1081-1085A condensation reaction between 1-cyclobutyl-3,4-dihydroisoquinoline and thioglycolic acid has been carried out using
DCC yields 10b-cyclobutyl-5,6-dihydro-2H-thiazolo [2, 3-a] isoquinolin-3(10bH)-one [CBIQTGA]. The synthesized
compound is characterised by using spectroscopic techniques. The interaction of CBIQTGA with bovine serum albumin has
been studied using UV spectroscopy, spectrofluorimetry and circular dichroism techniques

Kulal, Dnyaneshwar K

Navale, Dinesh N

Ramana, M M V

Ranade, Prasanna B

Sheikh, Mohmad V

Zote, Santosh W

NOPR

Indian Journal of Chemistry Vol. 60A, August 2021, pp. 1081-1085 Synthesis and spectroscopic investigation of binding of novel Thiazolo [2, 3-a] isoquinoline analog with bovine serum albumin Prasanna B Ranadea, Dinesh N Navalea, Santosh W Zoteb, Dnyaneshwar K Kulalc, Mohmad V Sheikhc & M M V Ramanad,* aDepartment of Chemistry, Vivekanand Education Society’s College of Arts, Science and Commerce, Chembur, Mumbai 400 071, India bDepartment of Chemistry, Sathaye College, Vile Parle (East), Mumbai 400 057, IndiacDepartment of Chemistry, Ramnarian Ruia Autonomous College, Matunga (East) Mumbai 400 019, India dDepartment of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai 400 098, India*E-mail: prasannaranade@yahoo.comReceived 05 February 2021; revised and accepted 16 July 2021 A condensation reaction between 1-cyclobutyl-3,4-dihydroisoquinoline and thioglycolic acid has been carried out using DCC yields 10b-cyclobutyl-5,6-dihydro-2H-thiazolo [2, 3-a] isoquinolin-3(10bH)-one [CBIQTGA]. The synthesized compound is characterised by using spectroscopic techniques. The interaction of CBIQTGA with bovine serum albumin has been studied using UV spectroscopy, spectrofluorimetry and circular dichroism techniques. Keywords: DCC, Cyclisation, BSA, Fluorescence, Circular dichroism Bovine serum albumin (BSA) is the most abundant and carrier protein which lies in blood plasma1. BSA consists of an amino acid chain of 537 residues. BSA gets divided into nine loops due to disulfide bonds into three structurally homologous domains. Intrinsic fluorescence is produced by tryptophan 134 which is located on the protein surface and tryptophan 212 lies in a hydrophobic binding pocket of protein2-4. Hence quenching of fluorescence is made useful in determining the binding affinities. Heterocyclic compounds are well known for their versatile biological activites5. Nitrogen and sulphur containing heterocycle are profoundly used as antimalarial6 and antibacterial7 agents, respectively.  Thiazolidinone8 is a five member heterocycle found in various drugs and exhibits several biological activites. Thiazolidinones are known to exhibit anti HIV9, anticancer10, antimalarial11, antitubercular12 activites. Besides these they are also reported for their anticonvulsant13, antihypnotic14, antifungal15, antibacterial16 and anti-inflammatory17 activites. Some of thiazolidinones are known to possess insecticidal activity18. Recently thiazolidinone derivatives were reported for their antiproliferative19 activity. Moreover thiazolidinone-peptide hybrids were reported for their antidengue activity20. Thiazolidinone is the main functionality in known diabetic drugs including Rosiglitazone and Pioglitazone21. Rosiglitazone was investigated for their interaction with BSA22.  