Claims:WE CLAIM
1. A compound of formula I,for photodynamic therapy of cancer

Formula I
2. The compound as claimed in claim1, wherein the compound possesses dual chemo and photodynamic for photodynamic therapy of canceractivity when exposed to near infrared light in the range of 700-820 nm.

3. The compound as claimed in claim 1, wherein the compoundexhibits a cellular response of 4h and is protonated in an acidic environment.
4. The compound as claimed in claim 1, wherein the compound selectively targets the mitochondria of cancer cell.
5. The compound as claimed in claim 1, wherein the compound produces reactive oxygen species when exposed to near infrared light.
6. The compound as claimed in claim 1, wherein the compound exhibits high molar absorption coefficient value in the range of 40,000-100,000 M-1 cm-1.

7. A process for synthesis of compound of formula I, comprising the steps of-

a) synthesizing compound L1; and

L1
b) coupling compound L1 with cis-diamminedichloroplatinum(II) in presence of Silver nitrate in a solvent to obtain compound of formula I.
8. The method of synthesis as claimed in claim 7, wherein the compound L1 is synthesized by acts comprising-
a)refluxing 4-(dimethylamino)benzaldehyde with1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4-difluoroboradiaza-indacene in a solvent; and
b) cooling and separating to obtain the compound L1.
9. The method as claimed in claim 8, wherein the refluxing is carried out for a period of 24hr.

10. The method as claimed in claims 7 and 8, wherein the solvent is selected from a group comprising Toluene and Dimethylformamide.
, Description:TECHNICAL FIELD
The present invention relates to a novel compoundfor photodynamic therapy of cancer.Specifically, the present invention relates toaPlatinum based Boron dipyrromethene;compound of formula I for photodynamic therapy of cancer. The present invention also relates to a process for preparation of said platinum-basedcompound.
BACKGROUND
The limitations of surgery and radiation therapy for cancer treatment have resulted in the emergence of “Chemotherapy” where a drug can interact with the cancer cells at the molecular level causing cellular death. Nearly 60-70% cancer patients are being treated with the platinum-based anticancer drugs, viz.Cisplatin (CP) as the most used one because of their clinical successes. Treatment of cancer using Cisplatin had become a boon in the era of chemotherapeutic anticancer agents due to its excellent DNA binding abilities, but it lacks in selectivity and causes severe side effects of chemotherapy on normal tissue. Earlier investigations in the field of platinum chemotherapeutics have led to a class of monofunctional platinum drugs like phenanthriplatin, imidazoplatin, pyriplatin. Pyriplatin with greater cellular uptake and similar property of Cisplatin act as a transcription inhibitor and has been studied in detail.Various attempts are made to increase the selectivity of these drugs along with reducing its side effects.
This has led to the emergence of a new methodology based on photo-irradiation, known as “Photodynamic Therapy” (PDT).PDT is a non-invasive treatment modality based on the efficiency of the photosensitizers in generating reactive oxygen species (ROS). In “Photodynamic Therapy”, cancer cell death results from light-activated generation of reactive oxygen species, wherein the drug remains dormant and gets activated only on exposure to light. Porfimer sodium (commonly known as Photofrin®) is an efficient PDT photosensitizer, approved by FDA for the treatment of skin cancer, oral cancer etc., but it suffers serious limitations due to its lower molar absorption coefficient values of the Q-bands in the near-IR (NIR) region, presence of oligomer mixture, long-term skin photosensitivity, hepatotoxicity and low efficacy. Recent studies have shown that the NIR light active BODIPY compounds are the emerging class of NIR-PDT agents that can overcome the limitations of Photofrin. It is necessary for a photo-therapeutic drug to be activated in NIR light with higher molar absorption coefficient as the NIR red light (600-800 nm) possesses the highest tissue penetration ability in the visible region due to which deeper tumors can be treated effectively.
Combinatorial investigations based on photodynamic therapy and chemotherapy have been conducted with the Boron dipyrromethene (BODIPY) conjugated chemotherapeutic drugs. There have been a few reports in literature disclosing platinum-derived photosensitizers that have been studied as Photodynamic Therapy agents. Pt-BODIPY conjugates containing OMe pendant have been disclosed in Dyes Pigm. 2017, 141, 5-12 wherein it has been disclosed that though the said compounds get activated in light, they exhibit more dark toxicity than the required photocytotoxic effect and hence are not suitable for therapeutic usage. Chinese patent application no. CN105198934 A discloses conjugates that exhibit micromolar photocytotoxicity (IC50 = 1.5 µM, 24 h incubation) indicating slower uptake of the compounds inside the cell.
There is an unmet need to developfurther alternate approach conjugates that can overcome deficiencies associated with the known arts. Need is also felt for novel platinum-based compound for photodynamic therapy of cancer thatcombine platinum-based chemotherapy with photodynamic therapy into a single molecular system, are highly efficacious, exhibit a faster cellular response and do not show any dark toxicity.
OBJECTS OF THE INVENTION
An object of the present invention is to provide a platinum-basedborondipyrromethene compound for photodynamic therapy of cancer, capable of selectively killing the tumor cells via light activation without harming the healthy cells.
Still another object of the present invention is to provide platinum-basedboron-dipyrromethene compound for photodynamic therapy of cancer that possess dual chemo- and photodynamic therapy activity in near infrared light.
Another object of the present invention is to provideplatinum-basedboron-dipyrromethene compound for photodynamic therapy of cancer,having low doses requirements and cytotoxicity at submicromolar level.
Another object of the present invention is to provideplatinum-basedboron-dipyrromethene compound for photodynamic therapy of cancerthatare highly efficacious, exhibit a faster cellular response and do not show any dark toxicity.
Yet another object of the present invention is to provide method for the synthesis of aplatinum-basedboron dipyrromethenecompound.
The other objects and preferred embodiments and advantages of the present invention will become more apparent from the following description of the present invention when read in conjunction with the accompanying examples and figures, which are not intended to limit scope of the present invention in any manner.
SUMMARY OF THE INVENTION
The present invention relates to novel compound of formula I for photodynamic therapy of cancer.Specifically, the present invention relates toplatinum-basedboron dipyrromethene for photodynamic therapy of cancer.

