Archives of Natural and Medicinal Chemistry Synthesis of Potent Cytotoxic Analogs of Boswellic Acids

Synthesis of a new group of boswellic acid analogs (3-9) has been accomplished through modification of enone functional group of 11-keto-β-boswellic acid (KBA, 1) and 3-O-acetyl-11-keto-β-boswellic acid (AKBA, 2) to chloro diene moiety. The structures of the analogs were confirmed by using 1H, 13C NMR and mass spectral data. These com pounds exhibited potent brine shrimp lethality and strong cytotoxicity against human cancer cell lines. The analogue 5 was found to be the most potent among the group as indicated by its efficacy against brine shrimp and MCF-7 cancer cell line with half inhibitory concentration (IC 50 ) values of 3.88 and 13.05 µM respectively.


Result and discussion
A few semi synthetic analogs of AKBA were developed with modifications done in variations functional groups. However, the 11-keto function has not been properly explored to generate potential new analogs of AKBA. Organochloro compounds, such as vancomycine, loratadine, setraline etc, are well known to be used as important medicines to combat different diseases. This observation encouraged the authors to prepare some chloro analogs of boswellic acids. Hence, the authors of present investigation were interested on the development of new analogs of AKBA through modification of 11-keto function (more specifically to convert the enone functional group to chlorodiene moiety).
As per classical chemistry, the displacement of carbonyl function by gem-dichloride function is generally achieved in the presence of phosphorous pentachloride [16]. The said displacement reaction in conjunction with E 2 elimination in presence of an inorganic base is employed to prepare alkenyl halides, allenes and alkynes. Cycloalkenylhalides can also be formed using a similar approach, but formation of cycloalkynes and cycloallenes can be a difficult task due to ring strain. The enone function in 3-O-acetyl-11-ketoβλ-boswellic acid (AKBA, 2) has been found to be resistant towards many chemical reagents, such as peroxy acids and reducing agents. As part of developing new analogs of AKBA, the authors intended to explore the feasibility of chlorinating the 11-keto group of AKBA. To achieve this objective, AKBA was dissolved in DMF and treated with three equivalents of PCl5 at room temperature. The TLC monitoring of the reaction mixture showed complete consumption of AKBA and appearance of a prominent new TLC spot. The product was originally perceived to be 3-O-acetyl-11, 11-dichloro-β-boswellic acid. However, the chromatographic purification of the reaction mixture yielded pure compound (4), which showed λ max at 248 nm in the UV spectrum. The characteristic 12-H proton (δ 5.57) of the enone moiety of AKBA was absent in the 1H NMR spectrum of 4. Instead, a sharp singlet was observed at δ 5.40. Careful analysis of 1H NMR and 13C NMR spectral data revealed that the structure of the new analog is 3-O-acetyl-11chloro-9-ene boswellic acid (4). It is presumed to be de-hydrochlorination product of initially formed dichloro precursor, wherein the transformation could have been facilitated by the acedic proton α to the gem-dichloro function. Similar treatment of 11-ketoboswellic acid (KBA) under identical conditions yielded 11-chloro-9 -ene boswellic acid (3). The chlorodiene compounds 3 and 4 were evaluated for their anti-tumor activity in comparison with AKBA (2) in breast cancer cell line MDA-MB-231. MBA-MD-231 is a triple negative breast cancer cell line, which has high propensity to develop metastasis. Human epidermal growth factor receptor (Her 2) tumors of this type are highly aggressive, resistant to chemotherapy and prognosis is very poor. The natural compound AKBA has shown potent cytotoxicity against MBA-MD-231 with an IC 50 value of 55.26µM. Interestingly, the chlorodiene compounds both 3 and 4 showed significantly better efficacy when compared to AKBA with IC 50 values of 42.85 and 35.72µM respectively. This unexpected outcome encouraged the authors, to select compound 4 as a lead compound and to conduct further analog development through modification of other key functional group, i.e. an acid function. The acid function was converted to an amide moiety using the following strategy [17].
Compound 4 was treated with thionylchloride at reflux temperature to obtain its acid chloride. Without purification, the crude acid chloride was subjected to amidification separately with diamino compounds selected from hydrazine, 1,2-diaminoethane and piperazine in THF at ambient temperature and each of the corresponding crude reaction mixtures were subjected to column chromatography over silica gel using hexane/ethyl acetate mixture as eluents to yield the amide compounds 5, 6 and 7 respectively in 60-70% over all yield. The structures of the analogs were confirmed by using 1 H, 13 C NMR, mass and IR spectra. For comparison, 3-O-acetyl-9-ene-β-boswellic acid (9) was prepared by subjecting KBA to reduction using excess lithium aluminum hydride followed by purification and acetylation of the intermediate 9-eneβ-boswellic acid (8a) with pyridine/acetic anhydride. During the reduction step 11-hydroxy-β-boswellic acid (8) was also formed as one of the product.
All the analogs (3 to 9) were tested in vitro for brine shrimp lethality and cytotoxicity against breast cancer cell lines, MCF-7 and MDA-MB-231. The Brine shrimp lethality has been shown to have correlation to the cytotoxicity by a comparative proliferation study in 9KB and 9PS cells [18][19]. The Brine shrimp lethality has been evaluated in comparison with a positive standard podophyllotoxin. The amides of chlorodiene showed potent brine shrimp lethality. The efficacies shown by the analogs 5, 6 and 7 in brine shrimp assay are superior as indicated by their IC 50 values 3.88, 3.69 and 4.01μM respectively, when compared to that exhibited by the positive control podophyllotoxin (IC 50 : 8.15μM). MCF-7 is an estrogen positive human breast adenocarcinoma cell line. The chlorodienes 3 and 4 exhibited relatively poor cytotoxicities against MCF-7 cell with IC 50 values 70.57 and 60.89 μM respectively when compared to that of AKBA (IC 50 : 49.52 μM), but the amide analogs 5, 6 and 7 of AKBA showed significantly improved efficacy with IC 50 values at 13.05, 27.18, and 19.84 μM respectively. In addition to the chlorodiene compounds 3 and 4, the amide analogs 5, 6 and 7 also showed superior cytotoxic activity, when compared with AKBA in MDA-MB-231 cell line as summarized Table 1, with ethylenediamine amide being the most potent of all analogs with an IC 50 value of 20.89μM. The analogs 3-O-acetyl-9-ene-μ-boswellic acid (9) and 11-hydroxy-μ-boswellic acid (8) showed inferior activity (IC 50 values > 100 μM). The improved efficacy for chloro analog 4 in conjunction with complete lack of efficacy for its closest cousin 8 indicates potential contribution of chloride group to the efficacy. Over all the chlorodiene analogs and their amides may offer potential benefit against the solid tumors, more particularly against estrogen negative adenocarcinoma ANMC-103.

