Development of Phortress Analogues: Design, Synthesis and Anticancer Screening of Benzothiazole Fused Acetamides

INTRODUCTION

Combination of two or more drugs is a common strategy for the treatment of cancer in unresponsive patients.¹ This encourages researchers globally for the development of ligands comprising of two or more pharmacophores in asingle molecule to modulate the multiple targets. This has led to a promising concept in drug design and development to produce new hybrid pharmacophores with improved affinity and efficacy in comparison to parent drug. The molecular hybridization is one of the new strategies in rational design of prototypes or ligands, which are generally synthesized based on the identification of pharmacophoric subunit structures of two or more known bioactive molecules and fused them in to a single molecular motif that maintains pre-selected characteristics of the original templates.² Molecular hybridization is believed to be analogues to the conventional combination therapy, with the exception that two molecules are fused or joined together with covalent bond to form single entity.^3-4 Over the last two decades, the privileged hybridized motifs were proved to be a fruitful approach for the discovery and are rapidly emerging theme in medicinal chemistry.

Despite increase in sophisticated approach in designing cancer chemotherapy, there is still no curative treatment that is 100% effective. Currently cancer chemotherapeutic agents lack selectivity and specificity against cancerous cells/tissueswhich are significant setbacks. Consequently, cancer chemotherapy targeting tumor represents one of the most significant challenges for medicinal chemists and oncologist.⁵ Heterocyclic skeletons serve as ideal scaffolds on which pharmacophores can be appended to yield potent and selective drugs.⁶

In the past two decades, 2-substituted benzo [d] thiazole analogues have been extensively studied for their anticancer activity and continued to magnetize considerable attention in anticancer research.^7-9 Literature reports suggest that 2-(4-Amino phenyl) benzo [d] thiazoles¹⁰ and their lysyl-amide derivative (Phortress)¹¹ possess remarkable in vitro anticancer activity by inhibiting EGFR tyrosine kinase especially against breast, colon, and ovarian cell lines.

The Fig.1 Iillustrates the development of Phortress analogues. This versatile and potent scaffold has provided an impetus for the anticancer drug discovery in recent years. 

As exemplified above, exploiting “chemistrydriven” approach to anticancer drug discovery,^12-14 a rich lode of novel bioactive agents had strucked and now report the synthesis and properties of a further new, but different, series of simple 2-phenylbenzothiazoles bearing acetamide substitution on phenyl moiety.

MATERIALS AND METHODS

Computational methods

The computational work was performed on Pentium IV workstation using molecular operating environment (MOE-2004.03), developed by Chemical Computing Group Inc, Canada. Structurally similar derivatives of 45 phortress analogues^15-16 were selected for the present study. All the compounds were drawn in builder (structural) module and then subjected to conformational analysis and energy minimization using stochastic conformer search with Root Mean Square (RMS) gradient of 0.001 and iteration limit 10000 employing MMFF94 force field Fig. 2.

The X ray crystallographic data of EGFR bound benzothiazole is not known publicly and a molecule can have various conformations and may exhibit variations in biological activity and reactivity. Hence, lowest energy conformer among energy minimized conformer was chosen for QSAR study. Lowest energy conformer was transferred to data base viewer and molecular descriptors were calculated. MOE calculates 213 sub descriptors from three major classes of descriptors; 2D descriptor (uses the atoms and connection information of the molecule for the calculation); i3D, internal 3D descriptors (uses 3D coordinate information about each molecule and they are invariant to rotation and translation of the conformer) and x3D, external 3D descriptors, which use 3D coordinate information.¹⁷

The 46 benzothiazole derivatives, chosen were divided in to two sets. The 35 compounds were taken in to training set and 11 compounds were taken in to test set. GI50 values for EGFR inhibition, were transformed in to –log (GI50 X 10- 6) i.e., pGI50. QSAR model was developed using pGI50 as activity field and partial least square (PLS) method for performing regression analysis. PLS regression method was used to analyze the internal interaction between the data matrices including the independent variable matrix of molecular descriptors of MOE and the dependent variable matrix of pGI50. Using the prediction fit performed in training set model; predicted pGI50 values of test set were evaluated. Model developed was optimized using LOO (leave one out method) and $Z score values. These values provide absolute difference between the value of model and activity field divided by square root of the mean square error of data set. Graphical interpretation of cross validated model to identify the outlier was performed by plotting the $Z score values. Further, outliers identified a $Z score plot were only two, (i.e., the residual value that exceeds twice the standard error of estimate of the model) indicating that the descriptors pruned were adequate. The correlation graphs of EGFR activity of training set and test set are depicted in Fig. 3.1 and Fig.3.2, respectively.

