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Article

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

by
Toni C. Denner
,
Niels Heise
,
Julian Zacharias
,
Oliver Kraft
,
Sophie Hoenke
and
René Csuk
*
Organic Chemistry, Martin-Luther University Halle-Wittenberg, Kurt-Mothes, Str. 2, D-06120 Halle (Saale), Germany
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(3), 1009; https://doi.org/10.3390/molecules28031009
Submission received: 19 December 2022 / Revised: 12 January 2023 / Accepted: 17 January 2023 / Published: 19 January 2023
(This article belongs to the Special Issue Small Molecule Hybrids for Anticancer and Antiviral Therapy)
Browse Figures
Versions Notes

Abstract

:
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.

Graphical Abstract

1. Introduction

The ubiquitous metalloenzymes carbonic anhydrases (CAs) [1,2,3,4,5,6] are present in bacteria [7,8] and fungi [9,10,11,12], plants and animals. Inhibitors of these enzymes have been clinically exploited for decades, and the discovery of multiple human isoforms [13,14,15,16,17,18] has led to many new applications and the development of new therapeutic principles, among them antiglaucoma [19,20,21] and antitumor drugs but also antiepileptic [22,23,24,25,26] and antiobesity drugs [27,28,29] as well as agents for the management of Alzheimer’s disease [30,31], neuropathic pain, cerebral ischemia, and some forms of arthritis [32,33,34]. Furthermore, the development of inhibitors for bacterial carbonic anhydrases is thought as a new concept to develop antibacterial drugs [35,36,37,38,39,40,41,42]. In addition, drug conjugates were investigated for their ability to treat a variety of disorders in a multitargeting approach [43,44,45,46,47]. The most investigated compound, however, is SLC-0111 (U-104, Figure 1) [48,49,50,51,52,53] for the management of advanced, metastatic solid tumors; this compound is now in Phase Ib/II clinical trials [54].
For many years, especially CA IX and CA XII were in the focus of scientific interest to combat cancer. Recently, CA II in the endothelium of glial tumors became a potential target for therapy [55,56,57,58,59,60,61,62,63,64]. Furthermore, the CA II inhibitor acetazolamide was suggested as a chemosensitizer for treating temozolomide resistant gliomas [65,66,67,68,69]. In addition, CA II was found among up-regulated genes and as a candidate gene correlated with glioma malignancy and patient survival. [70]
The association of CA II with several pathological diseases prompted us to synthesize novel CA II inhibitors. Recently, it was revealed that methyl sulfamates, [71] derived from methyl triterpenoates such as oleanolic acid, ursolic acid, and glycyrrhetinic acid or betulinic acid, are effective and competitive inhibitors of CA II and several cycloartane derivatives [72] held significant activity for CA II, too. Euphol [73] was also shown to induce autophagy and to sensitize temozolomide cytotoxicity in glioblastoma cells. Several 1H-1,2,3-triazoles derived from pentacyclic triterpenoid 3-O-acetyl-(11-keto)-boswellic acids [74] were inhibitors CA II. Furthermore, hybrids of bile acids [75,76,77,78] have been shown to act as inhibitors of CAs.
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.

2. 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 (1H NMR δ = 1.99 ppm, and in 13C NMR δ = 171.9 and 21.0 ppm). The succinyl spacer is characterized by the two CH2 groups (in 13C NMR at δ = 30.0 and 30.9 ppm). The heterocycle shows in its 13C NMR spectrum the characteristic signals at δ = 161.0 and 164.3 ppm; the sulfamate group held in the 1H NMR spectrum the signal for the NH2 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 LiAlH4 (Scheme 2). This allowed the corresponding diols 7 and 8 to be obtained in good yields.
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 un-isolated 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.
The coupling of 13, 14, and 17 with 5 (Scheme 4) gave the products 18–20, respectively.
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 Ki 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 Ki = 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 (EC50 > 30 μM; cut-off of the assay) was found for parent triterpenoic acids.

3. 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 Ki = 0.129 μM and an EC50 = 8.5 μM for human A375 melanoma cells.

4. 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-IR-spectrometer 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.
A 96-well microplate spectrometer BMG Labtech Spectrostar Omega working in the slow kinetics mode and measuring the absorbance at a distinct wavelength of λ = 415 nm with center scanning was used for the enzymatic studies. In short: A mixture of 4-NA solution (125 µL, 6 mM in 50 mM Tris-HCl buffer, pH 8), enzyme solution (25 µL, 0.3 mg/mL), and compounds solutions (25 µL, 3 different concentrations and water as a blank) was incubated at 37 °C for 20 min. The substrate (25 µL, [4-NA] = 0.75 mM, 0.50 mM, 0.25 mM, 0.15 mM) was added to start the enzymatic reaction. The absorbance data was recorded under a controlled temperature of 37 °C for 30 min at 1 min intervals at λ = 415 nm. The relative inhibition was determined as the quotient of the slopes (compound divided by blank) of the linear ranges.

4.1. Di-O-Acetyl-betulin

Compound 1 was prepared from BN (15.0 g, 34 mmol) by acetylation with acetic anhydride as previously described; re-crystallization from ethanol gave 1 (15.8 g, 90%) as a white solid; RF = 0.73 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 221 °C (lit.: [85] 216–218 °C); [α]D = +16.6° (c = 0.061, MeOH), [lit.: [85] [α]D = +19.7° (CHCl3)]; MS (ESI, MeOH): m/z = 467.5 (100%, [M + H-HOAc]+).

4.2. 3-O-Acetyl-betulin

Selective deacetylation of 1 (8.0 g, 15.2 mmol) with cat. amounts of CaH2 in MeOH/THF (100 mL, 1:1 v:v) for 12 h at 20 °C followed by usual aqueous workup and chromatography (silica gel, hexanes/ethyl acetate, 8:2) gave 2 (6.1 g, 83%) as a colorless solid; RF = 0.40 (silica gel, hexanes/ethyl acetate, 8:2); m.p.: 256–259 °C (lit.: [85] 258–260 °C); [α]D = +28.8° (c = 0.039, CHCl3), [lit.: [85] [α]D = +25.7° (CHCl3)]; MS (ESI, MeOH): m/z = 992,0 (100%, [2M+Na]+).

