DESC:CONJUGATES OF BF2-OXA-SMARAGDYRIN AND METHOD OF PREPARATION THEREOF

FIELD OF THE INVENTION

[0001] The present invention generally relates to a process for synthesizing agents for bio-imaging and photothermal therapy, and more particularly relates to conjugates of BF2-Oxasmaragdyrin and method of preparation thereof.

BACKGROUND OF THE INVENTION

[0002] Bioimaging allows in vivo imaging of biological processes, to detect changes in receptor kinetics, molecular and cellular signalling and interactions and the movement of molecules through membranes. The present invention focuses on synthesis of stable bio-imaging agents. Being mostly non-invasive, bioimaging offers precise tracking of metabolites that can be used as biomarkers for disease identification, progress and treatment response. Recently, nanoparticles have provided significant progress that can be simultaneously used for cancer diagnosis and therapy. Theragnostics aims to provide “image-guided cancer therapy” by integrating therapeutic and imaging agents in a single platform. In addition, molecular imaging techniques facilitate “image-guided surgery” enabling maximization of tumour excision and minimization of side effects. The optical signals generated by fluorescence nanoparticles offer the possibility to distinguish tumour sites and normal tissues.
[0003] Optical imaging has emerged as a non-invasive diagnostic technique which can be used to visualise cellular functions with high sensitivity, at low cost and less toxicity. Dyes which absorb and fluoresce in visible region has been employed extensively. These dyes have inherent disadvantages to image the mammalian cells/tissues as mammalian cells absorb and auto-fluoresce in this region leading to less signal to noise ratio. As mammalian cells are highly transparent in the region of 650–900 nm, dyes which fluoresce in this region are therefore very useful for Near-Infrared Fluorescence (NIRF) imaging and therapy. Hence there is a growing need for dyes that can be easily prepared to absorb and emit strongly in the NIR region. There are only few dyes that are fluorescent in this region; these include cyanine dyes, squaraine dyes, BODIPY analogues benzo[c]heterocycles, xanthenes, phthalocyanines, and a small section of porphyrin derivatives.
[0004] One such molecule which absorb and emit in far-red region of NIR-I biological window is smaragdyrin which belongs to the class of expanded porphyrins. Smaragdyrins are 22p aromatic expanded pentapyrrolic macrocycles in which the five pyrroles are connected by three meso carbons and two direct pyrrole–pyrrole bonds. Until recently, it was believed that the presence of two direct pyrrole–pyrrole bonds makes the smaragdyrin macrocycle highly strained and not very stable for further studies. However, Chandrashekar and co-workers found a way that the smaragdyrins can be stabilized if one of the pyrrole rings is replaced with a furan ring. The resultant 25-oxasmaragdyrins are stable and show interesting spectral and electrochemical properties. The 25-oxasmaragdyrins absorb and emit in the visible to far-red region with moderate quantum yields and singlet state lifetimes. Figure 34 of the present invention discloses various complexes of oxasmaragdyrin known in the art with different functional group which exhibit NIR fluorescence and can also be used for NIR Fluorescence imaging as well as photothermal therapy. The stability and photophysical properties can be further enhanced when the 25-oxasmaragdyrin is complexed with BF2 unit to form BF2-oxasmaragdyrins.
[0005] Various targeting molecules have been used in prior arts to internalise drugs and nanoparticles such as transferrin, pluronic, folic acid, biotin and polysaccharides. Glucosamine is one such molecule which easily permeates through the cell membrane as glucose is essential for the synthesis of highly glycosylated lysosomal proteins. Glunde and coworkers have demonstrated that fluorophore bound at C6 position of glucosamine can be used to optically image lysosomes in cells.
[0006] One of the methods for treating cancer involves photothermal therapy (PTT). Photothermal therapy is a kind of therapy based on increasing the temperature of tumoral cells above normal body temperature by 4°C to 5°C. To aim this temperature, cells must be illuminated with a laser, and the energy of the radiation is transformed into heat. The employed radiations are in the near-infrared radiation range. Further, magnetic nanoparticles are also used in photothermal therapy. However, molar absorption coefficient of the magnetic nanoparticles in the near infrared region is low. The success of PTT relies momentously on photothermal agents that convert light energy to localised heat and that being utilized for tumor ablation. To date, various materials have been screened for their application in PTT, and some of these include gold nanoparticles, graphene and graphene oxide, black phosphorus, palladium, copper sulphide, copper selenide, molybdenum oxide. The non-biodegradability and long-term toxicity of inorganic materials, however, are of grave concern. Though polymer-based materials were developed and their photothermal potential was evaluated, such materials are often encumbered with lengthy synthesis and complicated processing techniques. To overcome these hurdles and the need to develop efficient photothermal agents has resulted in propelling research towards organic dyes. The materials possessing absorption/fluorescence in the biological tissue window (650-950 nm), possess excellent potential as a Near-Infrared Fluorescence (NIRF) imaging and/or therapeutic agent. Hence, there is a need for a stable and non-toxic bioimaging and photothermal agents.

SUMMARY OF THE INVENTION

[0007] This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention. This summary is not intended to identify key or essential inventive concepts of the claimed subject matter, nor is it intended for determining the scope of the claimed subject matter.
[0008] The present invention discloses a compound with formula (I) and formula (II)
(I) (II)
wherein R1 comprises of 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine, L,L-diphenylalanine, or cell penetrating polypeptides and R comprises of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA), (tert-butoxycarbonyl)-L,L-glycylglycine conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB), N-acetyl-L-tryptophan conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT), wherein these conjugates are used for bioimaging and photothermal therapy.

[0009] An embodiment of the present invention also discloses a process to synthesize conjugates of BF2-oxasmaragdyrins such as diphenylalanine conjugated BF2-oxasmaragdyrin, Glucosamine conjugated BF2-Oxasmargdyrin, Polypeptide conjugated BF2-Oxasmargdyrin and amino acid conjugated BF2-Oxasmaragdyrin. The process for synthesizing glucosamine conjugated BF2-Oxasmargdyrin comprises synthesizing BF2-25-Oxasmaragdyrin containing p-carboxy phenyl; adding BF2-25-oxasmaragdyrin (102) and a coupling reagent (HATU, HBTU or HCTU) in a solution of 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine (104) under nitrogen atmosphere to form a mixture wherein BF2-25-oxasmaragdyrin (102) and the coupling reagent are dissolved in Dichloromethane (CH2Cl2). The mixture is stirred for at least 24 hours to obtain crude tert-butoxycarbonyl (Boc) protected glucosamine conjugated BF2-25-oxasmaragdyrin. Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin is purified by basic alumina column chromatography to obtain a green solid. Purified Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin is further treated with a mixture of organic solvents for at least 15 minutes followed by neutralisation with methanolic ammonia. The progress of the reaction is monitored by TLC analysis. Once the reaction is complete solvent is removed under reduced pressure to obtain crude BF2-25-oxasmaragdyrin-glucosamine conjugate (108). The crude compound is washed with water to obtain purified BF2-25-oxasmaragdyrin-glucosamine conjugate (108).
[00010] Another embodiment of the present invention further discloses a process to synthesize L, L-diphenylalanine conjugated BF2-oxasmaragdyrin self-assemblies. The process comprises of synthesizing BF2-25-oxasmaragdyrin synthesizing methyl L-phenylalanyl-L-phenylalaninate. BF2-25-oxasmaragdyrin is hydrolysed with Lithium hydroxide (LiOH) in Tetrahydrofuran (THF/H2O) to obtain (meso-(4-carboxy phenyl) BF2-25-oxasmaragdyrin) which is further reacted with protected L,L-diphenylalanine in presence of 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIEA) in dichloromethane under inert atmosphere for 8 hours to obtain a reaction mixture. Further, methyl L, L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) is obtained by basic alumina column chromatography and methyl L, L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) is hydrolysed to obtain L, L-diphenylalanine conjugated BF2-oxasmaragdyrin (120).
[00011] Yet another embodiment of the present invention disloces a process to synthesize Poly-arginine (R9) conjugated BF2-Oxasmargdyrin and Cysteine-Arginine-Glycine-Aspartic acid-Lysine (CRGDK) comprises synthesizing BF2-25-oxasmaragdyrin containing p-carboxy methylphenyl substituent at meso position (122) simultaneously preparing a solid supported (Rink amide resin) peptides with free N-terminal (124, 134) by automated microwave-accelerated solid phase peptide synthesis. A mixture of BF2-25-oxasmaragdyrin, HATU, HOBt and DIEA in an organic solvent is added to the free N-terminal solid supported peptide, this mixture is shaken at room temperature followed by removal of solvent via vacuum filtration to obtain a residue. The residue is washed with Dimethylformamide (DMF) and (CH2Cl2) followed by drying under vacuum. Crude polypeptide BF2-25-Oxasmaragdyrin conjugate is obtained by cleavage of the resin with Trifluoroacetic acid/ Triisopropylsilane/water (TFA/TIPS/H2O) followed by precipitating the crude polypeptide BF2-25-Oxasmaragdyrin conjugate in cold diethyl ether. Crude polypeptide BF2-25-Oxasmaragdyrin conjugate is precipitated in cold Diethyl ether (Et2O).
[00012] Yet another embodiment of the present invention discloses a process to synthesize amino acid conjugated BF2-25-Oxasmaragdyrin. The process comprises of adding Hydrazine Hydrate to a pre-heated solution of meso(4-nitrophenyl) BF2-oxasmaragdyrin and 10% Pd/C in ethanol to obtain a mixture. The mixture is then refluxed for a pre-set time and filtered after completion of the reaction. The mixture is further processed in a rotary evaporator to obtain a crude solid which is dissolved in an organic solvent. Dissolved crude solid is washed with an acid solution. The organic fractions of are collected, rotary evaporated and purified to obtain pure meso(4-aminophenyl) BF2-oxasmaragdyrin. N,N-diisopropylethylamine is added dropwise to a stirred solution of N-protected amino acid, meso-(4-amino phenyl) BF2-oxasmaragdyrin (141) and a coupling reagent in dry dichloromethane at 0 °C under nitrogen atmosphere to obtain a second mixture, progress of the reaction is monitored by TLC, the second mixture is diluted with dichloromethane and the organic phase is washed with 1N HCl, saturated Na2CO3¬solution and water. The organic phase is dried over Na2SO4, filtered and rotary evaporated to obtain crude brown semisolid amino acid conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin. The crude compound is purified by column chromatography to obtain pure amino acid conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (142).

BRIEF DESCRIPTION OF THE DRAWINGS

[00013] Reference will be made to embodiments of the invention, examples of which may be illustrated in the accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
[00014] The objects, features, and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[00015] Figure 1 illustrates (a) Biocompatibility of glucosamine conjugated BF2-25-oxasmaragdyrin on fibroblasts L929 cells (n = 5). CLSM images showing cellular intake of glucosamine conjugated BF2-25-oxasmaragdyrin in MDA-MB-231 cells (b) Differential Interference Contrast (DIC) image, (c) Fluorescence channel with 680-720 nm and (d) merged image of all channels.
[00016] Figure 2 illustrates characterisation of L, L-diphenylalanine conjugated BF2-25-oxasmaragdyrin self-assemblies (a, b) Scanning Electron Microscope (SEM) images of self-assemblies. (c) Transmission Electron Microscope (TEM) image of self-assemblies. (d) Size distribution of self-assemblies Absorption (e) and fluorescence (f) comparison of self-assembled particles to that of the single-molecular entities of L, L-diphenylalanine conjugated BF2-25-oxasmaragdyrin
[00017] Figure 3 illustrates the photothermal evaluation of L, L-diphenylalanine conjugated BF2-25-oxasmaragdyrin self-assemblies (a, b, c) simulated data curves for concentration of self-assembles (mg/mL), laser irradiation power (mW), laser irradiation time and rise in temperature. (d) Simultaneous heating and cooling curves for nanoparticles irradiated with 650 mW power of 750 nm laser. (e, f) SEM images of nanoparticles after four cycles of heating and cooling.
[00018] Figure 4 illustrates CLSM images of MDA-MB-231 cells treated with Poly-arginine/CRGDK conjugated BF2-Oxasmargdyrin.
[00019] Figure 5 illustrates schematic representation of synthesis of glucosamine conjugated BF2-oxasmaragdyrin.
[00020] Figure 5a illustrates 1H, 1H-1H COSY NMR of compound (110).
