Alkylidene branched lupane derivatives: Synthesis and antitumor activity

European Journal of Medicinal Chemistry 53 (2012) 337e345

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Alkylidene branched lupane derivatives: Synthesis and antitumor activity René Csuk*, Sebastian Stark, Christoph Nitsche, Alexander Barthel, Bianka Siewert Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle (Saale), Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2012 Received in revised form 16 April 2012 Accepted 17 April 2012 Available online 25 April 2012

Several novel alkylidene branched lupane derivatives have been prepared. Many of these compounds showed a significant cytotoxicity. The most active compound, 2-methylene-betulonic acid, showed IC50 values between 0.2 and 0.6 mM for 15 different human cancer cell lines. Cytotoxicity can be improved by encapsulation in liposomes. These compounds act by triggering apoptotic cell death as shown by DNAladdering experiments and acridine orange/ethidium bromide staining. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Betulonic acid Methylene compounds Antitumor activity Apoptosis

1. Introduction The natural occurring lupane-type triterpene betulin (1, Scheme 1) is widely spread in plants. Betulin and derivatives show antiviral [1,2], antiplasmodial [3] and anti-inflammatory [1] activity. Many of its derivatives [4e7] inhibit the growth of human tumor cell lines by triggering apoptosis [8e10]. Several structureeactivity relationships have been established for betulin derivatives. Thus, the presence of a carboxylic or carbonylic group at carbon C28 seems mandatory for high cytotoxicity as well as the oxidation of hydroxyl group at carbon C3 afforded derivatives showing higher cytotoxicity. The same is true for introducing an unsaturation between carbons C20/C29. Acylation and carbon C3 and/or esterification at C28 diminish cytotoxicity. Oxidation of betulin either at position C3 and/or at C30 affects its binding to topoisomerases I/II, and hence to derivatives providing higher cytotoxicity [11]. Previously we could show that betulin derivatives holding an extra a,b-unsaturated functional group display stronger cytotoxicity toward human tumor cell lines than their parent compounds [12,13]. It is assumed that this improved cytotoxic effect is connected to the reaction of the a,b-unsaturated functional group in Michael addition reactions with suitable intracellular targets. Further, to improve the solubility of betulin (which is a notorious problem in screening and administration of betulin) we

* Corresponding author. Tel.: þ49 (0) 345 55 25660; fax: þ 49 (0) 345 55 27030. E-mail address: [email protected] (R. Csuk). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2012.04.023

decided to incorporate the a,b-unsaturated functional group within the triterpenoid skeleton. A compound meeting these requirements can be obtained by an oxidation of the secondary hydroxyl group at carbon C(3) in betulin followed by a subsequent methylenation reaction at the adjacent carbon C(2). 2. Results 2.1. Chemistry Commercially available betulin (1) was oxidized to betulonal (2) using Swern oxidation conditions (Scheme 1), and 2 was obtained in >90% yield. Betulonic acid (3) was obtained from 1 by Jones oxidation. Jones oxidation of 1 followed by an esterification yielded methyl betulonate (4) [12,13]. Previously, methylenation in a-position of a cyclohexanone has been performed by several different approaches, among them cascade syntheses [14], the use of silyl enolethers [15e17], fragmentation of g-oxosulfonium methylides [18] and the use of several zirconocene [19] or palladium catalysts [20,21]. One of the oldest approaches to a-alkylidene cycloalkanones, however, has been described as early as 1920 by Carl Mannich [22,23]. Recently, an interesting variation has been described using ionic liquids [24]. There are no precedences for Mannich-type reactions using the CeH acidic protons at C(2) of a triterpenoid skeleton. Treatment of 2 with dimethylammonium hydrochloride, aq. formalin in tBuOH at 60
C for 5 days did not result in forming the corresponding Mannich base. Formation of this product was invariably followed by a b-elimination reaction, and 2-methylene compound 5 was

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R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345

Scheme 1. a) (COCl)2, DCM, DMSO, NEt3, 90%; b) CrO3, H2SO4, 1 h, 25
C, 52%; c) K2CO3, acetone, MeI, 24 h, 25
C, 94%, d) NHMe2.HCl, aq. HCHO, t.BuOH, 60
C, 5 days, 75%; e) NHMe2.HCl, aq. HCHO, t.BuOH, 60
C, 3 days, 60%; f) K2CO3, paraformaldehyde, DMF, 90
C, 1 h, 59%; g) NHMe2.HCl, aq. HCHO, t.BuOH, 60
C, 6 days, 54%; h) paraformaldehyde, K2CO3, DMF, 90
C, 1 h, 71%.

obtained. Similar treatment of acid 3 or ester 4 invariably gave the C-2 methylene substituted compounds 6 and 7, respectively. Treatment of 4 with dimethylammonium hydrochloride and paraformaldehyde in glacial acetic acid, however, allowed to isolate the Mannich base 8. On standing in water or phosphate buffer, compound 8 decomposes rapidly, and 7 is formed by a b-elimination reaction. The absolute configuration at carbon C(2) in compound 8 was determined to be (S) with an axially oriented hydrogen substituent at C(2) by extensive 1H NMR NOESY experiments (Fig. 1). In the 1H NOESY spectra HeC(2) shows three longrange couplings with the proton HeC(1) and protons HaxeC(23) and HaxeC(25).

Fig. 1. Assignment of the absolute configuration at C(2) from experiments.

1

H NOESY NMR

From these experiments we assumed the aldol condensation might be a useful method for obtaining C-2 alkylidene branched derivatives. Previously, it has been shown that for the reaction of 4 with benzaldehyde or furfural [25] the products of an aldol condensation could be obtained. These products, however, did not show any significant cytotoxicity (IC50 > 30 mmol). Treating 3 with paraformaldehyde in the presence of finely grounded potassium carbonate gave the methylene compound 6. Similarly, from 4 compound 7 was obtained. Treatment of 4 with acetaldehyde/LDA gave (2E)-2-ethylidene 9 (Scheme 2) in 67% yield; from 4 and acrolein/LDA (2Z)-2-prop-2-enylidene 10 was obtained. The assignment of the absolute configuration of the alkenic bond was performed again using 1H NMR NOESY spectra. Thus, in 9 HeqeC(1) shows a long-range coupling with the methyl protons of the ethylidene moiety, whereas in 10 no long-range couplings for HeqeC(1) can be found e therefore paralleling previous assignments made for the respective benzylidene or furfurylidene derivatives [25]. Treatment of 6 with oxalyl chloride followed by the reaction with thiomorpholine yielded the ester 11 (Scheme 3). Michael reaction of 7 with 2-mercaptoethanol in the presence of DBU gave the 2-hydroxyethylmethylthio compound 12 whose oxidation with hydrogen peroxide in glacial acetic acid yielded the sulfonyl compound 13. Similarly, by a Michael reaction of 7 with nitromethane/DBU compound 14 was obtained. The absolute configuration of the newly created stereogenic center C(2) in compounds 11e13 was again deduced from the 1H

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339

Fig. 2. Left: Results from the AO/EB test: treatment of A549 lung carcinoma cells with 6 (0.8 mM); right: DNA-laddering (colon cancer cell line SW480; after treatment with IC90 concentration of 6 for 24 h).