Chemistry

The 2-chloro-N-(substituted-phenyl) acetamide(1a-j)wasobtained by condensation of commercially available substituted anilines and chloroacetyl chloride at 0-5°C under constant stirring. The synthesis of 4-(benzo[d] thiazol-2-yl)benzenamine(2a) was obtained by condensation of substituted 2-aminothiophenol with 4-aminobenzoic acid in presence of polyphosphoric acid (PPA).¹⁸ The 4-(benzo[d] thiazol-2-yl)benzenamineup on condensation with series of 2-chloro-N-(substituted-phenyl) acetamide in presence of glacial acetic acid yields 2-(4-(6-substituted-benzo[d]thiazol-2-yl) phenylamino)-N-(substituted-phenyl)acetamide (3a-j).

Synthesis of 4-(benzo [d] thiazol-2-yl) benzenamine(2a):

The starting material 4-(benzo[d]thiazol-2-yl) benzenaminewas prepared by mixing substituted 2-aminothiophenol with p-aminobenzoic acid in presence of polyphosphoric acid (PPA) to get steerable (semisolid) paste.This mixture was heated initially at 110◦C for 1 h then the temperature was raised slowly to 220◦C and the reaction mixture was heated for another 4 h with continuous stirring. Then the reaction mixture was cooled to 100◦C and poured into ice cold 10 % sodium carbonate solution. The alkaline slurry was filtered off and the product was washed thoroughly with water and dried at 60◦C. It was purified by recrystallisation from aqueous methanol as yellow solid.

Characterization of 4-(benzo[d]thiazol-2-yl) benzenamine (2a):

IR (KBr): 1308.96 cm-1 (C-N stre), 3191.58 cm^-1 (C-H stre), 3447.58 cm-1 (N-HStre). ¹ H NMR (400MHz, DMSO d6) δ (ppm): 5.73-5.74 (s, 2H, NH₂ ), 6.67 – 7.92 (m, 8H, Ar-H). 13C NMR (400MHz, DMSO d6) δ (ppm): 113.54, 113.58, 120.31, 121.52, 121.64, 124.07, 125.92, 128.62, 133.72, 151.85, 151.91, 153.86, (aromatic carbons), 168.12 (C-2). Mass:m/z 227.1; Calcd. 226.06.

Synthesis of 2-chloro-N-(substituted-phenyl) acetamide (1a-j):

Substituted anilines (0.05mol) were dissolved in a mixture of glacial acetic acid (25mL) and saturated solution of sodium acetate (25mL). This mixture was cooled to 0 to 5 ^o C then chloroacetyl chloride (6.2mL, 0.075mol) was added drop wise at 0-5 ^o C under constant stirring then it was left at room temperature for 5-6 h and crude precipitate was filtered, washed with 50% acetic acid and cold water. The product was recrystallised from ethanol.¹⁹

Synthesis of 2-(4-(6-substituted-benzo[d] thiazol-2-yl)phenylamino)-N-(substituted-phenyl)acetamide (3a-j):

The 4-(6-substituted-benzo [d] thiazol-2- yl) benzenamine (0.0025mol) was condensed with 2-chloro-N-substitued-phenylacetamide (0.0025 mol) in presence of glacial acetic acid (20mL), the reaction mixture was refluxed for 24 h and the obtained clear reaction mixture then allowed to cool to the room temperature and poured into ice cold water, the precipitate was filtered, washed with water and recrystallised by using ethanol. The physicochemical data of series of 2-(4-(benzo[d]thiazol-2-yl)phenylamino)-N-(substituted-phenyl) acetamides (3a-j) are depicted in Table 1 and spectral data areshown in Table 2.

In vitro cytotoxic activity

Cell lines: Human cancer cell lines, MCF-7 and HeLa cells, were cultured in MEM medium supplemented with 10% FBS, 1% l glutamine, and 50 mg/mL gentamicin sulphate in a CO₂ incubator in a humidified atmosphere of 5% CO₂ and 95% air. The EAC cells were maintained for 12-14 days in the peritoneal cavity of Swiss albino mice. The tumor cell cultures were started from mouse Ehrlich Ascites with at least one passage in vitro prior to use.

MTT assay: In vitro cytotoxicity was determined using a standard MTT assay²⁰ as mentioned in the literature. The percentage cytotoxicity and IC₅₀ values were determined at 24, 48, and 72 h of drug incubation.

