Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates

Acetylated triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were converted into their succinyl-spacered acetazolamide conjugates. These conjugates were screened for their inhibitory activity onto carbonic anhydrase II and their cytotoxicity employing several human tumor cell lines and non-malignant fibroblasts. As a result, the best inhibitors were derived from betulin and glycyrrhetinic acid while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A betulin-derived conjugate held a Ki = 0.129 μM and an EC50 = 8.5 μM for human A375 melanoma cells.

Consequently, we became interested in the design, synthesis and CA II screening of conjugates derived from pentacyclic triterpenes such as betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid ( Figure 2) with well-known inhibitor acetazolamide.

Results
Based on our previous results [71], triterpenoids ( Figure 2) betulin (BN), oleanolic acid (OA), ursolic acid (UA), and glycyrrhetinic acid (GA) appeared to us as suitable and representative starting materials. BN was converted (Scheme 1) to the diacetate 1 according to known procedures, the selective mono-deacylation of which with CaH2 in a methanol/water mixture gave the 3-O-acetate 2. Reaction of 2 with succinic anhydride in pyridine in the presence of DMAP (cat.) furnished the succinyl derivative 3.
Commercial acetazolamide (4) was deacetylated with conc. HCl under reflux and compound 5 was obtained. The reaction of 3 with 5 proved somewhat difficult in the beginning, as both an in situ generation of the corresponding carboxylic acid chloride and its coupling with 5 failed, as was the use of coupling reagents such as EDC, DCC, PyBOP, or T3P. However, a good yield of 6 was obtained by first reacting 3 with ethyl chloroformate in the presence of 4-methyl-morpholine in THF to yield a mixed anhydride in situ, whose reaction with 5 then gave 88% of 6.
Compound 6 shows the basic carbon skeleton unchanged from the starting material BN, and it is further characterized by the presence of the acetyl group at position C-3 ( 1 H NMR δ = 1.99 ppm, and in 13 C NMR δ = 171.9 and 21.0 ppm). The succinyl spacer is characterized by the two CH2 groups (in 13 C NMR at δ = 30.0 and 30.9 ppm). The heterocycle shows in its 13 C NMR spectrum the characteristic signals at δ = 161.0 and 164.3 ppm; the sulfamate group held in the 1 H NMR spectrum the signal for the NH2 group at δ = 8.30 ppm.

