl. The "alkyl" group may be optionally substituted.

As used herein, the term "alkoxy" refers to a straight or branched, saturated aliphatic hydrocarbon radical bonded to an oxygen atom that is attached to a core structure. Alkoxy groups may have one to six carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy and tert-butoxy.

“Aryl" refers to monocyclic or fused bicyclic or polycyclic ring system having the well-known characteristics of aromaticity, wherein at least one ring contains a completely conjugated pi-electron system. Typically, aryl groups contain 6 to 20 carbon atoms ("C ₆ -C ₂₀ aryl") as ring members, preferably 6 to 14 carbon atoms ("C ₆ -C ₁₄ aryl") or more preferably, 6 to 12 carbon atoms ("C ₆ -C ₁₂ aryl"). Fused aryl groups may include an aryl ring (e.g., a phenyl ring) fused to another aryl or heteroaryl ring or fused to a saturated or partially unsaturated carbocyclic or heterocyclic ring, provided the point of attachment to the base molecule on such fused ring systems is an atom of the aromatic portion of the ring system. Examples, without limitation, of aryl groups include phenyl, biphenyl, naphthyl, anthracenyl, indanyl, indenyl, phenanthrenyl, and tetrahydronaphthyl.

As used herein, the term "cyano" refers to -CN group.

As used herein, the term "halo" or“halogen" refers to fluoro (fluorine), chloro (chlorine), bromo (bromine) and iodo (iodine).

As used herein, the term "haloalkyl" means alkyl substituted with one or more halogen atoms, wherein the term“halo” and“alkyl” are as defined above. Examples of "haloalkyl" groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl and 2,2,2-trifluoroethyl.

"Heteroaryl" or "heteroaromatic" refer to monocyclic or fused bicyclic or polycyclic ring systems having the well-known characteristics of aromaticity that contain the specified number of ring atoms and include at least one heteroatom selected from N, O and S as a ring member in an aromatic ring. The inclusion of a heteroatom permits aromaticity in 5- membered rings as well as 6-membered rings. Typically, heteroaryl groups contain 5 to 20 ring atoms ("5-20 membered heteroaryl"), preferably 5 to 14 ring atoms ("5-14 membered heteroaryl"), and more preferably 5 to 12 ring atoms ("5-12 membered heteroaryl"). Heteroaryl rings are attached to the base molecule via a ring atom of the heteroaromatic ring, such that aromaticity is maintained. Thus, 6-membered heteroaryl rings may be attached to the base molecule via a ring C atom, while 5-membered heteroaryl rings may be attached to the base molecule via a ring C or N atom. Heteroaryl groups may also be fused to another aryl or heteroaryl ring or fused to a saturated or partially unsaturated carbocyclic or heterocyclic ring, provided the point of attachment to the base molecule on such fused ring systems is an atom of the heteroaromatic portion of the ring system. Examples of unsubstituted heteroaryl groups often include, but are not limited to, pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, triazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, benzofuran, benzothiophene, indole, benzimidazole, indazole, quinoline, isoquinoline, purine, triazine, naphthryidine and carbazole. In frequent preferred embodiments, 5- or 6-membered heteroaryl groups are selected from the group consisting of thiophenyl, pyrrolyl, furanyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, pyridinyl and pyrimidinyl, pyrazinyl and pyridazinyl rings.

As used herein, the term "hydroxy" or "hydroxyl" refers to -OH group.

As used herein, the term "nitro" refers to -N0 ₂ group.

The term "stereoisomer" or "stereoisomers" refers to any enantiomers, diastereomers or geometrical isomers of the compounds of formula (I) or (II), wherever they are chiral or when they bear one or more double bond. When the compounds of the formula (I) or (II) and related formulae are chiral, they can exist in racemic or in optically active form. It should be understood that the disclosure encompasses all stereochemical isomeric forms, including diastereomeric, enantiomeric and epimeric forms, as well as -isomers and /-isomers and mixtures thereof. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art. Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art. Additionally, the compounds of the present disclosure may exist as geometric isomers. The present disclosure includes all cis, trans, syn, anti, entgegen E and zusammen (Z) isomers as well as the appropriate mixtures thereof.

The abbreviations used in the entire specification may be summarized herein below with their particular meaning.

^U B NMR: Boron- 11 nuclear magnetic resonance spectroscopy; calcd: Calculated; DMSO: dimethyl sulfoxide; DCM: dichloromethane; equiv.: equvivalent(s); HRMS: high- resolution mass spectrometry; Na ₂ S0 ₄ : sodium sulphate; g: gram(s); h: hour(s); ¹ H NMR: proton nuclear magnetic resonance; ¹³ C NMR: carbon- 13 nuclear magnetic resonance; ¹⁹ F NMR: Fluorine- 19 nuclear magnetic resonance spectroscopy; M: molar; MeOH: methanol; mL; millilitre; pF: Microlitre; mg: milligram; Mp: melting point; degree Celsius (°C); mF: millilitre; [M+H] ⁺ : protonated molecular ion; m/z: mass-to-charge ratio; rt: room temperature; THF: tetrahydrofuran; TFC: thin-layer chromatography.

Another embodiment of the present disclosure provides process for preparation of the compounds of general formula (I) and formula (II) are set forth in the below examples and generalized scheme. One of skill in the art will recognize that scheme can be adapted to produce the compounds of general structure (I) and structure (II) and their pharmaceutically acceptable salts or stereo isomers according to the present disclosure.