NCI-60 DTP Human tumor cell line screen^21-24

The National Cancer Institute (NCI) has served the researchers throughout the world since 20 years. Among the compounds synthesized, two compounds were selected for 60 cell line screening. This screen is unique in that the complexity of a 60 cell line dose response produced by a given compound results in a biological response pattern which can be utilized in pattern recognition algorithms.

Using these algorithms, it is possible to assign a putative mechanism of action to a test compound, or to determine that the response pattern is unique and not similar to that of any of the standard prototype compounds included in the NCI database.

The RPMI 1640 medium was used in cancer screening panel which contains 5% fetal bovine serum and 2 mM L-glutamine. Generally cells were inoculated in microtiter plates (5000 to 40000 cells /well).After inoculation plates were kept for 24 hr in an incubator at 37°C, with 5 % CO2, 95 % air, and 100 % relative humidity. After 24 hr, cell population were measured by mixing two plates in Trichloroacetic acid (TCA) followed by drug addition . Drugs were solubilised in DMSO at different concentration and they were stored in frozen condition till their use. The detailed procedure is mentioned in the Development Therapeutics Programme.25 Using the absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as: 

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for 

which Ti>/=Tz

[(Ti-Tz)/Tz] x 100 for concentrations for which 

Ti<Tz.

Three dose response parameters werecalculated for each experimental agent. Growth inhibition of 50 % (GI50) wascalculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which wasthe drug concentration resulting in a 50% reduction in the net protein increase [as measured by sulforhodamine B (SRB) staining] in control cells during the drug incubation.

In vitro EGFR inhibition assay²⁶

Over-expression of EGFR kinase was linked with development of cancer such as glioblastoma, epithelian cancers, etc. The present therapeutic regimen developed for prevention of cancer targets EGFR. The EGFR Kinase Assay Kit was available to measure EGFR Kinase activity for screening and profiling applications using Kinase-Glo® MAX as a detection reagent. The reagent composed of EGFR 2 µg, Kinase buffer 1.5 mL, ATP 100 µL, and PTK substrate 100 µL. The assay was carried out using Erlotinib as standard.

The positive control, test inhibitor, and blank were prepared by adding these components only in test inhibitor standard inhibitor 5 µLwas added and blank did not contain EGFR. The EGFR required for assay 1ng/µl was diluted with kinase buffer. The EGFR activity was initiated by adding 20 µL of diluted EGFR to 96 well plate designated with positive control and test inhibitor control and incubated at 30 °C for 45 min. After the 40 min reaction, 50 µL of Kinase-Glo Max reagent was added to each well. The plate was covered with aluminium foil and incubated the plate at room temperature for 15 min, finally the density was measured using the microplate reader.

RESULTS AND DISCUSSION

Chemistry

The 4-(benzo [d] thiazol-2yl) benzamine was characterized by spectral studies, which includes IR, ¹ H NMR, 13C NMR, and Mass. The IR spectrum (2a) showed characteristic peaks at 3447.58 cm^-1 which was attributed for N-H stretching, C-H stretching was observed at 2191.58 cm^-1 and it showed peak at 1308.96 cm^-1 for C-N stretching which confirmed the formation of benzothiazole moiety. In ¹ H NMR spectrum of (2a) peaks at δ 5.73-5.74 ppm are attributed to two protons of NH₂ functional group and multiplet peaks of aromatic protons were observed at 6.67 – 7.92. The 13C NMR spectrum showed characteristic peaks at δ 113.54-168.12 ppm are attributed for aromatic carbon atoms. The compound 4-(benzo[d]thiazol2yl) benzamine (2a) was treated with 2-chloro-Nsubstituted phenylacetamides (1a-j) in glacial acetic acid to yield the final derivative 2-(4-(benzo[d] thiazol-2-yl)phenylamino)-N-(substituted phenyl) acetamides (3a-j).

The 2-(4- (benzo [d] thiazol-2yl) phenylamine)- N-phenylacetamide (3a) was confirmed by physical and spectral data. In IR spectrum, characteristic peak at 3281.55 cm^-1 was attributed for N-H stretching, peak at 3104.50 cm^-1 for C-H stretching, peak at 1662.18 cm^-1 for C=O and it showed peak at 1315.83 cm^-1 for C-N stretching. Here it was observed that the disappearance of characteristic peak at 3447.58 cm^-1 corresponding to the NH₂ group which was present in IR spectrum of (2a), instead of that the peak at 3281.55 cm^-1 for N-H stretching was obtained. This confirmed the formation of desired new compound (3a).

