Synthesis of new boswellic acid derivatives as potential antiproliferative agents

Abstract In the current investigation, a series of heterocyclic derivatives of boswellic acids were prepared along with new monomers of 3-O-acetyl-11-keto-β-boswellic acid (AKBA, 1) 11-keto-β-boswellic acid (KBA, 2) and several new bis-AKBA and KBA homodimers and AKBA-KBA heterodimers. The effects of these compounds on the proliferation of different human cancer cell lines, viz., FaDu (pharynx carcinoma), A2780 (ovarian carcinoma), HT29 (colon adenocarcinoma), and A375 (malignant melanoma), have been evaluated. Thus, KBA homodimer 21 effectively inhibited the growth of FaDu, A2780, HT29, and A375 cells with EC50 values below 9 μM. In addition, compounds 7, 8, 11, 12, 15, 16, and 17 also exhibited cytotoxic effects for A2780, HT29, and A375 cancer cells. In particular, the pyrazine analog 8 was highly cytotoxic for A375 cancer cells with an EC50 value of 2.1 μM. Graphical Abstract


Introduction
Cancer is considered one of the most dreadful diseases in the world. This disease caused nearly 8.2 million deaths in 2012 and around 14.1 million new cases (Roy et al. 2016) were repoted. Unfortunately, current chemotherapeutic agents are often linked with various side effects, and especially the development of chemo-resistance towards these clinical drugs impedes an efficient and sustainable treatment of cancer (Roy et al. 2016). Frankincense has traditionally been used to treat cancer and several inflammatory diseases such as chronic pain syndrome, asthma, cerebral edema, arthritis, and chronic bowel disease (Takahashi et al. 2012;Roy et al. 2016). In addition, the gum resin of Boswellia sacra (frankincense) has also traditionally been used, for example, in Oman to treat severe muscle pain, colds, cough, dental infections, and stomach aches but also fever (Al-Ghassany 2008).

Synthesis
The concentration of AKBA (1) in B. sacra is very low while the concentration of KBA (2), BA, (3), or ABA, (4) is relatively higher. To prepare KBA in larger quantities we followed the procedure reported by Jauch and Bergmann (2003) to obtain AKBA. All boswellic acids 1-4 can easily be interconverted (Wolfram et al. 2017).
KBA (2) was esterified with BnBr/K 2 CO 3 in DMF, and ester 5 was obtained in 91% yield (Scheme 2). Jones oxidation (CrO 3 /aq. H 2 SO 4 ) in acetone of the C-3 hydroxyl group gave 3-keto 6 in 87% yield. Compound 6 served as a valuable starting material for the syntheses to follow. Thus, synthesis of the pyrazine derivatives (Csuk et al. 2011;Zorina et al. 2018) of AKBA (compounds 7 and 8) was accomplished with 45% and 42% yield by reacting ketone 6 with ethylene diamine in morpholine as the solvent (Bhandari et al. 2014). Furthermore, ketone 6 gave upon reaction with potassium tert-butoxide in the presence of aniline not the corresponding indole but this reaction ended up with an opening of ring A and provided aldehyde 9 in 48% yield. The synthesis of semithiocarbazone 10 was accomplished by reacting 6 with semicarbazide. Monomers of boswellic acids 11-16 (Scheme 3) were prepared by esterifying boswellic acids 1 and 2 with three equivalents of the linkers 1,2-dibromoethane, 1,3-dibromopropane, and 1,4-dibromobutane in the presence of K 2 CO 3 in DMF, respectively (Saeed et al. 2018). The bis and hetero dimers of boswellic acids 17-25 (ester-linked) were obtained in good yields by reacting the monomers 11-16 in the presence of K 2 CO 3 in DMF with compounds 1 and 2.