[00021] Figure 5b illustrates Photophysical and electrochemical data for Glucosamine Conjugated BF2-Oxasmaragdyrin, Boc protected glucosamine conjugated BF2-25-Oxasmaragdyrin and compound (110).
[00022] Figure 6 illustrates Comparison of (a) absorption, (b) fluorescence spectra of compound (108), compound (106) and compound (110) (conc.: 1×10-5 M). (c) Fluorescence decay profile and the weighted residuals distribution fit of fluorescence decay of Glucosamine conjugated BF2-25-Oxasmaragdyrin in DMSO. The ?ex used was 440 nm and emission was detected at 710 nm. (d) Comparison of cyclic Voltammograms and Differential Pulse Voltammograms of Compound (106) and compound (110) in CH2Cl2 containing 0.1M TBAP as supporting electrolyte using scan rate of 50 mV/sec.
[00023] Figure 7 illustrates Biocompatibility of compound (108) on fibroblasts L929 cells (n = 5), (positive control (PC) being 1% Triton X-100, negative control (NC) being complete media).
[00024] Figure 8 illustrates CLSM images showing L929 Control cells treated with DAPI (a) Differential Interference Contrast (DIC) image, (b) DAPI channel with 410-454 nm emission filter,(c) Fluorescence channel with 680-730 nm emission filter,(d) merged image of all channels and L929 cells treated with DAPI and compound (108), (e) Differential Interference Contrast (DIC) image, (f) DAPI channel with 410-454 nm emission filter, (g) Fluorescence channel with 680-732 nm emission filter and (h) merged image of all channels.
[00025] Figure 9 CLSM images of L929 controls cells (a) Differential Interference Contrast (DIC) image, (b) Fluorescence channel with 680-720 nm and (c) merged image of all channels and L929 cells treated with compound (108) (d) Differential Interference Contrast (DIC) image, (e) Fluorescence channel with 680-740 nm emission filter and(f) merged image of all channels.
[00026] Figure 10 CLSM images of controls cells (a) Differential Interference Contrast (DIC) image, (b) Fluorescence channel with 680-720 nm and (c) merged image of all channels and cells treated with compound 108 (d) Differential Interference Contrast (DIC) image, (e) Fluorescence channel with 680-720 nm and (f) merged image of all channels.
[00027] Figure 11 illustrates mean fluorescence intensity per cell in control and compound 1 treated cells.
[00028] Figure 12 illustrates schematic representation synthesis of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin.
[00029] Figure 13 illustrates Schematic representation for the formation of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin self-assemblies.
[00030] Figure 14 illustrates Synthesis and characterisation of FF-BSC NP. (a)Schematic representation for the formation of L,L-diphenylalanine conjugated BF2-oxasmaragdyrin self-assemblies (FF-BSC NP). Absorption (b) and fluorescence (c) comparison of self-assembled particles to that of the single-molecular entities (1.6 × 10-6 M). (d) FEG-SEM images of FF-BSC NP (inset: higher magnification image) and (e) FEG-TEM image of self-assemblies.
[00031] Figure 15 illustrates pH and lyophilisation stability of FF-BSC NP. Absorbance spectrum of FF-BSC NP maintained at pH (a) 5.5, (b) 6.5, (c) 7.4; Fluorescence spectrum of FF-BSC NP maintained at pH (d) 5.5, (e) 6.5, (f) 7.4.SEM images of FF-BSC NP after lyophilization: (g) without cryo protectant, (h) F-68 (0.5%) + sucrose (1%), and (i) F-68 (0.5%) + trehalose (1%).
[00032] Figure 16 illustrates Types of variables, levels and selected ranges of factors for constructing the design.
[00033] Figure 16a CCD design layout along with recorded response.
[00034] Figure 16b ANOVA for the response, temperature for the selected quadratic model.
[00035] Figure 17 illustrates Photothermal transduction efficiency of FF-BSC NP. (a) 3D Plot and (b) Design space for the response, temperature evaluated with CCD; (c) time vs. temperature profile of FF-BSC NP, (d) multi-cycle temperature changes upon exposure of FF-BSC NP to a750 nm laser, comparison of (a) absorption and (b) fluorescence spectra of FF-BSC NP before and after 750 nm laser exposure. (Conc.: 15 µg/mL).
[00036] Figure 18 illustrates confirmatory trials for the obtained design space.
[00037] Figure 19 illustrates Biocompatibility and Hemocompatibility Studies. Biocompatibility and hemocompatibility of FF-BSC Np (a) In vitro biocompatibility of FF-BSC NP in L929 cells, (b) digital image of various samples of haemocompatibility study (FF-BSC NP in µg/mL), ESEM images of RBC treated with (c) positive control (PC)Triton X-100, (d) negative control (NC) PBS, (e) 300 µg/mL FF-BSC NP, (f) graph depicting the haemocompatibility study of FF-BSC NP.
[00038] Figure 20 illustrates CLSM images showing control cells and FF-BSC NP incubated cells, Control cells (a) Differential Interference Contrast (DIC) image, (b) DAPI channel with 410-454 nm emission filter, (c) Fluorescence channel with 680-730 nm emission filter, (d) merged image of all channels, FF-BSC NP incubated cells (e) Differential Interference Contrast (DIC) image, (f) DAPI channel with 410-454 nm emission filter, (g) Fluorescence channel with 680-730 nm emission filter and (h) merged image of all channels, In vitro cellular uptake and photothermal efficacy, (i) uptake of FF-BSC NP in 4T1 cells by flow cytometry, (j) graph depicting in vitro photothermal efficacy in 4T1 cells and (k) FACS scatter plot of Annexin-V PI staining in control and treated cells; C (cells), L (laser exposure).
[00039] Figure 21 Whole body fluorescence imaging and tumor homing. (a) Whole body fluorescence imaging of control and FF-BSC NP injected mice 24 h post material administration (b) Quantification of total fluorescence from the whole body of control and FF-BSC NP injected mice (c) Fold change in fluorescence output in FF-BSC NP injected mice as compared to control mice (d) Fluorescence images showing fractional tumor homing of nanoparticle in FF-BSC NP injected mice (e) Absolute quantification of the amount of FF-BSC NP in the tumor tissue of control and FF-BSC NP injected mice.
[00040] Figure 22 illustrates biodistribution and single dose toxicity studies. Ex vivo fluorescence imaging of vital organs; spleen (S), lungs (Lu), liver (Li), kidney (Ki) and heart (H) collected from control (a) and FF-BSC NP injected mice (b), (c) Qualitative evaluation of fluorescence output from organ samples of the control and FF-BSC NP injected mice, (d) absolute quantification of the amount of FF-BSC NP in organ samples collected from the control and FF-BSC NP injected mice, Toxicity evaluation of FF-BSC NP under preclinical settings (e and f) Hematoxylin and Eosin staining of the organ samples of heart, kidney, liver, spleen and lung sections of the control and FF-BSC NP injected mice (g) serum biochemical analysis of alanine transaminase (ALT), creatinine (CRE), urea (BUN) and aspartate aminotransferase (AST) in control and FF-BSC NP injected mice.
[00041] Figure 23 In vivo photothermal ablation by FF-BSC NP in 4T1-Luc2 xenograft mouse model. (a) in vivo bioluminescence images of a representative mouse each from various control groups and the PTT treatment group, (b) quantitative assessment of bioluminescence tumor signal as an indicator of live tumor cell mass present at the site for all groups during 30 days follow-up (***indicates p = 0.0001) and (c) Kaplan-Meier survival plot of various groups (** means p = 0.006).
[00042] Figure 24illustrates schematic representation for synthesis of peptide conjugated BF2-25-oxasmaragdyrin.
[00043] Figure 25a illustrates HPLC of crude CRGDK- peptide intermediate.
[00044] Figure 25b illustrates HPLC trace of crude R9-peptide intermediate.
[00045] Figure 25c LRMS of crude CRGDK- peptide intermediate.
[00046] Figure 25d illustrates LRMS of crude R9-peptide intermediate.
[00047] Figure 25e illustrates RP-HPLC of crude CRGDK- BF2-oxasmaragdyrin conjugate (128).
[00048] Figure 25f illustrates HPLC trace of crude R9- BF2-oxasmaragdyrin conjugate (128).
[00049] Figure 25g illustrates LRMS of crude CRGDK- BF2-oxasmaragdyrin conjugate (128) extracted at 29.39 min from HPLC.
[00050] Figure 25h illustrates HRMS of R9- BF2-oxasmaragdyrin conjugate (132) collected at 31.5 min from the analytical HPLC trace chromatogram.
[00051] Figure 26 CLSM images of control cells (a) Differential Interference Contrast (DIC) channel, (b) DAPI channel (c) fluorescence channel (d) merged image of all channels, cells treated with compound (132) (e) DIC channel, (f) DAPI channel, (g) fluorescence channel, (h) merged image of all channels. The fluorescence channel was recorded with a 680-740 nm emission filter.
[00052] Figure 27 CLSM images of control cells (a) Differential Interference Contrast (DIC) channel, (b) fluorescence channel, (c) merged image of all channels, cells treated with CRGDK- BF2-oxasmaragdyrin conjugate (128), (d) DIC channel, (e) fluorescence channel, (f) merged image of all channels. Cells treated with compound (128) (h) 3D view of CRGDK- BF2-oxasmaragdyrin conjugate (128) showing localization in MDA-MB-231 cells. The fluorescence channel was recorded with a 680-740 nm emission filter.
[00053] Figure 28 illustrates Types of variables, levels and selected ranges of factors for constructing the design.
[00054] Figure 28b illustrates Design matrix with observed temperature.
[00055] Figure 28c illustrates ANOVA for reduced quadratic model for response, temperature.
[00056] Figure 29 illustrates (a) 2D contour plot, (b) 3D response surface plot,(c) graphical optimization plot (Design Space) for the selected response, temperature (LT indicates laser irradiation time), (d) multi-cycle photothermal transduction potential of compound (132) upon NIR laser irradiation (ON indicates laser on and OFF indicates laser was off during that period). (e) Temperature change corresponding to laser ON and OFF of compound (132), and (f) time versus ln? from the cooling stage of compound (132).
[00057] Figure 30 illustrates Confirmation trials for the evaluation of design space.
[00058] Figure 31 illustrates (a) Absorption (b) fluorescence (c) TCSPC of CRGDK- conjugated BF2-oxasmaragdyrin 128 in different solvents. (d) Absorption (e) fluorescence (f) TCSPC of R9- conjugated BF2-oxasmaragdyrin 132 in different solvents.
[00059] Figure 32 illustrates schematic representation of Synthesis of meso(4-aminophenyl) BF2-oxasmaragdyrin and its amino acid/peptide conjugates.
[00060] Figure 33 illustrates different complexes of oxasmaragdyrins along with possible structural/functional modifications.
[00061] Figure 34 illustrates chemical structures known in the art which exhibit NIR fluorescence and which can be employed in Photothermal therapy.
[00062] It should be appreciated by those skilled in the art that any diagrams herein represent conceptual views of illustrative systems embodying the principles of the present invention.

DETAILED DESCRIPTION
[00063] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.
[00064] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” or “in an exemplary embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[00065] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.
[00066] The present invention generally relates to conjugates of BF2-Oxasmaragdyrin and method of preparation thereof. The precursor, BF2-Oxasmaragdyrin, required to synthesize the conjugates, may be prepared using a method reported in Narayanan, S. J.; Sridevi, B.; Chandrashekar, T. K.; Englich, U.; Ruhlandt-Senge, K., Org. Lett. 1999, 1, 587-590. The method comprises dissolving 1.263 mmol of Tripyrrane and 1.263 mmol dipyrromethene in 500 ml dry dichloromethane and stirring the mixture under nitrogen atmosphere for 5 min; adding 0.1263 mmol of Trifluoroacetic acid and stirring is continued for further 90 minutes; adding 3.789 mmol of chloranil and heating the reaction mixture at reflux for an additional 90 minutes; removing the solvent; purifying the residue by chromatography on basic alumina; identifying the pink fraction eluted with petroleum ether/dichloromethane. Oxasmaragdyrin is eluted as a green band when the eluent is dichloromethane. Further, preparing BF2 Complex of oxasmaragdyrin by a method reported in M. Rajeswara Rao, M. Ravikanth, J. Org. Chem. 2011, 76, 3582-3587; the method comprises mixing 0.157 mmol of oxasmaragdyrin in 30 ml dichloromethane (CH2Cl2) to form a solution; subsequently adding triethylamine (6.28 mmol) at room temperature to the solution; adding 7.85 mmol of Boron trifluoride diethyl etherate after 5 minutes and stirring the resultant mixture for 30 minutes; diluting the reaction mixture with CH2Cl2; washing the reaction mixture with 0.1 M NaOH and water; combining the organic layers; drying the organic layers over Na2SO4;filtering the resultant mixture; evaporating the remaining solvent on a rotary evaporator under vacuum; purifying the resulting crude product by column chromatography on alumina by using petroleum ether/dichloromethane (70:30). A green powder of Meso-aryl-BF2-Oxasmaragdyrin is obtained after purification. Figure 33 illustrates different complexes of oxasmaragdyrins along with related functional groups.