Formula I
In another aspect, the present invention relates to platinum-basedboron dipyrromethenethat possess dual chemo- and photodynamic therapy activity in near infrared light.
Other aspects of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learnt by the practice of the invention.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1: presents the mass spectrum of the compound of formulaI.
Figure 2: provides proton NMR spectra of compound of formula I.
Figure 3: shows the absorption and emission spectra of compound of formulaI.
Figure 4: highlights the light stability of compound of formula I.
Figure5: shows(a) Spectral changes of DPBF (1,3-diphenylisobenzofuran,50 µM) at 415 nm with time on treatment with compound of formula Iin air saturated DMF (dimethylformamide). (b) Plots showing the change in absorbance of DPBF at ~415 nm vs photoirradiation time in the presence of methylene purple (MB), compound of formula I(blue) and organic compound L1(red). The results suggest significant generation of singlet oxygen on photo-irradiation.
Figure 6: Photocytotoxicity of compound of formula I and the compound L1(IC50 values in µM) in cancerous cell lines, which are, BT474 (breast cancer), MCF-7 (breast cancer), A549 (human lung adenocarcinoma) cells and a normal cell line HPL1D (immortalized lung epithelial). Pre-incubation = 4 h (600-720 nm; light dose, 30 J cm-2; Waldmann PDT 1200 L), post-incubation = 19 h. Specifically, the figure presents data on the cytotoxicity of compound of formula Iin multiple normal and cancerous cell lines on irradiation with red light
Figure7: Quantitative analysis of cellular uptake of compound of formula I and the compound L1 in BT474(human breast carcinoma) cells (1 µM, 4 h incubation) with the cells untreated were used as control.
Figure 8: shows confocal images of BT474 (human breast carcinoma)cells incubated with redemitting compound of formula I(5 µM) for 4 h at 37 °C along with mito-tracker green (MTG). Scale bar: 20 µm. PCC is Pearson’s Correlation Coefficient. The PCC value of ~0.9 indicates primarily mitochondrial localization of the compound of formula I thus indicating the high selectivity of this PDT agent.
Figure 9: presents data showing in vivo anti-tumor potential of compound of formula1 using immunocompromised mice models.
Figure10: presents the elemental analysis of cis-diamminedichloroplatinum(II)giving bio-distribution of the compound of formula Iin bar diagrams
Figure 11: presents representative images of histological analysis of the tumor sections using Haematoxylin and Eosin staining
DETAILED DESCRIPTION
The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
The present invention relates to novel compound of formula I for photodynamic therapy of cancer.Specifically, the present invention relates toplatinum-basedboron dipyrromethene for photodynamic therapy of cancer.
In another embodiment, the present invention relates toplatinum-based conjugates that possess dual chemo- and photodynamic therapy activity when exposed to near infrared light.
In another embodiment, the present invention relates to platinum-basedborondipyrromethene (BODIPY) of formula I, wherein the compound has dual use for bioimaging and therapeutic action.
In one embodiment, the present invention relates to platinum based borondipyrromethene of formula I, comprising compound L1.