4.3. 4-{[(3β)-3-(Acetyloxy)lup-20(29)-en-28-yl]oxy}-4-oxobutanoic Acid

To a solution of 2 (4.0 g, 8.2 mmol) in dry pyridine (50 mL), DMAP (cat.) and succinic anhydride (1.64 g, 16.4 mmol) were added. The reaction mixture was stirred under reflux for 15 h. Usual aqueous work up and chromatography (silica gel, hexanes/ethyl acetate, 7:3) gave 3 [86,87,88,89,90,91] (3.4 g, 71%) as a white solid; m.p. 189–191 °C (lit.: [86,87,88] 190–191 °C); [α]D = +12.1° (c = 0.198, MeOH); RF = 0.15 (silica gel, hexanes/ethyl acetate, 8:2); IR (ATR): ν = 2943m, 2871w, 1732s, 1713s, 1455w, 1361w, 1366m, 1244s, 1159m, 1027w, 979m, 883w, 754m cm−1; 1H NMR (500 MHz, CDCl3): δ = 4.68 (s, 1H, 29-Ha), 4.58 (s, 1H, 29-Hb), 4.46 (dd, J = 10.6, 5.6 Hz, 1H, 3-Ha), 4.30 (d, J = 11.0 Hz, 1H, 28-Ha), 3.88 (d, J = 11.1 Hz, 1H, 28-Hb), 2.75–2.54 (m, 4H, 34-H, 35-H), 2.42 (td, J = 11.0, 5.8 Hz, 1H, 19-H), 2.03 (s, 3H, 32-H), 2.01–1.88 (m, 1H, 21-Ha), 1.85–1.79 (m, 1H, 16-Ha), 1.75 (dd, J = 12.5, 7.9 Hz, 1H, 22-Ha), 1.68 (s, 3H, 30-H), 1.72–1.54 (m, 7H, 1-Ha, 13-H, 15-Ha, 12-Ha, 2-H, 9-H), 1.50 (s, 1H, 6-Ha), 1.44–1.35 (m, 5H, 6-Hb, 11-Ha, 21-Hb, 7-H), 1.34–1.14 (m, 3H, 16-Hb, 18-H, 11-Hb), 1.02 (s, 3H, 23-H), 1.12–0.90 (m, 4H, 22-Hb, 12-Hb, 15-Hb, 1-Hb), 0.96 (s, 3H, 27-H), 0.84 (s, 3H, 24-H), 0.84 (s, 3H, 26-H), 0.83 (s, 3H, 25-H), 0.78 (m, 1H, 5-H). ppm; 13C NMR (126 MHz, CDCl3): δ = 177.8 (C-36), 172.6 (C-33), 171.3 (C-31), 150.2 (C-20), 110.0 (C-29), 81.1 (C-3), 63.3 (C-28), 55.5 (C-5), 50.4 (C-18), 48.9 (C-9), 47.9 (C-19), 46.6 (C-17), 42.8 (C-14), 41.0 (C-8), 38.5 (C-1), 37.9 (C-4), 37.7 (C-13), 37.2 (C-10), 34.2 (C-22), 29.9 (C-16), 29.1 (C-35), 28.1 (C-24), 27.2 (C-15), 25.3 (C-12), 23.8 (C-2), 21.4 (C-32), 20.9 (C-11), 19.3 (C-30), 18.3 (C-6), 16.6 (C-25), 16.2 (C-23), 14.9 (C-27) ppm; MS (ESI, MeOH): m/z = 583.6 (100%, [M-H]-); analysis calcd for C36H56O6 (584.83): C 73.93, H 9.65; found: C 73.67, H 9.88.

4.4. 5-Amino-1,3,4-thiadiazole-2-sulfonamide

A solution of acetazolamide (4, 9.0 g, 40.7 mmol) in conc. HCl (60 mL) was heated under reflux for 3 h. After neutralization with NaOH, saturation with NaCl and extraction with THF (4 × 100 mL) followed by removal of the organic solvent, 5 (6.9 g, 94 %) was obtained as a white solid; m.p. 195 °C decomp. (lit.: [92] 215.5–216); RF = 0.3 (silica gel, CHCl3/MeOH, 9:1); UV-Vis (MeOH): λmax (log ε) = 278 nm (3.80) IR (ATR): ν = 3427w, 3321m, 3173w, 2870w, 2636w, 1601s, 1496s, 1448m, 1338s, 1172m, 1139m, 1098w, 1058w, 941m, 647s, 581s, 484w cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 8.04 (s, 2H, NH2), 7.84 (s, 2H, NH2) ppm; 13C NMR (126 MHz, DMSO-d6): δ = 171.7, 157.9. ppm; MS (ESI, MeOH): m/z = 179.0 (100%, [M-H]-).

4.5. (3β)-3-(Acetyloxy)lup-20(29)-en-28-yl 4-{[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]amino}-4-oxobutanoate

Compound 3 (500 mg, 0.85 mmol) was dissolved in dry THF (50 mL), 4-methylmorpholine (172 mg, 1.7 mmol) and ethyl chloroformate (185 mg, 1.7 mmol.) were added. The reaction mixture was stirred at 20 °C for 15 min. Compound 5 (184 mg, 1.02 mmol) was added, and the mixture was heated under reflux for another 48 h. The solvent was removed, the residue dissolved in CHCl3, washed with 2 M NaOH, water and brine and dried (MgSO4). Chromatography (silica gel, CHCl3/MeOH, 9:1) gave 4 (560 mg, 88%) as a white solid; m.p. 161–164°C; RF = 0.55 (silica gel, hexanes/ethyl acetate, 7:3); UV-Vis (CHCl3): λmax (log ε) = 264 nm (3.92) IR (ATR): ν = 2944m, 1733m, 1701m, 1531w, 1371m, 1245s, 1173s, 1018w, 979m, 882w, 609m, 504w cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 8.30 (s, 2H, NH2), 4.69 (s, 1H, 29-Ha), 4.55 (s, 1H, 29-Hb), 4.36 (dd, J = 11.4, 4.7 Hz, 1H, 3-H), 4.23 (d, J = 10.9 Hz, 1H, 28-Ha), 3.78 (d, J = 11.1 Hz, 1H, 28-Hb), 2.87–2.78 (m, 2H, 35-H), 2.76–2.65 (m, 2H, 34-H), 2.43 (s, 1H, 19-H), 1.99 (s, 3H, 32-H), 1.85 (m, 1H, 21-Ha), 1.63 (s, 3H, 30-H), 1.76–1.43 (m, 9H, 16-Ha, 22-Ha, 12-Ha, 13-H, 1-Ha, 9-H, 15-Ha, 2-H), 1.42–1.12 (m, 9H, 6-H, 11-Ha, 21-Hb, 7-H, 18-H, 16-Hb, 11-Hb), 0.95 (s, 3H, 23-H), 0.93 (s, 3H, 27-H), 1.09–0.73 (m, 5H, 22-Hb, 12-Hb, 1Hb, 15-Hb, 5-H), 0.79 (s, 9H, 24-H, 25-H, 26-H) ppm; 13C NMR (126 MHz, DMSO-d6): δ = 171.9 (C-31), 171.3 (C-33), 170.1 (C-36), 164.3 (C-38), 161.0 (C-37), 149.8 (C-20), 110.0 (C-29), 79.9 (C-3), 61.9 (C-28), 54.6 (C-5), 49.5 (C-18), 48.1 (C-9), 47.0 (C-19), 46.0 (C-17), 42.2 (C-14), 40.4 (C-8), 37.4 (C-4), 37.0 (C-13), 36.6 (10), 34.2, 34.0 (22), 33.5 (7), 30.9 (34), 30.0 (35), 29.1 (16), 28.9, 28.5 (21), 27.6 (24), 26.6 (C-15), (C-12), 23.4 (C-2), 21.0 (C-32), 20.3 (C-11), 18.7 (C-30), 17.7 (C-6), 16.4 (C-25), 15.8 (C-26), 15.5 (C-23), 14.5 (C-27) ppm; MS (ESI, MeOH): m/z = 745.7 (100%, [M-H]-); analysis calcd for C38H58N4O7S2 (747.03): C 61.10, H 7.83, N 7.50; found: C 60.85, H 8.03, N 7.33.