[00067] The present invention discloses conjugates of BF2-oxasmaragdyrin for bio-imaging and photothermal therapy and method of preparation thereof. In one of the embodiments, conjugates of BF2-oxasmaragdyrin such as, Glucosamine conjugated BF2-Oxasmargdyrin, Poly-arginine conjugated BF2-Oxasmargdyrin, CRGDK conjugated BF2-Oxasmargdyrin, (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA), (tert-butoxycarbonyl)-L,L-glycylglycine conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB), N-acetyl-L-tryptophan conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT) are synthesized by using the compounds as disclosed in formula I and formula II.

[00068] BF2-25-oxasmaragdyrin-glucosamine conjugate: One of the embodiments of the present invention discloses Glucosamine conjugated BF2-25-oxasmaragdyrin (108) and a process to synthesise the same. As illustrated in figure 5, the process comprises of synthesizing precursors BF2-25-oxasmaragdyrin (102) containing p-carboxyphenyl substituent at meso position and a 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine (104) by a reported method as described in Narayanan, S. J.; Sridevi, B.; Chandrashekar, T. K.; Englich, U.; Ruhlandt-Senge, K., Org. Lett. 1999, 1, 587-59. Said precursors are then reacted with 6-amine-6-deoxy-1-Boc-2-N-Boc-ß-D-glucosamine (104) in presence of a coupling reagent and N,N-diisopropylethylamine (DIEA) in dichloromethane (CH2Cl2) under inert atmosphere for at least 24 hours. The coupling reagent may be selected from 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium Hexafluorophosphate (HCTU). This is followed by basic alumina column chromatography to obtain Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106) as a green solid with yield of around 50%-60%. Subsequently, Boc protected glucosamine conjugated BF2-25oxasmaragdyrin is treated with Trifluoroacetic acid (TFA)/CH2Cl2 followed by removing solvent under reduced pressure to yield BF2-25-oxasmaragdyrin-glucosamine conjugate (108) as green solid with yield of around 60%-80%, which may be used as it is without further purification. All the intermediates and the target compounds are characterized by 1H, 1H-1H COSY, 13C, 11B, 19F NMR spectroscopy and High Resolution Mass Spectrometry (HRMS).
[00069] Another aspect of the present invention discloses a process to synthesize Glucosamine conjugated BF2-Oxasmargdyrin. The process for synthesizing Glucosamine conjugated BF2-Oxasmargdyrin comprises synthesizing precursors BF2-25-Oxasmaragdyrin containing p-carboxy phenyl by a reported method. Adding BF2-25-oxasmaragdyrin (102) and HATU in a solution of 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine (104) under nitrogen atmosphere to form a mixture wherein BF2-25-oxasmaragdyrin (102) and HATU are dissolved in Dichloromethane (CH2Cl2). The mixture is stirred for at least 24 hours to obtain crude Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106). Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106) is purified by basic alumina column chromatography to obtain a green solid. Purified Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin is further treated with a mixture of organic solvents wherein the mixture of organic solvent is composed of Dichloromethane (CH2Cl2) and Trifluoroacetic Acid (TFA) mixed in a ratio of 3:1 for at least 15 minutes followed by neutralisation with methanolic ammonia. The progress of the reaction is monitored by TLC analysis. Once the reaction is complete solvent is removed under reduced pressure to obtain crude BF2-25-oxasmaragdyrin-glucosamine conjugate (108). The crude compound is washed with water (3 x 50 ml) to obtain purified BF2-25-oxasmaragdyrin-glucosamine conjugate (108).
[00070] To confirm the presence of intact glucosamine moiety in BF2-25-oxasmaragdyrin-glucosamine conjugate (108), the acetylation reaction was performed by treating BF2-25-oxasmaragdyrin-glucosamine conjugate (108) in dichloromethane with an excess of acetic anhydride in presence of triethylamine and catalytic amount of 4-N,N-dimethylaminopyridine for 4 hours at room temperature followed by workup and silica gel column chromatography to afford tetraacylated glucosamine conjugated BF2-oxasmaragdyrin (110) as a green solid (70% yield). Compound (110) was characterised in detail by 1H, 1H-1H COSY, 13C, 11B, 19F NMR and all the resonances were assigned based on their location, integration, coupling constant, and cross-peak connectivity in 1H-1H COSY NMR spectroscopy. In compound (110), BF2-oxasmaragdyrin core protons appear as five signals (four doublets and one singlet) in the region of 9.0 -10.5 ppm hence these peaks were identified as protons a-e. Further, Peaks 8.72, 8.36, 8.30, 7.70 ppm were identified as aryl protons f, g, i, j respectively by their integration and cross peak correlation in 1H-1H COSY. The four acyl protons appeared as four distinct singlets at 2.29, 2.25, 2.18 and 2.00 ppm. The peak at 6.92 ppm appeared as a triplet which was identified as ‘k’ proton showed correlation with peaks at 4.05 ppm and 3.77 ppm which were assigned as ‘l’ and ‘l`’ respectively. Further proton l’ shows a correlation with peak at 4.19 ppm which was designated as proton ‘m’. Proton ‘m’ shows a cross peak correlation with peak 5.27 ppm which was identified as proton ‘n’ which in turn showed a cross peak correlation with peak at 5.38 ppm assigned as proton ‘o’. Furthermore, proton ‘o’ showed a cross peak correlation with peak at 4.59 ppm identified as proton ‘p’ which in turn shows cross peak correlation with peak 6.29 ppm and 5.69 ppm which were assigned as protons ‘q’ and ‘r’ respectively. Thus, 1D and 2D NMR spectroscopy were very useful in deducing the molecular structure of compound (110).
[00071] ILLUSTRATIVE EXAMPLE
a. Synthesis of Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106):To a solution of 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine (104) (31.8 mg, 0.084 mmol), BF2-25-oxasmaragdyrin (102) (30 mg, 0.042 mmol) and HATU (15.2 mg, 0.040mmol) in CH2Cl2 (5 mL), N,N-Diisopropylethylamine (DIEA) (45 µl, 0.252 mmol) was added under nitrogen atmosphere and stirred for 24 hours at room temperature. The formation of the product was monitored by TLC which showed single less polar spot. After completion of the reaction, the reaction mixture was diluted with CH2Cl2 (20 mL) and washed with 1N HCl (2 × 30 mL), 1N NaOH (2 × 30 mL), brine solution (2 × 30mL). The organic layer was dried on anhydrous sodium sulphate and the solvent was removed under reduced pressure to give crude product. The crude product was purified by neutral alumina column chromatography (CH2Cl2/MeOH: 95/5) to obtain pure compound as green solid (35mg, yield: 78%). 1H NMR (400 MHz, CDCl3) d 10.34 (d, J = 4.5 Hz, 2H), 10.24 (dd, J = 4.5, 2.0 Hz, 2H), 9.58 (d, J = 4.5 Hz, 2H), 9.52 (s, 2H), 9.03 (dd, J = 4.4, 1.8 Hz, 2H), 8.72 (d, J = 8.2 Hz, 2H), 8.39 (d, J = 8.2 Hz, 2H), 8.30 (d, J = 7.9 Hz, 4H), 7.70 (d, J = 7.7 Hz, 4H), 7.05 (dd, J = 8.2, 4.5 Hz, 1H), 6.13 (d, J = 3.5 Hz, 1H), 4.85 (s, 1H), 4.59 – 4.40 (m, 1H), 4.07 – 3.83 (m, 2H), 3.57 (q, J = 9.6, 8.3 Hz, 2H), 2.80 (s, 6H), 1.49 (s, 9H), 1.26 (s, 9H).13C NMR (101 MHz, CDCl3) d 169.8, 152.2, 150.0, 143.5, 139.7, 138.1, 135.2, 134.4, 132.5, 131.6, 131.0, 130.8, 130.6, 128.4, 127.3, 125.1, 124.1, 123.9, 122.3, 121.1, 120.6, 107.2, 94.6, 83.6, 80.5, 73.3, 71.1, 54.3, 40.5, 32.1, 29.8, 28.5, 28.5, 27.9, 27.8, 22.8, 21.8.19F NMR (377 MHz, CDCl3) d -149.16.11B NMR (128 MHz, CDCl3) d -12.80. HRMS: calcd. for C60H59BF2N6O10 [M + H]+ 1072.4358; found 1072.4329.
b. BF2-25-oxasmaragdyrin-glucosamine conjugate (108) Compound 1: Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106) (50 mg, mmol) was dissolved in 3ml of CH2Cl2 /TFA (3:1 v/v) and stirred for 15 min under inert atmosphere. The progress of the reaction was monitored by TLC analysis. The solvent was removed under reduced pressure and the residue was neutralised by methanolic ammonia (10 ml). The solvent was removed on rotary evaporator under reduced vacuo to obtain the crude compound. The crude compound was washed several times with water (3 × 50 ml) to remove impurities and give BF2-25-oxasmaragdyrin-glucosamine conjugate (108) as green solid which was used without further purification (50 mg, yield: 73.7% ) 1H NMR (400 MHz, DMSO-d6) d 10.77 (d, J = 4.5 Hz, 2H), 10.69 (s, 2H), 9.70 (s, 2H), 9.57 (s, 2H), 9.01 (s, 2H), 8.76 (d, J = 7.7 Hz, 2H), 8.55 (d, J = 7.6 Hz, 2H), 8.34 (d, J = 7.2 Hz, 4H), 7.79 (d, J = 7.6 Hz, 4H), 2.77 (s, 6H), -4.19 (s, 2H).19F NMR (377 MHz, DMSO-d6) d -147.62.11B NMR (128 MHz, DMSO-d6) d -12.54.HRMS: calcd. for C50H43BF2N6O6 [M + H]+ 872.3308; found 872.3292.
c. tetraacylated glucosamine conjugated BF2-oxasmaragdyrin: To the mixture of triethylamine (0.50 ml, 0.458 mmol), 4-dimethylaminopyridine (1.4 mg, 0.011 mmol) and BF2-25-oxasmaragdyrin-glucosamine conjugate (108) (20 mg, 0.023 mmol) in dichloromethane at 0 °C under inert atmosphere, acetic anhydride (0.23 ml, 0.229 mmol) was added dropwise and stirred for 2 hours. After completion of reaction, the reaction mixture was quenched with 1N NaOH and compound was extracted with CH2Cl2 (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and solvent was evaporated to obtain crude product as green solid. The crude product was further purified by silica gel column chromatography (50% CH2Cl2/petroleum ether) to obtain compound (110) as green solid ( 18.0 mg, yield: 75%).1H NMR (500 MHz, CDCl3) d 10.35 (d, J = 4.4 Hz, 2H), 10.25 (dd, J = 4.5, 2.0 Hz, 2H), 9.60 (d, J = 4.4 Hz, 2H), 9.50 (s, 2H), 9.03 (dd, J = 4.5, 1.8 Hz, 2H), 8.72 (d, J = 8.0 Hz, 2H), 8.36 (d, J = 7.9 Hz, 2H), 8.30 (d, J = 7.5 Hz, 4H), 7.70 (d, J = 7.5 Hz, 4H), 6.92 (t, J = 5.9 Hz, 1H), 6.29 (d, J = 3.6 Hz, 1H), 5.69 (d, J = 8.9 Hz, 1H), 5.41 – 5.35 (m, 1H), 5.31 – 5.24 (m, 1H), 4.59 (ddd, J = 10.7, 8.9, 3.7 Hz, 1H), 4.19 (ddd, J = 9.6, 6.1, 2.7 Hz, 1H), 4.05 (ddd, J = 14.2, 6.4, 2.8 Hz, 1H), 3.86 – 3.72 (m, 1H), 2.80 (s, 6H), 2.29 (s, 3H), 2.25 (s, 3H), 2.13 (s, 3H), 2.00 (s, 3H), -3.95 (t, J = 5.7 Hz, 1H). 19F NMR (471 MHz, CDCl3) d -149.30. 11B NMR (160 MHz, CDCl3) d -12.26.13C NMR (126 MHz, CDCl3) d 172.0, 170.3, 169.9, 169.1, 167.9, 150.0, 142.9, 139.7, 138.1, 135.2, 134.4, 133.6, 131.6, 130.8, 130.7, 129.9, 129.6, 128.9, 128.4, 127.6, 127.1, 125.6, 125.1, 124.2, 123.9, 122.3, 121.0, 120.5, 117.4, 107.2, 91.0, 70.9, 70.9, 70.8, 70.7, 69.3, 68.8, 63.7, 51.6, 39.8, 29.8, 29.5, 23.3, 22.8, 21.8, 21.2, 21.1, 21.0, 20.9, 14.3.HRMS: calcd. for C58H51BF2N6O10 [M + H]+ 1040.3732; found 1040.3709.