Further the interaction of various thiazolo derivatives with BSA is also reported.23-27 The study of interaction of proteins with small molecules is important in the field of pharmacokinetics and pharmacodynamics28. BSA has structural analogy to human serum albumin29. The present study deals with the synthesis of new thiazolo derivative with its characterization by using different spectroscopic methods30. The synthesized derivative 10b-cyclobutyl-5,6-dihydro-2H-thiazolo [2, 3-a] isoquinolin-3(10bH)-one [CBIQTGA] wasfurther studied for its binding interaction with BSAusing fluorescence spectroscopy, circular dichroism(CD) spectroscopy.31Materials and Methods Materials All required chemicals and BSA was purchased from Sigma-Aldrich India and used without further purification31. BSA stock solution was prepared by dissolving BSA in Tris-HCl buffer with pH= 7.2. In all the experiments, double distilled water was used for dilution. In 5% ethanol, 10b-cyclobutyl-5,6-dihydro-2H-thiazolo[2,3-a] isoquinolin-3 (10bH) -one (CBIQTGA) stock solution (2.0×10-3 mol L-1) was prepared. All required solvents were of analytical grade and purchased from Sigma Aldrich, India. INDIAN J CHEM, SEC A, AUGUST 2021   1082Synthesis of CBIQTGA A mixture of 1-cyclobutyl-3,4-dihydroisoquinoline (0.1 mol), thioglycolic acid (0.3 mol) were stirred in tetrahydrofuran at 0-5 °C for 15 min and then  N,N-dicyclohexylcarbodiimide (0.4 mol) was added30. The reaction mixture was stirred for additional 50 min  at room temperature. The dicyclohexylurea was removed by filtration and filtrate was concentrated to dryness. The residue was taken in ethyl acetate and it was washed with 10% aq. citric acid, water and  10% aq. sodium hydrogen carbonate. Ethyl acetate extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue obtained was purified by column chromatography over silica gel (petroleum ether: chloroform 70:30) to obtain white solid. The reaction mechanism is shown in Scheme 1. The characterization spectra are given in Supplementary Data, Figs. S1-S4. Characterisation: Yield: 71%; melting point: 91 °C; IR (cm-1): 2971.41, 1667.28, 1411.92, 1330.27, 1293.09, 751.94; 1H NMR (CDCl3): 7.04-7.25 (m, 4H aromatic), 1.72-1.85 (m, 2H, aliphatic), 1.91-2.02 (m, 4H, aliphatic), 2.27-2.33 (m, 1H, aliphatic), 2.80-2.82 (d, 1H, aliphatic  J=6 Hz), 3.00-3.12 (m, 1H, aliphatic), 3.23-3.25 (d, 1H, aliphatic J=6 Hz), 3.56-3.61 (dd, 1H, aliphatic J=15 Hz), 3.75-3.80 (dd, 1H, aliphatic  J=15 Hz), 4.39-4.46 (m, 1H, aliphatic); 13C NMR (CDCl3): 16.75, 23.90, 25.42, 27.97, 34.26, 37.42, 46.44, 74.05, 125.66, 126.51, 127.48, 129.27, 131.42, 139.71, 169.24; Mass spectra: M+ at m/z: 260.57.  Preparation of BSA and CBIQTGA solution BSA solution (10 µM) was prepared based on  a molecular weight of 66,000 Dalton in 0.1 M phosphate buffer31. Initially a stock solution (50 µM) of CBIQTGA was prepared and different concentrations (10, 20, 30, 40 and 50 µM) were prepared by serial dilution of the stock solution.  Characterisation techinques Shimadzu RF-5301 PC spectrofluorophotometer was used to record the fluorescence. At 280 nm the excitation wavelength was set and emission was recorded in the range of 300-450 nm. Slit widths were kept at 5 nm each throughout the experiment.  At 293, 298, and 310 K temperatures with different concentrations of CBIQTGA solution in a 1 cm  quartz cell, the fluorescence emission of BSA was recorded. An incubation time of 30 min was set  for all fluorescence measurements for different temperatures31-32. Shimadzu UV-2401 PC spectro-photometer was used to record UV-visible spectra in the absence and presence of CBIQTGA at 298 K in the range of 250-310 nm. The quartz cuvette with  1 cm path length was used. The absorbance Tris-HCl buffer solution was recorded and subtracted. Jasco,  J-815, CD spectrometer was used to record the circular dichroism measurements. CD spectrum measurements for BSA were recorded in the far UV region with scan speed of 10 nm/min, in presence and absence of CBIQTGA. The band width was fixed at 5 nm31-33.  