NOESY NMR spectra. A [2 þ 3] cycloaddition reaction of 7 with diazomethane gave spiro-compound 15. 2.2. Biology The compounds 5e15 were tested for their cytotoxicity against 15 human cancer cell lines and mouse fibroblasts NiH3T3 using the colorimetric SRB protocol [26e28]. The summarized IC50 values in Table 1 were obtained from the doseeresponse curves. Compound 14 showed no sufficient solubility in the test, compound 8 was too unstable, and 15 was regarded inactive (IC50 > 30 mmol). All the C(2)-methylene derivatives 5e7 showed

an excellent cytotoxicity with the ester 7 being weakest in activity and the acid 5 showing IC50 values between 0.2 and 0.6 mmol for the different tumor cell lines. Cytotoxicity drops for the alkylidene compounds 9 and 10 significantly. The weaker activity of 9 and 10 compared to 7 might be explained by steric hindrance, the higher activity of acid 6 compared to ester 7 parallels previous findings showing that betulinic esters show a reduced cytotoxicity compared to the free acid. Betulin and derivatives often show only poor solubility in water; hence, their bioavailability is reduced. To overcome this limit we encapsulated ester 7 using a commercially available liposome

Scheme 2. a) LDA, MeCHO, THF, 78 / 25
C, 67%; b) NHMe2.HCl, paraformaldehyde, HOAc, 80
C, 30 min, 47%; c) LDA, acrolein, THF, 78 / 25
C, 63%; d) H2O, 40
C, 6 h, quant.

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Scheme 3. a) (COCl)2, toluene, NEt3, DMF, 30 min, 25
C, then thiomorpholine, NEt3, 100
C, 3 h, 55%; b) HSeCH2eCH2eOH, DBU, DCM, 25
C, 48 h, 60%; c) MeNO2, DBU, DCM, 25
C, 5 days, 66%; d) CH2N2, Et2O, 25
C, 3 days, 32%; e) H2O2, AcOH, 25
C, 30 min, 83%.

formulation [29]. Best results were obtained with soybean lecithin (Lipoid S75). Subsequent extrusion through a polycarbonate membrane [30] with a pore size of 100 nm using a LiposoFast system gave liposomes with a hydrodynamic diameter between 70 and 120 nm (Z-average 105 nm) as determined by dynamic light

Table 1 Cytotoxicity in a panel of various human cancer cell lines. cell line

518A2 A431 A253 FADU A549 A2780 DLD-1 HCT-8 HCT-116 HT-29 SW480 8505C SW1736 MCF-7 Lipo NiH3T3

IC50 values in mM for cancer cell lines 5

6

7

7L

9

10

11

12

13

3.8 3.0 1.0 5.1 1.8 1.3 3.7 7.1 3.2 7.1 2.7 1.3 2.1 2.1 2.7 4.7

0.5 0.2 0.4 0.5 0.6 0.6 0.4 0.2 0.2 0.4 0.4 0.6 0.4 0.3 0.6 0.8

4.2 4.9 4.0 7.7 5.1 5.1 6.4 9.0 4.5 5.9 4.5 5.0 4.9 4.4 4.6 4.9

1.6 1.6 1.5 2.6 2.3 2.5 2.6 2.1 2.2 2.2 1.5 2.3 1.9 1.8 1.9 2.3

>30 26.4 21.6 22.4 >30 16.1 >30 17.2 24.0 >30 16.0 26.4 21.9 27.2 27.8 29.1

11.5 13.0 10.4 17.0 13.0 11.2 14.2 14.0 14.6 10.9 10.5 11.3 15.0 11.2 11.6 13.1

1.5 1.4 1.4 1.6 1.5 1.0 1.5 1.5 1.3 1.6 1.5 1.5 1.5 1.4 1.5 1.9

1.5 1.7 1.5 2.5 2.1 1.6 2.5 2.5 1.9 1.4 1.5 2.3 2.1 1.5 1.8 2.1

2.6 2.8 2.5 3.8 3.3 2.6 4.4 4.0 2.8 2.5 2.6 3.4 3.3 2.6 2.7 3.2

Values are derived from doseeresponse curves obtained by measuring the percentage of viable cells relative to untreated controls after 96 h exposure of the test compounds to the cell line using an SRB assay for melanoma (518A2), zervic cancer (A431), head and neck tumor (A253, FADU), lung carcinoma (A549), ovarian cancer (A2780), colon cancer (DLD-1, HCT-8, HCT-116, HT-29, SW480), anaplastic thyroid cancer (8505c, SW1736), mamma carcinoma (MCF-7), liposarcoma (LIPO) and mouse fibroblasts (NiH3T3). Values are the average from at least three independent experiments. Variation was generally  10%; 7L corresponds to compound 7 liposome encapsulated.

scattering. Encapsulation was >96% as determined by HPLC. Their physical stability exceeds 4 weeks. Applicating compound 7 in liposomes in the SRB assay revealed an extra benefit from encapsulation by observing an increased cytotoxicity for all the cell lines. For the products resulting from the Michael addition reactions only compound 12 showed a good cytotoxicity; this compound is roughly three times more active than its parent compound 7. Cytoxicity becomes weaker for oxidized 13. Hence, the presence of an a,b-unsaturated system in ring A is not a prerequisite for high cytotoxicity, However, it cannot ruled out that 12 is transformed in the cells back into compound 7, and the higher cytotoxicity associated with compound 12 is indicative for a higher intracellular concentration of 12. In consequence, compound 12 would be able to act as a pro-drug for ester 7 or acid 6. Compounds 6, 7 and 12 were investigated regarding their potential to induce apoptosis. All of these compounds showed DNA-laddering (using the colon cancer cell line SW480), hence giving a good indication for apoptosis [31]. These compounds were also tested in an acridine orange/ethidium bromide (AO/EB) assay; all compounds showed green fluorescence in the AO/EB tests, which indicate apoptosis (Fig. 2). 3. Conclusions We have examinated betulonates bearing an exo-alkylidene moiety at C(2). These compounds are easily synthesized either by Mannich reactions followed by b-elimination or by aldol condensation reactions. These compounds showed promising cytotoxicity for 15 human tumor cell lines; they are also valuable starting materials for following Michael addition reactions. Encapsulation of one of these compounds increased cytotoxicity by a factor of three. The presence of an a,b-unsaturated system in ring A is not