[00072] Photophysical and Electrochemical properties: The absorption and fluorescence spectra of compounds were recorded, and data are presented in Figure 5b. The absorption and fluorescence studies revealed that the compounds absorb in visible far-red region and emit in far-red region. The absorption spectra of compounds (108), (106) and (110) in DMSO showed that all the molecules have similar absorption features (figure 6) with subtle differences in extinction coefficients (Figure 5b) since there is not much change in the electronic structure of the compounds. Similarly, the fluorescence spectra of compounds (108), (106) and (110) in DMSO show that all the compounds have similar fluorescence spectral features (figure 6) with slight change in quantum yields. Compound (108) has relatively lower quantum yield (ff = 0.12) compared to that of compounds (106) and (110) (ff = 0.19).
[00073] Furthermore, Time-correlated single photon counting (TCSPC) experiments was carried out on compounds (106) and (110) by exciting the compounds at 440 nm and detecting emission at 710 nm. All the compounds showed single exponential decay profile (Figure 6c) with similar excited state lifetime (~4.9 ns). The electrochemical properties of compounds (106) and (110) were studied in CH2Cl2 by cyclic voltammetry at a scan rate of 50 mV/s by using tetrabutylammonium perchlorate (TBAP) as the supporting electrolyte (Figure 6d). Compounds (106) and (110) showed three reversible oxidations and one reversible reduction (Figure 6b) unlike BF2-25-oxasmaragdyrin which shows two reversible oxidations and one reversible reduction. The electrochemical data indicate that these compounds are easier to oxidize and reduce and compounds are stable under electrochemical conditions. Since the compounds are chiral, we recordedcircular dichroism spectrum of compound (108) in DMSO and observed broad negative bands for the absorptions at 450 nm and 480 nm (Figure S19) supporting its chiral nature. However, detail studies are needed to understand the chiroptical properties of these macrocycles.
[00074] In vitro studies:
a. Biocompatibility: The synthesized material, compound (108) is intended to be used as an imaging agent and evaluation of biocompatibility is a pre-requisite. The compound (108) was evaluated for biocompatibility in L929 cells with varying concentrations, ranging from 10 µg/mL to 150 µg/mL and presented in Figure 3. The compound (108) was biocompatible in the evaluated range and the morphology was intact after 24 h exposure and the % cell viability was>90% with the highest concentration. From this study, it can be concluded that the synthesized material is highly biocompatible and further studies could be evaluated.
b. Cellular uptake: Qualitative in vitro cellular uptake of the compound (108) was performed in L929 and MDA MB 231 cells. It is necessary to evaluate the behaviour of material in the presence of cells i.e. the inherent property of fluorescence. The L929 and MDA MB 231 cells were incubated with compound (108) (10 µg/well) for 24 hours. CLSM data (Figure 8, 9, 10 and 11) revealed that the compound (108) was internalized in the cells and was able to exhibit strong fluorescence in far-red region. The mean fluorescence intensity per cell was calculated and it was observed that there was a significant difference (p<0.0001) between the compound (108) treated cells and control cells (Figure 11). DAPI staining experiments were carried out to understand the localisation of compound (108) in the cells. DAPI staining analysis showed (Figure 8) that the compound (108) was localised in cytoplasm of the cells with very good emission in far-red region and not present inside the nucleus.
[00075] Confocal Laser Scanning Microscopy (CLSM) was performed to evaluate the cellular uptake in L929 and MDA MB 231 cells. Briefly, the cells were seeded on sterile cover slips in a 12-well plate at a density of 25000 cells/well and incubated for 24 hours. BF2-25-oxasmaragdyrin-glucosamine conjugate (108) dissolved in DMSO was diluted appropriately and added to each well such that the final amount of compound in each well is10 µg/well. The cells were incubated for 24 h, followed careful washing with PBS and fixation. The fixation was carried out with 4% paraformaldehyde (15 min) at room temperature, followed by PBS wash. CLSM imaging (Olympus (IX 81) & Fluoview 500 and LEICA TCS SP8) was performed on fixed cells by using 488 nm excitation and 680-740 nm emission filter respectively.
[00076] 4', 6-diamidino-2-phenylindole (DAPI) staining: The fixed cells were stained with DAPI (10 µg/well) for 30 seconds and thoroughly washed with PBS. CLSM imaging was performed on fixed cells using 405 nm excitation and 410-454 nm emission filter for DAPI channel; 458 nm excitation and 680-732 nm emission filter for fluorescence channel of BF2-25-oxasmaragdyrin-glucosamine conjugate (108).
[00077] L, L-diphenylalanine conjugated BF2-oxasmaragdyrin self-assemblies: Another embodiment of the present invention discloses L, L-diphenylalanine conjugated BF2-oxasmaragdyrin and a process to synthesize said conjugate. As illustrated in figure 12, The process comprises synthesizing 25-oxasmaragdyrin containing p-carboxyphenyl (114) substituent at meso position using a reported method; hydrolysing 25-oxasmaragdyrin with Lithium hydroxide (LiOH) in Tetrahydrofuran (THF/H2O) to obtain (meso-(4-carboxy phenyl) BF2-25-oxasmaragdyrin) which is further reacted with protected L,L-diphenylalanine in presence of a coupling reagent 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIEA) in dichloromethane under inert atmosphere for at least 8 hours to obtain a reaction mixture to obtain methyl L,L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) which is further purified by basic alumina column chromatography; hydrolysing methyl L,L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) to obtain L,L-diphenylalanine conjugated BF2-oxasmaragdyrin (120).
[00078] Formation of self-assemblies: As illustrated in figure 13, to prepare spherical self-assemblies, compound (120) is dissolved in different water miscible solvents including acetonitrile, hexafluoro-2-propanol, acetone, DMSO and is diluted with water. The solvent is then removed either by evaporation or dialysis. Particles with various morphologies (spheres/rods/tubes/mixtures) and sizes are formed; aggregates are also observed under most conditions. An acetone-water mixture (1:1 ratio) results in monodisperse nanoparticles with spherical morphology (Fig. 14). The acetone is removed from the above solution under reduced pressure at 55°C to obtain self-assembled spherical particles (FF-BSC NP). DLS analysis revealed that the FF-BSC NP had a mean hydrodynamic diameter of 152.1 ± 13.7 nm and polydispersity (PDI) of 0.09 ± 0.05. The nanoparticles had a zeta potential of -22 ± 5 mV. Further, FEG-SEM and FEG-TEM analysis of the FF-BSC NP revealed spherical and controlled morphology with diameter of 123±33 nm as shown in Fig.1 and S30, respectively. These results indicate that the FF-BSC NP were monodisperse and exhibits spherical morphology.
[00079] ILLUSTRATIVE EXAMPLE
a. Synthesis of N-tert-butyl-L-phenyl alanine: Di-tert-butyl dicarbonate (0.68 mL, 3 mmol) was added dropwise to an ice cold suspension of L-phenyl alanine (0.165 g, 1 mmol) and K2CO3 (0.28g, 2 mmol) was suspended in 50 mL of dioxane-water (1:1 v/v) until a clear solution is formed. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic extract was dried on anhydrous Na2SO4 and rotary evaporated to give N-tert-butyl-L-phenyl alanine as a colourless solid (200 mg, 75% yield). 1H NMR (400 MHz, DMSO-d6) d 7.37 – 7.15 (m, 5H), 7.05 (d, J = 8.4 Hz, 1H), 4.16 – 4.02 (m, 1H), 3.01 (dd, J = 13.8, 4.6 Hz, 1H), 2.82 (dd, J = 13.8, 10.2 Hz, 1H), 1.31 (s, 9H). 13C NMR (101 MHz, DMSO-d6) d 174.0, 155.8, 138.4, 129.5, 128.5, 126.7, 78.5, 55.5, 36.9, 28.6.
b. L-Phenyalanine Methyl ester: L-Phenylalanine (0.5 g, 3.0 mmol) was dissolved in MeOH (50 mL), and SOCl2 (2 mL) was added dropwise at 0 °C under an inert atmosphere. The mixture was stirred for 6 h and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography to give the target L-Phenyalanine methyl ester as white solid (0.4 g, 73% yield). 1H NMR (500 MHz, D2O) d 7.58 – 7.39 (m, 3H), 7.38 – 7.25 (m, 2H), 4.45 (dd, J = 7.4, 5.9 Hz, 1H), 3.85 (s, 3H), 3.36 (dd, J = 14.6, 5.7 Hz, 1H), 3.25 (dd, J = 14.6, 7.6 Hz, 1H). 13C NMR (126 MHz, D2O) d 170.0, 133.7, 129.43, 129.40, 129.29, 129.24, 128.1, 128.0, 54.1, 53.6, 35.5.
c. Synthesis of methyl (tert-butoxycarbonyl)-L-phenylalanyl-L-phenylalaninate: N-(Tert-butoxycarbonyl)-L-phenylalanine (1.0 g, 5.58 mmol), methyl-L-phenylalaninate (1.47 g, 5.58 mmol) and HBTU (2.1 g, 1.5 mmol) were dissolved in 50 mL of dry dichloromethane. To the above solution, N,N-diisopropylethylamine (2.3 mL, 3 mmol) was added dropwise at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred for 6 hours. The progress of the reaction was monitored by TLC which showed the development of a less polar spot corresponding to the desired product. After the completion of reaction, the mixture was diluted with dichloromethane (50 mL) and washed with 1N HCl (3 x 30 mL) followed by saturated Na2CO3 solution (3 x 30 mL) and water (2 x 30 mL). The organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure to give the crude product as off-white semisolid. The crude product was purified by silica gel column chromatography (1:10 EtOAc/petroleum ether) to afford pure product as a colourless solid (1.66 g, 70% yield). 1H NMR (400 MHz, CDCl3) d 7.34 – 7.24 (m, 2H), 7.27 – 7.15 (m, 6H), 6.98 (dd, J = 7.3, 2.2 Hz, 2H), 6.24 (d, J = 7.7 Hz, 1H), 4.91 (s, 1H), 4.78 (q, J = 6.4 Hz, 1H), 4.32 (d, J = 8.1 Hz, 1H), 3.67 (s, 3H), 3.16 – 2.74 (m, 4H), 1.40 (s, 9H). 13C NMR (101 MHz, CDCl3) d 171.5, 170.70, 170.69, 135.8, 129.5, 129.4, 128.8, 128.7, 127.3, 127.1, 77.4, 53.4, 52.4, 38.4, 38.1, 28.4.
d. Synthesis of methyl L-phenylalanyl-L-phenylalaninate (116): A sample of methyl (tert-butoxycarbonyl)-L-phenylalanyl-L-phenylalaninate (35 mg, 0.9 mmol) was stirred in a mixture of trifluoroacetic acid/dichloromethane (1:1) for 30 minutes under nitrogen atmosphere at room temperature. The solvent was evaporated under reduced pressure and afforded methyl L-phenylalanyl-L-phenylalaninate (116) as a colourless solid and used without purification.