The schemes are given for the purpose of illustrating the disclosure and are not intended to limit the scope or spirit of the disclosure in any way. Starting materials shown in the schemes can be obtained from commercial sources or prepared based on procedures described in the literature. Furthermore, in the following schemes, where specific acids, bases, reagents, coupling agents, solvents, etc. are mentioned, it is understood that other suitable acids, bases, reagents, coupling agents etc. may be used and are included within the scope of the present disclosure. Modifications to reaction conditions, for example, temperature, duration of the reaction or combinations thereof, are envisioned as part of the present disclosure. All possible stereoisomers are envisioned within the scope of this disclosure.

The intermediates required for the synthesis are commercially available or alternatively, these intermediates can be prepared using known literature methods. The disclosure is described in greater detail by way of specific examples. All the chemicals and solvents were purchased from various commercial sources. Solvents THF and toluene were dried over sodium metal before use. Fresh dichloromethane was used for reaction after distilled over CaH ₂ . MeOH was dried over using Mg/I ₂ . ¹ H and ¹³ C spectra were recorded on a Bruker Avance 400 MHz and 500 MHz instruments in CDCb as a solvent with tetramethylsilane (TMS) as internal standard. IR spectra were recorded on JASCO FT/IR-5300 spectrometer. UV-Vis spectra were recorded on a Shimadzu UV-3600. Fluorescence spectra were recorded on Fluoromax-4 spectrometer at room temperature. High-resolution mass spectra (HRMS) were recorded on micro mass ESI-TOF MS. The progress of the reaction was monitored by TLC and visualized under UV and/or Iodine chamber. Column chromatography was performed on silica gel (100-200 mesh size), using ethyl acetate and hexanes mixture as eluent.

It is contemplated that some of the intermediates disclosed in the present disclosure may be used for the next step without any characterization data.

It is meant to be understood that the order of the steps in the processes may be varied, that reagents, solvents and reaction conditions may be substituted for those specifically mentioned, and that vulnerable moieties may be protected and protected, as necessary. Ring A and Ring B independently represents all the possible substitutions as disclosed in compound of formula (I) and formula (II).

General Synthetic Schemes:

Scheme-1:

Some compounds of formula (I) and formula (II) can be obtained as shown in Scheme- 1. Reacting compound of formula 1 with a compound of formula 2 by using appropriate reducing agent like NaH in appropriate solvent like toluene or benzene gives a compound of formula 3 which on further reaction with compound of formula 4 in presence of appropriate Pd catalyst like Pd(PPh ₃ ) ₄ in a suitable solvent like toluene and benzene yields some compounds of formula (I).

Compounds of formula (II) can be obtained by treating compound of formula (I) with BF ₃ OEt ₂ in presence of suitable solvent like DCM at room temperature.

Scheme-2:

Some compounds of formula (I) and formula (II) can be obtained as shown in Scheme-2. Reacting compound of formula 5 with a compound of formula 6 by using appropriate reducing agent like NaH in appropriate solvent like toluene or benzene gives a compound of formula 7 which on further hydrolysis or deprotection followed by reaction with malononitrile yields compounds of formula (I) which on further reaction with BF ₃ .OEt ₂ in presence of suitable solvent like DCM yields compound of formula (II).

EXAMPLES

The following examples are given by way of illustration of the working of the disclosure in actual practice and therefore should not be construed to limit the scope of present disclosure. All the chemicals and solvents were purchased from various commercial sources. Solvents THF and toluene were dried over sodium metal before use. Fresh dichloromethane was used for reaction after distilled over CaH ₂ . MeOH was dried over using Mg/I ₂ . ¹ H and ¹³ C spectra were recorded on a Bruker Avance 400 MHz and 500 MHz instruments in CDCl ₃ as a solvent with tetramethylsilane (TMS) as internal standard. IR spectra were recorded on JASCO FT/IR-5300 spectrometer. UV-Vis spectra were recorded on a Shimadzu UV-3600. Fluorescence spectra were recorded on Fluoromax-4 spectrometer at room temperature. High-resolution mass spectra (HRMS) were recorded on micro mass ESI-TOF MS. The progress of the reaction was monitored by TLC and visualized under UV and/or Iodine chamber. Column chromatography was performed on silica gel (100-200 mesh size), using ethyl acetate and hexanes mixture as eluent.

Intermediates

Synthesis of 4-(diphenylamino)benzaldehyde (1):

POCI3 (1.3 mL, 8.15 mmol) was added dropwise to a solution of triphenylamine (1 g, 4.0 mmol) in dry DMF (10 mL) at 0 °C and allowed to stir at 50-55 °C for 14 h. The progress of the reaction was monitored by TLC using 20% EtOAc/hexanes mixture as a mobile phase. After completion of the reaction, the reaction mixture was poured into ice-cold water and extracted with EtOAc (2 x 50 mL). The organic layer was washed with H ₂ 0 (50 mL) and brine solution (10 mL). The organic layer was dried over NaSCL and evaporated using a rotary evaporator under vacuum. The crude product was purified by column chromatography using 1:5 EtOAc/hexanes to get the desired product as a pale-yellow solid (0.8 g, yield = 72%). R/= 0.80. MP: 149-151 °C; 1H NMR (400 MHz, CDCL): d 9.80 (s, 1H), 7.67 (d, / =

8.8 Hz, 2H), 7.34 (t, J = 7.6 Hz, 4H), 7.17 (t, J = 6.8 Hz, 6H), 7.01 (d, J = 8.8 Hz, 2H); ¹³ C NMR (100 MHz, CDCL): d 190.5, 153.4, 146.2, 131.4, 129.8, 129.2, 126.4, 126.2, 119.4.

