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):

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.

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):

V/A W. L/11U1111UVVUUVU11Y U-W/VLyiU-l/l. V JUll V/A H JlVl VVlJUlllVl UIVIVXJI, VY11V1V111,
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.
T-n /"»ot-i-ai-n amKr\ni mo-n+0 -fno rrimnrtiinn r\f mrnrnla /TTl 10 a rrimnrtiinn AT mrmn a iT\/"V
or a pharmaceutical^ 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

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

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
10

"alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec -butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-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 ("C6-C20 aryl") as ring members, preferably 6 to 14 carbon atoms ("C6-C14 aryl") or more preferably, 6 to 12 carbon atoms ("C6-C12 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 -NO2 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.
UB NMR: Boron-11 nuclear magnetic resonance spectroscopy; calcd: Calculated; DMSO: dimethyl sulfoxide; DCM: dichloromethane; equiv.: equvivalent(s); HRMS: high-resolution mass spectrometry; Na2S04: sodium sulphate; g: gram(s); h: hour(s); JH NMR: proton nuclear magnetic resonance; 13C NMR: carbon-13 nuclear magnetic resonance; 19F NMR: Fluorine-19 nuclear magnetic resonance spectroscopy; M: molar; MeOH: methanol; mL; millilitre; uL: Microlitre; mg: milligram; Mp: melting point; degree Celsius (°C); mL: millilitre; [M+H]+: protonated molecular ion; m/z: mass-to-charge ratio; rt: room temperature; THF: tetrahydrofuran; TLC: 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 CaH2. MeOH was dried over using Mg/b. *H and 13C spectra were recorded on a Bruker Avance 400 MHz and 500 MHz instruments in CDCh 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:

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(PPh3)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 BF3 OEt2in 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 BF3.0Et2 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 CaFb. MeOH was dried over using Mg/b. *H and 13C spectra were recorded on a Bruker Avance 400 MHz and 500 MHz instruments in CDCh 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 (FIRMS) 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):
POCb (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 H2O (50 mL) and brine solution (10 mL). The organic layer was dried over NaS04 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; !HNMR (400 MHz, CDCb): S 9.80 (s, 1H), 7.67 (d,J = 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); 13C NMR(100MHz, CDCb): S 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-H20 (4:1 v/v) was added KMn04 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/= 0.22 in 1:5 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 7.90 (d, J= 8.8 Hz, 2H), 7.32 (t, J= 8.4 Hz, 4H), 7.17-7.12 (m, 6H), 6.99 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CDCb): 5 152.8, 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 IN NaOH solution followed by extraction with EtOAc. 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; lU NMR (400 MHz, CDCb): 5 7.85 (d, J= 8.8 Hz, 2H), 7.30 (d, J= 7.6 Hz, 4H), 7.15-7.10 (m, 6H), 6.99 (d, J= 8.8 Hz, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCb): 5 152.8, 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-dione (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 EtOAc/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; lU NMR (400 MHz, CDCb): 5 17.07 (s, 1H), 7.82 (dd, J = 19.2, 8.4 Hz, 4H), 7.66 (d, J= 8.0 Hz, 2H), 7.34 (t, J= 6.8 Hz, 4H), 7.20-7.16 (m, 6H), 7.05 (d, J= 8.4 Hz, 2H), 6.73 (s, 1H); 13C NMR (100 MHz, CDCb): 5 185.8, 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
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; *H NMR (400 MHz, CDCb): 5 16.43 (s, 1H), 7.77 (d, J= 8.8 Hz, 2H), 7.48 (d, J= 3.6 Hz, 1H), 7.33 (t, J= 8.4 Hz, 4H), 7.28-7.15 (m, 7H), 7.03 (d, J= 8.4 Hz, 2H), 6.48