e. Synthesis of meso-(4-carboxy methyl ester phenyl) BF2-oxasmaragdyrin (112): meso-(4-Carboxy methyl ester phenyl) BF2-oxasmaragdyrin (112) was synthesized by the previously reported method. 1H NMR (400 MHz, CDCl3) d 10.26 (d, J = 4.5 Hz, 2H), 10.17 (dd, J = 4.5, 2.0 Hz, 2H), 9.50 (d, J = 4.4 Hz, 2H), 9.44 (s, 2H), 8.96 (dd, J = 4.5, 1.8 Hz, 2H), 8.63 (d, J = 8.3 Hz, 2H), 8.57 (d, J = 8.3 Hz, 2H), 8.22 (d, J = 7.9 Hz, 4H), 7.63 (d, J = 7.6 Hz, 4H), 4.09 (s, 3H), 2.72 (s, 6H), -4.04 (t, J = 10.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) d 167.6, 150.0, 144.2, 139.7, 138.1, 135.0, 134.5, 130.9, 129.8, 129.7, 128.5, 125.2, 124.1, 124.0, 122.3, 121.1, 120.6, 107.3, 52.6, 21.8. 11B NMR (128 MHz, CDCl3) d -12.6. 19F NMR (377 MHz, CDCl3) d -149.2. HRMS C45H33BF2N4O3 Calculated mass: 726.2616 [M+H]+ Measured Mass: 726.2605.
f. Synthesis of meso-(4-carboxyphenyl) BF2-oxasmaragdyrin (114): 1 N LiOH (15 mL) was added to a solution of meso-(4-carboxy methyl ester phenyl) BF2-oxasmaragdyrin (112) (60 mg, 0.8 mmol) in THF (15 mL). The reaction mixture was stirred for 6 hours before being quenched with 1N HCl and extracted with dichloromethane (50 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and rotary evaporated to afford meso-(4-carboxyphenyl) BF2-oxasmaragdyrin (114) (50 mg, 80% yield) which was used without further purification. 1H NMR (400 MHz, DMSO-d6) d 10.78 (d, J = 4.5 Hz, 2H), 10.71 (dd, J = 4.5, 2.0 Hz, 2H), 9.73 (d, J = 4.5 Hz, 2H), 9.59 (s, 2H), 9.02 (dd, J = 4.5, 1.8 Hz, 2H), 8.81 (d, J = 8.1 Hz, 2H), 8.61 (d, J = 8.2 Hz, 2H), 8.42 – 8.31 (m, 4H), 7.79 (d, J = 7.5 Hz, 4H), 2.77 (s, 6H), -3.77 – -4.53 (m, 2H). 13C NMR (101 MHz, DMSO-d6) d 168.0, 149.5, 142.9, 139.0, 138.3, 135.4, 134.4, 131.2, 130.9, 130.2, 130.0, 128.9, 126.0, 124.9, 123.9, 123.6, 122.3, 121.4, 117.4, 107.1, 66.3, 34.4, 29.7, 21.7.11B NMR (128 MHz, DMSO-d6) d -12.81. 19F NMR (377 MHz, DMSO-d6) d -147.61. HRMS C44H31BF2N4O3 Calculated mass: 712.2459 [M+H]+ Measured Mass: 712.2443.
g. Synthesis of methyl-L,L-diphenylalaninate conjugated BF2-oxasmaragdyrin (5):N, N-diisopropylethylamine (30 µL, 2.1 mmol) was added dropwise to a stirred solution of methyl-L-phenylalanyl-L-phenylalaninate (116) (22 mg, 0.7 mmol), meso-(4-carboxyphenyl) BF2-oxasmaragdyrin (114) (50 mg, 0.7 mmol) and HBTU (40 mg, 1.05 mmol) in dry dichoromethane (15 mL) at 0 °C under nitrogen atmosphere. The progress of the reaction was monitored by TLC. After 6 h, the reaction mixture was diluted with dichloromethane (50 mL) and the organic phase was successively washed with 1N HCl (3 x 30 mL), saturated Na2CO3 solution (3 x 30 mL) and water (2 x 30 mL). The organic phase was then dried over anhydrous Na2SO4, filtered and rotary evaporated to give a brown semisolid. The crude product was subjected to column chromatographic purification (SiO2; 7:10 dichloromethane/petroleum ether) to afford the target product (118) (40 mg) in 55% yield. 1H NMR (500 MHz, CDCl3) d in ppm: 10.34 (d, J = 4.4 Hz, 2H), 10.25 (d, J = 2.6 Hz, 2H), 9.57 (d, J = 4.3 Hz, 2H), 9.52 (s, 2H), 9.04 (s, 2H), 8.69 (d, J = 7.9 Hz, 2H), 8.30 (d, J = 7.6 Hz, 6H), 7.71 (d, J = 7.6 Hz, 4H), 7.50 – 7.40 (m, 4H), 7.30 (d, J = 6.7 Hz, 2H), 7.13 (dd, J = 13.5, 7.6 Hz, 2H), 6.46 (d, J = 7.5 Hz, 1H), 5.07 (dd, J = 14.0, 7.3 Hz, 1H), 4.91 (dd, J = 13.3, 6.5 Hz, 1H), 3.78 (s, 3H), 3.42 (dd, J = 13.9, 6.2 Hz, 1H), 3.32 (dd, J = 13.9, 7.6 Hz, 1H), 3.23 (dd, J = 13.8, 5.5 Hz, 1H), 3.10 (dd, J = 13.9, 6.6 Hz, 1H), 2.80 (s, 6H), -3.97 (s, 2H). 13C NMR (101 MHz, CDCl3) d in ppm: 171.4, 170.5, 167.2, 149.8, 139.5, 137.9, 136.5, 135.6, 135.0, 134.3, 130.7, 129.6, 129.3, 128.9, 128.7, 128.3, 127.2, 127.0, 125.0, 123.8, 122.1, 120.9, 120.4, 107.1, 54.8, 53.6, 52.4, 38.3, 38.0, 21.6. 11B NMR (128 MHz, CDCl3) d -12.77. 19F NMR (377 MHz, CDCl3) d -149.32. HRMS C63H51BF2N6O5 Calculated mass: 1020.3987 [M+H]+ Measured Mass: 1020.3985.
h. Synthesis of compound (120):1N LiOH (15 mL) was added to a solution of methyl-L,L-diphenylalaninate conjugated BF2-oxasmaragdyrin (118) (30 mg, 29 µmol) in THF (15 mL). The reaction mixture was stirred for 6 hours before being quenched with 1N HCl and extracted using ethylacetate (50 mL). All the organic extracts were combined, dried over anhydrous Na2SO4, filtered and rotary evaporated to afford compound (120) (25 mg, 84% yield) which was used without further purification. 1H NMR (600 MHz, Acetone-d6) d 10.51 (d, J = 4.3 Hz, 2H), 10.45 (dd, J = 4.3, 2.0 Hz, 2H), 9.60 (d, J = 4.4 Hz, 2H), 9.54 (s, 2H), 8.97 (dd, J = 4.4, 1.9 Hz, 2H), 8.66 (d, J = 7.9 Hz, 2H), 8.46 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 8.2 Hz, 1H), 8.32 – 8.26 (m, 4H), 7.99 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.3 Hz, 4H), 7.53 (d, J = 7.0 Hz, 2H), 7.41 – 7.35 (m, 4H), 7.31 – 7.20 (m, 4H), 5.22 (ddd, J = 9.7, 8.0, 5.1 Hz, 1H), 4.90 (td, J = 7.6, 5.2 Hz, 1H), 3.48 (dd, J = 14.4, 5.0 Hz, 1H), 3.36 (dd, J = 13.8, 5.2 Hz, 1H), 3.29 (dd, J = 14.5, 9.7 Hz, 1H), 3.21 (dd, J = 14.0, 7.5 Hz, 1H), 2.76 (s, 6H), -3.95 (t, J = 10.1 Hz, 2H). 13C NMR (151 MHz, Acetone-d6) d 172.7, 171.6, 167.1, 167.0, 150.1, 147.3, 145.4, 142.5, 142.1, 139.5, 139.3, 138.6, 138.5, 137.7, 135.0, 134.4, 134.3, 131.7, 130.9, 130.7, 129.9, 129.8, 129.7, 129.7, 129.1, 128.7, 128.7, 128.5, 127.9, 126.9, 126.8, 125.5, 124.7, 124.0, 123.0, 121.4, 121.2, 118.1, 114.1, 107.3, 78.7, 78.5, 78.3, 78.0, 55.5, 54.3, 37.7, 37.7, 34.7, 33.9, 32.1, 22.8, 21.1, 13.8. 11B NMR (128 MHz, Acetone-d6) d -12.77. 19F NMR (377 MHz, Acetone-d6) d -148.89. HRMS C45H33BF2N4O3 Calculated mass: 1006.3830 [M+H]+ Measured Mass:1006.3827.
[00080] Significant changes have been observed in absorption and fluorescence properties of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin (Figure 15e, 15f) after formation of self-assemblies. The absorption spectrum was broadened, and the corresponding absorption coefficients were reduced. Similarly, there was complete quenching of fluorescence due to aggregation induced quenching phenomena. To assess whether the particles have formed upon addition of water to L, L-diphenylalanine conjugated BF2-oxasmaragdyrin (120) in acetone or during the evaporation of acetone from the acetone/water mixture of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin (120), the absorption and fluorescence spectra (Figure 15e, 15f) of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin were analysed in acetone, acetone/water (1:1 v/v) and water (evaporation of acetone from acetone/water mixture). It was found that there is no change in absorption and fluorescence spectra from acetone to acetone/water mixture indicating the molecular behaviour of L, L-diphenylalanine conjugated BF2-oxasmaragdyrin. However, significant changes were observed in absorption and fluorescence spectra of L, L-diphenylalanine conjugated BF2-oxasmaragdyrindue to formation of self-assemblies upon evaporation of acetone from the mixture of acetone/water.
[00081] Photo-thermal transduction: The FF-BSC NP exhibited good photo-thermal properties upon irradiation with a 750 nm laser. The photo-thermal transduction potential of the nanoparticles was evaluated by constructing central composite design (CCD) using the three independent variables, i.e., sample concentration, laser power, and laser irradiation time. Response surface the interaction among the factors. The design layout, along with the independent and dependent variables is provided in figure 16, experimental results tabulated in Figure 16a. A quadratic model was selected (p = 0.0025), and the ANOVA revealed the model significant (p < 0.0001)and lack of fit insignificant (p = 0.9433), as shown in Figure 16b.Further, the diagnostic plots did not reveal any abnormalities. The coded equation for the model is provided below:
Y = +54.21 + 3.65A + 5.64B + 6.02C + 0.35AB + 0.97AC + 1.87BC + 2.51A2 – 3.12B2 + 0.98C
where A, B, C, and Y represent the concentration of the sample, laser irradiation time, power of laser irradiation, and temperature, respectively. The positive and negative sign indicates the synergistic and antagonistic effect on the factors, respectively. Coefficients with single factors represent the effect of that factor, whereas coefficients with two factors and second-order represent the effects of interaction among those factors and quadratic nature, respectively. The above equation depicts that the power of laser has the highest impact, followed by irradiation time and concentration of sample on the photothermal transduction potential of FF-BSC NP.
[00082] A 3D plot was constructed by setting the laser power at 750mW for the response, temperature (Fig. 17a). Further, numerical and graphical optimization was carried out, and parameters were selected to maintain a temperature of 52-60°C (Fig.17b). The design space was validated by selecting three random points, and the experimental results were consistent with predicted ranges (figure 18).
[00083] In vitro biocompatibility and haemocompatibility study: External agents like nanoparticles should be biocompatible in order to be further utilized as a therapeutic agent. Assessment of the in vitro biocompatibility of FF-BSC NP was carried out in L929 cells. Fluorescence-based Alamar blue assay was performed wherein viable cells convert non-fluorescent blue-colored resazurin to fluorescent red-colored resorufin. Evaluation of the fluorescence intensity to determine the % cell viability revealed that FF-BSC NP was biocompatible up to a concentration of 250 µg/mL. This result indicated that these nanoparticles possess excellent biocompatibility within the desired therapeutic concentration (Fig. 19a).