Formula I Compound L1
In another embodiment, the compound of formula Iexhibits anticancer activity in near infrared red light(700-820 nm) and displays excellent photophysical properties.
In still another embodiment, the compound of formula I exhibits a faster cellular response of 4h and can easily be protonated in an acidic environment. On the other hand, the similar compounds exhibit a slow cellular response of 24 h leading to dark toxicity(Table1).
The compound of formula I has low molecular weight (less than 1000) which enhances the solubility and governs a better cellular uptake.
In another embodiment, the compound of formula I, selectively kill the tumor cells via light activation without harming the unexposed healthy cells. The compound selectively targets the organelle, especially mitochondria. In PDT, the compound goes inside the cells and gets accumulated at the tumor sites. The accumulated drug gets activated only on exposure to the suitable wavelength of light.
In another embodiment, the compound of formula I produces reactive oxygen species (ROS, singlet oxygen) at the cancer sites in presence of the light, thereby selectively killing the infected cells without hampering the normal living cells.Figure 6 provides the evidence of singlet oxygen generation by compound of formula I in the line diagram of the target compound of formula I and others excluding cis-diamminedichloroplatinum(II) which does not generate any ROS. Figure 6a) illustrates Spectral changes of DPBF (1,3-diphenylisobenzofuran, 50 µM) at 415 nm with time on treatment with compound of formula Iin air saturated DMF (dimethylformamide) whereas Figure 6b) illustrates plots showing the change in absorbance of DPBF at ~415 nm vs photoirradiation time in the presence of methylene purple (MB), compound of formula I(blue) and organic compound L1(red). The results suggest significant generation of singlet oxygen on photo-irradiation.
According to the embodiments of the present invention, the analysis for bio-distribution and cellular uptake of the compound of formula I is carried out with the cancer cell lines by using ICP-MS, i.e., inductively coupled plasma mass spectrometry. The time dependent ICP-MS gave the total cellular uptake of the target compound of formula I after several intervals of the incubation time. The core of compound of formula Ihas more influencing properties for significant cellular uptake as a basic requirement in PDT. Quantitative analysis of cellular uptake is carried out for compound of formulaI and the free compound L1 in BT474 (human breast carcinoma) cells (1 µM, 4 h incubation) wherein the untreated cells are used as control. Figure 7 shows the cellular uptake of compound of formula Iand compound L1.Bio-distribution is of importance in evaluating the efficiency of the PDT agent because of short lifetime and restricted diffusion range of singlet oxygen as the ROS.
Figure 10 illustrates the bio-distribution of the compound of formula I in bar diagrams. Biodistribution of Platinum as per ICP-MS in the primary organs of mice at 6 h, 12 h and 24 h after injection [Compound of formula I injected (5 mg/Kg body weight)] is determined. It is concluded that compound of formula I has significantly higher localization in the tumor compared to other organs and thus is therapeutically suitable to act as an anti-cancer PDT agent.
In yet another embodiment, the compound of formula I displays better solubility and efficacy(table 1) as compared with similar compounds.