(s, 1H); 13C NMR (100 MHz, CDCb): 5 181.3, 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): i) 2998, 1587, 1484, 1272, 1174, 1004, 787, 756, 694 cm"1; HRMS (ESI): calcd for C25Hi8BrN02S [M+H]+ 498.0134, found 498.0132.
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.H2O (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 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 7.35 (d, J= 12.4 Hz, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.12 (s, 1H), 4.12 (t, J= 1.6 Hz, 2H), 4.01 (t, J= 3.2 Hz, 2H); 13C NMR (100 MHz, CDCb): 5 141.8, 128.3, 126.7,126.3, 126.2, 100.3, 65.2.
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 Bu3SnCl (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 (2x30 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 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 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); 13C NMR (100 MHz, CDCb): 5 182.0, 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 Ci7H30SSn [M+Na]+ 425.0932, found 425.0933.
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 PPI13 (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 (2x5 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 EtOAc/Hexane mixture to yield brownish yellow viscus liquid (37 mg, yield = 65%); R/= 0.55 in 1:20 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 7.85 (s, 2H), 7.30 (t, J= 4.0 Hz, 1H), 1.60-1.52 (m, 6H), 1.38-129 (m, 7H), 1.20-1.16 (m, 5H), 0.89 (t, J= 7.2 Hz, 9H) ; 13C NMR (100 MHz, CDCb): 5 156.3, 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 C2oH30SSn [M+H]+ 451.1224, found 451.1224.
Synthesis of l-(5-bromothiophen-2-yl)ethan-l-one (9):
To a stirred solution of 2-bromothiophene (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% Na2C03 and extracted with dichloromethane (2 x 30 mL). The organic layer was combined and dried over with anhydrous Na2S04. 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; lU NMR (400 MHz, CDCb): 5 7.29 (q, J= 4.0 Hz, 2H), 2.50 (s, 3H); 13C NMR (100 MHz, CDCb): 5 189.3, 150.4, 138.2, 133.3, 85.5, 26.6.
To a stirred solution of triphenylamine (1 g, 4.0 mmol) in dry dichloromethane (20 mL) anhydrous AlCb (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 NaHCCb solution followed by extracted with dichloromethane (2 x 50 mL). The organic layer was combined and dried over with anhydrous Na2S04. 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; *H NMR (400 MHz, CDCb): 5 7.79 (d, J= 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); 13C NMR (100 MHz, CDCb): 5 196.6, 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 IN 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; *H NMR (400 MHz, CDCb): 5 10.08 (s, 1H), 8.18 (d, J= 6.8 Hz, 2H), 7.93 (d, J= 8.4 Hz, 2H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCb): 5 191.8, 166.1, 139.2, 135.1, 130.3, 129.6, 52.7; IR (neat): v 1722, 1680, 1500, 1386, 1112, 1014, 813, 761 cm"1; HRMS (ESI): calcd for C9H802
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; !HNMR (400 MHz, CDCb): 5 8.04 (t, J = 6.8 Hz, 2H), 7.54 (t, J= 6.8 Hz, 2H), 5.82 (s, 1H), 4.10-4.07 (m, 2H), 4.02-4.01 (m, 2H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCb): 5 166.7, 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"1; 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)propane-l,3-

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; ^NMR (500 MHz, CDCb): 5 17.01 (s, 1H), 7.97 (d, J= 8.5 Hz, 2H), 7.84 (d, J= 9.0 Hz, 2H), 7.58 (d, J= 8.5 Hz, 2H), 7.34-7.31 (m, 4H), 7.71-7.14 (m, 6H), 7.03 (d, J = 9.0 Hz, 2H), 6.76 (s, 1H), 5.87 (s, 1H), 4.13-4.12 (m, 2H), 4.07-4.06 (m, 2H); 13C NMR (125 MHz, CDCb): 5 186.1, 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 NaHCCb (20 mL) and extracted with EtOAc (2x15 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; 'HNMR (500 MHz, CDCb): 5 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, J= 10.5 Hz, 2H), 6.80 (s, 1H); 13C NMR (125 MHz, CDCb): 5 191.6, 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(PPh3)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; ^NMR (500 MHz, CDCb): 5 17.03 (s, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.85 (d, J= 9.0 Hz, 2H), 7.81 (s, 1H), 7.76 (d, J= 8.0 Hz, 2H), 7.74 (d, J= 4.0 Hz, 1H), 7.52 (d, J= 4.0 Hz, 1H), 7.33 (t, J= 8.0 Hz, 5H), 7.17 (t, J= 7.5 Hz, 6H), 7.04 (d, J= 9.0 Hz, 2H), 6.77 (s, 1H); 13C NMR (125 MHz, CDCb): 5 186.5, 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,
124.8, 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'-bithiophen]-5-yl)methylene)malononitrile (16)
Compound 5 (0.25 g, 0.52 mmol), compound 8 (0.47 g, 1.05 mmol) and Pd(PPh3)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 EtOAc/hexanes; *H NMR (500 MHz, CDCb): 5 16.48 (s, 1H), 7.79 (d, J= 8.0 Hz, 2H), 7.68 (d, J= 3.5 Hz, 2H), 7.42 (d, J= 3.0 Hz, 1H), 7.38 (d, J= 2.0 Hz, 1H), 7.33 (t, J= 7.5 Hz, 5H), 7.18-7.15 (m, 6H), 7.02 (d, J= 8.5 Hz, 2H), 6.58 (s, 1H); 13CNMR(125 MHz, CDCb): 5 182.0, 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)benzylidene)malononitrile (17)
Compound 14 (0.350 g, 0.834 mmol) andmalononitrile (0.067 ul, 1.00 mmol), and PPh3 (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 EtOAc (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 EtOAc/hexanes mixture. Red solid; MP: 179°C; Yield = 57%, R/= 0.51 in 1:3 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 16.90 (s, 1H), 8.07 (d, J= 8.4 Hz, 2H), 7.98 (d, J= 8.4 Hz, 2H), 7.83 (t, J = 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); 13C NMR (100 MHz, CDCb): 5 187.5, 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 BF3.0Et2 (1.2 equiv.) dropwise at room temperature and maintained for another 0.5 h. The reaction colour was changed instantly