The structure (3a) was ascertained by ¹ H NMR spectrum, whichshowed characteristic peak at δ 10.29 was attributed for a proton of NH, peak at δ 2.11 corresponding to two protons of aliphatic CH₂ group and multiplets of aromatic protons were observed between δ 6.75-8.10 which correspond to aromatic peaks. In ¹³C NMR spectrum, characteristic peak at δ 56.04 attributed for carbon of CH₂ group, peaks at δ 119.10-166.99 and peak at δ 168.77 correspond to carbon of carbonyl group (C=O) which was not observed in the compound (2a). The presence of respective peaks confirms the formation of the desired (3a) compound.

All the synthesized compounds were confirmed by physical and spectral data similarly as that of the derivative (3a). The physico-chemical data of series of 2-(4-(benzo[d]thiazol-2-yl)phenylamino)- N-(substituted-phenyl)acetamides (3a-j) are depicted in Table 3.

Cytotoxic activity of synthesized compounds by MTT assay

The synthesized novel compounds 2-(4-(benzo[d] thiazol-2-yl)phenylamino)-N-(substitutedphenyl)acetamides (3a-j) were screened for in vitro cytotoxicity against two cancer cell lines (HT 29 and MCF-7) by standard MTT assay. In preliminary cytotoxicity study, benzothiazoleacetamide hybrid derivatives were found more effective on both HT-29 and MCF-7 cell lines. On the HT 29, the IC50 for the title compounds (3a-j) were in the range of 18.30 - 76.54 µM at 24 h of drug exposure, whereas on MCF-7 cell lines, the IC50 values were in the range of 11.70 - 61.24 µM at 24 h of drug exposure (Table 4).

This preliminary cytotoxicity revealed that MCF-7 cells were more sensitive to all the tested compounds than HT 29 cells. The electronegative halogen substituent generally increases the cytotoxicity in MTT bioassay as these electronegative substituent on the para position of aromatic ring increases the lipophilicity of the molecules. It was substantiated that the result of preliminary MTT bioassay showed excellent activity with the halogen substituted derivatives. Hence these molecules were taken up to assess the cytotoxic potency at different time intervals in MCF-7 cells and HT 29 cells.

In vitro anti-cancer NCI 60 cell line penal screening: The developmental therapeutics program (DTP) one dose mean graph of 2-(4-(benzo[d]thiazol-2-yl) phenylamino)-N-(4-fluorophenyl)acetamide(3b) NSC: D-781559 and 2-(4-(benzo[d]thiazol-2-yl) phenylamino)-N-(4-methoxyphenyl) acetamide (3d) NSC: D-781560 were showed in Fig. 4.1 and 4.2, respectively. The growth percentage of (3d) was found to be more compared to (3b) in most of the cell lines. The mean growth percentage of 60 cell line penal in (3b) was found to be 95.76, whereas compound (3d) had shown mean growth percentage of 96.73.

In vitro EGFR tyrosine kinase inhibition assay

The synthesized compounds were dissolved in 100 µL DMSO and tested for EGFR tyrosine kinase inhibition activity. Kinase activity data werereported as the percent remaining enzyme activity after subtraction of enzyme inhibitory activity of control reactions as background. Compound 3d showed the highest inhibitory activity against EGFR-TK with percentage enzyme inhibition value of 80.65.

#Results are presented as % enzyme inhibition & compared to Erlotinib as a reference standard.

CONCLUSION

In the present study, synthesis of QSAR study was conducted to understand binding forces involved in EGFR and benzothiazoleacetamide hybrid pharmacophore. Syntheses of ten such pharmacophores were achieved and further structures of the synthesized compounds were characterized by spectral evidences. The synthesized derivatives show promising cytotoxic activity against MCF-7 and HT-29 cancer cells. Among the synthesized derivatives, compounds 3b and 3d were screened at NCI against 60 cell lines. Further to understand specificity of the compounds, in-vitro EGFR inhibition were carried out and results shown remarkable inhibition of EGFR activity.

ACKNOWLEDGMENTS

The work was supported by research grant of Rajiv Gandhi University of Health Sciences, Karnataka, No. P-141:2015-16 date: January 06, 2016. Authors are thankful to Chemical Computing Group for providing MOE software. Authorsare also thankful to CDRI, Lucknow and SAIF, Punjab University for spectral studies.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.