4.6. (3β) Olean-12-en3-3,28-diol (Erythrodiol)

To a solution of OA (5.0 g, 10.7 mmol, 1.00 eq.) in dry THF (150 mL), LiAlH4 (2.0 g, 53.6 mmol, 5.00 eq.,) was slowly added. Stirring under reflux was continued for another 2 h. After cooling to 20 °C, the reaction was quenched (slow addition of 20 mL MeOH), and aq. HCl (6 M, 50 mL) was added. The reaction mixture was extracted with ethyl acetate (3 × 75 mL); the combined organic phases were washed with aq. NaOH (1 M, 2 × 50 mL), brine (50 mL) and dried (MgSO4). The solvent was removed under reduced pressure, and the residue was subjected to chromatography (silica gel, chloroform/hexanes/ethyl acetate, 10:8:2) to yield 7 (4.61 g, 97%) as a colorless solid; m.p. 218–219 °C (lit.: [93] 217–219 °C); RF = 0.17 (silica gel, chloroform/hexanes/ethyl acetate, 10:8:2); [α]D = +72.1° (c = 0.113, MeOH) (lit.: [94] [α]D = +75.0° (c = 0.325, CHCl3)).

4.7. (3β) Urs-12-ene-3,28-diol (Uvaol, 8)

Following the procedure given for the synthesis of 7, from UA (5.00 g, 10.7 mmol), dry THF (150 mL) and LiAlH4 (2.0 g, 53.6 mmol) followed by chromatography (silica gel, (chloroform/hexanes/ethyl acetate, 10:8:2) 8 (4.36 g, 90%) was obtained as a colorless solid; m.p. 227–229 °C (lit.: [93] 225–227 °C); RF = 0.18 (silica gel, chloroform/hexanes/ethyl acetate, 10:8:2); [α]D = +60.5° (c = 0.109, MeOH); (lit.: [95] [α]D = +62.6° (c = 0.62, CHCl3)).

4.8. (3β)-Olean-12-ene-3,28-diyl Diacetate

Acetylation of 7 (4.00 g, 9.04 mmol) in dry pyridine (16 mL) with acetic anhydride (2.6 mL, 27.1 mmol) for 15 h at 20 °C followed by usual aqueous work up and chromatography (silica gel, (hexanes/ethyl acetate, 9:1) gave 9 (4.37 g, 92%) as a colorless solid; m.p. 184-186 °C (lit.: [96] 184-186 °C); RF = 0.43 (silica gel, hexanes/ethyl acetate, 9:1); [α]D = +62.2° (c = 0.124, CHCl3); (lit.: [97] [α]D = +56.0° (c = 1.0CHCl3)).

4.9. (3β)-Urs-12-ene-3,28-diyl Diacetate

Acetylation of 8 (3.48 g, 7.86 mmol) as described above for the synthesis of 9 gave 10 (3.66 g, 88%) as a colorless solid; m.p. 151–153 °C (lit.: [98] 150-151 °C); RF = 0.41 (silica gel, hexanes/ethyl acetate, 9:1); [α]D = +51.4° (c = 0.115, CHCl3).

4.10. (3β)-28-Hydroxyolean-12-en-3-yl Acetate

A solution of 9 (3.1 g, 5.89 mmol) and aluminum isopropoxide (12.3 g, 58.8 mmol) in isopropanol (150 mL) was heated under reflux for 4 h. Usual aqu. work-up followed by chromatography (silica gel, hexanes/ethyl acetate, 8:2) gave 11 (1.71 g, 60%) as a colorless solid; m.p. 234–236 °C (lit.: [99] 233–234 °C); RF = 0.55 (silica gel, hexanes/ethyl acetate, 8:2); [α]D = +67.4° (c = 0.122, CHCl3) (lit.: [100] [α]D = +71° (c = 0.70, CHCl3)).

4.11. (3β)-28-Hydroxyolean-12-en-3-yl Acetate

As described above for the synthesis of 11, from 10 (2.7 g, 5.13 mmol) and aluminum propoxide (10.7 g, 51.3 mmol) in isopropanol (150 mL) followed by chromatography (silica gel, hexanes/ethyl acetate, 8:2) 12 (1.92 g, 77%) was obtained as a colorless solid; m.p. 265–267 °C (lit.: [101] 258–261 °C); RF = 0.51 (silica gel, hexanes/ethyl acetate, 8:2); [α]D = +63.1° (c = 0.139, CHCl3) (lit.: [102] [α]D = +70.5° (c = 0.145, CHCl3)).