upon addition of BF3.0Et2. 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,dioxaborinin-4-yl)phenyl)thiophen-2-yl)methylene)malononitrile (18)
Red solid; MP: 268°C; Yield = 36%, R/ = 0.13 in 1:3 EtOAc/hexanes; *H NMR (400 MHz, CDCb): 5 8.14 (d, J= 7.6 Hz, 1H), 8.00-7.98 (m, 2H), 7.83-7.75 (m, 5H), 7.39 (t, J= 7.6 Hz, 5H), 7.02 (s, 1H), 6.98 (d, J= 8.8 Hz, 2H); UB NMR: 5 1.28; 19F NMR: 5 -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,dioxaborinin-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 EtOAc/hexanes; *H NMR (500 MHz, CDCb): 5 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, J= 3.5 Hz, 1H), 7.47 (d, J= 4.0 Hz, 1H), 7.43 (d, J= 4.0 Hz, 1H), 7.39 (t, J= 8.0 Hz, 5H), 7.21 (d, J= 7.5 Hz, 4H), 6.96 (d, J= 9.0 Hz, 2H), 6.81 (s, 1H); UB NMR: 5 0.95; 19FNMR: 5 -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-l,dioxaborinin-4-yl)benzylidene)malononitrile (20):
Red solid; MP: 257.9°C; Yield = 44%, R/= 0.13 in 1:3 EtOAc/hexanes; *H NMR (500 MHz, CDCb): 5 8.15 (d, J= 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, J= 8.0 Hz, 5H), 7.00 (s, 1H), 6.93 (d, J= 9.0 Hz, 2H); UB NMR: 5 1.20; 19F NMR: 5 -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.
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%
97

heat-inactivated fetal bovine serum (FBS), 1 mM NaHCCb, 2 mM -glutamine, 100 units/mL penicillin and 100 ug/mL streptomycin. All cell lines were maintained in culture at 37 °C in an atmosphere of 5% CO2.
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 uL of stock was diluted to 1 mL in culture medium to obtain experimental stock concentration of 200 ug/mL. This solution was further serially diluted with media to generate a dilution series of 0.001 ug to 100 ug/ml. Exactly 100 uL of each diluent was added to 100 uL of cell suspension (total assay volume of 200 uL) and incubated for 24 h at 37 °C in 5% CO2. 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 103) were seeded in each well containing 0.1 mL of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO2, the cells were treated with 100 uL 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 ug/mL or 0.001 to 100 ppm. The cell viability was assessed after 24 h, by adding 10 uL 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 uL 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.

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.
bDoxorubicin was employed as positive control. NA indicates that the derivatives are not active at 100 jug/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 ug 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 uL 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 uL 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.

1. A compound of formula (I) 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.
2. The compound as claimed in claim 1, wherein ring A is phenyl or thiophenyl.
3. The compound as claimed in claim 1, wherein ring B is phenyl or thiophenyl.
4. The compound as claimed in claim 1, wherein D is
5. The compound as claimed in claim 1 is a compound of formula (III) or 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.
6. The compound as claimed in claim 5, wherein 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.
7. The compound as claimed in claim 6, wherein ring A is phenyl or thiophenyl.
8. The compound as claimed in claim 5, wherein compound of formula (IV) is a compound of formula (IVA):

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.
9. The compound as claimed in claim 8, wherein ring A is phenyl or thiophenyl.
10. The compound as claimed in claim 5, wherein ring A is absent and ring B is phenyl.
11. The compound as claimed in any of the claims 1 to 10, wherein the compound is

12. A composition comprising at least one compound as claimed in any of the claims 1 to 11, or a stereoisomer thereof.
13. The compound as claimed in any of the claims 1 to 11, wherein the compound is useful as fluorescence probe in detecting tubulin in a sample.
14. A method of detecting tubulin in a sample, wherein the method comprises contacting the compound as claimed in any of the claims 1 to 11 with the sample and detecting the fluorescence generated due to the reaction of the compound with the sample.
15. A kit comprising at least one compound as claimed in any of the claims 1 to 11, and/or instructions for carrying out a method of detecting tubulin in a sample.