24-Nor-allobetulins possess strong α-glucosidase inhibitory activity

Abstract A series of 24-nor-allobetulin derivatives holding 3β-hydroxy-, oxime, methoxyoxime, lactame and 4-bromobenzylidene substituents have been synthesized and their differences in the NMR spectra were studied in detail. It was revealed that 3-oxo-24-nor-allobetulin loses selectivity in the reaction of oximation and forms a mixture of Z/E oximes (and methoxyoximes) in contract to the related derivatives of native scaffold (that forms only E-isomers). The screening of α-glucosidase inhibitory activity revealed that 24-nor-allobetulins are more active than allobetulins. The lead 3-oxo-24-nor-allobetulin with IC50 0.49 µM was more than 60-fold and 500-fold active than acarbose and 3-oxo-allobetulin, respectively. We can conclude that the removal of the C-24 methyl group significantly increased the antidiabetic effect and 24-nor-allobetulins should be identified as the new and promising scaffolds as α-glucosidase inhibitors on the basis of triterpenoids. Graphical Abstract


Introduction
Pentacyclic triterpenoids are natural compounds which are widely represented among the plant metabolites and characterized by various biological activities (Dra sar 2022).Synthetic possibility of the triterpene skeleton combined with unsaturated bond and functional groups allows to obtain numerous derivatives that often are more biologically active as compared with native triterpenoids.
Taking into accounts these data, we have synthesized a series of 24-nor-allobetulin derivatives, investigated their NMR differences and a-glucosidase inhibitory activity in the comparison with the native allobetulin related compounds.