In yet another embodiment, the compound of formula I selectively targets mito-DNA of cells and therefore exhibits maximum efficacy of PDT anticancer effect. No such effect has been reported for previously known compounds. Most of the Platinum based anticancer drugs have several limitations and cannot be used directly for tracking the drugs inside the body, and they mainly target nuclear DNA, which reduce their efficacy due to nucleotide excision repair (NER) machinery. In comparison, the compound of formula I shows emissive property which is explored for the cellular tracking of the drug inside the body. Said compound selectively targets mito-DNA of cell and increases prodrug efficacy to the maximum extent as mito-DNA lacks the NER machinery. This has been demonstrated in Figure 9 illustratingthe determination of the organelle targeting properties of compound of formulaI by confocal laser microscopy in cancer cells. Confocal images of BT474 (human breast carcinoma) cells incubated with red emitting compound of formula I(5 µM) for 4 h at 37 °C along with mito-tracker green (MTG) are represented wherein Scale bar is 20 µm and PCC is Pearson’s Correlation Coefficient. The PCC value of ~0.9 indicates primarily mitochondrial localization of the compound of formula I, thus indicating the high selectivity of this PDT agent.
According to the embodiments of the present invention, the anticancer potential of compound of formula Ihas been evaluated in both light and dark.Figure 4 highlights the light stability of compound of formula I.Time dependent spectral traces of compound of formula I are evaluatedin dark (blue) and in red light (600-720 nm); light dose, 30 J cm-2; Waldmann PDT 1200 L) (red) in 1% DMSO/DMEM solutions. The resulting data indicate significant photo-stability of the compound of formula I.
In yet another embodiment, the compound of present invention demonstrateshigh molar absorption coefficient value in the range of40,000-100,000 M-1 cm-1. The same has been demonstrated in Figure 5.The absorption spectral properties for compound of formula I are responsible for the PDT activity with high efficacy with additional ability to exhibit an intense emission band at ~710-740 nm which is suitable for cellular imaging.The incorporation of heavy metal like platinum in the BODIPY-based compound makes the intersystem crossing (ISC) facile and hence quenches the fluorescence quantum yield (?F) with an enhancement of singlet oxygen quantum yield. The values for the corresponding substituted compound L1 also decreased to a similar extent. ISC promotes the type-II process which in turn leads to the generation of singlet oxygen species following the energy transfer pathway from triplet molecular oxygen photosensitizer and thus making it feasible for doing required PDT study in red light. Also, the figure 6 provides the evidence of singlet oxygen generation by compound of formula I in the line diagram of the target compound of formula I and others excluding Cis-diamminedichloroplatinum(II) which does not generate any ROS. Cis-diamminedichloroplatinum(II) mainly absorbs in UV region and therefore not able to generate any ROS in visible light and alone it cannot serve as photodynamic therapeutic agent.
According to the embodiments of the present invention, the efficacy of compound of formula I apoptosis is also analyzed. It is known that the positively charged metal compounds are more prone to accumulation in the mitochondria because of negative potential difference across the membrane of mitochondria. Current analysis is performed using Annexin-V-FITC/PI assessment, which determines the mode of cell death with the help of flow cytometry. Annexin V-FITC dye and red fluorescence of propidium iodide (PI) are used for the investigation on the cellular apoptosis (early apoptosis- high annexin, low PI; late apoptosis- low annexin and high PI), viable cell (unstained, showing auto fluorescence) and necrosis (stained by PI). The investigations of the compound of formula I have revealed the apoptotic nature of cell death.
In yet another embodiment, the compound of present invention facilitates the detection of localization of drug molecule inside the cell organelles by increasing the photoluminescence of the target compound of formula I.
A comparison of the performance of compound of present invention with known compounds in the prior art are given in table I.