4.12. 4-{[(3β)-3-(Acetyloxy)-olean-12-en-28-yl]oxy}-4-oxobutanoic Acid

To a solution of 11 (0.45 g, 0.928 mmol) in dry pyridine (15 mL) succinic anhydride (0.188 g, 1.86 mmol) and cat. DMAP were added, and the mixture was stirred for 1 day under reflux. Usual aq. work-up followed by chromatography (silica gel, hexanes/ethyl acetate (1% HCOOH), 8:2 → 7:3) gave 13 (0.460 g, 85%) as a colorless solid; m.p. 124–126 °C; RF = 0.47 (silica gel, hexanes/ethyl acetate, 7:3); [α]D = +49.6° (c = 0.126, CHCl3); IR (ATR): ν = 2946m, 2864 w, 1734s, 1712s, 1463w, 1432w, 1387m, 1364m, 1244s, 1160s, 1095w, 1027m, 1004m, 986m, 967m cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.01 (brs, 1H, COO-H), 5.19 (t, J = 3.6 Hz, 1H, 12-H), 4.55–4.44 (m, 1H, 3-H), 4.07 (d, J = 11.0 Hz, 1H, 28-Ha), 3.72 (d, J = 11.0 Hz, 1H, 28-Hb), 2.71–2.59 (m, 4H, 34-H + 35-H), 2.04 (s, 3H, 32-H), 2.01–1.99 (m, 1H, 18-H), 1.97–1.78 (m, 3H, 16-Ha + 11-H), 1.77–1.46 (m, 9H, 19-Ha + 15-Ha + 2-H + 1-Ha + 9-H + 6-Ha + 7-Ha + 22-Ha), 1.45–1.21 (m, 4H, 6-Hb + 22-Hb + 7-Hb + 21-Ha), 1.19–1.10 (m, 5H, 16-Hb + 21-Hb + 27-H), 1.12–0.95 (m, 3H, 19-Hb + 1-Hb + 15-Hb), 0.94 (s, 3H, 25-H), 0.93 (s, 3H, 26-H), 0.88 (s, 3H, 30-H), 0.86 (s, 6H, 29-H + 24-H), 0.85 (s, 3H, 23-H), 0.84–0.80 (m, 1H, 5-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 177.9 (C-36), 172.2 (C-33), 171.3 (C-31), 143.7 (C-13), 123.0 (C-12), 81.1 (C-3), 71.3 (C-28), 55.4 (C-5), 47.6 (C-9), 46.3 (C-19), 42.7 (C-18), 41.8 (C-14), 39.9 (C-8), 38.4 (C-1), 37.8 (C-4), 36.9 (C-10), 36.0 (C-17), 34.1 (C-21), 33.3 (C-30), 32.6 (C-7), 31.6 (C-22), 31.0 (C-20), 29.1 (C-34), 29.1 (C-35), 28.2 (C-24), 26.1 (C-27), 25.7 (C-15), 23.7 (C-29), 23.7 (C-11), 23.7 (C-2), 22.3 (C-16), 21.4 (C-32), 18.4 (C-6), 16.8 (C-26), 16.8 (C-23), 15.7 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 583.9 (100%, [M-H]-); analysis calcd for C36H56O6 (584.84): C 73.93, H 9.65; found: C 73.71, H 9.86.

4.13. 4-{[(3β)-3-(Acetyloxy)urs-12-en-28-yl]oxy}-4-oxobutanoic Acid

Following the procedure given for 11, from 12 (1.35 g, 2.79 mmol), 14 (1.12 g, 69%) was obtained as a colorless solid; m.p. 112–114 °C; RF = 0.50 (silica gel, hexanes/ethyl acetate, 7:3); [α]D = +42.7° (c = 0.131, CHCl3); IR (ATR): ν = 2948m, 2925m, 1734s, 1712s, 1456m, 1432w, 1388m, 1370m, 1269m, 1244s, 1158s, 1095w, 1025m, 1006m, 985m, 967m cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.01 (brs, 1H, COO-H), 5.13 (dd, J = 3.7 Hz, 1H, 12-H), 4.53–4.44 (m, 1H, 3-H), 4.10 (d, J = 11.0 Hz, 1H, 28-Ha), 3.64 (d, J = 11.0 Hz, 1H, 28-Hb), 2.70–2.60 (m, 4H, 34-H + 35-H), 2.04 (s, 3H, 32-H), 1.98–1.88 (m, 2H, 16-Ha + 11-H), 1.76 – 1.68 (m, 1H, 15-Ha), 1.67–1.59 (m, 2H, 1-Ha + 2-H), 1.59–1.48 (m, 4H, 22-Ha + 7-Ha + 9-H + 6-Ha), 1.47–1.28 (m, 6H, 21-Ha + 18-H + 6-Hb + 19-H + 7-Hb + 22-Hb), 1.27–1.13 (m, 2H, 21-Hb + 16-Hb), 1.08 (s, 3H, 27-H), 1.08–1.06 (m, 1H, 1-Hb), 0.99–0.96 (m, 1H, 15-Hb), 0.97 (s, 3H, 26-H), 0.96 (s, 3H, 25-H), 0.93 (d, J = 5.8 Hz, 3H, 29-H), 0.91–0.88 (m, 1H, 20-H), 0.86 (s, 3H, 24-H), 0.86 (s, 3H, 23-H), 0.85–0.83 (m, 1H, 5-H), 0.80 (d, J = 5.3 Hz, 3H, 30-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 178.0 (C-36), 172.2 (C-33), 171.3 (C-31), 138.3 (C-13), 125.7 (C-12), 81.1 (C-3), 71.8 (C-28), 55.4 (C-5), 54.4 (C-18), 47.7 (C-9), 42.1 (C-14), 40.1 (C-8), 39.5 (C-20), 39.3 (C-19), 38.6 (C-1), 37.8 (C-17), 37.1 (C-4), 36.9 (C-10), 35.8 (C-22), 32.8 (C-7), 30.6 (C-21), 29.2 (C-34), 29.2 (C-35), 28.2 (C-24), 26.1 (C-15), 23.7 (C-2), 23.5 (C-11), 23.5 (C-16), 23.5 (C-27), 21.4 (C-32), 21.4 (C-29), 18.3 (C-6), 17.4 (C-30), 16.9 (C-26), 16.8 (C-23), 15.9 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 607.9 (100%, [M + Na]+); analysis calcd for C36H56O6 (584.84): C 73.93, H 9.65; found: C 73.68, H 9.91.