R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345

a prerequisite for an excellent cytotoxicity. Increased cytotoxicity was observed also for a 2-hydroxyethylthio-derivative; the activity of this compound to induce cell death might result from a higher intracellular concentration of the compound. The results from DNA-laddering experiments and an AO/EB assay provided evidence for an apoptotic cell death. 4. Experimental 4.1. General Instrumentation, cell lines and culture conditions, SRB cytotoxicity assay, AO/EB staining and DNA fragmentation assay as described previously [26e28,31]. 4.2. Preparation of the liposomes Unilamellar liposomes of approximately 100 nm diameter were obtained by the method of Olson [30] employing a laboratory extruder (LiposoFast, Avestin Inc.). In a typical experiment for preparing 5 ml dispersion of liposomes, 5 mg of the compound were mixed with an excess (125 mg) of phosphatidylcholine formulation (Lipoid S75) in chloroform (5 ml) and evaporated to a film. The lipid film was hydrated with H2O (5 ml) for 24 h at room temperature. The solution obtained was extruded through a polycarbonate filter of 100 nm pore size. Twenty-one cycles were applied and concentration of the compound was determined by HPLC (RP18, 4.6  250, l ¼ 230 nm, methanol, 1.3 ml). Liposomes were characterized by DLS. 4.3. 2-Methylene-3-oxolup-20(29)en-28-al (5) A solution of 2 (097 g, 2.21 mmol), dimethylammonium hydrochloride (200 mg, 2.45 mmol) and aq. formalin (37%, 0.3 ml, 3.76 mmol) in t-butanol (15 ml) was stirred at 60
C for 5 days. The solvents were removed under reduced pressure, and the residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 5 (975 mg, 75%) as a colorless foam; m.p. 205
C (decomp.); RF ¼ 0.78 (n-hexane/ethyl acetate, 4:1); [a]D ¼ þ56.6
(c ¼ 5.70, CHCl3); IR (KBr): n ¼ 3432 m, 2945 s, 2868 m, 1718 m, 1687 m, 1642 m, 1454 m, 1377 m, 1246 m, 1141 w, 1056 m, 967 w, 884 m cm1; UVevis (MeOH): lmax (nm) (log ε) ¼ 232 (0.44); 1H NMR (500 MHz, CDCl3): d ¼ 9.67 (d, 1 H, J ¼ 1.5 Hz, CH (28)), 5.96 (m, 1 H, CHa (31)), 5.12 (m, 1 H, CHb (31)), 4.76 (m, 1 H, CHa (29)), 4.64 (m, 1 H, CHb (29)), 2.88 (ddd, 1 H, J ¼ 11.4, 11.2, 5.8 Hz, CH (19)), 2.69 (d, 1 H, J ¼ 15.1 Hz, CHa (1)), 2.11e2.03 (m, 3 H, CHb (1) þ CH (13) þ CHa (21)), 1.93e1.18 (m, 17 H, CHa (16) þ CHa (15) þ CHa (12) þ CHb (16) þ CH (18) þ CH (9) þ CHa (22) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (21) þ CHb (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CH (5)), 1.70 (s, 3 H, CH3 (30)), 1.12e1.03 (m, 1 H, CHb (12)), 1.10 (s, 3 H, CH3 (24)), 1.04 (s, 3 H, CH3 (23)), 1.00 (s, 3 H, CH3 (27)), 0.96 (s, 3 H, CH3 (25)), 0.83 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.5 (C3, CO), 206.4 (C28, CHO), 149.6 (C20, C]CH2), 142.2 (C2, C]CH2), 123.6 (C31, C] CH2), 110.2 (C29, C]CH2), 59.3 (C17, Cquart.), 54.0 (C5, CH), 48.3 (C9, CH), 47.9 (C18, CH), 47.4 (C19, CH), 47.1 (C1, CH2), 45.8 (C4, Cquart.), 42.6 (C14, Cquart.), 40.7 (C8, Cquart.), 38.7 (C13, CH), 36.9 (C10, Cquart.), 33.4 (C7, CH2), 33.1 (C16, CH2), 29.8 (C15, CH2), 29.1 (C21, CH2), 28.8 (C22, CH2), 28.1 (C24, CH3), 25.5 (C12, CH2), 22.4 (C23, CH3), 21.3 (C11, CH2), 20.1 (C6, CH2), 19.0 (C30, CH3), 15.6 (C25, CH3), 15.5 (C26, CH3), 14.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 923.4 (86% [2M þ Na]þ), 901.5 (12% [2M þ H]þ), 505.1 (77% [M þ Na þ MeOH]þ), 451.4 (18% [M þ H]þ); analysis for C31H46O2 (450.70): C, 82.61; H, 10.29; found: C, 82.52; H, 10.37.

341

4.4. 2-Methylene-3-oxolup-20(29)en-28-oic acid (6) 4.4.1. Mannich reaction A solution of 3 (1.35 g, 2.90 mmol), dimethylammonium hydrochloride (550 mg, 6.75 mmol) and aq. formalin (37%, 0.7 ml, 8.6 mmol) in t-butanol (15 ml) was stirred at 60
C for 3 days. The solvents were removed under reduced pressure, and the residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 6 (810 mg, 60%) as colorless solid. 4.4.2. Aldol condensation A mixture of finely grounded K2CO3 (7.0 g, 54.6 mmol), paraformaldehyde (7.0 g, 0.23 mol) and 3 (3.3 g, 7.26 mmol) was suspended in dry DMF (80 ml) and heated for 1 h at 90
C. After cooling to 25
C, the mixture was poured into cold water (200 ml) and extracted with ethyl acetate (3  100 ml). The extracts were washed with brine (30 ml), dried (Na2SO4), and the solvents were removed under diminished pressure. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 4:1) to yield 6 (2 g, 59.0%) as a colorless solid. 4.4.3. Data for 6 M.p. 242e245
C; RF ¼ 0.47 (n-hexane/ethyl acetate, 8:2); [a]D ¼ þ39.3
(c ¼ 5.40, CHCl3); IR (KBr): n ¼ 3069 m, 2943 s, 2872 s, 1690 s, 1639 w, 1602 m, 1459 m, 1398 w, 1380 m, 1358 w, 1316 w, 1290 w, 1229 m, 1196 w, 1152 w, 1111 w, 1084 w, 1060 m cm1; UVevis (methanol): lmax (log ε) ¼ 218 (4.15), 252 (3.85) nm; 1H NMR (500 MHz, CDCl3): d ¼ 10.76 (br s, 1 H, COOH (28)), 5.95 (br m, 1 H, CHa (31)), 5.11 (br m, 1 H, CHb (31)), 4.73 (br m, 1 H, CHa (29)), 4.60 (br s, 1 H, CHb (29)), 3.00 (ddd, 1 H, J ¼ 10.6, 10.6, 4.5 Hz, CH (19)), 2.68 (d, 1 H, J ¼ 15.3 Hz, CHa (1)), 2.31e2.18 (m, 2 H, CH 13 þ CHa (16)), 2.05 (d, 1 H, J ¼ 15.3 Hz, CHb (1)), 2.04e1.91 (m, 2 H, CHa (21) þ CHb (22)), 1.77e1.66 (m, 1 H, CHa (12)), 1.68 (s, 3 H, CH3 (30)), 1.63 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.57e1.15 (m, 12 H, CH (5) þ CH (9) þ CHa (15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11)), 1.14e1.02 (m, 2 H, CHb (12) þ CHb (15)), 1.09 (s, 3 H, CH3 (23)), 1.01 (s, 3 H, CH3 (24)), 0.98 (s, 3 H, CH3 (27)), 0.97 (s, 3 H, CH3 (26)), 0.82 (s, 3 H, CH3 (25)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.6 (C3, C]O), 182.5 (C28, COOH), 150.3 (C20, C]CH2), 142.2 (C2, C]CH2), 123.7 (C31, CH2), 109.8 (C29, CH2), 56.4 (C17, Cquart.), 54.0 (C5, CH), 49.2 (C9, CH), 48.4 (C18, CH), 47.1 (C1, CH2), 46.9 (C19, CH), 45.8 (C4, Cquart.), 42.5 (C14, Cquart.), 40.5 (C8, Cquart.), 38.5 (C13, CH), 37.0 (C10, Cquart.), 36.9 (C22, CH2), 33.4 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.7 (C15, CH2), 28.1 (C23, CH3), 25.5 (C12, CH2), 22.3 (C24, CH3), 21.4 (C11, CH2), 20.1 (C6, CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.5 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 956.1 (40% [2M þ Na]þ), 521.3 (75% [M þ Na þ MeOH]þ); 1399.5 (15% [3MeH]), 954.3 (65% [2M þ Nae2H]), 931.7 (75% [2MeH]), 497.8 (20% [M þ MeOHeH]), 465.6 (100% [M-H]); analysis for C31H46O3 (466.70): C, 79.78; H, 9.93; found: C, 79.54; H, 10.11. 4.5. Methyl 2-methylene-3-oxolup-20(29)en-28-oate (7) 4.5.1. Mannich reaction A solution of 4 (938 mg, 2.06 mmol), dimethylammonium hydrochloride (150 mg, 1.84 mmol) and aq. formalin (37%, 0.4 ml, 4.9 mmol) in t-butanol (15 ml) was stirred at 60
C for 6 days. The solvents were removed under reduced pressure, and the residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 7 (540 mg, 54%) as a colorless foam. 4.5.2. Aldol condensation A mixture of finely grounded K2CO3 (2.5 g, 18.1 mmol), paraformaldehyde (2.5 g, 83.3 mmol) and 4 (2.0 g, 4.3 mmol) was