Results
Based on our previous results [71], triterpenoids ( Figure 2) betulin (BN), oleanolic acid (OA), ursolic acid (UA), and glycyrrhetinic acid (GA) appeared to us as suitable and representative starting materials. BN was converted (Scheme 1) to the diacetate 1 according to known procedures, the selective mono-deacylation of which with CaH 2 in a methanol/water mixture gave the 3-O-acetate 2. Reaction of 2 with succinic anhydride in pyridine in the presence of DMAP (cat.) furnished the succinyl derivative 3.
Commercial acetazolamide (4) was deacetylated with conc. HCl under reflux and compound 5 was obtained. The reaction of 3 with 5 proved somewhat difficult in the beginning, as both an in situ generation of the corresponding carboxylic acid chloride and its coupling with 5 failed, as was the use of coupling reagents such as EDC, DCC, PyBOP, or T3P. However, a good yield of 6 was obtained by first reacting 3 with ethyl chloroformate in the presence of 4-methyl-morpholine in THF to yield a mixed anhydride in situ, whose reaction with 5 then gave 88% of 6.
Compound 6 shows the basic carbon skeleton unchanged from the starting material BN, and it is further characterized by the presence of the acetyl group at position C-3 ( 1 H NMR δ = 1.99 ppm, and in 13 C NMR δ = 171.9 and 21.0 ppm). The succinyl spacer is characterized by the two CH 2 groups (in 13 C NMR at δ = 30.0 and 30.9 ppm). The heterocycle shows in its 13 C NMR spectrum the characteristic signals at δ = 161.0 and 164.3 ppm; the sulfamate group held in the 1 H NMR spectrum the signal for the NH 2 group at δ = 8.30 ppm.
For the preparation of the corresponding analogous compounds derived from OA, UA, or GA, the commercially relatively inexpensive triterpene carboxylic acids OA, UA first had to be reduced using LiAlH 4 (Scheme 2). This allowed the corresponding diols 7 and 8 to be obtained in good yields. For the preparation of the corresponding analogous compounds derived from OA, UA, or GA, the commercially relatively inexpensive triterpene carboxylic acids OA, UA first had to be reduced using LiAlH4 (Scheme 2). This allowed the corresponding diols 7 and 8 to be obtained in good yields.   For the preparation of the corresponding analogous compounds derived from OA, UA, or GA, the commercially relatively inexpensive triterpene carboxylic acids OA, UA first had to be reduced using LiAlH4 (Scheme 2). This allowed the corresponding diols 7 and 8 to be obtained in good yields.
Since the reduction of GA by LiAlH 4 failed to give good yields, GA was first converted into acetate 15, the reaction of which with ethyl chloroformate/TEA gave an un-isolated mixed anhydride, the reduction of which with NaBH 4 at room temperature afforded compound 16 in good yields within a few minutes. Its reaction with succinyl anhydride yielded 17.
The coupling of 13, 14, and 17 with 5 (Scheme 4) gave the products 18-20, respectively. Compounds 7 and 8 were converted into the corresponding diacetates 9 and 10, respectively, whose selective de-acetylation gave compounds 11 and 12. Analogous conditions as described above could now be carried out for the subsequent reactions to yield the target compounds.
Thus, the mono-acetates 11 and 12 were converted (Scheme 3) to the succinyl derivatives 13 and 14.
Since the reduction of GA by LiAlH4 failed to give good yields, GA was first converted into acetate 15, the reaction of which with ethyl chloroformate/TEA gave an unisolated mixed anhydride, the reduction of which with NaBH4 at room temperature afforded compound 16 in good yields within a few minutes. Its reaction with succinyl anhydride yielded 17. Screening of compounds 6, 18-20 for their activity was performed with CA II as previously described; the results from the assays are compiled in Table 1. Acetazolamide (4) was used as a positive control. These assays showed glycyrrhetinic acid-derived conjugate 20 as the best inhibitor for this enzyme followed by betulin-derived 6. These compounds were even better inhibitors than gold standard acetazolamide (4). Oleanolic and ursolic-derived conjugates showed a diminished ability to inhibit CA II. Parent compounds, i.e., betulin, betulinic acid, ursolic acid, oleanolic acid, and glycyrrhetinic acid did not inhibit the enzyme under the conditions of the assay at all. Compounds 2, 3, 7-17 showed inhibition rates less than 10%.
For compounds with the highest inhibition percentage, i.e., 6 and 19 and 20, some extra measurements were performed to determine their respective inhibition constants K i values. The results from these experiments are summarized in Table 2; Figure 3 shows the Dixon plot for compound 6; this compound acts as a competitive inhibitor for the enzyme and holds a rather low K i = 0.129 µM. Initial molecular modelling calculations were performed to get some insights in the mode of action of the conjugates. These calculations, however, did not provide any reasonable explanation for the different ability of the conjugates to inhibit the enzyme. While it seems plausible that the acetazolamide moiety interacts with the active site of the enzyme in a manner like parent acetazolamide, it cannot be excluded; however, that the conjugates also act as non-zinc binding inhibitors, thus paralleling previous findings for structurally similar pentacyclic triterpenoid arjunolic acid [79].
Previously especially CA IX was extensively studied in the process of tumorigenesis, [15,80] and several derivatives of pentacyclic triterpenoids have been revealed as inhibitors of this isoform, too [81]. The selectivity of the triterpenoid investigated so far toward individual isoforms of CA, however, was not particularly pronounced.
Compounds 6 and 18-20 were screened for their cytotoxic activity in sulforhodamine B assays (SRB), employing several human tumor cell lines. The results from these assays are summarized in Table 3. Expression of CA II and its involvement cancer has previously been established for A375 [82], HT29 [83] as well as for MCF-7 cells [84]. Cell line A2780 and non-malignant fibroblasts (NIH 3T3) were employed for comparison.
As a result, the highest cytotoxicity was established for botulin-derived 6 followed by glycyrrhetinic acid-derived 20. This parallels the finding for the inhibition rates for CA II established for these compounds. A significantly lower cytotoxicity was determined for oleanolic or ursolic acid-derived compounds 18 and 19, respectively. The malignant/non-malignant cell selectivity, however, was low for all compounds. No cytotoxicity (EC 50 > 30 µM; cut-off of the assay) was found for parent triterpenoic acids.