[00084] A nano-particulate formulation of FF-BSC NP is expected to encounter blood upon intravenous administration. Hence, the assessment of haemocompatibility is an absolute requirement. Hemo-incompatibility is often influenced by the physicochemical properties of the material e.g., size, charge, and shape. The blood sample treated with Triton X-100 resulted in complete hemolysis, and no RBC pellet was observed, as shown in Fig. 4b. Further, ESEM analysis of FF-BSC NP treated samples revealed that the morphology of RBCs was intact, and no significant difference was observed (Fig. 4c-e). The RBCs treated with varying concentrations of FF-BSC NP did not exhibit hemolysis, and the percentage of hemolysis was well below 5%, in accordance with the American Society for Testing and Materials (ASTM) E2524 guidance (Fig. 19f)
[00085] Cellular uptake, photothermal efficacy testing and mode of cell death: The optimal uptake of nanoparticles by the cells is a crucial factor, as inefficient uptake can compromise the therapeutic efficacy. Hence, the cellular uptake of FF-BSC NP was rigorously evaluated in 4T1 cells (murine breast cancer). Confocal laser scanning microscopy (CLSM) study revealed that FF-BSC NP was sufficiently internalized and the NIR fluorescence of the nanoparticles (680-740 nm) can be efficiently utilized for cellular imaging. Further, DAPI staining experiments confirmed that the FF-BSC NP were localized in the cytoplasm of the cells (Fig. 20 a-h). Additional validation of the nanoparticles uptake by the cells was performed with flow cytometry at different time-points in 4T1 cells. Flow cytometric analysis indicated that there was time-dependent uptake of nanoparticles by the cells (starting as early as one hour); this was evident from shift in the median fluorescence intensity peak of the nanoparticle incubated cells with respect to the control cells. Following 12-24 h of incubation, nanoparticle internalization was found to be almost 100% in comparison to the control cells (Fig. 20i). The efficient fluorescence and uptake of the nanoparticles displayed by the cells post nanoparticles administration forms the basis for utilization of these nano assemblies for further in vitro and in vivo efficacy and whole body imaging studies.
[00086] In vitro therapeutic efficacy evaluation is a prerequisite for in vivo efficacy testing. Thus, in vitro photothermal efficacy testing of FF-BSC NP was carried out in 4T1 cells. A combination of different nanoparticle concentrations (5, 10, and 15µg of NPs) coupled with varied laser exposure duration (3, 5 and 7 min) revealed that 5µg of NPs and seven min of laser exposure reduces cell viability by more than 50% as evaluated by Alamar assay. Increasing the nanoparticle concentration to 10µg showed a dose dependent increase in cytotoxicity even at lower laser exposure times (i.e., viability was <60% at 5 min of laser exposure). A further increase in the FF-BSC NP concentration to 15µg enabled reduced laser exposure (3 min) to be utilized to enhance cell death to >90% (p=0.0002). Cells exposed to material and laser alone did not exhibit changes in cell viability (>95%), Fig. 5j. Hence, the in vitro study revealed that the developed formulation has excellent photothermal ablation efficacy and potential for in vivo tumor ablation.
[00087] Apoptosis-inducing therapeutic agents are clinically more preferred as apoptosis is regarded as a cleaner mechanism of cell death as opposed to necrosis that might induce non-specific inflammation. To evaluate this, gold standard Annexin-V PI staining was performed to determine the mode of cell death after photothermal treatment with FF-BSC NP. Fractional positivity for Annexin-V, PI single stain, and Annexin-V PI dual staining was seen across all control groups (Cells only, cells+laser, cells+FF-BSC NP) as expected indicating basal cell death. On the other hand, the PTT treatment group showed >75% increase in the dual stained population and an 18% increase in the PI stained population indicating that the majority of cells are in the late apoptotic stage while a second fraction of the treated population was undergoing necrosis (Fig. 20k). This suggests that PTT using FF-BSC NP induces a cleaner mechanism of cell death with little evidence of necrosis, making the treatment procedure using the designed formulation more appealing for clinical application.
[00088] Whole-body imaging and in vivo tumor homing: FF-BSC NP retained the fluorescence, even after cellular uptake (Fig. 20a-h) indicating that the designed formulation can serve as an efficient cellular imaging agent. This provides a very strong base for possible utilization of FF-BSC NP as a whole-body imaging agent. In this context, non-invasive live animal fluorescence imaging was performed post intravenous (IV) administration of the nano-formulation in mice. Upon 24h post material administration, significantly (p=0.0001) strong fluorescence could be seen throughout the body of the mice, which is >100 fold-higher than the background body signal. This clearly suggests that such material holds a very strong potential for further exploration as a targeted imaging agent (Fig. 21a-c). Additionally, a desirable feature of any novel non-targeted nano-formulation is the capacity to passively accumulate in the tumor tissue taking advantage of the leaky tumor vasculature via enhanced permeability and retention (EPR) effect. As FF-BSC NP exhibited sufficient fluorescence, the tumor homing ability of FF-BSC NP was evaluated by qualitative time-dependent fluorescence imaging. Fluorescence imaging upto 72h after IV administration demonstrate that during initial time points (from 2-24h) FF-BSC NP fluorescence could be seen throughout the body. After 24h, at 48 and 72h, specific homing of a portion of nanoparticles was observed in the tumor (Fig. 21d). Further, to investigate the amount of material accumulated, the tumor tissue was harvested, homogenized, and the compound was extracted in DMSO. The fluorescence of the extract was recorded and the amount of FF-BSC was quantified. Absolute quantification clearly indicated that out of 300 µg of the FF-BSC NP injected, a portion (6-7 µg/g) of the compound was deposited in the tumor tissue (Fig. 21e). Though the quantity accumulated was significant (p=0.001), it may not be sufficient to perform effective photothermal tumor ablation via IV administration of the nanomaterial. Hence, photothermal ablation studies in the preclinical settings was done with intratumoral injection of FF-BSC NP.
[00089] Biodistribution and Single-Dose Toxicity Testing: A detailed understanding of the in vivo biodistribution and toxicity of the FF-BSC NP in preclinical animal models like mice, which possess close physiologic resemblance to that of humans, is imperative. Bio-distribution and toxicity assessment of FF-BSC NP was carried out by IV administering the nano formulation in swiss mice. Fourteen days post-administration of FF-BSC NP, ex vivo fluorescence imaging of the vital organs like lungs, liver, kidney, spleen, and heart was performed to reveal that the majority of the nanoparticles got deposited in these organs. Absolute quantification of the material deposited in these vital organs was also performed by extracting the compound from tissue homogenate in DMSO. The analysis indicated that that the nanoparticles had accumulated in liver, lung and spleen, and a lesser quantity accumulated in kidney and heart. These results suggested that the major portion of the injected material could not avoid opsonization by the components of the reticuloendothelial system (RES). This hypothesis also accounts for the inefficient passive tumor homing observed in this study. The data also correlates with the ex vivo fluorescence imaging data, further confirming the deposition of the injected compound in these organs. The presence of a portion of injected material in the kidney, also hints towards the possibility of renal clearance of FF-BSC NP (Fig. 22a-d). Further, histological examination of H&E (Hematoxylin and Eosin) stained section of the vital organs showed no visible pathological change in these organs as compared to the normal tissues (Fig. 22e-f). Additionally, the acute toxicity of the injected material was evaluated by serum biochemical analysis 72h after nanomaterial injection. Biochemical analysis revealed that the serum levels of alanine transaminase, aspartate aminotransferase, creatinine, and urea (a key determinant of the normal functioning of liver and kidney) in the material injected mice were in-line with the control mice (Fig. 22g). Hence, the formulated nanoparticles are highly biocompatible and non-toxic in these preclinical settings. These encouraging preclinical datasets on FF-BSC NP will serve the purpose to fast track bench to bedside translation of this prospective material.
[00090] In vivo photothermal ablation efficacy of FF-BSC NP: The in vivo photothermal efficacy of FF-BSC NP was tested in a 4T1-Luc2 xenograft breast tumor model using non-invasive bioluminescence imaging in mice. The FF-BSC NP (300µg) was injected intratumorally as it could be more relevant strategy for local application of PTT in a clinical setting. Upon exposure to laser beam on the tumor site the material showed excellent efficacy in vivo. In comparison to the control groups, a dramatic reduction in bioluminescence signal post PTT (p=0.0001), indicate drastic tumor mass ablation, which was further associated with no signs of weight loss, suggesting minimal or no therapy burden. However, the bioluminescence signal of control, laser control as well the material control groups showed progressive increase till day 5, indicating the non-toxic nature of laser/material used in those sets of mice. Post day 5, bioluminescence signal for these groups reached saturation of CCD camera detection capacity, therefore beyond this time point no further imaging was carried out for these animals. However, the animals were followed for survival analysis. Owing to permissible tumor size limitations well as progressive tumor necrosis beyond day 5, the mice in these groups were sacrificed by day 10. PTT treated group of animals were followed-up till day 30 and showed no enhancement in the bioluminescence signal (signal at ablated tumor site was equivalent to the background), highlighting effective and complete tumor ablation and no signs of tumor relapse post PTT. This effect has prolonged the overall survival of mice in the PTT treated group (Fig. 23a-c). Altogether, the in vivo data sufficiently indicate biocompatibility, NIRF imaging potential, and preclinical efficacy of nanoformulation (FF-BSC NP) for effective photothermal ablation of tumor.
[00091] Peptide conjugated BF2-oxasmaragdyrin: Another embodiment of the present invention discloses Peptide conjugated BF2-oxasmaragdyrin and a process to synthesize the same. Peptide conjugated BF2-Oxasmaragdyrin includes Poly-arginine (R9) conjugated BF2-Oxasmargdyrin (132) and CRGDK conjugated BF2-25-Oxasmargdyrin (128). In an aspect of the present invention
[00092] The target CRGDK-conjugated BF2-25-oxasmaragdyrin (128) and R9-conjugated BF2-25-oxasmaragdyrin (132) are prepared over a sequence of steps, as shown in Figure 24. The starting BF2-25-oxasmaragdyrin (122) containing a p-carboxy phenyl substituent at the meso position is synthesized by a reported method as described earlier. Solid supported (Rink amide) peptides with free N-terminal (124, 134) are prepared by automated microwave-accelerated solid phase peptide synthesis with fmoc-protected amino acid precursors (Troy J. Attard; Neil M. O’Brien-Simpson; Eric C. Reynolds, Int. J.Pept. Res.Ther. (2009) 15:69–79). The formation of the desired sequence is determined by sample cleavage of a resin aliquot (30 mg) with Trifluroacetic acid/Triisopropylsilane/water (TFA/TIPS/H2O) (3 mL, 95:3:2) followed by evaporation of solvent, precipitation of the peptide by cold Et2O (3 mL) and analysis of the crude precipitate by analytical RP-HPLC (Figure 25a and 25b) and mass spectrometry (Figure 25c and 25d). To obtain solid supported BF2-oxasmaragdyrin-peptide conjugates, corresponding solid supported (Rink amide) peptides with free N-termini are treated with a mixture containing BF2-25-oxasmaragdyrin (122), HATU, HOBt and DIEA in DMF (3 mL). After 24 h, the resin is washed with DMF and CH2Cl2 before being cleaved. Respective resins are cleaved, precipitated in cold Et2O and centrifugation provides the target crude peptide-BF2-oxasmaragdyrin constructs (128) and (132) as green solids. RP-HPLC and mass spectrometry analysis of crude CPP-conjugated BF2-oxasmaragdyrin (128) and (132) (Figure 25eand 25f) showed that the target conjugates (major) together with unidentified side products (Figure 25g and 25h). To obtain pure compounds, preparative RP-HPLC is performed on compounds (128), (132) using C4, C18 columns respectively under linear gradients of 0.1% TFA in MeCN. The fractions corresponding to the desired products are identified, combined together and lyophilized to give pure target peptide conjugated BF2-oxasmaragdyrins (128) and (132) as green foamy solids in 11% and 5% yields, respectively. The peptide conjugated BF2-oxasmaragdyrins (128, 132) were characterised by HRMS: The spectrum of CRGDK-conjugated BF2-oxasmaragdyrin (128) showed a molecular ion ([M+H]+) and R9-conjugated BF2-oxasmaragdyrin (132) presented as the triprotonated species ([M+3H]3+/3) (Figure 25h).