Table 1: Comparison of performance of compound of formula I with known compounds
Comparison CN105198934 Dyes Pigm. Compound of present invention
Structure

Electronic factor -OR functional group -OMe (-OR) functional group -N(Me)2 functional group, electron rich system
in vitro analysis
CN105198934 Dyes Pigm. Compound of present invention
Cellular Uptake 24 h pre-incubation 48 h incubation 4 hpre-incubation
6X higher uptake than CN105198934
Anticancer-PDT Effect Not tested on normal cells Exhibit more dark toxicity than the required PDT effect Tested on normal cells showed nominal dark toxicity
Organelle Targeting Nucleus targeting, having NER drawback (NER is “nuclear excision repair” mechanism that reduces the drug efficacy) Mito-targeting Mitochondria targeting
Avoid NER machinery
in vivo analysis
CN105198934 Dyes Pigm. Compound of present invention
in vivo PDT effect No clear demonstration Dark cytotoxicity> light cytotoxicity Inhibiting the tumor growth
Bio-distribution Accumulated in Tumor as well as Lungs Not reported Mostly accumulated in tumor
H & E Analysis Not reported Not reported Targeting primarily the tumor, Negligible toxicity in different organs

In another embodiment, the present invention relates to a facile process for the synthesis of compound of formula Icomprising the steps of:
a) Synthesizing compound L1; and
b) Coupling compound L1 with Cis-diamminedichloroplatinum(II)) to obtain compound of formula I.

Wherein the compound L1 is synthesized by coupling 4-(dimethylamino)benzaldehyde with Pyridyl boron-dipyrromethene precursor (1,3,5,7-tetramethyl-8-(4-pyridyl)-4,40-difluoroboradiaza-indacene). The method of synthesis is given in scheme I.