Synthesis of 4-(diphenylamino)benzoic acid (2):

To a stirred solution of 4-(diphenylamino)benzaldehyde 1 (2 g, 7.34 mmol) in 100 mL of acetone-H ₂ 0 (4:1 v/v) was added KMn0 ₄ portion wise. The reaction mixture was allowed to stir at 60 °C for 4 h. The reaction mixture was concentrated under vacuum, filtered under suction and washed with water. The filtrate was acidified with dil. HC1 (50 mL) to give a white precipitate which was filtered using a suction pump. It was washed with water and dried under vacuum to get the titled compound as a white solid. MP: 210-212 °C; yield = 71%, R _f = 0.22 in 1:5 EtO Ac/hexanes; 1H NMR (400 MHz, CDCb): d 7.90 (d, / = 8.8 Hz,

2H), 7.32 (t, / = 8.4 Hz, 4H), 7.17-7.12 (m, 6H), 6.99 (d, / = 8.8 Hz, 2H); ¹³ C NMR (100 MHz, CDCb): d 152.8, 146.5, 131.7, 129.7, 126.2, 124.8, 120.9, 119.6.

Synthesis of methyl 4-(diphenylamino)benzoate (3):

To a stirred solution of 4-(diphenylamino)benzoic acid 2 (1 g, 2.56 mmol) in MeOH (40 mL) was added acetyl chloride (0.9 mL, 12.84 mmol) dropwise at 0 °C. The reaction mixture was allowed to stir at room temperature for 16 h. The reaction was monitored by TLC using 20% EtOAc/hexanes mixture. After completion of reaction, the reaction mixture was poured into water and neutralized with 1N NaOH solution followed by extraction with EtO Ac. The organic layer was dried over sodium sulphate and concentrated to get a residue. The residue was passed through a plug of short column using 1:5 EtOAc/hexanes to afford the titled compound as a white solid (0.6 g, yield = 74%). MP: 100-103 °C; R/ = 0.80 in 1:5 EtOAc/hexanes; 1H NMR (400 MHz, CDCb): d 7.85 (d, / = 8.8 Hz, 2H), 7.30 (d, / = 7.6 Hz, 4H), 7.15-7.10 (m, 6H), 6.99 (d, / = 8.8 Hz, 2H), 3.87 (s, 3H); ¹³ C NMR (100 MHz, CDCb): d 152.8, 146.5, 131.7, 129.7, 126.2, 124.8, 120.9, 119.6; HRMS (ESI): calcd for C20H17NO2 [M+H] ⁺ 304.1332, found 304.1331.

Synthesis of l-(4-(diphenylamino)phenyl)-3-(4-iodophenyl)propane-l,3-dion e (4):

A solution of methyl 4-(diphenylamino)benzoate 3 (0.5 g, 1.65 mmol) in toluene (3 mL) was added to a stirred solution of NaH (0.2 g, 8.24 mmol) in dry toluene (20 mL) at room temperature under nitrogen atmosphere. The solution was allowed to stir at room temperature for 0.5 h. Then, 4-iodo acetophenone (0.4 g, 1.63 mmol) was added to the reaction mixture and stirred at refluxed condition for 21 h. The reaction mixture was allowed to cool to room temperature then neutralized with dil. HC1 and extracted with EtOAc (2x20 mL). The organic phase was combined and dried over anhydrous sodium sulphate to get residue. The residue was purified by column chromatography using 10% of EtO Ac/Hexane mixture to yield the titled compound as a yellow solid (0.4 g, yield = 46%,). MP: 124-128 °C; R/ = 0.51 in 1:10 EtOAc/hexanes; 1H NMR (400 MHz, CDCL): d 17.07 (s, 1H), 7.82 (dd, / = 19.2, 8.4 Hz, 4H), 7.66 (d, / = 8.0 Hz, 2H), 7.34 (t, / = 6.8 Hz, 4H), 7.20-7.16 (m, 6H), 7.05 (d, / = 8.4 Hz, 2H), 6.73 (s, 1H); ¹³ C NMR (100 MHz, CDCL): d 185.8, 182.6, 152.0, 146.4, 137.8, 135.1, 129.6, 128.8, 128.4, 127.3, 125.9, 124.7, 120.0, 99.3, 92.1; IR (neat): u 3409, 1584, 1487, 1230, 1179, 1003, 786, 750, 698 cm ¹ ; HRMS (ESI): calcd for C27H20INO2 [M+H] ⁺ 518.0611, found 518.0613.

Synthesis of l-(5-bromothiophen-2-yl)-3-(4-(diphenylamino)phenyl)propane- l,3-dione

(5):

Methyl 4-(diphenylamino)benzoate 3 (0.5 g, 1.65 mmol) was added to a stirred solution of NaH (0.2g, 8.24 mmol) in dry toluene (20 mL) at room temperature for 0.5 h. l-(5- bromothiophen-2-yl)ethan-l-one (0.34 g, 1.66 mmol) was added to the reaction mixture and was stirred at reflux condition under atmosphere of nitrogen for 21 h. The reaction mixture was allowed to cool to room temperature then neutralized with dil. HC1 and extracted with EtOAc (2 x 20 mL). The organic phase was combined and dried with anhydrous sodium sulphate. The product was purified by column chromatography using 10% of EtOAc/hexanes mixture. Yellow solid (0.27 g, yield = 35%); MP: 137-139 °C; R/ = 0.61 in 1:5 EtOAc/hexanes; 1H NMR (400 MHz, CDCL): d 16.43 (s, 1H), 7.77 (d, / = 8.8 Hz, 2H), 7.48 (d, / = 3.6 Hz, 1H), 7.33 (t, / = 8.4 Hz, 4H), 7.28-7.15 (m, 7H), 7.03 (d, / = 8.4 Hz, 2H), 6.48 (s, 1H); ¹³ C NMR (100 MHz, CDCb): d 181.3, 180.1, 151.9, 148.3, 146.5, 138.2, 130.9, 130.6, 129.7, 129.6, 128.4, 126.0, 125.9, 124.7, 124.5, 120.2, 120.1, 91.6, 82.6; IR (neat): u 2998, 1587, 1484, 1272, 1174, 1004, 787, 756, 694 cm ¹ ; HRMS (ESI): calcd for C ₂₅ Hi ₈ BrN0 ₂ S [M+H] ⁺ 498.0134, found 498.0132.