Conclusions
Pentacyclic triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were acetylated at position C-3 and converted into their succinyl-spacered acetazolamide conjugates. Their screening for their inhibitory activity onto carbonic anhydrase II and screening for their cytotoxicity in SRB assays employing several human tumor cell lines and non-malignant fibroblasts showed the conjugates derived from betulin and glycyrrhetinic acid to be the best inhibitors while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A botulin-derived conjugate held a K i = 0.129 µM and an EC 50 = 8.5 µM for human A375 melanoma cells.

Experimental
NMR spectra were recorded using the Varian spectrometers (Darmstadt, Germany) DD2 and VNMRS (400 and 500 MHz, respectively). MS spectra were taken on a Advion expressionL CMS mass spectrometer (Ithaca, USA; positive ion polarity mode, solvent: methanol, solvent flow: 0.2 mL/min, spray voltage: 5.17 kV, source voltage: 77 V, APCI corona discharge: 4.2 µA, capillary temperature: 250 • C, capillary voltage: 180 V, sheath gas: N2). Thin-layer chromatography was performed on pre-coated silica gel plates supplied by Macherey-Nagel (Düren, Germany). IR spectra were recorded on a Spectrum 1000 FT-IRspectrometer from Perkin Elmer (Rodgau, Germany). The UV/Vis-spectra were recorded on a Lambda 14 spectrometer from Perkin Elmer (Rodgau, Germany); optical rotations were measured using a JASCO-P2000 instrument (JASCO Germany GmbH, Pfungstadt, Germany) The melting points were determined using the Leica hot stage microscope Galen III (Leica Biosystems, Nussloch, Germany) and are uncorrected. The solvents were dried according to usual procedures. Microanalyses were performed with an Elementar Vario EL (CHNS) instrument (Elementar Analysensysteme GmbH, Elementar-Straße 1, D-63505 Langenselbold, Germany). All dry solvents were distilled over respective drying agents except for DMF which was distilled and stored under argon and molecular sieve. Reactions using air-or moisture-sensitive reagents were carried out under argon atmosphere in dried glassware. Triethylamine was stored over potassium hydroxide. Biological assays were performed as previously reported employing cell lines obtained from the Department of Oncology [Martin-Luther-University Halle Wittenberg; they were bought from ATCC: malignant: A 375, HT29, MCF7, and A2780; non-malignant: NIH 3T3]. Oleanolic and ursolic acid were obtained from Betulinines (Strbrna Skalice, Czech Republic) and used as received. Glycyrrhetinic acid was bought from Orgentis Chemicals GmbH (Gatersleben).
For the SRB assay: cells were seeded into 96-well plates on day zero at appropriate cell densities to prevent confluence of the cells during the period of the experiment. After 24 h, the cells were treated with different concentrations (1, 3, 7, 12, 20, and 30 µM), but the final concentration of DMSO/DMF never exceeded 0.5%, which was non-toxic to the cells. After 72 h of treatment, the supernatant media from the 96-well plates were discarded, then the cells were fixed with 10% trichloroacetic acid and allowed to rest at 4 • C. After 24 h of fixation, the cells were washed in a strip washer and then dyed with SRB solution (200 µL, 10 mM) for 20 min. Then the plates were washed four times with 1% acetic acid to remove the excess of the dye and allowed to air-dry overnight. Tris base solution (200 µL, 10 mM) was added to each well. The absorbance was measured with a 96-well plate reader from Tecan Spectra.
For the CA II assay: Carbonic anhydrase II (bCA II, ≥3000 W-A units/mg from bovine erythrocytes) as well as 4-nitrophenyl acetate (4-NA) were purchased from Sigma.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.