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suspended in dry DMF (30 ml) and heated for 1 h at 90
C. After cooling to 25
C, the mixture was poured into cold water (200 ml) and extracted with ethyl acetate (3  100 ml). The extracts were washed with brine (30 ml), dried (Na2SO4), and the solvents were removed under diminished pressure. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 7 (1.45 g, 70.7%) as a colorless solid. 4.5.3. Data for 7 M.p. 152e155
C (decomp.); RF ¼ 0.83 (n-hexane/ethyl acetate, 4:1); [a]D ¼ þ69.5
(c ¼ 2.6, CHCl3); IR (KBr): n ¼ 3435 m, 2949 s, 2869 m, 1729 s, 1690 m, 1643 m, 1453 m, 1377 m, 1318 m, 1266 w, 1188 m, 1155 s, 1135 s, 1061 m, 1010 m, 985 m, 883 m cm1; UVevis (methanol): lmax (nm) (log ε) ¼ 238 (0.40), 205 (1.90); 1H NMR (500 MHz, CDCl3): d ¼ 5.95 (m, 1 H, CHa (32)), 5.10 (m, 1 H, CHb (32)), 4.72 (m, 1 H, CHa (29)), 4.58 (m, 1 H, CHb (29)), 3.66 (s, 3 H, CH3 (31)), 2.99 (m, 1 H, CH (19)), 2.67 (d, 1 H, J ¼ 15.3 Hz, CHa (1)), 2.29e2.19 (m, 2 H, CHa (16) þ CH (13)), 2.05 (d, 1 H, J ¼ 15.3 Hz, CHb (1)), 1.93e1.81 (m, 2 H, CHa (21) þ CHa (22)), 1.76e1.64 (m, 1 H, CHa (12)), 1.68 (s, 3 H, CH3 (30)), 1.60 (dd, 1 H, J ¼ 11.5, 11.5 Hz, CH (18)), 1.53e1.14 (m, 13 H, CH (9) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CHb (15) þ CH (5)), 1.12e1.01 (m, 1 H, CHb (12)), 1.08 (s, 3 H, CH3 (24)), 1.03 (s, 3 H, CH3 (23)), 0.97 (s, 3 H, CH3 (27)), 0.95 (s, 3 H, CH3 (25)), 0.83 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.5 (C3, CO), 176.6 (C28, CO), 150.3 (C20, C]CH2), 142.3 (C2, C]CH2), 123.6 (C31, C]CH2), 109.8 (C29, C]CH2), 56.5 (C17, Cquart.), 53.9 (C5, CH), 51.2 (C31, CH3), 49.4 (C18, CH), 48.4 (C9, CH), 47.1 (C1, CH2), 46.9 (C19, CH), 45.8 (C4, Cquart.), 42.4 (C14, Cquart.), 40.6 (C8, Cquart.), 38.3 (C13, CH), 36.9 (C10, CH2), 36.9 (C22, Cquart.), 33.5 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.0 (C24, CH3), 25.5 (C12, CH2), 22.4 (C23, CH3), 21.4 (C11, CH2), 20.0 (C6, CH2), 19.3 (C30, CH3), 15.6 (C25, CH3), 15.4 (C26, CH3), 14.6 (C27, CH3); MS (ESI, MeOH): m/z ¼ 983.2 (72% [2M þ Na]þ), 535.3 (60% [M þ H þ MeOH]þ), 481.4 (100% [M þ H]þ); analysis for C32H48O3 (480.72): C, 79.95; H, 10.06; found: C, 79.73; H, 10.16. 4.6. Methyl (2a)-2-[(dimethylamino)methyl]-3-oxolup-20(29)en28-oate (8) A solution of 4 (1.01 g, 2.15 mmol), dimethylammonium hydrochloride (800 mg, 9.81 mmol) and paraformaldehyde (300 mg, 10.0 mmol) in glacial acetic acid (5 ml) was stirred at 80
C for 30 min. After cooling to 0
C, an aq. solution of Na2CO3 (satd., 50 ml) was added dropwise, and the mixture was extracted with ethyl acetate (3  75 ml). The solvent was stripped off under reduced pressure, and the remaining yellowish product dissolved in hot pentane (15 ml), insoluble material was filtered off, and the solvent was removed. Compound 8 (1.06 g, 94%) was obtained as a colorless, amorphous solid; m.p: 76e79
C; RF ¼ 0.07 (n-hexane/ ethyl acetate, 4:1); [a]D ¼ 5.8
(c ¼ 4.10, CHCl3); IR (KBr): n ¼ 3432 m, 2947 s, 2869 m, 2764 m, 1729 s, 1703 m, 1641 w, 1458 m, 1378 m, 1158 m, 963 w cm1; UVevis (methanol): lmax (nm) (log ε) ¼ 300 (0.05), 258 (0.30), 225 (1.65); 1H NMR (500 MHz, CDCl3): d ¼ 4.66 (m, 1 H, CHa (29)), 4.52 (m, 1 H, CHb (29)), 3.60 (s, 3 H, CH3 (31)), 2.93 (ddd, 1 H, J ¼ 10.8, 10.8, 4.3 Hz, CH (19)), 2.73 (m, 1 H, CH (2)), 2.58 (dd, 1 H, J ¼ 12.4, 5.0 Hz, CHa (32)), 2.21e2.09 (m, 4 H, CH (13) þ CHa (16) þ CHb (32) þ CHa (1)), 2.10 (s, 6 H, 2  CH3 (33) þ (34)), 1.86e1.77 (m, 2 H, CHa (21) þ CHa (22)), 1.69e1.15 (m, 12 H, CH (18) þ CHa (12) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.60 (s, 3 H, CH3 (30)), 1.50 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (9)), 1.10e1.01 (m, 3 H, CH (5) þ CHb (15) þ CHb (12)), 0.89e0.80 (m, 1 H, CHb (1)), 1.02 (s, 3 H, CH3 (25)), 0.99 (s, 3 H, CH3 (23)), 0.97 (s, 3 H, CH3 (24)), 0.91 (s, 3 H, CH3 (26)), 0.87 (s, 3 H, CH3 (27)) ppm; 13C