Synthesis of 2-(thiophen-2-yl)-l,3-dioxolane (6):

Compound 6 was synthesized using a literature procedure. Thiophene-2-carbaldehyde (3 g, 26.75 mmol), ethylene glycol (3 ml, 53.50 mmol) and PTSA.H ₂ 0 (0.5 g, 2.67 mmol) were taken in benzene (50 mL) and allowed to stir vigorously for 16 h under reflux condition using a Dean-Stark setup. The reaction mixture was then poured into 10% aqueous NaOH (200 mL) solution and extracted with EtOAc (2 x 50 mL) and washed with water. The organic layer was combined and dried over anhydrous sodium sulphate. The organic phase was concentrated under vacuum. The crude was directly used for next step without further purification. Brown liquid (3.8 g, yield = 90%), R/ = 0.51 in 1:10 EtO Ac/hexanes; ¹ H NMR (400 MHz, CDCb): d 7.35 (d, / = 12.4 Hz, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.12 (s, 1H), 4.12

(t, / = 1.6 Hz, 2H), 4.01 (t, / = 3.2 Hz, 2H); ¹³ C NMR (100 MHz, CDCb): d 141.8, 128.3, 126.7,126.3, 126.2, 100.3, 65.2.

Synthesis of 5-(tributylstannyl)thiophene-2-carbaldehyde (7):

To a stirred solution compound 6 (2 g, 12.80 mmol) in dry THF (50 mL) was added n-BuLi (12 mL, 19.2 mmol) at -78 °C under nitrogen atmosphere at -78 °C for 1 h. Then the reaction mixture was brought to -40 °C and stirred for 4 h. Then Bu ₃ SnCl (3.8 mL, 14.08 mmol) was added slowly to the reaction mixture at -78°C and the reaction mixture was brought to room temperature and stirred for 24 h. After the reaction was complete, the reaction mixture was neutralized with 1M HC1 (50 mL) and extracted with diethyl ether (2 x 30 mL). The organic phase was dried with anhydrous sodium sulphate and concentrated under vacuum. The crude was purified with column chromatography using 33 % of EtOAc/hexanes mixture. Brownish yellow viscous liquid (3.4 g, yield = 66%); R/ = 0.74 in 1:5 EtO Ac/hexanes; 1H NMR (400 MHz, CDCb): d 9.95 (s, 1H), 7.86 (s, 4H), 7.29 (s, 1H), 1.60-1.57 (m, 6H), 1.38-1.33 (m, 6H), 1.20-1.16 (m, 6H), 0.94-0.90 (m, 9H); ¹³ C NMR (100 MHz, CDCb): d 182.0, 151.6, 149.2, 136.8, 136.3, 28.9, 27.3, 13.7, 11.1; IR (neat): u 3352, 2957, 2871, 2726, 1670, 1461, 1377, 1251, 1074, 873, 757, 662 cm ¹ ; HRMS (ESI): calcd for Ci ₇ H ₃₀ SSn [M+Na] ⁺ 425.0932, found 425.0933.

Synthesis of 2-((5-(tributylstannyl)thiophen-2-yl)methylene)malononitrile (8):

Compound 8 was prepared according to the literature procedure. Malononitrile (10 mg, 0.15 mmol), 5-(tributylstannyl)thiophene-2-carbaldehyde 7 (50 mg, 0.125 mmol), and PPh ₃ (7 mg, 20 mol %) were stirred without solvent at 80 °C for 2 h. The reaction was monitor by TLC and the reaction was completed, the reaction mixture was diluted with water and extracted with EtOAc (2 x 5 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under vacuum. The crude product was purified by column chromatography using 1: 10 EtO Ac/Hexane mixture to yield brownish yellow viscus liquid (37 mg, yield = 65%); R/ = 0.55 in 1:20 EtO Ac/hexanes; 1H NMR (400 MHz, CDCb): d 7.85 (s, 2H), 7.30 (t, 7 = 4.0 Hz, 1H), 1.60-1.52 (m, 6H), 1.38-129 (m, 7H), 1.20-1.16 (m, 5H), 0.89 (t, 7 = 7.2 Hz, 9H) ; ¹³ C NMR (100 MHz, CDCb): d 156.3, 149.9, 140.8, 138.3, 136.9, 114.4, 113.5, 76.7, 28.9, 27.2, 13.7, 11.3; IR (neat): u 2951, 2915, 2217, 1571, 1406, 1313, 1272, 1065, 874, 750 cm ¹ ; HRMS (ESI): calcd for C ₂ oH ₃ oSSn [M+H] ⁺ 451.1224, found 451.1224.

Synthesis of l-(5-bromothiophen-2-yl)ethan-l-one (9):

To a stirred solution of 2 -bromo thiophene (2 g, 12.27 mmol) in dry dichloromethane (30 mL) was added acetyl chloride (1.05 mL, 14.72 mmol) drop wise at 0 °C for 0.5 h. Anhydrous AlCb (2 g, 14.72 mmol) was added portions wise. Then, the reaction was warmed to room temperature and stirred for 12 h. The reaction mixture was poured into an ice cooled water, neutralized with 10% Na ₂ C0 ₃ and extracted with dichloromethane (2 x 30 mL). The organic layer was combined and dried over with anhydrous Na ₂ S0 ₄ . The solvent was concentrated using a rotavap. The crude was purified by column chromatography using 20% EtOAc/hexanes to yield brown solid (1.65 g, yield = 66%); MP: 111-113 °C; R/ = 0.43 in 1:5 EtOAc/hexanes; 1H NMR (400 MHz, CDCL): d 7.29 (q, / = 4.0 Hz, 2H), 2.50 (s, 3H); ¹³ C NMR (100 MHz, CDCL): d 189.3, 150.4, 138.2, 133.3, 85.5, 26.6.

