Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

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Synthesis of triterpenoid triazine derivatives from allobetulone and betulonic acid with biological activities Thuc Dinh Ngoc a, Nico Moons a, Youngju Kim a, Wim De Borggraeve a, Anastassiya Mashentseva a, , Graciela Andrei b, Robert Snoeck b, Jan Balzarini b, Wim Dehaen a,⇑ a b

Molecular Design and Synthesis, Department of Chemistry, University of Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 24 February 2014 Accepted 28 April 2014 Available online xxxx Keywords: Triterpenoid triazine Diels–Alder reaction Murine leukemia Cervix carcinoma HeLa Lymphoblast CEM tumor cells

a b s t r a c t The synthetic transformation and modification of natural products with the aim to improve the biological properties is an area of current interest. The triterpenoids betulin and betulinic acid are very abundant in nature and now are commercially available. In our study, starting from betulin and betulinic acid, we obtained allobetulone and betulonic acid in a few synthetic steps. The ketone function at the A-ring was used as the starting point for the synthesis of a series of 1,2,4-triazine-fused triterpenoids. The alkylation and Liebeskind–Srogl coupling were used for further substitution of 1,2,4-triazines, and the intramolecular hetero Diels–Alder reaction leads to interesting fused thienopyridine derivatives. All new compounds were tested for their cytostatic activities against murine leukemia L1210, human cervix carcinoma HeLa and human lymphoblast CEM tumor cells. The results show that some triterpenoid triazine betulonic acid derivatives have a promising cytostatic activity in vitro and could be used as potential leads for the development of new type of anti-cancer agents. Several compounds were also endowed with anti-HCMV activity in the low micromolar range. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Natural products have played an important role for the design of novel therapeutics due to their enormous structural diversity1 providing high screening hit rates against various diseases.2 The synthetic transformation of natural compounds to improve their biological activity is an area of current interest in organic synthesis and medicinal chemistry. Nevertheless, the scarcity of accessible material has been obstructing the synthetic exploration of the chemical space of natural products. However, betulin (1) (the trivial name for lup-20(29)-ene-3b,28-diol), which is abundant in nature and commercially available, has been extensively studied over many years.3 It is well known that, 1 and its analogues exhibit a wide range of biological activities, such as anticancer,4 antibacterial,5 anti-inflammatory6 and antiviral7 activities. Betulin (1), a pentacyclic triterpene, is found as a major compound in the bark of the birch tree (12–30% of dry weight) along with trace amounts of allobetulin (2), betulinic acid (4), betulinic aldehyde and lupeol.3 Betulin (1) itself has only few synthetic ⇑ Corresponding author. Tel.: +32 16 32 74 39. E-mail address: [email protected] (W. Dehaen). Now affiliated to L.N. Gumileyov Eurasian National University, Astana, Kazakhstan.  

applications. On the other hand, numerous transformations of allobetulin (2) and betulinic acid (4) have been reported.8 Allobetulin (2) can be easily prepared from betulin (1) via an acid-catalyzed isomerization, which belongs to the class of Wagner–Meerweintype rearrangements (Fig. 1).3 Although a number of derivatives of 2 and 4 can be found in the literature,9 1,2,4-Triazine derivatives of these compounds using 2 and 4 as scaffold have not been reported. Triazine compounds are nitrogen containing heterocycles, which represent a pharmaceutically important class of compounds. They have attracted attention due to their antimicrobial,10 antiviral,11 anticancer,12 or other biological activities Therefore, we were interested in the preparation of triazine derivatives of 2 and 4 after converting them to the ketone analogues allobetulone (3) and betulonic acid (5), respectively. Allobetulone 3 can be prepared from commercially available 1 via 2 by acid-catalyzed isomerization and subsequent oxidation.13 On the other hand, 5 can be obtained from commercially available 4 or 1 after simple oxidation (Scheme 1).14 Previously, we have reported the preparation of the isomeric 2-oxoallobetulin from 3 and the subsequent selective functionalization of the A-ring.15 In a continuing study on the modification of the A-ring, we have prepared sixteen kinds of new derivatives from 2 and 4, including the first synthetic examples of triterpenoid-derived 1,2,4-triazines. Herein, we describe the

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H

O

H

O

H

OH

HO

HO

1

H

O

2

H

3

H

Figure 1. Structure of compounds 1–3.