[00093] Absorption and fluorescence studies were performed on peptide conjugated BF2-oxasmaragdyrins (128) and (132) and the data are tabulated in Table S2. CRGDK- conjugated BF2-oxasmaragdyrin 128 absorbs in the visible region and emits in the NIR region (~717 nm) with moderate quantum yield (ff = 0.23) and an excited state lifetime of 4.74 ns (Figure 31 a-c). Conjugate 128 aggregates in the presence of water leading to a red shift with significant broadening in absorption spectra (Figure 31a) and complete quenching of fluorescence (Figure 31b). Similarly, R9-conjugated BF2-oxasmaragdyrin (132) also absorbs in the visible region and emits in NIR region (~719 nm) with a moderate quantum yield (ff = 0.24) and an excited state lifetime of 5.23 ns (Figure 31d-f). Conjugate (132) was also found to aggregate in water with concomitant broadening of the absorption spectrum with slight red shift. The quenching of fluorescence of conjugate (132) (Figure 31e) with a decrease in quantum yield in water (ff = 0.04) was observed compared to that in DMSO (ff = 0.24) due to aggregation induced quenching phenomena. A decrease in an excited state lifetime was also observed for conjugate (132) in water (ts = 3.32 ns) compared to that in DMSO (ts = 5.23 ns).
[00094] ILLUSTRATIVE EXAMPLE
a. CRGDK-peptide (124): The desired free N-terminal solid supported peptide was prepared by automated microwave-accelerated solid-phase peptide synthesis as described above. The formation of the desired product was confirmed by performing a sample cleavage of the solid supported peptide (30 mg) with TFA/TIPS/H2O (95:3:2) followed by precipitating the peptide in cold Et2O (2 mL). The precipitate was centrifuged, and the residue was analyzed by analytical HPLC with a gradient of 0-100% buffer B/buffer A over 40 min. A single peak at 9.9 min retention time (Figure S1) was observed. LRMS C21H39N9O8S Mass Calculated: 577.65 [M+H]+ Mass Observed: 577.3.
b. R9-peptide (134): The desired free N-terminal solid supported peptide was prepared by automated microwave-accelerated solid-phase peptide synthesis as described above with minor modification. The coupling was carried out at room temperature for 40 min followed by a 25W microwave burst at 75 °C for 10 min. The formation of the desired product was confirmed by performing a sample cleavage of the solid supported peptide (30 mg) by TFA/H2O (97:3) followed by precipitating the peptide by cold Et2O (2 mL). The precipitate was centrifuged, and the residue was analysed by analytical HPLC with a gradient of 0-40% buffer B/buffer A over 40 min. A single peak at 13.1 min retention time (Figure S7) was observed. LRMS C54H111N37O9 Mass Calculated: 1054.42 [(M+H+6CF3COOH)/2]+ Mass Observed: 1054.1.
c. CRGDK-conjugated BF2-oxasmaragdyrin (128): To the free N-terminal solid supported peptide a, a mixture of compound (122) (107 mg, 0.15 mmol), HATU (56.7 mg, 0.145 mmol), HOBt (20 mg, 0.145 mmol) and DIEA (80 µL, 0.45 mmol) in DMF (3 mL) was added. The mixture was shaken at room temperature for 24 hours and the solvent was removed via vacuum filtration. The residue was washed with DMF (8 × 10 mL), CH2Cl2 (8 × 10 mL) and dried under vacuum. The crude conjugate 1a was obtained by cleavage of the resin by with 10 mL of TFA/TIPS/H2O (95:3:2) followed by precipitating the crude conjugate (128) in cold Et2O (20 mL). The precipitate was centrifuged, and the residue was analyzed by analytical HPLC on a C4 column with a gradient of 10-100% buffer A/buffer B over 30 min. The desired conjugate (128) eluted at 27.2 min and was confirmed by mass spectrometry. Conjugate (128) was purified by preparative HPLC on a C4 column and the desired fractions were collected, lyophilized to give pure CRGDK-conjugated BF2-oxasmaragdyrin (128) as green solid. (14 mg, yield: 11%). HRMS C65H70BF2N14O9S Calculated mass: 1271.5237 [M+H]+ Measured Mass: 1271.5101.
d. R9-conjugated BF2-oxasmaragdyrin (132): To the free N-terminal solid supported peptide (134), a mixture of compound (122) (107 mg, 0.15 mmol), HATU (56.7 mg, 0.145 mmol), HOBt (20 mg, 0.145 mmol) and DIEA (80 µL, 0.45 mmol) in DMF (3 mL) was added. The mixture was shaken at room temperature for 24 hours and the solvent was removed via vacuum filtration. The residue was washed with DMF (8 × 10 mL), CH2Cl2 (8 × 10 mL) and dried under vacuum. The crude conjugate (132) was obtained by cleavage of the resin with 10 mL of TFA/TIPS/H2O (95:3:2) followed by precipitating the crude compound (132) in cold Et2O (20 mL). The precipitate was centrifuged, and the residue was analyzed by analytical HPLC on a C18 column with a gradient of 0-100% buffer A/buffer B over 45 min. The desired compound (132) was eluted at 23.6 min which was confirmed by mass spectrometry (Figure S9 and S10). Conjugate (132) was purified by preparative HPLC on a C18 column and the desired fractions were collected, lyophilized to give pure R9-conjugated BF2-oxasmaragdyrin (132) as green solid (11 mg, yield: 5%). HRMS C98H140BF2N41O11 Calculated mass: 706.7343 [(M+3H)/3]+, Measured Mass: 706.7313.
[00095] Biocompatibility: The conjugates (128, 132) were designed to be used as NIRF imaging agents; hence, evaluation of biocompatibility was essential. The CPP conjugated BF2-oxasmaragdyrins (128, 132) were evaluated for biocompatibility in L929 cells under varying concentrations, ranging from 10 µg/mL to 125 µg/mL. The results are presented in Figure S12. CRGDK-conjugated BF2-oxasmaragdyrin (128) was found to be biocompatible across the evaluated range and cell morphology remained intact at the end of study. Additionally, the percentage of cell viability was ~90% up to a concentration of 125 ug/mL of conjugate 128. Similarly, R9-conjugated BF2-oxasmaragdyrin (132) showed good biocompatibility up to 80 µg/ml (>80% cell viability). The lower cell viability beyond 80 µg/ml is attributed to the high positive charge of conjugate (132) due to the presence of multiple arginine residues. As seen from Figure S12b, the cell viability of conjugate (132) follows similar trend as that of the linear polyarginine sequence (132) indicating that the lower cell viability is to manifest from the polyarginine sequence. Successful completion of the biocompatibility studies with conjugates (128, 132) allowed further biological evaluation.
[00096] Cellular uptake: To evaluate the fluorescence behaviour of the materials under biological conditions, qualitative in vitro cellular uptake of the conjugates 1a-b was performed in MDA-MB-231 cells. CLSM data (Figure 26 and Figure 27) revealed that the material was internalized and localized in cytoplasm of cells in 24 h. A strong fluorescence in the NIR region was observed indicating the NIRF imaging potential of compounds.
[00097] Photothermal transduction studies: Photothermal transduction studies were performed to evaluate the photothermal efficacy of compound (132). The concentration of sample, laser power, and laser exposure time plays a crucial role in the photothermal transduction of a material. By utilizing a central composite design (CCD), it was possible to evaluate the impact of individual and interaction factors on the selected response. The design layout along with the experimental results are presented in Figure 28a and Figure 28b. Quadratic model was selected (p <0.05) based on sequential sum of squares, as shown in Table S5. ANOVA for the selected model indicates the model significant (p<0.0001), and the lack of fit insignificant (p>0.05), as shown in Figure 28c.The equation used for evaluating the relative impact of various factors on response is provided below
Temperature = +51.77 + 5.54A + 4.21B + 8.26C + 2.11AC - 6.87C2
A, B, and C indicates concentration of sample, laser power and laser irradiation time, respectively. From this equation, it can be understood that the factor C has the highest impact on response, followed by A and B. Further, diagnostics revealed no anomalies with the selected model, and the contour as well as 3D plots are presented in Figure 29a-b. Design space (yellow region) for the selected model was constructed by selecting the response range as 52-65 °C (Figure 29c). The design space was evaluated by selecting three random points, and the experimental results were within the predicted ranges (Figure 30). From the results, it can be concluded that R9-conjugated BF2-oxasmaragdyrin (132) possesses excellent photothermal properties and could be utilized for photothermal cancer therapy
[00098] Photothermal conversion efficiency: Photothermal conversion efficiency of the material was calculated using equation S1. The sample was irradiated with NIR laser and the temperature increment was recorded. The temperature drop was recorded after turning off the laser and data was plotted as shown in Figure29e-f. Based on the calculations, the photothermal conversion efficiency of compound (132) was found to be 72.3%. These results indicate that the conjugate (132) possesses excellent photothermal properties and conversion efficiency, which could potentially be exploited for photothermal cancer therapy.
[00099] Multi-cycle photothermal transduction potential: Most organic dyes are sensitive to light and possess poor thermal stability which limits their use for PTT. The multi-cycle photothermal transduction potential and stability of conjugate (132) was evaluated by irradiating the same sample with a 750 nm laser over several cycles. Conjugate (132) showed consistent temperature increment over four cycles of exposure (5 min cycle each) (Figure 29). Photobleaching results in loss of fluorescence and photothermal transduction potential of organic compounds and R9-conjugated BF2-oxasmaragdyrin (132) exhibited no signs of photobleaching and excellent stability.
[000100] The photophysical properties of the conjugates were thoroughly evaluated and both conjugates were found to be stable, absorb in the UV-Vis and emit in the NIR region, possess moderate quantum yields with singlet state lifetimes, and be biocompatible. CLSM experiments also revealed that the conjugates (128) and (132) were internalized into a MDA-MB-231 cell line within 24 hours and emitted strong NIR fluorescence inside the cells. The property of NIR emission could be exploited for in vivo whole-body or site-specific imaging. Additionally, the R9-conjugated BF2-oxasmaragdyrin (132) when irradiated with a 750 nm laser exhibited photothermal efficiency of 72.3% and possessed high stability with no concomitant bleaching observed over multiple cycles of irradiation. R9-conjugated BF2-oxasmaragdyrin (132) could be used as a potential theragnostic.
[000101] Yet another embodiment of the present invention discloses meso(4-aminophenyl) BF2-oxasmaragdyrin and its amino acid conjugates. As illustrated in figure 32 synthesis of meso(4-aminophenyl) BF2-oxasmaragdyrin is carried out by adding Hydrazine Hydrate (5 mL) to a pre-heated solution of meso(4-nitrophenyl) BF2-oxasmaragdyrin (100 mg, 1.3µmol), 10% Pd/C (30mg) in ethanol (10 mL) and it is refluxed for 6 hours. After completion of the reaction, the mixture is filtered, and the solution is rotary evaporated to give crude solid. The crude solid is then dissolved in chloroform and washed with 10% HCl (2 × 20 mL). The organic fractions are collected, rotary evaporated, followed by basic alumina column chromatographic purification to yield pure meso(4-aminophenyl) BF2-oxasmaragdyrin. Yield: 95 mg; 90%. 1H NMR (400 MHz, Chloroform-d) d 10.18 (d, J = 4.5 Hz, 2H), 10.09 (dd, J = 4.4, 2.1 Hz, 2H), 9.55 (d, J = 4.4 Hz, 2H), 9.36 (s, 2H), 8.87 (dd, J = 4.4, 2.0 Hz, 2H), 8.39 (d, J = 8.4 Hz, 2H), 8.26 (d, J = 8.0 Hz, 4H), 7.71 – 7.58 (m, 4H), 7.27 (d, J = 5.7 Hz, 2H), 4.13 (s, 1H), 2.78 (s, 6H), -3.28 – -3.36 (m, 2H).HRMS C43H32BF2N5O, Calculated mass [M+H]+ = 683.2670, Observed mass = 683.2670.
[000102] As illustrated in figure 32 The amino acid conjugates of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSN) are synthesized by adding dropwise N,N-diisopropylethylamine (30 µL, 2.1 mmol) to a stirred solution of N-protected amino acid, meso-(4-amino phenyl) BF2-oxasmaragdyrin (141) (50 mg, 0.7 mmol) and HBTU (40 mg, 1.05 mmol) in dry dichloromethane (15 mL) at 0 °C under nitrogen atmosphere. The amino acids are selected from alanine, glycylglycine and tryptophan. The progress of the reaction is monitored by TLC. After 6 h, the reaction mixture is diluted with dichloromethane (50 mL) and the organic phase is successively washed with 1N HCl (3 x 30 mL), saturated Na2CO3 solution (3 x 30 mL) and water (2 x 30 mL). The organic phase is then dried over anhydrous Na2SO4, filtered and rotary evaporated to give a brown semisolid. The crude product is subjected to column chromatographic purification (SiO2; 7:10 dichloromethane/petroleum ether) to afford the target product (142) (40 mg) in 10-15% yield. The amino acid conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin synthesized are (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA), (tert-butoxycarbonyl)-L,L-glycylglycine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB) and N-acetyl-L-tryptophan conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT).