Synthesis of l-(4-(diphenylamino)phenyl)ethan-l-one (10):

To a stirred solution of triphenylamine (1 g, 4.0 mmol) in dry dichloromethane (20 mL) anhydrous A1CL (0.65 g, 4.9 mmol) was added portion wise into the solution at 0 °C for 0.5 h. Acetyl chloride (0.35 mL,4.9 mmol) was added dropwise to the reaction mixture. Then, the reaction was changed the temperature 25 °C for 12 h. The reaction mixture was poured into the ice cooled water and basified 1M NaHCCL solution followed by extracted with dichloromethane (2 x 50 mL). The organic layer was combined and dried over with anhydrous Na ₂ S0 ₄ . The solvent was concentrated with rotavapor. The crude was purified 20% EtOAc/Hexane combination with column chromatography. Pale yellow solid; Yield = 76%, R/ = 0.76 in 1:5 EtOAc/hexanes; 1H NMR (400 MHz, CDCL): d 7.79 (d, / = 8.8 Hz, 2H), 7.31 (t, J = 8.4 Hz, 4H), 7.16-7.10 (m, 6H), 6.98 (d, J = 8.8 Hz, 2H), 2.52 (s, 3H); ¹³ C NMR (100 MHz, CDCL): d 196.6, 152.2, 146.5, 130.7, 129.7, 126.0, 124.7, 119.7, 26.4. Synthesis of methyl 4-formyl benzoate (11):

To a stirred solution of 4-formyl benzoic acid (2 g, 13.32 mmol) in MeOH (30 mL) was added acetyl chloride (4.8 mL, 66.61 mmol) dropwise at 0 °C. The reaction mixture was allowed to stir at room temperature for 16 h. The reaction was monitored by TLC and the solvent was removed under reduced vacuum. The reaction mixture was diluted with EtOAc and neutralized with 1N NaOH solution followed by extraction with ethyl acetate (2 x 20 mL). The organic layer was dried over sodium sulphate and the filtrate was concentrated using rotavap. The crude was purified using 20% EtOAc/hexanes in a silica gel column. White solid (1.8 g, yield = 85%); MP: 83.0 °C; R/ = 0.46 in 1:5 EtOAc/hexanes; 1H NMR (400 MHz, CDCb): d 10.08 (s, 1H), 8.18 (d, J = 6.8 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 3.94 (s, 3H); ¹³ C NMR (100 MHz, CDCb): d 191.8, 166.1, 139.2, 135.1, 130.3, 129.6, 52.7; IR (neat): u 1722, 1680, 1500, 1386, 1112, 1014, 813, 761 cm ; HRMS (ESI): calcd for C ₉ H ₈ 0 ₂ [M+H] ⁺ 165.0546, found 165.0542.

Synthesis of methyl 4-(l,3-dioxolan-2-yl)benzoate (12):

Methyl 4-formylbenzoate 11 (2 g, 12.19 mmol), ethylene glycol (1.4 ml, 24.38 mmol) and PTSA.H2O (0.47 g, 2.44 mmol) in toluene (50 mL) all mixed together and allowed for vigorous stirring for 16 h under reflux condition in a Dean-Stark setup. The reaction mixture was poured into 10% NaOH (200 mL) solution and extracted with EtOAc (2 x 50 mL) and washed with water. The organic layer was combined and dried over anhydrous sodium sulphate. The organic phase was concentrated under vacuum. The crude was purified with column chromatography using 20% EtOAc/hexanes solvent mixture. Pale yellow liquid (2.3 g, yield = 90%), R/= 0.43 in 1:10 EtOAc/hexanes; 1H NMR (400 MHz, CDCb): d 8.04 (t, / = 6.8 Hz, 2H), 7.54 (t, / = 6.8 Hz, 2H), 5.82 (s, 1H), 4.10-4.07 (m, 2H), 4.02-4.01 (m, 2H), 3.88 (s, 3H); ¹³ C NMR (100 MHz, CDCb): d 166.7, 142.8, 130.8, 129.6, 126.4, 103.0, 65.3, 52.1; IR (neat): u 3436, 2954, 2879, 1717, 1577, 1436, 1277, 1117, 849, 763, 732 cm ⁴ ; HRMS (ESI): calcd for C11H12O2 [M+H] ⁺ 209.0808, found 209.0800

Synthesis of l-(4-(l,3-dioxolan-2-yl)phenyl)-3-(4-(diphenylamino)phenyl)p ropane-l,3- dione (13):

l-(4-(diphenylamino)phenyl)ethan-l-one 10 (1 g, 3.48 mmol) was added to a stirred suspension of NaH (0.696 g, 17.42 mmol) in dry toluene (20 mL). The solution was allowed to stir at room temperature for 0.5 h. Methyl 4-(l,3-dioxolan-2-yl) benzoate 12 (0.725 g, 3.48 mmol) was added to the reaction mixture and refluxed under an atmosphere of nitrogen for 21 h. The reaction mixture was allowed to cool at room temperature, neutralized with dil.