H

H

OH

H OH

CrO3/H2SO4 HO

acetone, 0oC

1

H

O O

acetone, 0oC

5

H

OH CrO3/H2SO4

O HO

4

H

Scheme 1. Synthesis of betulonic acid from betulin and betulinic acid.

synthesis of all of these derivatives and evaluation of their cytostatic activities against two human cancer cell lines in cell culture.

H

2. Results and discussion Betulin (1) was converted to allobetulone (3) by isomerization, followed by oxidation.12 2-Oxoallobetulone (6) was obtained from allobetulone (3) in the presence of t-BuOK and oxygen in t-BuOH according to the literature.16 The reaction conditions were optimized by using pure oxygen and raising the reaction temperature from 20 to 40 °C. The product (6) mostly existed in the enol form as apparent by 1H NMR spectroscopic analysis in CDCl3. 2-Oxoallobetulone (6) was used as a precursor for triazine heterocyclic ring systems. Although 1,2-dicarbonyl compounds have been commonly used for the synthesis of 1,2,4-triazines and their derivatives, only a couple of examples of triazines based on terpenoid structures have been reported.17,18 The condensation of 2-oxoallobetulone (6) with thiosemicarbazide in ethanol with K2CO3 afforded the desired 1,2,4-triazine allobetulin derivative (7).19 This compound was confirmed by 2D-NMR analysis of the coupling of both C-2 with C-1 hydrogen and C-3 with C-23 hydrogen. According to HMBC the signal of two protons on the C-1 at 3.0 ppm and 2.24 ppm have correlations with C-2 at 145.6 ppm and the signal of proton on the C-23 at 1.32 ppm has a correlation with C-3 at 173.1 ppm (see Supplementary information). Probably due to the steric hindrance of two methyl groups located at C-4, the first nucleophilic attack of thiosemicarbazide occur at the C-2 position, and ring closure happened afterwards (Scheme 2). Further modification of 1,2,4-triazine (7) can be applied using various synthetic methods. Firstly, the possibility of S-alkylation of the 3-thione at the triazine moiety in 7 with various substituents was investigated. By using triethylamine as base in THF, various S-alkylated 1,2,4-triazine derivatives (8–10) were obtained with good to excellent yield (51–94%, Scheme 3, Table 1).

H

O

H

3

t-BuOH, t-BuOK 40oC

N

RS

H

Table 1 Substituted triazine 7 Nr

R

Yield (%)

8

Me

86

9

51

10

94

An alternative route to the substituted triazines was accomplished by Liebeskind–Srogl cross-coupling. This transitionmetal-catalyzed coupling of organosulfur compounds and boronic acids was firstly reported by Liebeskind et al. in 2000 as the coupling of thioesters with several boronic acids.20 Later the scope of this reaction was extended to aromatic thioethers.21 Although simple 1,2,4-triazines were used for Liebeskind–Srogl coupling,22 there have been no attempt with terpene derivatives. In this study, phenylboronic acid and 4-t-butylphenylboronic acid were coupled to 8 with Pd(PPh3)4 and CuTC to give the triazines 11 and 12, respectively, in good yields (76–78%, Scheme 4).

O

6

O

H HN NH2 S C NH2

H

N

Scheme 3. Alkylation of allobetulin triazine.

HO O

N

Et3N, THF, 18 - 22h

H O2

O

RX

7

O

K2CO3, EtOH reflux

HN S

N N

H

7

Scheme 2. Synthesis of triazine.