Results and Discussion  UV-visible spectra of BSA-CBIQTGA complex UV-visible spectra of BSA in absence and presence of CBIQTGA show absorption peak at 280 nm, which increases with the increase in concentration of CBIQTGA as shown in Fig. 1. The shifting of absorption position to lower wavelength region may be due to formation of ground state complex BSA-CBIQTGA31-32.  Fluorescence quenching of BSA in presence of CBIQTGA The fluorescence of BSA is due to tryptophan (Trp) moiety which is very sensitive. The fluorescence spectra (Fig. 2a) were recorded for the free BSA and mixed with different concentrations of CBIQTGA to study the interaction between them. The spectra indicate that the BSA has a strong emission band observed between 345-380 nm when excited at  280 nm31-32. The intensity of this fluorescence band gradually decreases with increase in concentration of quencher i.e., CBIQTGA. This gradual decrease in the   Scheme 1 — Reaction mechanism for the synthesis of CBIQTGA RANAfluorescenceCBIQTGA aThe fluodynamic or of interactiofluorescenceas shown inStern-Volme = 1 +KSV Q where, F0 anintensities inrespectively.constant andKq is the qumacromolecmolecule wplot of F0/F this plot yieThe quenthe temperaquenching obetween CBwhich are dvalue of Kq lifetime of tscattering cquenchers wThe value increasing tbetween CBwhich resultpresent caseorder of 10quenching bFig. 1 — U DE et al.: BIND of BSA indnd BSA to forescence qustatic mechann between C quenching d Figs. 2b &r equation35 a=1+Kq0Q d F denote t the absence  KSV is th [Q] is the cenching rateule, τ0 is thithout any qvs. [Q] is prelds the Sternching coture thus inccurs becausIQTGA and estabilized awas calculatehe biopolymollision quenith the biopoof quenchinemperature; IQTGA and s in success the quench11. This indietween CBIQV-visible spectrING OF THIAZicates the brm a compleenching occism, dependBIQTGA anata at differe 2c, were as given belohe steady-staand presencee Stern-Voloncentration constant oe average uencher. Thsented in Fig-Volmer connstant dedicating the of some sBSA by formt higher temd by assumier of 10-8 s .ching constlymer is 2 x g constant this indicateBSA by comful quenchining constancates the preTGA and BS a of BSA-CBIQOLO [2, 3-a] Iinding betwex.  urs either ing on the wd BSA33-34. Tnt temperatunalyzed by w. te fluorescen of CBIQTGmer quench of CBIQTGf the biologilifetime of e Stern-Volm. 3 and slopestant (Table creases wat fluorescenpecific binding complexperatures. Tng fluorescen The maximuant of vario1010 L mol-1 decreases wd that bindplex formatg. While in t Kq is of sence of staA34-35. TGA complex SOQUINOLINE  en by ay he res the ce A, ing A, cal the er  of 1). ith  ce ing es he ce m us s-1. ith ing ion the the  tic BindinCBIQTThebindinwere o 𝑙𝑜𝑔𝐹 TheFig. 4the slare subindin  Fig. 2 CBIQT  ANALOG WIg constant anGA complex  binding cg sites (n) bbtained from0 𝐹𝐹log𝐾 plot of log and the valope and Y-ammarized ing sites betw— FluorescenGA at (a) 298 KTH BOVINE SEd number ofonstant (K) etween the  the equation𝑛𝑙𝑜𝑔 𝑄 (F0-F)/F vsue of n andxis intercept Table 1. Theen BSA andce quenching , (b) 293K and RUM ALBUM binding sitesand the nuCBIQTGA  as follows:   . log [Q] is  K are obtai, respectivelye value of n CBIQTGAof BSA in p(c) 310 K IN 1083 of BSA-mber of and BSA shown in  ned from  34-35, and umber of  is nearly  resence of10841. Thereforethat these cothe protein aThis result complex of decompose a Mode of bindiUsually vhydrophobic electrostatic small molecchange ΔH0 avan’t Hoff’s ebe obtained.  