HC1 and extracted with EtOAc (2 x 20 mL). The organic phase was combined and dried over with anhydrous sodium sulphate. The product was purified by column chromatography using 10% of EtOAc/hexanes mixture. Yellow solid; MP: 122.5°C; Yield = 62%, R/ = 0.35 in 1:3 EtOAc/hexanes; 1H NMR (500 MHz, CDCb): d 17.01 (s, 1H), 7.97 (d, / = 8.5 Hz, 2H), 7.84 (d, / = 9.0 Hz, 2H), 7.58 (d, / = 8.5 Hz, 2H), 7.34-7.31 (m, 4H), 7.71-7.14 (m, 6H), 7.03 (d, /

= 9.0 Hz, 2H), 6.76 (s, 1H), 5.87 (s, 1H), 4.13-4.12 (m, 2H), 4.07-4.06 (m, 2H); ¹³ C NMR (125 MHz, CDCb): d 186.1, 183.3, 152.1, 146.6, 146.5, 142.0, 136.5, 129.7, 128.8, 127.1, 126.8, 126.1, 126.0, 124.7, 120.3, 103.2, 92.6, 65.5; IR (neat): u 3000, 2920, 1722, 1670, 1484, 1272, 1169, 1107, 1024, 813, 699 cm ¹ ; HRMS (ESI): calcd for C30H25NO4 [M+H] ⁺ 464.1856, found 464.1858.

Synthesis of4-(3-(4-(diphenylamino)phenyl)-3-oxopropanoyl)benzaldehyde (14):

To a stirred solution of substituted compound 13 (0.750 g, 1.617 mmol) in acetone (15 mL), was added PTSA.H2O (0.062 g, 0.33 mmol) and stirred at room temperature for 3-4 h under nitrogen atmosphere condition. The reaction progress was monitored by TLC using 20% EtOAc/hexanes mixture. The reaction mixture was neutralized with NaHC0 ₃ (20 mL) and extracted with EtOAc (2 x 15 mL). The solvent was evaporated under vacuum, the crude product was used for next reaction without further purifications. Yellow solid; MP: 130.9 °C; Yield = 45%, R/= 0.56 in 1:3 EtOAc/hexanes; 1H NMR (500 MHz, CDCb): d 16.93 (s, 1H), 10.08 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 9.0 Hz, 2H),

7.35-7.32 (m, 4H), 7.18-7.14 (m, 6H), 7.03 (d, / = 10.5 Hz, 2H), 6.80 (s, 1H); ¹³ C NMR (125 MHz, CDCb): d 191.6, 187.2, 180.9, 152.4, 146.4, 140.9, 138.4, 129.9, 129.0, 127.5, 126.1, 124.9, 119.9, 93.5; IR (neat): u 3000, 2925, 1701, 1587, 1484, 1288, 1231, 1190, 782, 761, 699 cm ¹ ; HRMS (ESI): calcd for C28H21NO3 [M+H] ⁺ 420.1594, found 420.1595. Example 1: 2-((5-(4-(3-(4-(Diphenylamino)phenyl)-3-oxopropanoyl)phenyl) thiophen-2- yl)methylene)malononitrile (15)

A mixture of compound 4 (0.7 g, 1.35 mmol), compound 8 (1.2 g, 2.70 mmol) and Pd(PPh ₃ )4 (0.32 g, 0.27 mmol) were heated in toluene (12 mL) at 80 °C for 22 h under nitrogen condition. The reaction was monitored by TLC using 33% of EtOAc/hexanes mixture. The solvent was removed under reduced pressure with a rotavap. The crude was purified with column chromatography using ethyl acetate and hexanes mixture. Red solid (0.26 g, Yield = 35%); MP: 211 °C; R/= 0.13 in 1:3 EtOAc/hexanes; 1H NMR (500 MHz, CDCb): d 17.03 (s, 1H), 8.00 (d, / = 8.0 Hz, 1H), 7.85 (d, / = 9.0 Hz, 2H), 7.81 (s, 1H), 7.76 (d, / = 8.0 Hz, 2H),

7.74 (d, / = 4.0 Hz, 1H), 7.52 (d, / = 4.0 Hz, 1H), 7.33 (t, / = 8.0 Hz, 5H), 7.17 (t, / = 7.5 Hz, 6H), 7.04 (d, / = 9.0 Hz, 2H), 6.77 (s, 1H); ¹³ C NMR (125 MHz, CDCb): d 186.5, 181.7,

154.8, 152.3, 150.5, 146.5, 139.9, 137.1, 135.2, 129.7, 128.9, 128.0, 126.8, 126.1, 125.6,

124.9, 120.0, 114.0, 113.3, 92.7; IR (neat): u 2920, 2849, 2224, 1660, 1572, 1424, 1276, 1189, 1013, 789 cm ¹ ; HRMS (ESI): calcd for C35H23N3O2S [M+H] ⁺ 550.1584, found

550.1582.

Example 2: 2-((5'-(3-(4-(Diphenylamino)phenyl)-3-oxopropanoyl)-[2,2'-bi thiophen]-5- yl)methylene)malononitrile (16)

Compound 5 (0.25 g, 0.52 mmol), compound 8 (0.47 g, 1.05 mmol) and Pd(PPh ₃ )4 (0.13 g, 0.10 mmol) were mixed together in toluene (5 mL) and heated at 80 °C for 22 h under nitrogen atmosphere. The reaction was monitored by TLC. After completion of the reaction solvent was removed under reduced pressure in a rotavap. The crude was purified with column chromatography using ethyl acetate with hexanes mixture. Red solid (0.092 g, Yield = 31%); MP: 236.3 °C; R/ = 0.43 in 1:3 EtO Ac/hexanes; 1H NMR (500 MHz, CDCb): d 16.48 (s, 1H), 7.79 (d, / = 8.0 Hz, 2H), 7.68 (d, / = 3.5 Hz, 2H), 7.42 (d, / = 3.0 Hz, 1H), 7.38 (d, / = 2.0 Hz, 1H), 7.33 (t, / = 7.5 Hz, 5H), 7.18-7.15 (m, 6H), 7.02 (d, / = 8.5 Hz, 2H), 6.58 (s, 1H); ¹³ C NMR (125 MHz, CDCb): d 182.0, 179.8, 152.2, 150.2, 147.8, 146.4, 144.4,

140.4, 134.8, 130.4, 129.7, 128.6, 127.7, 126.1, 124.9, 120.0, 113.2, 92.1; IR (neat): u 2920, 2850, 2220, 1574, 1488, 1333, 1189, 1060, 783 cm ¹ ; HRMS (ESI): calcd for C33H21N3O2S2 [M+H] ⁺ 556.1148, found 556.1149.