4.14. (3β, 20β)-3-Acetyloxy-11-oxoolean-12-en-29-oic Acid

Acetylation of GA (2.50 g, 5.31 mmol) as described above followed by chromatography (silica gel, hexanes/ethyl acetate, 8:2) gave 15 (2.15 g, 79%) as a colorless solid; m.p. 305–307 °C (lit.: [96] 316–318 °C); RF = 0.41 (silica gel, hexanes/ethyl acetate, 9:1); [α]D = +163.3° (c = 0.142, CHCl3); (lit.: [103] [α]D = +165.1° (c = 0.7, CHCl3)).

4.15. (3β, 20β) 3-Acetyloxy-29-hydroxyolean-12-en-11-one

To a solution of 15 (1.3 g, 2.76 mmol) and triethylamine (1.1 mL, 7.60 mmol) in dry THF (15 mL), at −12 °C, ethyl chloroformate (1.1 mL, 11.1 mmol) was added, and the mixture was stirred for 15 min. The precipitate was filtered off, and the filtrate was slowly added to a freshly prepared solution of sodium borohydride (0.522 g, 13.8 mol) in water (2.5 mL). Stirring at room temperature was continued for another 15 min followed by usual aq. work-up and chromatography (silica gel, CHCl3/Et2O/hexanes/HCOOH, 25:25:43:7) to yield 16 (1.09 g, 82%) as a colorless solid; m.p. 264–266 °C; RF = 0.46 (silica gel, CHCl3/Et2O/hexanes/HCOOH, 25:25:43:7); [α]D = +91.6° (c = 0.129, CHCl3); UV-Vis (CHCl3): λmax (log ε) = 249 nm (4.07); IR (ATR): ν = 3569w, 3550 w, 2925m, 2862w, 1725s, 1695m, 1651s, 1626m, 1465w, 1455m,1386m, 1366m, 1325w, 1279w, 1246s, 1209m, 1173s, 1143m, 1095w, 1048m, 1025s, 1001m, 985m cm−1; 1H NMR (500 MHz, CDCl3): δ = 5.58 (s, 1H, 12-H), 4.50 (dd, J = 11.8, 4.7 Hz, 1H, 3-H), 4.13 (d, J = 11.0 Hz, 1H, 30-Ha), 4.03 (d, J = 11.0 Hz, 1H, 30-Hb (30)), 2.78 (dt, J = 13.6, 3.6 Hz, 1H, 1-Ha), 2.35 (s, 1H, 9-H), 2.13–2.06 (m, 2H, 18-H + 16-Ha), 2.04 (s, 3H, 32-H), 1.86–1.76 (m, 1H, 15-Ha), 1.74–1.53 (m, 7H, 2-Ha + 22-Ha + 7-Ha + 19-Ha + 2-Hb + 21-Ha + 6-Ha) 1.50–1.36 (m, 3H, 6-Hb + 7-Hb + 22-Hb + 19-Hb), 1.36 (s, 3H, 27-H), 1.21– 1.16 (m, 1H, 15-Hb), 1.15 (s, 3H, 25-H), 1.12 (s, 3H, 26-H), 1.07–0.98 (m, 2H, 1-Hb + 16-Hb), 0.95 (s, 3H, 29-H), 0.87 (s, 6H, 28-H + 23-H), 0.86 (s, 3H, 24-H), 0.81–0.77 (m, 1H, 5-H), 0.80 (d, J = 5.7 Hz, 3H, 30-H) ppm; 13C NMR (126 MHz, CDCl3): δ = 200.4 (C-11), 171.4 (C-31), 169.8 (C-13), 128.5 (C-12), 80.9 (C-3), 67.0 (C-30), 61.8 (C-9), 55.2 (C-5), 47.0 (C-18), 45.6 (C-8), 43.5 (C-14), 40.2 (C-19), 38.9 (C-1), 38.2 (C-4), 37.1 (C-10), 36.0 (C-22), 34.3 (C-20), 32.8 (C-7), 32.4 (C-17), 30.2 (C-21), 28.6 (C-28), 28.2 (C-24), 27.9 (C-29), 26.7 (C-16), 26.5 (C-15), 23.7 (C-2), 23.5 (C-27), 21.4 (C-32), 18.8 (C-26), 17.5 (C-6), 16.8 (C-23), 16.5 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 497.9 (100%, [M-H]-); analysis calcd for C32H50O4 (498.74): C 77.06, H 10.10; found: C 76.81, H 10.35.

4.16. 4-{[(3β,20β)3-(Acetyloxy)-11-oxoolean-12-en-30-yl]oxy}-4-oxobutanoic Acid

Following the procedure described above, from 16 (0.270 g, 0.541 mmol) 17 (0.315 g, 97%) was obtained as a colorless solid; m.p. 109–111 °C; RF = 0.44 (silica gel, hexanes/ethyl acetate, 1:1); [α]D = +110.5° (c = 0.118, CHCl3); UV-Vis (CHCl3): λmax (log ε) = 254 nm (4.05); IR (ATR): ν = 2949m, 2928m, 2871w, 1730s, 1657m, 1465w, 1456w, 1388m, 1365m, 1322w, 1244s, 1209m, 1158m, 1091w, 1049w, 1028m, 1001m, 986 m cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.01 (brs, 1H, COO-H), 5.64 (s, 1H, 12-H), 4.70 (d, J = 10.9 Hz, 1H, 30-Ha), 4.51 (dd, J = 11.5, 4.9 Hz, 1H, 3-H), 3.46 (d, J = 10.9 Hz, 1H, 30-Hb), 2.85 (dt, J = 13.6, 3.5 Hz, 1H, 1-Ha), 2.79–2.71 (m, 2H, 34-H), 2.58–2.49 (m, 2H, 35-H), 2.37 (s, 1H, 9-H), 2.36–2.25 (m, 1H, 18-H), 2.12–2.06 (m, 1H, 16-Ha), 2.04 (s, 3H, 32-H), 1.87–1.75 (m, 1H, 15-Ha), 1.74–1.36 (m, 9H, 2-H + 7-Ha + 6-Ha + 19-Ha + 6-Hb + 22-Ha + 7-Hb + 21-Ha), 1.35 (s, 3H, 27-H), 1.33–1.14 (m, 4H, 21-Hb + 22-Hb + 19-Hb + 16-Hb), 1.12 (s, 6H, 25-H + 26-H), 1.10– 0.98 (m, 2H, 1-Hb + 15-Hb), 0.95 (s, 3H, 29-H), 0.87 (s, 9H, 28-H + 23-H + 24-H), 0.83–0.77 (m, 1H, 5-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 202.2 (C-11), 174.8 (C-36), 172.9 (C-33), 171.8 (C-13), 171.1 (C-31), 128.1 (C-12), 80.7 (C-3), 67.7 (C-30), 61.9 (C-9), 55.2 (C-5), 46.7 (C-18), 45.5 (C-8), 43.4 (C-14), 39.2 (C-19), 39.0 (C-1), 38.3 (C-4), 37.4 (C-10), 36.1 (C-22), 34.9 (C-20), 32.7 (C-7), 32.3 (C-17), 31.3 (C-21), 29.5 (C-35), 28.9 (C-34), 28.9 (C-28), 28.2 (C-24), 28.2 (C-29), 26.6 (C-16), 26.6 (C-15), 23.7 (C-2), 23.3 (C-27), 21.4 (C-32), 19.0 (C-26), 17.5 (C-6), 16.8 (C-23), 16.7 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1) m/z = 600.0 (96%, [M + H]+); analysis calcd for C36H54O7 (598.82): C 72.21, H 9.09; found: C 71.97, H 9.32.