Fig. 3 — Sterwith BSA at thr  Fig. 4 — DoubCBIQTGA  Table 1 — T (K) Ksv293 3.7298 3.1310 1.3  the value ofmpounds cas well as relesuggests theBSA with Ct higher tempng an der Waainteractions,interactions ules to macnd entropy chquation and cThe plot of lnn-Volmer plotsee different temle-logarithmic pBinding paramedifferent  (M-1) Kq (M3×103 3.732×103 3.121×103 1.3 binding conn easily formased in desir formation BIQTGA, wheratures36 ls force, hyd hydrophilic are responsibromolecule37ange ΔS0 wehange in freeK vs. 1/T is  for the interacperatures  lot for the quenters of CBIQTGtemperatures -1 S-1) K (×1011 4.1×1011 2.91×1011 1.9INDIAN J CHEstant sugges complex wed target areof an unstaich may parrogen bondiinteraction ale for bind. The enthare obtained fr energy ΔG0 cshown in Figtion of CBIQTching of BSA wA with BSA at L M-1) n 6×102 0.778×102 0.760×102 0.80M, SEC A, AU  ted ith as. ble tly ng, nd ing lpy om an . 5 and thslope (-60.6interacindicaforcesnegatiCBIQ Effect Maits speCBIQCBIQnegati GA  ith     Fig. 5 —CBIQT Fig. 6 — Table T (K)293 298 310  GUST 2021 e values of ∆and intercept 75 J mol-1 Ktion and negate that hydro are involvedve value of ∆TGA and BSAof CBIQTGA oin implicationed of analysiTGA interactTGA are shve bands at  Plot of ln K vGA at 293 K, 2 Circular dichr2 — Thermodynat 0H  (kJ mo -32.637  H0 and ∆S0 wof graph38. Th-1) as a prtive value ofgen bonding in the bindG suggests th occurred spn the conforma of circular ds, sensitivity ions. The CDown in Fig. 208 nm and s. 1/T of the in98 K and 310 K oism spectra ofamic Paramete different tempel-1) 0S  (J m -60.6 ere obtainede negative vaoof for a h ∆H0 (-32.637 and van ding39-40 (Tablat the bindingontaneously. tion of BSA  ichroism, tecin analyzing  spectra of B6, which sh222 nm, resteraction betwee  BSA with CBIQrs of CBIQTGAratures ol-1 K-1) 0G-175 -1-1 from the lue of ∆S0 ydrophilic  kJ mol-1) er Waals e 2). The  between hnique is the BSA-SA with ows two pectively.  n BSA and  TGA   with BSA (kJ mol-1) 4.693 4.089 3.523 RANADE et al.: BINDING OF THIAZOLO [2, 3-a] ISOQUINOLINE ANALOG WITH BOVINE SERUM ALBUMIN   1085These bands are characteristics of α helical structure of BSA39-40. The band intensity of BSA decreases with the addition of CBIQTGA. CD spectra of BSA reveal that BSA retains its α-helix structure after binding to the CBIQTGA40-41.   Conclusions A new derivative CBIQTGA was synthesized by simple method with good yield. UV study reveals the formation of CBIQTGA–BSA complex. Fluorescence spectra conclude the static quenching mechanism for binding between CBIQTGA and BSA. Negative  value of free energy indicates that CBIQTGA-BSA complex formation is spontaneous process and negative value of entropy indicates hydrophilic interaction between BSA and CBIQTGA. Circular dichroism spectra show the binding between CBIQTGA and BSA alters the conformation of BSA.  Supplementary Data Supplementary data associated with this  article are available in the electronic form at http://nopr.niscair.res.in/jinfo/ijca/IJCA_60A(08)1081-1085_SupplData.pdf.  Acknowledgement Prasanna Ranade and Dinesh Navale gratefully acknowledge the use of central instrumentation facilities at Vivekanand Education Society’s College of Arts, Science and Commerce funded by FIST-DST, Ministry of Science and Technology, Govt. of India and DBT STAR college scheme.   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Synthesis and spectroscopic investigation of binding of novel Thiazolo [2, 3-a] isoquinoline analog with bovine serum albumin

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Synthesis and spectroscopic investigation of binding of novel Thiazolo [2, 3-a] isoquinoline analog with bovine serum albumin

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