Example 3:

2-(4-(3-(4-(diphenylamino)phenyl)-3-oxopropanoyl)benzylid ene)malononitrile (17)

Compound 14 (0.350 g, 0.834 mmol) andmalononitrile (0.067 pl, 1.00 mmol), and PPI13 (0.053 g, 0.200 mmol) were stirred without solvent at 80 °C for 2-4 h. The reaction was monitored by TLC. The reaction was diluted with water and extracted with EtO Ac (2 x 20 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated using vacuum. The crude was purified with column chromatography using 1:5 EtO Ac/hexanes mixture. Red solid; MP: l79°C; Yield = 57%, R/= 0.51 in 1:3 EtO Ac/hexanes; Ή NMR (400 MHz, CDCb): d 16.90 (s, 1H), 8.07 (d, / = 8.4 Hz, 2H), 7.98 (d, / = 8.4 Hz, 2H), 7.83 (t, / = 8.8 Hz, 3H), 7.34 (t, J = 8.0 Hz, 4H), 7.18 (d, J = 8.8 Hz, 6H), 7.03 (d, J = 8.8 Hz, 2H), 6.58 (s, 1H); ¹³ C NMR (100 MHz, CDCb): d 187.5, 179.5, 158.6, 146.3, 140.8, 133.4, 130.9,

129.8, 129.1, 127.8, 127.1, 126.2, 125.0, 119.7, 113.5, 112.4, 93.6, 84.4; IR (neat): u 3063, 2920, 2224, 1589, 1331, 1232, 1189, 1030, 931, 794, 761 cm ¹ ; HRMS (ESI): calcd for C31H21N3O2 [M+H] ⁺ 468.1707, found 468.1707.

General procedure for formation of 1,3-diketone with BF2 Complexes:

To a stirred solution of the corresponding 1, 3-diketone substrate (1 equiv.) in dry dichloromethane (0.1 mmol, 5 mL) was added BF ₃ .0Et ₂ (1.2 equiv.) dropwise at room temperature and maintained for another 0.5 h. The reaction colour was changed instantly upon addition of BF ₃ .OEt ₂ . The reaction was quenched using 0.2 N NaOH solution in water and extracted using dichloromethane. The crude product was further purified by column chromatography .

Example 4: 2-((5-(4-(6-(4-(diphenylamino)phenyl)-2,2-difluoro-2H-l,diox aborinin-4- yl)phenyl)thiophen-2-yl)methylene)malononitrile (18)

Red solid; MP: 268°C; Yield = 36%, R/ = 0.13 in 1:3 EtO Ac/hexanes; Ή NMR (400 MHz, CDCb): d 8.14 (d, / = 7.6 Hz, 1H), 8.00-7.98 (m, 2H), 7.83-7.75 (m, 5H), 7.39 (t, / = 7.6 Hz, 5H), 7.02 (s, 1H), 6.98 (d, / = 8.8 Hz, 2H); ^U B NMR: d 1.28; ¹⁹ F NMR: d -140.7; IR (neat): u 2922, 2853, 2220, 1732, 1573, 1486, 1340, 1189, 1032, 796, 692 cm ¹ ; HRMS (ESI): calcd for C35H22BF2N3O2S [M+H] ⁺ 598.1567, found 598.1577.

Example 5: 2-((5'-(6-(4-(diphenylamino)phenyl)-2,2-difluoro-2H-l,dioxab orinin-4-yl)- [2,2'-bithiophen]-5-yl)methylene)malononitrile (19)

Dark violet solid; MP: 240.9°C; Yield = 35%, R/= 0.13 in 1:3 EtO Ac/hexanes; 1H NMR (500 MHz, CDCb): d 7.94 (d, J = 9.0 Hz, 2H), 7.89 (d, J = 9.5 Hz, 1H), 7.81 (d, J = 7.0 Hz, 2H), 7.69 (d, / = 3.5 Hz, 1H), 7.47 (d, / = 4.0 Hz, 1H), 7.43 (d, / = 4.0 Hz, 1H), 7.39 (t, / = 8.0 Hz, 5H), 7.21 (d, / = 7.5 Hz, 4H), 6.96 (d, / = 9.0 Hz, 2H), 6.81 (s, 1H); ^U B NMR: d 0.95; ¹⁹ F NMR: d -141.5; IR (neat): u 2946, 2925, 2223, 1577, 1531, 1427, 1278, 1200, 1138, 1009, 901, 870 cm ¹ ; HRMS (ESI): calcd for C33H20BF2N3O2S2 [M+Na] ⁺ 626.0950, found

626.0944. Example 6: 2-(4-(6- (4-(diphenylamino)phenyl) -2,2-difluoro-2H- 1 ,dioxaborinin-4- yl)benzylidene)malononitrile (20) :

Red solid; MP: 257.9°C; Yield = 44%, R/= 0.13 in 1:3 EtO Ac/hexanes; 1H NMR (500 MHz, CDCb): d 8.15 (d, 7 = 8.5 Hz, 2H), 7.99-7.95 (m, 4H), 7.79 (s, 1H), 7.38-7.36 (m, 4H), 7.23 (s, 1H), 7.19 (d, 7 = 8.0 Hz, 5H), 7.00 (s, 1H), 6.93 (d, 7 = 9.0 Hz, 2H); ^U B NMR: d 1.20; ¹⁹ F NMR: d -140.6; IR (neat): u 2921, 2858, 2224, 1720, 1489, 1338, 1251, 1131, 1094, 926, 802, 756, 696 cm ¹ ; HRMS (ESI): calcd for C31H20BF2N3O2 [M+H] ⁺ 516.1689, found 516.1687.