Experimental General experimental procedures:
IR spectra were recorded on Perkin-Elmer (model spectrum BX) FT-IR instrument using KBr disc. The 1H NMR spectra were recorded on BrukerAvance AV 400 MHz Spectrometer and 13 C NMR spectra were recorded on BrukerAvance AV 100 MHz Spectrometer. Mass studies were performed on LC-MS system equipped with Agilent 1100 series LC/ MSD detector and 1100 series Agilent HPLC pump. Normal phase silica gel (ACME, 100-200 mesh) was used for column chromatography. Silica gel pre-coated plates (AlugramSil G/UV 254 ) were used for thin layer chromatography. Brine shrimp (Artemiasalina Cysts) eggs were obtained from Argent Chemical Laboratories, Redmond (USA). The solvents and other chemicals used were of LR grade and were procured from Qualigens Fine Chemicals, Mumbai (India).

Brine Shrimp Lethality Bioassay
Brine shrimp (Artemia salina) nauplii were hatched using brine shrimp eggs [20]. In a conical shaped vessel (1L), filled with sterile artificial sea water (prepared using sea salt 38g/L and adjusted to pH 8.5 using 1N NaOH) under constant aeration for 48h. After hatching, 10 nauplii were drawn through a pipette and placed in each vial containing 4.5 mL brine solution and treated with various concentrations of test substances (2)(3)(4)(5)(6)(7)(8)(9) and the final volume was made up to 5 mL using brine solution. The cultures were maintained at 37°C for 24h under incandescent lamps. The surviving larvae were counted. Each experiment was conducted along with control (vehicle treated), at various concentrations of the test substance with each set containing 6 tubes and mean of the results were noted in the table. The percentage lethality was determined by comparing the mean surviving larvae of test and control tubes. The LC50 values were obtained from the plot drawn containing concentration (µM) verses percentage inhibition. Podophyllotoxin was used as a positive control. The results are summarized in (Table 1).

Determination of cytotoxicity using MTT assay
The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [21] was employed to evaluate the cytotoxicity of the boswellic acid analogs. The cell lines human breast adrenocarcinoma cell line (MCF-7) and breast cancer cell line (MDA-MB-231) were obtained from American Type Culture Collection (Rockville, MD), and cultured with complete RPMI-1640 medium (Gibco BRL) supplemented with 10% (V/V) heat-inactivated fetal calf serum, antibiotics, L-glutamine. The cell numbers were determined with a hemocytometer, and viabilities were assessed by try pan-blue dye exclusion. The known amount of test compounds (2-9) were dissolved in a few drops of DMSO and diluted with water to make the concentration of DMSO less than 0.1%. Various concentrations of test compounds were added to the test cultures (2x10 6 cells/mL) grown in a 96-well plate and the drug treated cultures were incubated for 24h. The control cultures were exposed to the solvent carrier only. MTT is a pale yellow substance that was reduced by living cells to yield a dark blue formazan product. This process requires active mitochondria only and dead cells, even the freshly dead ones do not significant reduce the amount of MTT. MTT in PBS (0.1 mg) was added into each well and then incubated at 37°C for 4h. The MTT formazan [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] crystals formed due to dye reduction by viable cells were dissolved using acidified isopropanol (0.1N HCl) and mixed at room temperature. After 20 min, index of cell viability was calculated by measuring the optical density (OD) of color produced by MTT dye reduction with a microplate reader (BIO-RAD, model 3550, USA) at 570 nm (OD 570-620 ) and expressing it as percentage of control. The mean OD of the content of four wells was used for assessing the cell viability. Viable cells (%) = [(total cells-dead cells)/total cells] × 100%. The IC50 values were obtained from the plot drawn containing concentration (µM) verses percentage inhibition. The data is summarized in (Table 1). Citation: Suryachandra RR, Lakshmareddy E, Machi Raju G, Trimurtulu G (2017)

Conclusion
Boswellic acids are indicated for the wide range of disease indications especially inflammation and cancer. Five new analogs of AKBA with chlorodiene functionality were synthesized and their cytotoxicity was evaluated. All the chlorodiene analogs and their amides offer potential benefit against the solid tumors, more particularly against estrogen negative adenocarcinoma of breast. This work justifies the need for further exploration of Boswellic acid analogs as anticancer agents.