NMR (125 MHz, CDCl3): d ¼ 217.3 (C3, CO), 176.6 (C28, CO), 150.4 (C20, C]CH2), 109.7 (C29, C]CH2), 59.9 (C32, CH2), 57.3 (C5, CH), 56.4 (C17, Cquart.), 51.2 (C31, CH3), 50.0 (C9, CH), 49.4 (C18, CH), 48.4 (C4, Cquart.), 47.0 (C19, CH), 46.2 (C1, CH2), 45.8 (C33 þ C34, 2  CH3), 42.5 (C14, Cquart.), 40.7(C8, Cquart.), 40.3 (C2, CH), 38.3 (C13, CH), 37.3 (C10, Cquart.), 36.9 (C22, CH2), 34.1 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 25.4 (C12, CH2), 25.3 (C24, CH3), 21.6 (C23, CH3), 21.2 (C11, CH2), 19.3 (C6, CH2), 19.2 (C30, CH3), 16.1 (C25, CH3), 16.0 (C26, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/ z ¼ 526.4 (100% [M þ H]þ); analysis for C34H55NO3 (525.81): C, 77.66; H, 10.54; N, 2.66; found: C, 77.57; H, 10.76; N, 2.42. 4.7. Methyl (2 E) 2-ethyliden-3-oxolup-20(29)en-28-oate (9) To a freshly prepared solution of LDA [from diisopropylamine (200 mg, 2.0 mmol) and n-butyllithium (1.0 ml, 1.6 mmol, 1.6 M in n-hexane) in dry THF (10 ml)] at 78
C a solution of 4 (300 mg, 0.64 mmol) in THF (5 ml) was added, and stirring was continued for another hour. A solution of acetaldehyde (150 mg, 3.41 mmol) in THF (5 ml) was slowly added, and the mixture was allowed to warm to 25
C. The reaction was quenched by the careful addition of aq. hydrochloric acid (150 ml, 1.0 M) and extracted with ethyl acetate (3  100 ml). The organic layers were washed with brine (100 ml), dried (Na2SO4), and the solvent was removed. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 9 (210 mg, 66.6%) as a colorless solid; m.p. 140e142
C; RF ¼ 0.75 (n-hexane/ethyl acetate, 8:2); [a]D ¼ þ 57.4
(c ¼ 4.65, CHCl3); IR (KBr): n ¼ 3432 s, 2949 s, 2869 m, 1728 s, 1683 m, 1618 m, 1457 m, 1380 m, 1188 m, 1153 m, 1133 m cm1; UVevis (methanol): lmax (log ε) ¼ 218 (3.96), 263 (3.78) nm; 1H NMR (500 MHz, CDCl3): d ¼ 6.65 (ddq, 1 H, J ¼ 7.5, 2.5, 1.7 Hz, CH (32)), 4.74 (d, 1 H, J ¼ 1.7 Hz, CHa (29)), 4.60 (br s, 1 H, CHb (29)), 3.66 (s, 3 H, CH3 (31)), 3.00 (ddd, 1 H, J ¼ 10.8, 10.8, 4.3 Hz, CH (19)), 2.65 (d, 1 H, J ¼ 16.1 Hz, CHa (1)), 2.32e2.19 (m, 2 H, CH (13) þ CHa (16)), 1.94e1.84 (m, 2 H, CHa (21) þ CHa (22)), 1.82 (d, 1 H, J ¼ 16.1 Hz, CHb (1)), 1.78e1.73 (m, 1 H, CHa (12)), 1.68 (s, 3 H, CH3 (30)), 1.66 (d, 3 H, J ¼ 7.5 Hz, CH3 (33)), 1.62 (dd, 1 H, J ¼ 11.5, 11.5 Hz, CH (18)), 1.58e1.21 (m, 12 H, CH (5) þ CH (9) þ CHa (15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11)), 1.20e1.01 (m, 2 H, CHb (12) þ CHb (15)), 1.06 (s, 3 H, CH3 (24)), 1.03 (s, 3 H, CH3 (23)), 0.99 (s, 3 H, CH3 (27)), 0.95 (s, 3 H, CH3 (25)), 0.76 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.6 (C3, C]O), 176.6 (C28, COOCH3), 150.5 (C20, C]CH2), 135.8 (C32, CH), 135.2 (C2, C]CH), 109.6 (C29, CH2), 56.5 (C17, Cquart.), 53.0 (C5, CH), 51.2 (C31, CH3), 49.4 (C18, CH), 48.5 (C9, CH), 46.9 (C19, CH), 44.9 (C4, Cquart.), 42.4 (C1, CH2), 42.4 (C14, Cquart.), 40.4 (C8, Cquart.), 38.4 (C13, CH), 36.9 (C22, CH2), 36.0 (C10, Cquart.), 33.2 (C7, CH2), 32.0(C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 29.1 (C23, CH3), 25.6 (C12, CH2), 22.1 (C24, CH3), 21.6 (C11, CH2), 20.3 (C6, CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.4 (C25, CH3), 14.6 (C27, CH3), 13.6 (C33, CH3) ppm; MS (ESI, MeOH): m/ z ¼ 549.0 (79% [M þ Na þ MeOH]þ), 495.4 (100% [M þ H]þ); analysis for C33H50O3 (494.75): C, 80.11; H, 10.19; found: C, 79.95; H, 10.26. 4.8. (2 Z) methyl 3-oxo-2-prop-2-en-1-ylid-lup-20(29)en-28-oate (10) To a freshly prepared solution of LDA [from diisopropylamine (200 mg, 2.0 mmol) and n-butyllithium (1.0 ml, 1.6 mmol, 1.6 M in n-hexane) in dry THF (10 ml)] at 78
C a solution of 4 (300 mg, 0.64 mmol) in THF (5 ml) was added, and stirring was continued for another hour. A solution of acrolein (150 mg, 2.7 mmol) in THF (5 ml) was slowly added, and the mixture was allowed to warm to 25
C. The reaction was quenched by the careful addition of aq. hydrochloric acid (150 ml, 1.0 M) and extracted with ethyl acetate