Absorption and emission characteristics (in DCM) of the compounds 15-20 were depicted in Table 1 below.

Table 1

Cytotoxic and fluorescence properties of diphenylamino-methylene malononitrile based compounds in HeLa cells

HeLa (human cervical cancer) cell line was obtained from the National Centre for Cellular Sciences (NCCS), Pune, India. Cells were cultured in DMEM media, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM NaHC0 ₃ , 2 mM -glutamine, 100 units/mL penicillin and 100 pg/mL streptomycin. All cell lines were maintained in culture at 37 °C in an atmosphere of 5% C0 ₂ .

Initially, stock solutions of each test substances were prepared in 100% dimethyl sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) with a final concentration of 10 mg/mL. Exactly 20 pL of stock was diluted to 1 mL in culture medium to obtain experimental stock concentration of 200 pg/mL. This solution was further serially diluted with media to generate a dilution series of O.OOlpg to 100 pg/ml. Exactly 100 pL of each diluent was added to 100 pL of cell suspension (total assay volume of 200 pL) and incubated for 24 h at 37 °C in 5% C0 ₂ . Respected volume of DMSO used as a control.

CYTOTOXICITY:

Cytotoxicty was measured using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the known method (Mosmann T, J Immunol Methods. 1983 Dec 16; 65(l-2):55-63). Briefly, the cells (3 x 10 ³ ) were seeded in each well containing 0.1 mL of medium in 96 well plates. After overnight incubation at 37 °C in 5% C0 ₂ , the cells were treated with 100 pL of different test concentrations of test compounds at identical conditions with five replicates each. The final test concentrations were equivalent to 0.001 to 100 pg/mL or 0.001 to 100 ppm. The cell viability was assessed after 24 h, by adding 10 pL of MTT (5 mg/mL) per well. The plates were incubated at 37 °C for additional three hours. The medium was discarded and the formazan blue, which formed in the cells, was dissolved with 100 pL of DMSO. The rate of color formation was measured at 570 nm in a spectrophotometer (Bio-rad). The percent inhibition of cell viability was determined with reference to the control values (without test compound). The data were subjected to linear regression analysis and the regression lines were plotted for the best straight-line fit. The IC50 (inhibition of cell viability) concentrations were calculated using the respective regression equation. IC50 values of the test compounds for 24 h on HeLa cell line were calculated and depicted in Table 2 below.

Table 2

Exponentially growing cells were treated with different concentrations of compounds ( Examples 1-6) for 24h and cell growth inhibition was analyzed through MTT assay.

The values represent the mean ± SE of three individual observations.

hDoxorubicin was employed as positive control. NA indicates that the derivatives are not active at 100 pg/mL concentration.

The effect of compounds of formula (I) and formula (II) on viability of HeLa cells is shown in FIG. 1.

FLUORESCENCE MICROSCOPY:

For microscopic evaluation, HeLa cells were cultured on cover slips in the 6-well plates to 70 % confluence and treated with 10 pg of compounds (Examples 1-6) in complete cell culture media for up to 12 h. In all experiments, a corresponding DMSO control was run in parallel for 12 h. After incubating for 12 h, HeLa cells were washed with PBS for three times and fixed with 4% paraformaldehyde for 20 min, mounted using with DAPI for visualization of nuclei, and incubated for 1 h in the dark. After incubation cells were visualized and captured by fluorescence microscopy (Olympus, USA) using excitation wavelengths between 400-418 for DAPI and 478-495 for compounds.

For reference, paralally HeLa cells were cultured on cover slips in the 6-well plates to 70 % confluence washed with PBS for three times and fixed with 4% paraformaldehyde for 20 min, permeabilized with cold methanol for another 10 min. After that, the cells were blocked with 5% BSA for 1 h. Subsequently, the cells were washed with PBS, and incubated with anti-tubulin antibody in 3% BSA (1:200, Sigma.) overnight at 4 °C. After being washed with PBS for three times, each cover slip was added 200 pL of Alexa Fluor 546 anti-mouse secondary antibody in 3% BSA (1:500, Molecular probes.) and incubated at room temperature for 1 h. At last, HeLa cells was stained with 20 pL of DAPI for 1 h and observed under confocal microscope (Olympus, USA).

Localization and fluorescence properties of compounds of formula (I) and formula (II) in HeLa cells:

The fluorescence properties of the compounds of formula (I) and formula (II) were evaluated in HeLa cell line. Preliminary fluorescence microscopy experimental results indicated that, these compounds were entered in to the cell, accumulated in cytoplasm and remarkably emitting green fluorescence. Then, continued to investigate the specific recognition of the compounds of formula (I) and formula (II) for tubulins in microtubules of living cancer cells by confocal microscopy. Confocal microscopy images are presented in FIG. 2, 3 and 4 correspond to Example 1, Example 2 and Example 6 respectively. Figure 5 corresponds to fluorescence images of Example 3, Example 4 and Example 5.