4.17. (3β) 3-(Acetyloxy)olean-12-en-28-yl-4-{[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]amino}-4-oxobutanoate

To a solution of 13 (0.250 g, 0.427 mmol) in dry THF (20 mL) at −15 °C, 4-methylmorpholine (0.1 mL, 0641 mmol) and ethyl chloroformate (0.05 mL, 0.513 mmol) were added, and the mixture was stirred for 10 min at this temperature. Then 5-amino-1,3,4-thiadiazole-2-sulfonamide 5 (0.092 g, 0.513 mmol) was added, and the mixture was heated under reflux for 4 h. The solvents were removed under diminished pressure, and the residue was subjected to chromatography (silica gel, (CHCl3/MeOH, 95:5) to yield 18 (0.166 g, 52%) as a colorless solid; m.p. 198–200 °C; RF = 0.36 (silica gel, CHCl3/MeOH, 9:1); [α]D = +19.2° (c = 0.122, CHCl3); UV-Vis (CHCl3): λmax (log ε) = 262 nm (4.45); IR (ATR): ν = 3332w, 3234w, 2947m, 2864m, 1734m, 1704s, 1545m, 1536m, 1463w, 1432w, 1422w, 1371s, 1329m, 1245s, 1216m, 1173s, 1092m, 1027m, 1004m, 985m, 967m, 755m, 651m, 637m, 606 s, 504m cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 13.07 (brs, 1H, CON-H), 8.30 (d, J = 6.2 Hz, 2H, SN-H), 5.15 (t, J = 3.7 Hz, 1H, 12-H), 4.38 (dd, J = 11.7, 4.5 Hz, 1H, 3-H), 3.96 (d, J = 10.9 Hz, 1H, 28-Ha), 3.60 (d, J = 10.9 Hz, 1H, 28-Hb), 2.89–2.76 (m, 2H, 34-H), 2.72–2.65 (m, 2H, 35-H), 2.02–1.97 (m, 1H, 18-H), 1.99 (s, 3H, 32-H), 1.86 (td, J = 13.9, 4.6 Hz, 1H, 16-Ha), 1.82–1.75 (m, 2H, 11-H), 1.70 (dd, J = 13.4, 13.4 Hz, 1H, 19-Ha), 1.62–1.13 (m, 11H, 15-Ha + 2-H + 1-Ha + 9-H + 6-Ha + 21-Ha + 22-Ha, 6-Hb + 22-Hb + 7-Hb), 1.11 (s, 3H, 27-H), 1.09–0.93 (m, 4H, 16-Hb + 7-Hb + 19-Hb + 1-Hb), 0.88 (s, 3H, 25-H), 0.85 (d, 3H, 30-H), 0.84 (d, 3H, 29-H), 0.83 (s, 3H, 24-H), 0.83–0.80 (m, 7H, 26-H + 23-H + 5-H); ppm; 13C NMR (126 MHz, DMSO-d6): δ = 171.6 (C-33), 171.2 (C-36), 170.1 (C-31), 164.3 (C-38), 161.0 (C-37), 143.4 (C-13), 122.2 (C-12), 79.9 (C-3), 70.0 (C-28), 54.5 (C-5), 46.7 (C-9), 45.7 (C-19), 41.8 (C-18), 41.1 (C-14), 39.2 (C-8), 37.7 (C-1), 37.3 (C-4), 36.3 (C-10), 35.4 (C-17), 33.4 (C-7), 32.9 (C-30), 31.8 (C-21), 31.0 (C-22), 30.5 (C-20), 30.0 (C-34), 28.4 (C-35), 27.7 (C-24), 25.7 (C-27), 25.1 (C-15), 23.4 (C-29), 23.2 (C-2), 23.0 (C-11), 21.5 (C-16), 20.9 (C-32), 17.7 (C-6), 16.6 (C-26), 16.2 (C-23), 15.2 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 770.1 (100%, [M + Na]+); analysis calcd for C38H58S2N4 (747.02): C 61.10, H 7.83, N 7.50; found: C 60.81, H 8.03, N 7.39.

4.18. (3β)-3-(Acetyloxy)urs-12-en-28-yl-4-{[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]amino}4-oxobutanoate