R. Csuk et al. / European Journal of Medicinal Chemistry 53 (2012) 337e345

(3  100 ml). The organic layers were washed with brine (100 ml), dried (Na2SO4), and the solvent was removed. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1) to yield 10 (320 mg, 63.2%) as a colorless solid; m.p. 175e180
C; RF ¼ 0.79 (n-hexane/ethyl acetate, 8:2); [a]D ¼ þ77.5
(c ¼ 5.11, CHCl3); IR (KBr): n ¼ 3435 m, 3072 w, 2950 s, 2869 s, 1728 s, 1684 m, 1642 w, 1456 m, 1380 m, 1318 w, 1188 s, 1156 s, 1133 s, 1082 m, 987 m cm1; UVevis (methanol): lmax (log ε) ¼ 218 (4.10), 259 (3.98) nm; 1H NMR (500 MHz, CDCl3): d ¼ 6.92 (d, 1 H, J ¼ 11.5 Hz, CH (32)), 6.45 (ddd, 1 H, J ¼ 16.8, 11.5, 10.2 Hz, CH (33)), 5.52 (br d, 1 H, J ¼ 16.8 Hz, CHa (34)), 5.41 (br d, 1 H, J ¼ 10.2 Hz, CHb (34)), 4.69 (s, 1 H, CHa (29)), 4.55 (s, 1 H, CHb (29)), 3.61 (s, 3 H, CH3 (31)), 2.95 (ddd, 1 H, J ¼ 10.8, 10.8, 4.4 Hz, CH (19)), 2.81 (d, 1 H, J ¼ 16.4 Hz, CHa (1)), 2.25e2.14 (m, 2 H, CH (13) þ CHa (16)), 1.91 (d, 1 H, J ¼ 16.4 Hz, CHb (1)), 1.88e1.78 (m, 2 H, CHa (21) þ CHa (22)), 1.75e1.67 (m, 1 H, CHa (12)), 1.63 (s, 3 H, CH3 (30)), 1.56 (dd, 1 H, J ¼ 11.3, 11.3 Hz, CH (18)), 1.53e1.16 (m, 12 H, CH (5) þ CH (9) þ CHa (15) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11)), 1.16e0.96 (m, 2 H, CHb (12) þ CHb (15)), 1.03 (s, 3 H, CH3 (24)), 1.00 (s, 3 H, CH3 (23)), 0.93 (s, 3 H, CH3 (27)), 0.90 (s, 3 H, CH3 (25)), 0.72 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.8 (C3, C]O), 176.6 (C28, COOCH3), 150.5 (C20, C]CH2), 136.7 (C32, CH), 133.8 (C2, Cquart.), 131.6 (C33, CH), 125.2 (C34, CH2), 109.6 (C29, CH2), 56.5 (C17, Cquart.), 53.0 (C5, CH), 51.3 (C31, CH3), 49.4 (C18, CH), 48.4 (C9, CH), 46.9 (C19, CH), 45.1 (C4, Cquart.), 42.6 (C14, Cquart.), 42.4 (C1, CH2), 40.5 (C8, Cquart.), 38.4 (C13, CH), 36.9 (C22, CH2), 36.1 (C10, Cquart.) 33.2 (C7, CH2), 32.1 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 29.2 (C23, CH3), 25.6 (C12, CH2), 22.4 (C24, CH3), 21.6 (C11, CH2), 20.3 (C6, CH2), 19.4 (C30, CH3), 15.7 (C26, CH3), 15.4 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 507.5 (100% [M þ H]þ); analysis for C34H50O3 (506.76): C, 80.58; H, 9.94; found: C, 80.41; H, 10.07. 4.9. 2-Methylene-28-oxo-28-thiomorpholin-4-yl-lup-20(29)en-3one (11) A solution of 6 (107 mg, 0.23 mmol) and oxalyl chloride (0.2 ml, 0.30 g, 2.36 mmol) in dry toluene (5 ml) containing triethylamine (0.1 ml, 73 mg, 0.72 mmol) and DMF (0.1 ml, 95 mg, 1.30 mmol) was stirred at 25
C for 30 min. The solvents were removed under reduced pressure, and the residue was suspended in dry toluene (5 ml), and thiomorpholine (0.5 ml, 513 mg, 4.97 mmol) and triethylamine (0.2 ml, 146 mg, 1.44 mmol) were added. After stirring at 100
C for an additional 30 min, the mixture was cooled to 25
C, diluted with water (200 ml) and extracted with ethyl acetate (3  100 ml). The extracts were washed with brine (100 ml), dried (Na2SO4), and the solvents were removed. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to yield 11 (69 mg, 54.5%) as a colorless solid; m.p. 250e255
C; RF ¼ 0.65 (n-hexane/ethyl acetate, 8:2); [a]D ¼ þ28.5
(c ¼ 5.25, CHCl3); IR (KBr): n ¼ 3442 m, 2948 s, 2867 m, 1687 m, 1636 s, 1457 w, 1403 m, 1288 w, 1247 w, 1167 m, 1056 w cm1; UVevis (methanol): lmax (log ε) ¼ 218 nm (4.28); 1H NMR (500 MHz, CDCl3): d ¼ 5.94 (s, 1 H, CHa (31)), 5.11 (s, 1 H, CHb (31)), 4.72 (s, 1 H, CHa (29)), 4.58 (s, 1 H, CHb (29)), 4.00e3.73 (br d, 4 H, CH2 (32)), 2.97 (ddd, 1 H, J ¼ 11.0, 10.9, 3.5 Hz, CH (19)), 2.90 (ddd, 1 H, J ¼ 13.0, 13.0, 3.4 Hz, CH (13)), 2.69 (d, 1 H, J ¼ 15.3 Hz, CHa (1)), 2.64e2.53 (m, 4 H, CH2 (33)), 2.11e2.00 (m, 2 H, CHa (16) þ CHb (1)), 1.96e1.90 (m, 1 H, CHa (22)), 1.88e1.80 (m, 1 H, CHa (21)), 1.77e1.72 (m, 1 H, CHa (12)), 1.67 (s, 3 H, CH3 (30)), 1.62e1.14 (m, 13 H, CH (5) þ CH (9) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11) þ CH2 (15)), 1.57 (dd, 1 H, J ¼ 11.3, 11.3 Hz, CH (18)), 1.09 (s, 3 H, CH3 (23)), 1.03 (s, 3 H, CH3 (24)), 1.01e0.88 (m, 1 H, CHb (12)), 0.97 (s, 3 H, CH3 (26)), 0.97 (s, 3 H, CH3 (27)), 0.83 (s, 3 H, CH3 (25)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 207.6 (C3, C]O), 173.6 (C28, CON), 151.1

343

(C20, C]CH2), 142.4 (C2, C]CH2), 123.5 (C31, CH2), 109.2 (C29, CH2), 54.8 (C17, Cquart.), 54.1 (C5, CH), 52.8 (C18, CH), 48.8 (C9, CH), 48.0 (br, C32, CH2), 47.1 (C1, CH2), 45.9 (C4, Cquart.), 45.6 (C19, CH), 42.0 (C14, Cquart.), 40.5 (C8, Cquart.), 37.0 (C13, CH), 36.9 (C10, Cquart.), 35.9 (C22, CH2), 33.4 (C7, CH2), 32.4 (C16, CH2), 31.3 (C21, CH2), 29.9 (C15, CH2), 28.0 (C23, CH3), 27.7 (C33, CH2), 25.6 (C12, CH2), 22.4 (C24, CH3), 21.7 (C11, CH2), 20.1 (C6, CH2), 19.7 (C30, CH3), 15.7 (C26, CH3), 15.5 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/ z ¼ 606.0 (46% [M þ Na þ MeOH]þ), 552.4 (100% [M þ H]þ); analysis for C35H53NO2S (551.87): C, 76.17; H, 9.68; N, 2.54; S, 5.81 found: C, 76.00; H, 9.79; N, 2.41; S, 5.69. 4.10. Methyl (2a) 2-{[(2-hydroxyethyl)thio]methyl}-3-oxolup20(29)en-28-oate (12) To a solution of 7 (480 mg, 1.0 mmol) and 2-mercaptoethanol (0.9 ml, 1.0 g, 12.8 mmol) in dichloromethane (10 ml), DBU (0.49 ml, 0.50 g, 3.3 mmol) was added, and the mixture was stirred for 48 h at 25
C. After dilution with water (200 ml) and extraction with ethyl acetate (3  100 ml), the organic phase was washed with aq. hydrochloric acid (100 ml, 0.5 M), aq. NaHCO3Lösung (100 ml) and brine (100 ml). The extract was dried (Na2SO4), the solvents were removed, and the residue subjected to chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to yield 12 (336 mg, 60.1%) as a colorless foam; RF ¼ 0.50 (n-hexane/ethyl acetate, 8:2); [a]D ¼ þ71.7
(c ¼ 5.00, CHCl3); IR (KBr): n ¼ 3447 m, 2949 s, 2868 s, 1727 s, 1642 w, 1458 m, 1386 m, 1160 m, 1064 w cm1; UVevis (methanol): lmax (log ε) ¼ 216 nm (4.01); 1H NMR (500 MHz, CDCl3): d ¼ 4.71 (s, 1 H, CHa (29)), 4.58 (s, 1 H, CHa (29)), 3.72 (t, 2 H, J ¼ 5.8 Hz, CH2 (34)), 3.64 (s, 3 H, CH3 (31)), 3.00e2.84 (m, 3 H, CH (2) þ CH (19) þ CHa (32)), 2.70 (t, 2 H, J ¼ 5.8 Hz, CH2 (33)), 2.35 (dd, 1 H, J ¼ 12.2, 6.0 Hz, CHb (32)), 2.25e2.17 (m, 2 H, CH (13) þ CHa (16)), 1.98 (dd, 1 H, J ¼ 13.1, 10.8 Hz, CHa (1)), 1.91e1.83 (m, 2 H, CHa (21) þ CHa (22)), 1.75e1.64 (m, 2 H, CH (5) þ CHa (12)), 1.67 (s, 3 H, CH3 (30)), 1.60 (dd, 1 H, J ¼ 11.4 Hz, 11.4 Hz, CH (18)), 1.50e1.20 (m, 12 H, CH (9) þ CHa (15) þ CHb (1) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11)), 1.19e0.93 (m, 2 H, CHb (12) þ CHb (15)), 1.06 (s, 3 H, CH3 (23)), 1.01 (s, 3 H, CH3 (24)), 0.99 (s, 3 H, CH3 (27)), 0.88 (s, 3 H, CH3 (25)), 0.66 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 218.8 (C3, C]O), 176.6 (C28, COOCH3), 150.4 (C20, C] CH2), 109.6 (C29, CH2), 60.5 (C34, CH2OH), 56.5 (C14, Cquart.), 52.3 (C5, CH), 51.2 (C31, CH3), 50.0 (C9, CH), 49.3 (C18, CH), 48.8 (C1, CH2), 46.9 (C19, CH), 46.7 (C4, Cquart.), 42.4 (C14, Cquart.), 42.1 (C2, CH), 40.6 (C8, Cquart.), 38.5 (C13, CH), 36.9 (C10, Cquart.), 36.9 (C22, CH2), 36.2 (C33, CH2), 32.9 (C7, CH2), 32.2 (C16, CH2), 32.0 (C32, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.9 (C23, CH3), 25.6 (C12, CH2), 22.0 (C11, CH2), 20.0 (C6, CH2), 19.3 (C24, CH3), 19.3 (C30, CH3), 18.5 (C26, CH3), 15.2 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 581.4 (100% [M þ Na]þ); analysis for C34H54SO4 (558.86): C, 73.07; H, 9.74; S, 5.74 found: C, 72.86; H, 9.99; S, 5.53. 4.11. Methyl (2a) 2-{[(2-hydroxyethyl)sulfonyl]methyl}-3-oxolup20(29)en-28-oate (13) To a solution of 12 (200 mg, 0.36 mmol) in glacial acetic acid (15 ml) hydrogen peroxide (10 ml, 0.10 mol, 35% in water) was added, and the mixture was stirred at 25
C for 30 min. The mixture was diluted with water (200 ml), extracted with ethyl acetate (3  100 ml), the extracts were washed with aq. NaHCO3 (satd., 100 ml), an aq. solution of (NH4)2Fe(SO4)2 [5.0 g, 12.75 mmol in water (100 ml)] and brine (100 ml). After drying (Na2SO4), the solvents were removed, and the residue was subjected to chromatography (silica gel, ethyl acetate/methanol, 95:5)