Chemistry
Starting from 24-nor-allobetulin 2 at first we have obtained 3b-hydroxy-derivative 3 by the reduction with NaBH 4 in methanol (Scheme 1).Oxime 4 and methoxyoxime 5 were synthesized by the interaction of 2 with hydroxylamine or methoxyhydroxylamine hydrochlorides in pyridine under reflux conditions with good yields.The Beckmann rearrangement of oxime 4 by treating with SOCl 2 in dioxane led to lactame 6 with a yield of 78% after purification.The similar derivatives 8-11 of native allobetulin 1 have also been synthesized according to ( Li et al. 1998;Dra c ınsk y et al. 2006;Flekhter et al. 2009;Heller, Obernauer et al. 2015;Kazakova et al. 2019).Benzylidene derivatives 7 and 12 were prepared by Claisen-Schmidt reaction of 3oxo-allobetulins 2 or 8 with 4-bromobenzaldehyde.
Since the study of structural differences between native triterpenoids and their 24nor-analogues are not presented in the literature, we conducted a detailed comparison of NMR spectra using a series of 24-nor 2-7 and native 1, 8-12 allobetulins (Figures S14-S20, for details see the supplementary material).Figure S20 showed an example of the NMR differences for compounds 2 and 8.For compound 2, the chemical shift at the C-4 atom decreased by d c 3.2 compared to compound 8, and the shift of the carbonyl group also decreased by d c 4.38.In the HMBC spectrum of compound 8 the interaction of protons at C-1 and C-2, at C-3 and C-4 with 3-oxo-group was observed.At the same time the interaction of protons at C-1 and C-2, at C-24 and H-4 proton (d H 2.20) with 3-oxo-group in the HMBC spectrum of compound 2 was registrated.The proton H-4 has a b-configuration, since it has a large J ¼ 12.3 Hz with protons at C-5, which proves their trans-axial arrangement.An additional proof of the b-configuration of the proton H-4 was the NOE interaction between C-4 and C-25.The proton at C-5 has a-configuration, which was proved by the spectral data of compound 8.
The analysis of the 13 NMR spectra of 24-nor-derivatives 2-7 showed no signals of the carbon of the methyl group -23 and a shift of the signals of atoms -4 and 24-CH 3 to a high field when compared with the compounds 1, 8-12 of native allobetulin.The proton signals H-4 atom in the 1 H NMR spectra of compounds 2-7 were found as a doublet quartet in the range of d H 1.27-2.20.The b-configuration H-4 is followed from the values of the spin-spin interaction constants of protons at C-4 and C-5.In addition, the interaction of H-4 with the protons of the methyl group at C-25, which is in the b-configuration, was observed in the NOESY NMR spectra (Figure S13, Supplementary Material).The b-configuration of the hydroxyl group of compound 3 was confirmed by the interaction of protons at C-2 (d H 1.58) and C-3 (d H 3.18) with J ¼ 11.4 Hz, which indicated their trans arrangement.No significant difference in the chemical shifts of the C-3 carbon atom in the 13 C NMR spectra of oximes 4, 9, methoxyoximes 5, 10 and lactams 6, 11 were observed (Figures S15-S17, Supplementary Material).Compound 5 is a mixture of E/Z-isomers in the ratio of 2:1 that was confirmed by the double signals of carbon atoms in the 13 C NMR spectra.The assignment to the Z and E isomers was made on the basis of NOESY spectra data (Figure S16, Supplementary Material).Thus, for the E-isomer a cross-peak of CH 3 ON-protons with an equatorial proton at position C-2 (d H 3.81/3.03)was observed.The assignment to the Z-configuration was made on the basis of the observed NOE interaction of CH 3 ON-group with the methine proton H-4 (d H 3.82/2.51).The 2:1 ratio of E/Z-isomers was confirmed by the data of integration of 1 H NMR signals.
Oxime 4 was also obtained as a mixture of E/Z-isomers.The broadened signal of the NOH-group did not allow us to observe NOESY interactions, which, as in the case of methoxyoxime 5, allowed us to assign to the Eor Z-isomer.However, the spectral pattern (the position of the signals and their SSCC) was similar to the methoxyoxime derivative 5 (Figure S19, Supplementary Material), which made it possible to attribute the signals in the 1 H and 13 C spectra to the Eand Zisomers of the oxime derivative 4 and determine the ratio of E/Z-isomers as 1.7:1.Noted that the allobetulin methoxyoxime 10 was isolated as E-isomer (Flekhter et al. 2009), which testifies to the different reactivity of the native and 24-nor-allobetulin due to steric factors.
In the NMR spectra of bromobenzylidene derivatives 7, 12 the absence of the -24 methyl group led to a shift in the signal of the 3-oxo-group by d H 3.49 in a high field, the signals of aromatic carbon atoms were found in the region of d c 122.70-134.99,and in the HSQC NMR spectra they correlated with the signals of aromatic protons at d H 7.24-7.51.
One can see that the removal of the C-24 methyl group significantly increased the antidiabetic effect up to 500 times (in the case of 3-oxo-allobetulins 2 and 8).Taking into account these results we can conclude that 24-nor-allobetulins are the new and promising scaffolds for evaluation of a-glucosidase inhibitors on the basis on triterpenoids.
For the lead compound 2 cytotoxic activity against some cancer cells was also evaluated (Table S1, see the supplementary material).

Conclusion
To sum up, the chemistry of triterpenoid allobetulin was developed and a set of 24-norderivarives have been synthesized.It was revealed that 3-oxo-24-nor-allobetulin loses selectivity in the reaction of oximation and forms a mixture of Z/E oximes in contract to the related derivatives of native scaffold (that forms only E-isomers).The comparison of NMR spectra of native and 24-nor-allobetulins was studied in detail.It was established that 24-nor-allobetulins are more strong a-glucosidase inhibitors than allobetulins.The lead 3-oxo-24-nor-allobetulin with IC 50 0.49 mM was more than 500-fold active than 3oxo-allobetulin.We can conclude that the removal of the C-24 methyl group significantly increased the antidiabetic effect and 24-nor-allobetulins should be identified as the new and promising scaffolds as a-glucosidase inhibitors on the basis on triterpenoids.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The synthetic part of the reported study was supported by Federal program No. 1021062311392-9-1.4.1.The study of biological activity of compounds 1-12 was supported by

Table 1 .
a-Glucosidase inhibitory activity for compounds 1-12.RFBRand by the joint project between Vietnam Academy of Science and Technology and Russian Foundation for Basic Research (Project No. QTRU01.05/20-21).