Scheme-I
EXAMPLES
The method of synthesis is explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
All the required chemicals are purchased from commercial suppliers. Cis-diamminedichloroplatinum(II)) is purchased from Arora Matthey, India. Mito-Tracker Green (MTG) are purchased from Invitrogen (USA). All animal experiments with mouse models are done in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Mice are maintained in pathogen-free facility with a 12-hour light dark cycle and ad libitum access to standard diet and water.
The cancer cells available inhouse at Indian Institute of Science are adopted for experimentation.
Example 1: Synthesis of Platinum based Boron dipyrromethenecompound of formula I
Step 1: Preparation of compound L1
Pyridyl boron-dipyrromethene precursor (1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4-difluoroboradiaza-indacene) (150 mg, 1equiv.) and commercially available 4-(Dimethylamino)benzaldehyde (412 mg, 6 equiv.) are dissolved in dry toluene (60 mL). After 15 min of stirring, (1.6 mL) glacial acetic acid and (1.92 mL) piperidine and is refluxed for 24 h with help of a Dean-stark apparatus. The solvent is pumped by cold trap to give the crude product, which is purified by column chromatography using EtOAc/hexane and methanol to affordcompound L1 as greenish blue solid (46% yield).
Step 2:Preparation of compound of formula I
Cis-diamminedichloroplatinum(II) (100 mg, 0.33 mmol, 1 equiv.)was dissolved in 5 mL N,N-dimethylformamide (DMF), and reacted with silver nitrate (AgNO3)(45 mg, 0.30 mmol, 0.9 equiv.) under inert nitrogen atmosphere. The reaction mixture is then stirred in dark for 24 h. Silver chloride thus precipitated is separated from the solution by filtration.Compound L1(175 mg, 0.30 mmol, 0.9 equiv.) is added to the filtrate and the reaction mixture is stirred for 24 h at 25 ?C. The solvent is removed by evaporation using a rotary evaporator, and the residue is dissolved in 40 mL of methanol. The unreacted yellow colored Cis-diamminedichloroplatinum(II) is separated by filtration. The product (220 mg (75%, 0.25 mmol) is precipitated out using diethyl ether (60 mL) followed by washing with 50 mL diethyl ether. The solid is dried in vacuum and stored in dark.
Example 2: Characterization studies of compound of formula I
The synthesis of the compoundL1and subsequent metal binding to the compound L1 framework resulting in compound of formula I are ascertained by various spectroscopic techniques, namely, HR-MS, NMR, UV-vis and single crystal XRD (for compound L1).
Mass Spectra:The characterization of compound of formula1 is done by HRMS recorded in positive mode where the spectra shows highest intensity peak at 852.2379 (m/z) assigned to [M-NO3]+(as illustrated in Figure 1) and the corresponding neutral compound L1appeared at 588.381 which is assigned to [M+H]+ value as the most abundant peak.The isotopic distribution pattern of the peaks corresponds to the monocationic part of the platinum compound of formula I. The high purity of the sample is ascertained from the single intense peak.
NMR studies for compound of formula I:
The purity along with the environment of the target compound of formula I is ascertained from the heteronuclear 13C NMR analysis besides 1H NMR spectral data.
Figure 2 provides the data authenticating the formation of the compound of formula Iby NMR spectroscopy. 1H NMR (400 MHz, DMSO-d6): d 8.90 (d, J = 7.44, 2H), 7.95 (s, 1H), 7.78 (d, J = 7.37, 2H), 7.46 (m, 6H), 7.33 (s, 1H), 7.29 (s, 1H), 6.94 (s, 2H), 6.79 (d, J = 7.52, 4H), 4.80 (s, 3H), 4.39 (s, 3H), 3.00 (s, 12H), 1.44 (s, 6H).
13C NMR (100 MHz, DMSO-d6): d 163.24, 153.54, 152.10, 146.49, 140.80, 138.70, 131.05, 129.80, 127.80, 124.76, 119.01, 113.04, 37.60, 15.30.
X-Ray (single crystal X-ray diffraction study for compound L1):
Single crystal X-ray diffraction study is done to ascertain the molecular structure of the compound L1.The compound L1 is neutral in nature and is crystallized in the space group C 2/c of the monoclinic crystal system. (?/Å = 8.1579(8), b/Å = 29.182(3), c/Å = 14.5046(14), (?/? = 90, ?/? =95.889(3), ?/? =90, V/ Å3 3434.8(6), Z = 4)
Absorption and Fluorescence Emission:
The compoundL1and compound of formula I are studied from their absorption and fluorescence emission properties using UV-visible absorption and fluorescence spectroscopy in 1% DMSO/DMEM buffer of pH = 7.2. Figure 3 shows the absorption and emission spectra of compound of formula I. The intense absorption band for the compoundcentered around 680-710 nm is due to the inherent characteristic properties of the BODIPY moiety. Accordingly, the compound of formula I also demonstrated intense and similar absorption bands due to the presence of the BODIPY core (e >104 M-1 cm-1) at ~700-730 nm for the compound of formula I.The absorption peak of compound of formula I is within the desired PDT spectral window and the molar extinction coefficient value of >42,000 is higher than that of Photofrin Q-bands indicating better therapeutic potential of compound of formula I.