Following the procedure given above for the synthesis of 18, from 14 (0.250 g, 0.427 mmol) 21 (0.217 g, 68%) was obtained as a colorless solid; m.p. 190–192 °C; RF = 0.33 (silica gel, CHCl3/MeOH, 9:1); [α]D = +27.4° (c = 0.129, CHCl3); UV-Vis (CHCl3): λmax (log ε) = 263 nm (4.98); IR (ATR): ν = 3244w, 2948m, 2925m, 2871m, 1733m, 1705s, 1531m, 1457w, 1431w; 1414w, 1367s, 1245s, 1174s, 1094m, 1027m, 1006m, 985m, 967m, 655m, 603s, 508m cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 13.07 (brs, 1H, CON-H), 8.30 (s, J = 2H, SN-H), 5.10 (t, J = 3.2 Hz, 1H, H-12), 4.38 (dd, J = 11.5, 4.7 Hz, 1H, H-3), 3.94 (d, J = 10.8 Hz, 1H, 28-Ha), 3.54 (d, J = 10.9 Hz, 1H, 28-Hb), 2.84–2.78 (m, 2H, 34-H), 2.71–2.65 (m, 2H, 35-H), 1.99 (s, 3H, 32-H), 1.92–1.80 (m, 3H, 16-Ha + 11-H), 1.65–1.53 (m, 3H, 15-Ha + 2-Ha + 1-Ha), 1.53–1.42 (m, 5H, 2-Ha + 9-H + 7-Ha + 6-Ha + 22-Ha), 1.40–1.06 (m, 7H, 18-H + 6-Hb + 21-Ha + 22-Hb + 7-Hb + 21-Hb + 16-Hb), 1.04 (s, 3H, 27-H), 1.01–0.96 (m, 1H, 1-Hb), 0.90 (s, 3H, 23-H), 0.87 (d, J = 6.7, 3H, 29-H), 0.86 (s, 3H, 26-H), 0.85–0.83 (m, 2H, 15-Hb + 1-Hb), 0.83 (s, 6H, 24-H + 25-H), 0.83–0.80 (m, 1H, 20-H), 0.87 (d, J = 5.3 Hz, 3H, 30-H) ppm; 13C NMR (126 MHz, DMSO-d6): δ = 171.4 (C-33), 171.2 (C-36), 170.1 (C-31), 164.3 (C-38), 161.0 (C-37), 138.0 (C-13), 124.8 (C-12), 79.9 (C-3), 70.4 (C-28), 54.5 (C-5), 53.4 (C-18), 46.8 (C-9), 41.4 (C-14), 39.4 (C-8), 38.7 (C-20), 38.5 (C-19), 37.9 (C-1), 37.3 (C-4), 36.4 (C-17), 36.3 (C-10), 35.1 (C-2), 32.1 (C-7), 30.0 (C-34), 29.9 (C-21), 28.4 (C-35), 27.7 (C-24), 25.5 (C-15), 23.3 (C-2), 23.0 (C-27), 22.9 (C-11), 22.8 (C-16), 21.0 (C-29), 20.9 (C-32), 17.1 (C-6), 17.1 (C-30), 16.6 (C-23), 16.3 (C-26), 15.2 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 770.1 (100%, [M + Na]+); analysis calcd for C38H58S2N4O7 (747.02): C 61.10, H 7.83, N 7.50; found: C 60.89, H 8.07, N 7.36.

4.19. (3β, 20β) 3-(Acetyloxy)-11-oxoolean-12-en-30-yl-4-{[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl] amino}-4-oxobutanoate

Following the procedure given above for the synthesis of 18, from 17 (0.250 g, 0.417 mmol) 20 (0.215 g, 68%) was obtained as a colorless solid; m.p. 179–181 °C; RF = 0.41 (silica gel, CHCl3/MeOH, 9:1); [α]D = +83.5° (c = 0.114, CHCl3); UV-Vis (CHCl3): λmax (log ε) = 256 nm (4.25); IR (ATR): ν = 3252w, 2950m, 2872w, 1727m, 1709m, 1643m, 1528m, 1466w, 1455w, 1365s, 1323m, 1247s, 1215m, 1173s, 1088w, 1049w, 1028m, 1001m, 985m, 754s, 667m, 654m, 604s, 509m cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 13.08 (brs, 1H, CON-H), 8.30 (s, J = 2H, SN-H), 5.48 (s, 1H, 12-H), 4.42 (dd, J = 11.8, 4.5 Hz, 1H, 3-H), 4.05 (d, J = 11.0 Hz, 1H, 30-Ha), 3.92 (d, J = 11.0 Hz, 1H, 30-Hb), 2.82 (dd, J = 7.5, 5.5 Hz 2H, 35-H), 2.71 (dd, J = 7.6, 5.5 Hz, 2H, 34-H), 2.61 (dt, J = 13.4, 3.6 Hz, 1H, 1-Ha), 2.38 (s, 1H, 9-H), 2.16–2.04 (m, 2H, 18-H + 16-Ha), 2.00 (s, 3H, 32-H), 1.79–1.35 (m, 7H, 16-Hb + 7-Ha + 19-Ha + 2-Ha + 6-Ha + 2-Hb + 6-Hb). 1.34 (s, 3H, 27-H), 1.34–1.31 (m, 3H, 7-Hb + 21-Hb + 22-Hb), 1.24–1.06 (m, 4H, 19-Hb + 22-Hb + 15-Hb + 1-Hb), 1.06 (s, 1H, 25-H) 1.04 (s, 1H, 26-H)), 0.86– 0.98 (m, 2H, 15-Hb + 5-H), 0.85 (s, 3H, 29-H), 0.82 (s, 6H, 23-H + 24-H), 0.80 (s, 3H, 28-H) ppm; 13C NMR (126 MHz, DMSO-d6): δ = 198.9 (C-11), 171.8 (C-36), 171.1 (C-33), 170.1 (C-31), 170.0 (C-13), 127.4 (C-12), 79.7 (C-3), 67.0 (C-30), 60.8 (C-9), 53.7 (C-5), 46.0 (C-18), 44.9 (C-8), 43.0 (C-14), 39.7 (C-19), 37.8 (C-1), 37.5 (C-4), 36.5 (C-10), 35.4 (C-22), 34.0 (C-20), 31.9 (C-7), 31.8 (C-17), 29.9 (C-35), 29.3 (C-21), 28.3 (C-34), 28.1 (C-28), 27.7 (C-24), 27.2 (C-29), 25.9 (C-16), 25.9 (C-15), 23.2 (C-2), 23.1 (C-27), 20.9 (C-32), 18.3 (C-26), 16.9 (C-6), 16.6 (C-23), 16.1 (C-25) ppm; MS (ESI, MeOH/CHCl3, 4:1): m/z = 784.0 (100%, [M + Na]+; analysis calcd for C38H56S2N4O8 (761.00): C 59.97, H 7.42, N 7.36; found: C 59.71, H 7.71, N 7.09.