[000103] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA) (143):10 mg, 15% Yield. 1H NMR (400 MHz, Chloroform-d) d 10.24 (d, J = 4.6 Hz, 2H), 10.15 (dd, J = 4.5, 2.1 Hz, 2H), 9.55 (d, J = 4.4 Hz, 2H), 9.44 (s, 2H), 8.95 (dd, J = 4.4, 2.0 Hz, 2H), 8.66 (s, 1H), 8.57 (d, J = 8.4 Hz, 2H), 8.28 (d, J = 8.3 Hz, 4H), 8.13 (d, J = 8.4 Hz, 2H), 7.82 (dd, J = 7.4, 1.0 Hz, 2H), 7.76 – 7.60 (m, 6H), 7.50 – 7.42 (m, 2H), 7.37 (td, J = 7.4, 1.3 Hz, 2H), 4.61 (t, J = 6.6 Hz, 2H), 4.34 (t, J = 6.8 Hz, 1H), 2.79 (s, 6H), -3.70 (s, 2H).HRMSC61H47BF2N6O4, Calculated mass [M+H]+ =976.3724, Observed mass = 976.3724.
[000104] (tert-butoxycarbonyl)-L,L-glycylglycine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB ) (144): 13 mg, 12% Yield. 1H NMR (400 MHz, Chloroform-d) d 10.25 (s, 2H), 10.15 (s, 2H), 9.55 (s, 2H), 9.44 (s, 2H), 8.95 (s, 2H), 8.88 (s, 1H), 8.56 (s, 2H), 8.28 (d, J = 6.1 Hz, 6H), 7.69 (d, J = 7.1 Hz, 4H), 5.31 (s, 2H), 4.34 (s, 2H), 3.98 (s, 2H), 3.65 (s, 2H), 2.79 (s, 6H), 1.56 (s, 9H), -3.69 (s, 2H).
[000105] N-acetyl-L-tryptophan conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT) (145): 8 mg, 10% Yield. 1H NMR (400 MHz, Chloroform-d) d 10.24 (d, J = 4.5 Hz, 2H), 10.16 (d, J = 3.9 Hz, 2H), 9.80 (q, J = 2.9 Hz, 2H), 9.52 (d, J = 4.4 Hz, 2H), 9.43 (s, 2H), 8.95 (dd, J = 4.5, 1.9 Hz, 2H), 8.49 (d, J = 8.2 Hz, 2H), 8.28 (d, J = 7.7 Hz, 4H), 8.22 (s, 2H), 7.93 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 7.5 Hz, 4H), 7.54 – 7.45 (m, 2H), 6.56 (s, 1H), 5.22 – 5.03 (m, 2H), 3.20 (q, J = 7.2 Hz, 3H), 2.79 (s, 6H), -3.70 (s, 2H).
[000106] Although the present disclosure has been described in the context of certain aspects and embodiments, it will be understood by those skilled in the art that the present disclosure extends beyond the specific embodiments to alternative embodiments and/or uses of the disclosure and obvious implementations.

,CLAIMS:We Claim:
1. A compound comprising, a compound of formula (I) or formula (II):
(I) (II)
wherein R1 comprises of6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine, L, L-diphenylalanine, or cell penetrating polypeptides; and
R comprises of (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated of meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA), (tert-butoxycarbonyl)-L,L-glycylglycine conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB), N-acetyl-L-tryptophan conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT), wherein these compounds are used for bioimaging and photothermal therapy.
2. The compound as claimed in claim 1, wherein cell penetrating polypeptides are selected from polyarginine (R9) and Cysteine-Arginine-Glycine-Aspartic Acid-Lysine (CRGDK).
3. A process for synthesizing BF2-25-Oxasmaragdyrin conjugates, the process comprising:
synthesizing BF2-25-Oxasmaragdyrin (102) containing p-carboxy phenyl;
adding BF2-25-oxasmaragdyrin (102) and a coupling reagent in a solution of 6-amine-6-deoxy-1-O-Boc-2-N-Boc-ß-D-glucosamine (104) under nitrogen atmosphere to form a mixture wherein BF2-25-oxasmaragdyrin (102) and the coupling reagent are dissolved in Dichloromethane (CH2Cl2);
stirring the mixture for at least 24 hours to obtain crude Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106);
obtaining purified Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106) by basic alumina column chromatography as a green solid;
treating purified Boc protected glucosamine conjugated BF2-25-oxasmaragdyrin (106) with a mixture of organic solvents for at least 15 minutesfollowed by neutralising with methanolic ammonia;
Monitoring the progress of the reaction by TLC analysis;
removing solvent under reduced pressure to obtain crude BF2-25-oxasmaragdyrin-glucosamine conjugate (108);
washing the crude compound with water to obtain purified BF2-25-oxasmaragdyrin-glucosamine conjugate (108);
4. The process as claimed in claim 3, wherein the mixture of organic solvent is composed of Dichloromethane (CH2Cl2) and Trifluoroacetic Acid (TFA) mixed in a ration of 3:1.
5. The process as claimed in claim 3, wherein the Glucosamine conjugated BF2-25-Oxasmaragdyrin is washed with water (3 x 50 ml) to remove side products and obtain a pure conjugate.
6. The process as claimed in claim 3, wherein yield of Glucosamine conjugated BF2-25-Oxasmaragdyrin is at least 50-80%.
7. The process as claimed in claim 3, wherein the coupling reagent can be selected from 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium Hexafluorophosphate (HCTU).
8. The process as claimed in claim 3, wherein BF2-25-oxasmaragdyrin-glucosamine conjugate (108) is further subjected to the process of acetylation to confirm the presence of intact glucosamine moiety.
9. The process as claimed in claim 8, wherein the acetylation process comprises treating BF2-25-oxasmaragdyrin-glucosamine conjugate (108) in dichloromethane with an excess of acetic anhydride in presence of triethylamine and catalytic amount of 4-N,N-dimethylaminopyridine for 4 hours at room temperature followed by workup and silica gel column chromatography to afford compound (110) as a green solid.
10. The process as claimed in claim 1, wherein the Glucosamine conjugated BF2-25-Oxasmaragdyrin absorb in visible far-red region and emit in far-red region.
11. The process as claimed in claim 1, wherein the Glucosamine conjugated BF2-25-Oxasmaragdyrin exhibitsfluorescence properties in NIR region (~710 nm).
12. A process for synthesizing BF2-25-Oxasmaragdyrin conjugates, the process comprising:
synthesizing BF2-25-oxasmaragdyrin;
synthesizing methyl L-phenylalanyl-L-phenylalante;
hydrolysing BF2-25-oxasmaragdyrin with Lithium hydroxide (LiOH) in Tetrahydrofuran (THF/H2O) to obtain (meso-(4-carboxy phenyl) BF2-25-oxasmaragdyrin) which is further reacted with protected L,L-diphenylalanine in presence of a coupling reagentand N,N-diisopropylethylamine (DIEA) in dichloromethane under inert atmosphere for 8 hours to obtain a reaction mixture;
obtaining methyl L, L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) by basic alumina column chromatography; and
Hydrolysing methyl L, L-diphenylalanate conjugated BF2-25-oxasmaragdyrin (118) to obtain L, L-diphenylalanine conjugated BF2-oxasmaragdyrin (120).
13. The process as claimed in claim 12, wherein the BF2-25-oxasmaragdyrin is synthesized using a reported method.
14. The process as claimed in claim 12, wherein L,L-diphenylalanine conjugated BF2-oxasmaragdyrin (120) is further processed to synthesize self assemblies.
15. The process as claimed in claim 12, wherein the coupling reagent is selected from 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium Hexafluorophosphate (HCTU).
16. The process as claimed in claim 13, wherein the self assemblies are synthesised by
dissolving the conjugate in acetone and adding water in the ratio of 1:0.1 to 1:25; removing acetone at room/elevated temperature under atmospheric/reduced pressure conditions to obtain a suspension of self-assembled spherical particles (121) in water with varying sizes.
Dissolving the conjugate in different solvents: Hexafluoroisopropanol (HFIP) or Dimethyl sulfoxide (DMSO) and diluting with water (1:1 to 1:25 ratios), removing the solvent by various methods such as evaporation and dialysis;
17. The process as claimed in claim 16, wherein the solvents are selected from acetonitrile or acetone.
18. The process as claimed in claim 16, wherein the solvent and water are mixed in a ratio of 1:0.1 to 1:25.
19. A process for synthesizing conjugates of BF2-Oxasmaragdyrin, the process comprising:
synthesizing BF2-25-oxasmaragdyrin containing p-carboxy methylphenyl substituent at meso position (122),
preparing a solid supported (Rink amide resin) peptides with free N-terminal (124, 134) by automated microwave-accelerated solid phase peptide synthesis;
adding a mixture of BF2-25-oxasmaragdyrin, a coupling reagent, HOBt and DIEA in an organic solvent to the free N-terminal solid supported peptide;
shaking the mixture at room temperature;
removing the solvent via vacuum filtration to obtain a residue;
washing the residue with Dimethylformamide (DMF) and (CH2Cl2) followed by drying under nitrogen atmosphere;
obtaining crude polypeptide BF2-25-Oxasmaragdyrin conjugate by cleavage of the resin with Trifluoroacetic acid/ Triisopropylsilane/water (TFA/TIPS/H2O) followed by precipitating the crude polypeptide conjugate 1a in cold Et2O;
precipitating the crude polypeptide BF2-25-Oxasmaragdyrin conjugate in cold Diethyl ether (Et2O).
20. The process as claimed in claim 19, wherein the coupling reagent is selected from 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium Hexafluorophosphate (HCTU).
21. The process as claimed in claim 19, wherein the organic solvent is Dimethylformamide (DMF).
22. The process as claimed in claim 19, wherein Trifluoroacetic acid, Triisopropylsilane, and water are mixed in a ratio of 95:3:2.
23. A process for synthesizing conjugates of BF2-Oxasmaragdyrin, the process comprising of:
adding Hydrazine Hydrate to a pre-heated solution of meso(4-nitrophenyl) BF2-oxasmaragdyrin, 10% Pd/C in ethanol to obtain a mixture;
refluxing the mixture for a pre-set time;
filtering the mixture after completion of the reaction and processing the mixture in a rotary evaporator to obtain a crude solid,
dissolving the crude solid in an organic solvent followed by washing with an acid solution;
collecting, rotary evaporating and purifying the organic fractions to obtain pure meso(4-aminophenyl) BF2-oxasmaragdyrin;
adding dropwise N,N-diisopropylethylamine to a stirred solution of N-protected amino acid, meso-(4-amino phenyl) BF2-oxasmaragdyrin (141) and a coupling reagent in dry dichloromethane at 0 °C under nitrogen atmosphere to obtain a second mixture;
monitoring the progress of the reaction by TLC;
diluting the second mixture with dichloromethane;
washing organic phase with 1N HCl, saturated Na2CO3¬ solution and water;
drying the organic phase over Na2SO4;
filtering and rotary evaporating the organic phase to obtain crude brown semisolid amino acid conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin;
purifying the crude compound by column chromatography to obtain pure amino acid conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (142).
24. The process as claimed in claim 23, wherein the preset time is atleast 6 hours.
25. The process as claimed in claim 23, wherein the organic solvent is chloroform.
26. The process as claimed in claim 23, wherein the acid solution is 10% HCl.
27. The process as claimed in claim 23, wherein the coupling reagent is selected from HATU, HBTU and HCTU.
28. The process as claimed in claim 23, wherein the amino acids are selected from alanine, glycine and tryptophan.
29. The process as claimed in claim 23, wherein the amino acid conjugates of meso(4-aminophenyl) BF2-oxasmaragdyrin are (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNA) (143), (tert-butoxycarbonyl)-L,L-glycylglycineconjugatedmeso(4-aminophenyl) BF2-oxasmaragdyrin (BSNGGB) (144) and N-acetyl-L-tryptophan conjugated meso(4-aminophenyl) BF2-oxasmaragdyrin (BSNT) (145).