Evaluation of the quantum yield of singlet oxygen generation:
UV-visible spectral titration using 1,3-diphenylisobenzofuran (DBPF) as a singlet oxygen quencher is performed. The appendage of heavy atom(s) on Borondipyrromethene moiety and/or suitable modification of its core structure enhance the quantum yield of singlet oxygen generation.Figure6 provides the evidence of singlet oxygen generation by compound of formula I in the line diagram of the target compound of formula I and others excluding cis-diamminedichloroplatinum(II) which does not generate any ROS. Figure 6(a) illustrates spectral changes of DPBF (1,3-diphenylisobenzofuran, 50 µM) at 415 nm with time on treatment with compound of formula I in air saturated DMF (dimethylformamide) whereas Figure 6(b)illustrates plots showing the change in absorbance of DPBF at ~415 nm vs. photoirradiation time in the presence of methylene purple (MB), compound of formula I(blue) and organic compound L1(red). The results suggest significant generation of singlet oxygen on photo-irradiation.
Investigation on the mitochondrial dysfunction in the PDT treatment:
The study is carried out by JC-1 assay and the data are interpreted according to the changes observed in mitochondrial membrane potential (MMP). JC-1, abbreviated for 5,5',6,6'-tetrachloro-1,1'-3,3'-tetraethyl-benzimidazolylcarbocyanine iodide, is a lipophilic cationic fluorescent dye with mitochondrial selectivity. JC-1 monomer exhibits green fluorescence observed mainly in apoptotic cells with low ??m value, and it forms aggregates in healthy cells with high ??m value showing red fluorescence. Herein, compound of formula I is found to be highly selective in targeting mitochondria. After 4 h pre-incubation of the compound of formula I in one of the cell lines, the sample is irradiated with broad band red light (Waldmann PDT 1200 L, 600-720 nm). A gradual decrease in the intensity of red fluorescence is observed with substantial growth of green signal of the JC-1 dye, thus confirming apoptotic cellular damage.
Example 3: In vivo anti-tumor potential of compound of formula Iusingimmune compromised mice models.
Athymic nu/nu male mice (4-6 weeks old) are used for xenografting studies. For subcutaneous tumors, 106 luciferases expressing BT474 cells are washed and harvested in DMEM and subsequently injected subcutaneously over the flank on each side in a volume of 0.1 mL. After all, tumors are about 5 mm in diameter, the mice are divided into two groups; a test group which is treated with compound of formula Iat 5 mg/kg body weight, and a control group. The mice are protected from light. For each mouse, the right tumor is photoirradiated with red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW) 4 hour after injection of compound of formula I and the left tumor is protected from any light exposure. The tumors are monitored after every two days by tumor diameter measurements for 15 days. Tumor volume is calculated as Volume = 0.5 × Width2 × Length. The same is illustrated in Figure 9. Figure 9(a) illustrates representative images of (Human Carcinoma cell line) BT474 tumors in mice after compound of formula I is injected (5 mg/Kg body weight) and subjected to red light radiation (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW). Figure 9(b)illustrates tumor volume measure of mice over a period of 15 days after injectingcompound of formula I, wherein only right side of tumor is irradiated in red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW).
Human mammary carcinoma cells are injected subcutaneously into the left and right flanks of 4 to 6-week-old male immunocompromised mice. After the tumors attained approximately 1 cm diameter, the mice are divided into two groups: a saline treated control group and compound of formula Itreated test group. Further, to test that compound of formula Iis activated upon light irradiation (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW), tumors on one of the flanks only are irradiated at constant intervals. No reduction in the size of the tumor is observed in the non-radiated control mice, thus ascertaining that irradiation with light alone has no significant role in the treatment of tumor. Whereas, the tumors exposed to the compound of formula I as well as red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW) of single wavelength, showed inhibition of further tumor growth as compared to the saline treated and the compound of formula I with non-irradiated control mice, as evident from the tumor diameter measurements. The observation reveals that there is an in vivo light-mediated activation of the drug (compound of formula I) which inhibits further growth of the subcutaneous tumors. Mechanistically, our in vitro experimental evidence suggests that compound of formula Itargets mitochondrion in the tumor cells, resulting apoptotic cell death.The same is depicted in Figure 8.