2.2.Cytotoxic activity and SAR studies
The compounds were subsequently screened for their cytotoxic activity for human cancer cell lines FaDu, A2780, HT29, and A375. The EC 50 values of the boswellic acid analogs and dimers for these cell lines are summarized in Table 1. Compounds having EC 50 values over 30 mM were considered as not active.
EC 50 ¼ 22.7 lM; 8: EC 50 ¼ 12.2 lM) and A375 (7: EC 50 ¼ 12.8 lM; 8: EC 50 ¼ 2.1 lM) tumor cells. It is interesting to note that compound 8 exhibited a much better activity than compound 7. In particular, AKBA analog 8 showed six times stronger cytotoxic effect for A375 cancer cells (8: EC 50 ¼ 2.1 lM vs 7: EC 50 ¼ 12.8 lM). These results suggest that an addition of a pyrazine group has a significant impact on the activity of boswellic acids. On the other hand, ring A modified seco-structure 9 was only active against cell lines A2780 (EC 50 ¼ 17.9 lM) and A375 (EC 50 ¼ 19.4 lM). This also indicated that a cleavage of ring A did not enhance activity. This parallels previous Scheme 3. Reagents and conditions: a) 1,2-dibromoethane (3 eq) or 1,3-dibromopropane (3 eq) or 1,4-dibromobutane (3 eq), K 2 CO 3 , DMF; b) compounds 1 or 2 (1 eq), 1,2-dibromoethane (1 eq) or 1,3-dibromopropane (1 eq) or 1,4-dibromobutane (1 eq), K 2 CO 3 , DMF. findings for other triterpenoic acids (Csuk et al. 2011). Compound 7 (being decarboxylated at position C-23 compared to its parent compound), and compound 9, which holds a modified ring A, have shown diminished activities as compared to compound 8. This indicates that the presence of a carboxyl group and of an intact ring A is crucial for retaining the cytotoxic effects of the boswellic acid analogs. Further, the semithiocarbazone 10 demonstrated good cytotoxic effects for the three cancer cell lines A2780 (EC 50 ¼ 9.3 lM), HT29 (EC 50 ¼ 11.3 lM) and A375 (EC 50 ¼ 10.8 lM). This result revealed that an incorporation of extra nitrogen into the AKBA skeleton enhances activity; this finding is in excellent agreement with previous findings (Wolfram et al. 2017). Cytotoxic effects of the three monomers of AKBA 11-13 were not impressive at all. Monomers 12 and 13, holding a bromopropyl or a bromobutyl group, were found to be inactive for all of the tested cancer cell lines (EC 50 > 30 lM). It was only monomer 11, carrying a bromoethyl group, that was found to be active against at least three types of cancer cells: A2780 (EC 50 ¼ 16.3 lM), HT29 (EC 50 ¼ 27.7 lM), and A375 (EC 50 ¼ 12.1 lM). On the other hand, the growth inhibitory activity of the three monomers of KBA viz., compounds 14-16 was very significant inasmuch as all these monomers were active against the tested tumor cell lines (FaDu, A2780, HT29 and A375) with EC 50 values ranging from 12.1 to 21.7 lM.
Homodimers of AKBA 17-19 holding an ethylene, a trimethylene or a tetramethylene spacer were found to be inactive (EC 50 > 30 lM) against all the four cancer cell lines tested. Similarly, the two homodimers of KBA 21 and 22 having a trimethylene or a tetramethylene linker also turned out to be inactive (EC 50 > 30 lM). However, the KBA homodimer 20, holding an ethylene group as linker, proved to be the most potent cytotoxic compound. This compound showed significant inhibitory effects on the proliferation of all the four types of cancer cells viz., FaDu (8.6 lM), A2780 (EC 50 ¼ 6.6 lM), HT29 (EC 50 ¼ 6.2 lM), and A375 (EC 50 ¼ 8.2 lM). Finally, it may be noted that among all the tested compounds, only compounds 14-16 and 20 were effective in inhibiting the proliferation of pharynx carcinoma (FaDu) cells.

Conclusion
We have prepared ring A modified AKBA analogs (compounds 7, 8, 9, and 10), AKBA monomers (11-13) and KBA monomers (14-16) as well as homo-and heterodimers of AKBA and KBA (17-24). The most remarkable anti-proliferative activity was displayed by the pyrazine analog 8 against A375 (malignant melanoma) cells with an EC 50 value as low as 2.1 lM. SAR studies revealed that the presence of an amine enhances the activity of boswellic acids. Moreover, it was also observed that decarboxylation or cleavage of ring A led to reduced cytotoxic activity. KBA homodimer 20 and KBA-AKBA heterodimer 23 exhibited cytotoxic effects while all the other dimers turned out to be inactive. In fact, 20 was the most potent compound showing significant cytotoxic effects against all the four cancer cell lines with EC 50 11.0 lM. It is interesting to note that compound 20 (holding an ethylene group as linker in its dimeric structure) was cytotoxic. This suggests that the length of the spacer plays a significant role in establishing the cytotoxic effects of KBA homodimers.