Author Contributions

Conceptualization, R.C.; validation, R.C.; investigation, T.C.D., N.H., O.K., J.Z., and S.H.; writing—original draft preparation, R.C. writing—review and editing, T.C.D., N.H., J.Z. and S.H R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We like to thank D. Ströhl, Y. Schiller and S. Ludwig for the NMR spectra; IR spectra, micro-analyses, and optical rotations were measured by M. Schneider.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

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Molecules 28 01009 g001 550
Figure 1. Structure of well-established CA inhibitors SLC-0111 and acetazolamide.
Figure 1. Structure of well-established CA inhibitors SLC-0111 and acetazolamide.
Molecules 28 01009 g001
Molecules 28 01009 g002 550
Figure 2. Structures of starting materials betulin (BN), oleanolic acid (OA), ursolic acid (UA), and glycyrrhetinic acid (GA).
Figure 2. Structures of starting materials betulin (BN), oleanolic acid (OA), ursolic acid (UA), and glycyrrhetinic acid (GA).
Molecules 28 01009 g002
Molecules 28 01009 sch001 550
Scheme 1. Reactions and conditions: (a) Ac2O, TEA, DMAP (cat.), DCM, 20 °C, 12 h, 90%; (b) CaH2, MeOH/THF, 20 °C, 12 h, 83%; (c) pyridine, DMAP, succinic anhydride, reflux, 15 h, 71%; (d) THF, 4-methyl-morpholine, ethyl chloroformate, 20 °C, 15 min, then 5, reflux, 48 h, 88%; (e) conc. HCl, reflux, 3 h, 94%.
Scheme 1. Reactions and conditions: (a) Ac2O, TEA, DMAP (cat.), DCM, 20 °C, 12 h, 90%; (b) CaH2, MeOH/THF, 20 °C, 12 h, 83%; (c) pyridine, DMAP, succinic anhydride, reflux, 15 h, 71%; (d) THF, 4-methyl-morpholine, ethyl chloroformate, 20 °C, 15 min, then 5, reflux, 48 h, 88%; (e) conc. HCl, reflux, 3 h, 94%.
Molecules 28 01009 sch001
Molecules 28 01009 sch002 550
Scheme 2. Reactions and conditions: (a) LiAlH4, THF, reflux, 2 h; (b) Ac2O, pyridine, 20 °C, 15 h; (c) Al(iPrO)3, iPrOH, reflux, 4 h.
Scheme 2. Reactions and conditions: (a) LiAlH4, THF, reflux, 2 h; (b) Ac2O, pyridine, 20 °C, 15 h; (c) Al(iPrO)3, iPrOH, reflux, 4 h.
Molecules 28 01009 sch002
Molecules 28 01009 sch003 550
Scheme 3. Reactions and conditions: (a) Pyridine, DMAP, succinic anhydride, reflux, 24 h; (b) Ac2O, pyridine, 20 °C, 15 h; (c) ethyl chloroformate, TEA, THF, −12 °C, 15 min, then sodium borohydride in water, 15 min; (d) pyridine, DMAP, succinic anhydride, reflux, 24 h.
Scheme 3. Reactions and conditions: (a) Pyridine, DMAP, succinic anhydride, reflux, 24 h; (b) Ac2O, pyridine, 20 °C, 15 h; (c) ethyl chloroformate, TEA, THF, −12 °C, 15 min, then sodium borohydride in water, 15 min; (d) pyridine, DMAP, succinic anhydride, reflux, 24 h.
Molecules 28 01009 sch003
Molecules 28 01009 sch004 550
Scheme 4. Reactions and conditions: (a,b) THF, 4-methyl-morpholine, ethyl chloroformate, 20 °C, 15 min, then 5, reflux, 48 h.
Scheme 4. Reactions and conditions: (a,b) THF, 4-methyl-morpholine, ethyl chloroformate, 20 °C, 15 min, then 5, reflux, 48 h.
Molecules 28 01009 sch004
Molecules 28 01009 g003 550
Figure 3. Dixon plot for compound 6 and CA II.
Figure 3. Dixon plot for compound 6 and CA II.
Molecules 28 01009 g003
Table
Table 1. Inhibition percentage of conjugates (at 10 µM concentration) and of standard acetazolamide (4).
Table 1. Inhibition percentage of conjugates (at 10 µM concentration) and of standard acetazolamide (4).
CompoundInhibition [%]
489.9 ± 0.6
693.0 ± 0.1
1849.4 ± 0.1
1970.8 ± 0.2
2096.8 ± 0.2
Table
Table 2. Ki values (in μM) for conjugates 6, 19, and 20.
Table 2. Ki values (in μM) for conjugates 6, 19, and 20.
CompoundKi (in μM)
60.129 ± 0.02
190.91 ± 0.17
205.22 ± 0.57
Table
Table 3. Cytotoxicity of acetazolamide (4) and conjugates 6, 18-20 assessed from SRB-assays (EC50 values [µM] after 72 h of treatment). Human cancer cell lines: A375 (epithelial melanoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma), A2780 (ovarian carcinoma); non-malignant: NIH 3T3 (fibroblasts); n.d. not determined; positive control: doxorubicin (DX).
Table 3. Cytotoxicity of acetazolamide (4) and conjugates 6, 18-20 assessed from SRB-assays (EC50 values [µM] after 72 h of treatment). Human cancer cell lines: A375 (epithelial melanoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast adenocarcinoma), A2780 (ovarian carcinoma); non-malignant: NIH 3T3 (fibroblasts); n.d. not determined; positive control: doxorubicin (DX).
A375HT29MCF-7A2780NIH 3T3
4>30>30>30>30>30
68.5 ± 0.710.2 ± 1.38.9 ± 0.79.3 ± 1.29.5 ± 1.0
1810.1 ± 0.814.2 ± 1.410.6 ± 1.411.8 ± 1.414.0 ± 1.5
1913.7 ± 1.115.0 ± 0.612.4 ± 0.812.5 ± 1.714.8 ± 1.5
209.2 ± 0.513.0 ± 1.310.5 ± 1.29.8 ± 0.811.9 ± 1.7
DXn.d.0.25 ± 0.020.1 ± 0.010.1 ± 0.010.01 ± 0.001
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Denner, T.C.; Heise, N.; Zacharias, J.; Kraft, O.; Hoenke, S.; Csuk, R. Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates. Molecules 2023, 28, 1009. https://doi.org/10.3390/molecules28031009

AMA Style

Denner TC, Heise N, Zacharias J, Kraft O, Hoenke S, Csuk R. Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates. Molecules. 2023; 28(3):1009. https://doi.org/10.3390/molecules28031009

Chicago/Turabian Style

Denner, Toni C., Niels Heise, Julian Zacharias, Oliver Kraft, Sophie Hoenke, and René Csuk. 2023. "Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates" Molecules 28, no. 3: 1009. https://doi.org/10.3390/molecules28031009

APA Style

Denner, T. C., Heise, N., Zacharias, J., Kraft, O., Hoenke, S., & Csuk, R. (2023). Small Structural Differences Govern the Carbonic Anhydrase II Inhibition Activity of Cytotoxic Triterpene Acetazolamide Conjugates. Molecules, 28(3), 1009. https://doi.org/10.3390/molecules28031009

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