Histological analysis:
Histological analysis of the tumor sections by H&E staining revealed that the tumors treated with the compound of formula I and also irradiated with red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW) showed significant tissue alterations indicating necrosis or apoptosis of the tumor cellsin the tumors that are treated with the compound of formula Iand irradiated with IR light but not in the case of the saline treated control group which showed normal tumor tissue architecture. Furthermore, histological analysis of the major organs, that is liver, kidney, spleen, heart, lung, brain, intestine, and ovaries revealed that none of these organs are affected by the drug injected which unequivocally ascertains no apparent toxicity of the invented moleculeto the healthy organs of the mice treated, which corroborates that the compound of formula I is activated by red light. Thus, localized activation of the compound of formula I in the affected area can be an effective treatment strategy. Histological examination is carried out on primary organs of mice allocated into two different treatments: (a) Physiological saline (control) + red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW). (b) compound of formula Iinjected (5 mg/Kg body weight) and subjected to red light (685 nm ± 10%, 20 mW/cm2, BTL 4110 premium with 50 mW). Scale bar 200 ?m.Figure 11 presents representative images of histological analysis of the tumor sections using Haematoxylin and Eosin staining.
Photocytotoxicity Studies:
The in-vitrophotocytotoxicity studies of the compound of formula I and the compoundL1 with concentrations from 0.08 to 50 µM in 1% DMSO/Dulbecco’s modified Eagle’s medium (DMEM) are investigated by MTT assay against human breast cancer (BTB474, MCF-7), lung cancer (A549) cells and immortalized lung epithelial normal cells (HPL1D) cells lines using red light (Waldmann PDT 1200 L, 600-720 nm, light dose = 30 J cm-2). After removing the DPBS buffer, fresh DMEM media are introduced and kept in dark for 19 h. MTT dye is 4 mg mL-1 are added and finally the formazan crystals are dissolved in DMSO solvents. Data are obtained with a TECAN microplate reader and fitted using GraphPad Prism 7 software after independent sets of experiments done in triplicate for each concentration. Confocal microscopy is used for subcellular investigation with compound of formula I (5 µM in 1% DMSO/DMEM) in the dark for 4 h. Cells are stained with DAPI (1 mg mL-1) for 5 min. Live cells are stained with Mito-tracker Green (MGR, 100 nM). Images are captured in a Zeiss microscope with an oil immersion lens and magnification of 63X. Cellular apoptosis assay is performed using Annexin- V/FITC/PI dye in 1% DMSO/ DMEM. Approximately 3 x 105 BT474 cells are seeded in six-well plates and cultured for 24 h. The cells are incubated with the compound of formula I for 4 h in the dark and then exposed to red light (Waldmann PDT 1200 L, 600-720 nm, light dose = 30 J cm-2). Cells are then kept for another 19 h in DMEM/ 10% fetal bovine serum (FBS) buffer in the dark, after which the medium is discarded and the cells are trypsinized and re-suspended in the binding buffer (300 mL, 1X). Annexin- V/FITC (0.5 mL) and PI (1 mL) are added to the cell suspensions and incubated for 5 min. Readings are taken with the FACS instrument. Cells are then processed and analyzed with a FACS Verse machine (BD Biosciences) and populations of cells are obtained from histograms generated by the Cell Quest Pro software (BD Biosciences). Figure 7 presents data on the cytotoxicity of compound of formula Iin multiple normal and cancerous cell lines on irradiation with red light (Waldmann PDT 1200 L, 600-720 nm). Photocytotoxicity of compound of formula I and the compoundL1(IC50 values in µM) in cancerous cell lines, which are, BT474(breast cancer), MCF-7 (breast cancer), A549 (human lung adenocarcinoma) cells and a normal cell line HPL1D (immortalized lung epithelial) is evaluated wherein the cells are pre-incubated for 4 h (600-720 nm; light dose, 30 J cm-2; Waldmann PDT 1200 L) and post-incubation is done for 19 h.The results of photocytoxicity study are depicted in Table 2.

Table 2. The Photocytotoxicity (IC50 in µM) Data for the compound of formula Iand L1
Cell lines used Compound of formula I Compound L1
BT474 La 0.9±0.07 17.1±0.5
BT474 Db >50 >50
MCF-7 La 1.3±0.1 15.4±0.3
MCF-7 Db >50 >50
A549 La 3.3±0.2 18.2±0.4
HPL1D La 47.8±0.9 >50
HPL1D Db >50 >50
a All the cells treated with the compound of formula Iand L1. Pre-incubation = 4 h (600-720 nm; light dose, 30 J cm-2; Waldmann PDT 1200 L). b Post incubation = 19 h in dark.
L = Light; D = Dark.

Thus, the present invention provides a potential compound of formula I for treatment of cancer. The economical process of preparation and the facile adoption of the compound in targeting cancer cells act as a boon in the medical field.