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to yield 13 (177 mg, 83.3%) as a colorless solid; m.p. 140e145
C; RF ¼ 0.74 (ethyl acetate/methanol, 9:1); [a]D ¼ þ51.5
(c ¼ 5.85, CHCl3); IR (KBr): n ¼ 3429 m, 3072 w, 2948 s, 2868 s, 1725 s, 1642 w, 1455 m, 1384 m, 1318 w, 1287 w, 1188 m, 1156 s, 1133 m, 1062 m, 1012 w cm1; UVevis (methanol): lmax (log ε) ¼ 214 nm (4.13); 1H NMR (500 MHz, CDCl3): d ¼ 4.70 (s, 1 H, CHa (29)), 4.57 (s, 1 H, CHb (29)), 4.16e4.05 (m, 2 H, CH2 (34)), 3.63 (s, 3 H, CH3 (31)), 3.49e3.38 (m, 1 H, CH (2)), 3.25e3.17 (m, 1 H, CHa (32)), 3.09e2.81 (m, 3 H, CH (19) þ CH2 (33)), 2.48 (dd, 1 H, J ¼ 12.9, 3.1 Hz, CHb (32)), 2.25e2.14 (m, 2 H, CH (13) þ CHa (16)), 2.40 (dd, 1 H, J ¼ 12.9, 11.2 Hz, CHa (1)), 1.91e1.81 (m, 2 H, CHa (21) þ CHa (22)), 1.80e1.74 (m, 1 H, CH (5)), 1.72e1.62 (m, 1 H, CHa (12)), 1.65 (s, 3 H, CH3 (30)), 1.58 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.53e1.10 (m, 13 H, CH (9) þ CHb (1) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11) þ CH2 (15)), 1.09 (s, 3 H, CH3 (23)), 1.05e0.96 (m, 1 H, CHb (12)), 1.01 (s, 3 H, CH3 (24)), 0.97 (s, 3 H, CH3 (27)), 0.87 (s, 3 H, CH3 (25)), 0.66 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 218.0 (C3, C]O), 176.6 (COOCH3), 150.3 (C20, C]CH2), 109.7 (C29, CH2), 56.8 (C34, CH2), 56.5 (C17, Cquart.), 54.5 (C33, CH2), 53.8 (C32, CH2), 52.2 (C5, CH), 51.2 (C31, CH3), 49.9 (C9, CH), 49.3 (C18, CH), 49.1 (C1, CH2), 46.8 (C19, CH), 46.8 (C4, Cquart.), 42.4 (C14, Cquart.), 40.6 (C8, Cquart.), 38.5 (C13, CH), 37.3 (C10, Cquart.), 36.9 (C22, CH2), 36.6 (C2, CH), 32.8 (C7, CH2), 32.0 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.7 (C23, CH3), 25.5 (C12, CH2), 22.0 (C11, CH2), 19.9 (C6, CH2), 19.3 (C30, CH3), 19.2 (C24, CH3), 18.8 (C26, CH3), 15.2 (C25, CH3), 14.5 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 613.3 (56% [M þ Na]þ), 591.3 (74% [M þ H]þ); analysis for C34H54SO6 (590.85): C, 69.11; H, 9.21; S, 5.43; found: C, 68.89; H, 9.07; S, 5.35. 4.12. Methyl (2a) 2-(2-nitroethyl)-3-oxolup-20(29)en-28-oate (14) To a solution of 7 (365 mg, 0.76 mmol) in nitromethane (10 ml) and dichloromethane (5 ml) DBU (2.0 ml, 2.04 g, 13.40 mmol) was added, and the mixture was stirred for 5 days at 25
C. After diluting with water (200 ml) and extraction with ethyl acetate (3  100 ml), the extracts were washed with brine (100 ml) and dried (Na2SO4). The solvents were removed, and the residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to yield 14 (273 mg, 66.3%) as a colorless solid; m.p. 165e168
C; RF ¼ 0.59 (nhexane/ethyl acetate, 8:2); [a]D ¼ þ1.7
(c ¼ 5.34, CHCl3); IR (KBr): n ¼ 3444 w, 2955 s, 2877 s, 1730 s, 1698 s, 1550 m, 1380 w, 1320 m, 1277 w, 1190 m, 1144 s, 1130 m, 1029 w, 980 w cm1; UVevis (methanol): lmax (log ε) ¼ 218 nm (4.00); 1H NMR (500 MHz, CDCl3): d ¼ 4.67 (s, 1 H, CHa (29)), 4.53 (br s, 1 H, CHb (29)), 4.48e4.28 (m, 2 H, CH2 (33)), 3.60 (s, 3 H, CH3 (31)), 2.92 (ddd, 1 H, J ¼ 10.9, 10.7, 4.5 Hz, CH (19)), 2.83e2.72 (m, 1 H, CH (2)), 2.30e2.11 (m, 3 H, CH (13) þ CHa (16) þ CHa (32)), 1.99e1.75 (m, 4 H, CHa (1) þ CHa (21) þ CHa (22) þ CHb (32)), 1.70e1.58 (m, 2 H, CH (5) þ CHa (12)), 1.62 (s, 3 H, CH3 (30)), 1.55 (dd, 1 H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.42e0.93 (m, 14 H, CH (9) þ CHb (1) þ CHb (12) þ CHb (16) þ CHb (21) þ CHb (22) þ CH2 (6) þ CH2 (7) þ CH2 (11) þ CH2 (15)), 0.99 (s, 3 H, CH3 (23)), 0.96 (s, 3 H, CH3 (24)), 0.94 (s, 3 H, CH3 (27)), 0.83 (s, 3 H, CH3 (25)), 0.61 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 219.0 (C3, C]O), 176.6 (C28, COOCH3), 150.4 (C20, C]CH2), 109.6 (C29, CH2), 73.8 (C33, CH2), 56.5 (C17, Cquart.), 52.2 (C5, CH), 51.3 (C31, CH3), 50.0 (C9, CH), 49.3 (C18, CH), 48.8 (C1, CH2), 46.9 (C19, CH), 46.8 (C4, Cquart.), 42.4 (C14, Cquart.), 40.6 (C8, Cquart.), 38.5 (C13, CH), 37.9 (C2, CH), 37.0 (C22, CH2), 36.9 (C10, Cquart.), 32.9 (C7, CH2), 32.0 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 28.9 (C23, CH3), 28.0 (C32, CH2), 25.6 (C12, CH2), 22.0 (C11, CH2), 21.3 (C24, CH3), 19.9 (C6, CH2), 19.2 (C30, CH3), 18.7 (C26, CH3), 15.2 (C25, CH3), 14.6 (C26, CH3) ppm; MS (ESI, MeOH): m/z ¼ 564.4 (100% [M þ Na]þ); analysis for C33H51NO5 (541.76): C, 73.16; H, 9.49; N, 2.59; found: C, 73.02; H, 9.65; N, 2.37.

4.13. (2 R)-Spiro[pyrazol-30 ,2-28-methoxy-3-oxolup-20(29)-en-28one] (15) To a solution of 7 (515 mg, 1.07 mmol) in dry THF (10 ml), a solution of diazomethane (15 ml, 15 mmol, 1 M in ether) was added, and the mixture was stirred for 3 days at 25
C. A drop of acetic acid was added, the mixture diluted with water (100 ml) and extracted with ether (3  100 ml). The organic phases were dried (Na2SO4), and the solvent was removed. The residue was subjected to chromatography (silica gel, n-hexane/ethyl acetate, 9:1), and 15 (180 mg, 32.2%) was obtained as a colorless solid; m.p. 97e100
C ¼ 0.79 (n-hexane/ethyl acetate, 8:2); (decomp.); RF [a]D ¼ þ128.4
(c ¼ 2.34, CHCl3); IR (KBr): n ¼ 3435 w, 2949 s, 2869 s, 1728 s, 1703 s, 1642 w, 1549 w, 1452 w, 1383 m, 1318 w, 1279 w, 1187 m, 1155 s, 1134 s, 1083 w, 1059 w, 1032 w cm1; UVevis (methanol): lmax (log ε) ¼ 215 nm (4.14); 1H NMR (500 MHz, CDCl3): d ¼ 4.70 (br s, 1 H, CHa (29)), 4.59 (ddd, 1 H, J ¼ 17.4, 9.4, 3.8 Hz, CHa (50 )), 4.56 (br s, 1 H, CHb (29)), 4.24 (ddd, 1 H, J ¼ 17.4, 9.0, 8.6 Hz, CHb (50 )), 3.65 (s, 3 H, CH3 (31)), 2.97 (ddd, 1 H, J ¼ 11.1, 11.0, 4.5 Hz, CH (19)), 2.60 (ddd, 1 H, J ¼ 12.6, 9.0, 3.8 Hz, CHa (40 )), 2.46 (dd, 1 H, J ¼ 10.3, 3.8 Hz, CH (5)), 2.28e2.18 (m, 2 H, CH (13) þ CHa (16)), 2.05 (d, 1 H, J ¼ 14.4 Hz, CHa (1)), 1.92e1.83 (m, 2 H, CHa (21) þ CHa (22)), 1.79 (d, 1 H, J ¼ 14.4 Hz, CHb (1)), 1.74e1.15 (m, 14 H, CH (9) þ CH (18) þ CHb (22) þ CH2 (7) þ CHb (16) þ CHb (21) þ CH2 (15) þ CHa (12) þ CH2 (11) þ CH2 (6)), 1.65 (s, 3 H, CH3 (30)), 1.10 (br s, 6 H, CH3 (23) þ CH3 (24)), 1.07e0.90 (m, 2 H, CHb (40 ), CHb (12)), 0.99 (s, 3 H, CH3 (27)), 0.94 (s, 3 H, CH3 (25)), 0.80 (s, 3 H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d ¼ 211.5 (C3, C] O), 176.6 (C28, COOCH3), 150.4 (C20, C]CH2), 109.6 (C29, CH2), 101.7 (C2/C30 , Cspiro), 76.7 (C50 , CH2), 56.5 (C17, Cquart.), 54.1 (C1, CH2), 51.2 (C31, CH3), 50.9 (C5, CH), 49.3 (C18, CH), 49.2 (C9, CH), 46.9 (C19, CH), 46.2 (C4, Cquart.), 42.5 (C14, Cquart.), 40.5 (C8, Cquart.), 38.4 (C13, CH), 36.9 (C22, CH2), 36.8 (C10, Cquart.), 32.8 (C7, CH2), 32.0 (C16, CH2), 30.5 (C21, CH2), 29.6 (C15, CH2), 29.3 (C40 , CH2), 27.5 (C23, CH3), 25.5 (C12, CH2), 22.1 (C24, CH3), 21.9 (C11, CH2), 20.1 (C6, CH2), 19.3 (C30, CH3), 18.4 (C26, CH3), 15.2 (C25, CH3), 14.6 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 545.3 (60% [M þ Na]þ), 495.4 (100% [M þ H-N2]þ); analysis for C33H50N2O3 (522.76): C, 75.82; H, 9.64; N, 5.36; found: C, 75.61; H, 9.88; N, 5.17. Acknowledgments We like to thank Dr. D. Ströhl for the NMR experiments, Dr. R. Kluge for the ESI-MS spectra and Mrs. F. Flemming (Organic Chemistry, Universität Halle-Wittenberg) for her help with some experiments. The cell lines were kindly provided by Dr. Thomas Müller (Dept. of Haematology/Oncology, Universität HalleWittenberg). Support by “Gründerwerkstatt e Biowissenschaften” is gratefully acknowledged. References [1] R.H. Cichewicz, S.A. Kouzi, Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection, Med. Res. Rev. 24 (2004) 90e114. [2] J.F. Mayaux, A. Bousseau, R. Pauwels, T. Huet, Y. Henin, N. Dereu, M. Evers, F. Soler, C. Poujade, E. Declercq, J.B. Lepecq, Triterpene derivatives that block entry of human-immunodeficiency-virus type-1 into cells, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 3564e3568. [3] H.L. Ziegler, H. Franzyk, M. Sairafianpour, M. Tabatabai, M.D. Tehrani, K. Bagherzadeh, H. Hagerstrand, D. Staerk, J.W. Jaroszewski, Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structureeactivity relationships for betulinic acid analogues, Bioorgan. Med. Chem. 12 (2004) 119e127. [4] E. Pisha, H. Chai, I.S. Lee, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell, C.W.W. Beecher, H.H.S. Fong, A.D. Kinghorn, D.M. Brown, M.C. Wani, M.E. Wall, T.J. Hieken, T.K. Dasgupta, J.M. Pezzuto, Discovery of betulinic acid as a selective inhibitor of human-melanoma that functions by induction of apoptosis, Nat. Med. 1 (1995) 1046e1051.

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