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T. Dinh Ngoc et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

O

H N

N N

MeS

H

N

THF, 18 - 22h

8

O

H

Pd(PPh3)4 CuTC ArB(OH)2

N N

11: R=H, 78% 12: R=t-Bu, 76%

H

R Scheme 4. Liebeskind–Srogl couplings on allobetulin triazine.

N

MeS

mCPBA, MgSO4

N

DCM, rt, 1h

N

H

N

MeO2S

N

HNuc N

8

H 13: 82%

N

Base

N N

Nuc

H 14-16

Scheme 5. Nucleophilic substitution on the methyl sulfonyl group.

Furthermore, we were also successful in oxidizing the methylated triazine 8 with mCPBA,23 thereby providing the possibility of nucleophilic substitution of the sulfonyl group of 13. Three nucleophiles were added resulting in products 14, 15 and 16 in reasonable yields (59–91%), (Scheme 5 and Table 2). The prepared but-3-yne-sulfanyl-1,2,4-triazine allobetulin (10) was in a next step submitted to an intramolecular hetero Diels–Alder reaction.24,25 The reaction was straightforward but needed elevated temperatures, and thienopyridine derivative (17) was achieved after refluxing (10) in xylene for 20 h with 49% yield. Also this thienopyrido A ring fusion is unique for complicated triterpenoid natural products (Scheme 6).

Table 2 Nucleophiles added to the sulfonyl triazine 13 Nr

Nucleophile

Nuc

Yield (%)

14 15 16

Phenol Diethyl malonate NaOH

PhO (C2H5OOC)2CH HO

59 61 91

H N

N N

S

10

H

2-Iodophenol was coupled via a Sonogashira reaction to trimethylsilylacetylene,26 and the resulting product 18 was added via SNAr to triazine 13. Surprisingly, during the addition reaction the formed phenol adduct immediately underwent ring-closure via intramolecular Diels–Alder reaction and subsequent removal of the TMS group under the reaction conditions resulted in the benzofuropyridine 19 with a satisfying yield of 62% (Scheme 7). Betulonic acid (5) can be isolated from nature, but can also be synthesized directly from betulin or betulinic acid.11–13 In this study, we prepared betulonic acid (5) from commercially available betulin 1 by oxidation (Scheme 1). Because the A-ring substructure of betulonic acid is similar to that of allobetulone, the procedures, used for allobetulone were also applied for betulonic acid. The E-ring of allobetulone (3) consists of a relatively stable oxabicyclic structure, while betulonic acid (5) has isopropenyl and carboxylic groups at this position that might have some reactivity. Fortunately, the reactions starting from 5 selectively occurred only at the A-ring. Therefore, it was not necessary to protect any functional groups at the E-ring of betulonic acid (5). 2-Oxobetulonic acid (20) was obtained analogously by oxidation of betulonic acid (5) with oxygen in the presence of t-BuOK in t-BuOH.27 The condensation reaction of (20) with thiosemicar-

O

O

H

xylene reflux

S

N

17

H

Scheme 6. Synthesis of thienopyridine derivative 17.

Si

O

H OH 18

+

13

K2CO3 18-crown-6 DMF, 70oC, 18h

O

N

H

19

Scheme 7. Diels–Alder reactions of allobetulin triazine.

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N

COOH

N N

S

22 c

COOH 5

O

COOH

HO

a

b

20

O

HN S

COOH

N 21

N

d

COOH e S

N

N S

24

COOH

N N

23

Scheme 8. Synthesis of betulonic acid derivatives. Reagents and conditions: (a) O2, t-BuOK, t-BuOH 40 °C, (b) thiosemicarbazide, K2CO3, EtOH, reflux; (c)) MeI, Et3N, THF, rt, (d) 4-bromobut-1-yne, Et3N, THF, rt; (e) xylene, reflux.

bazide in ethanol with potassium carbonate afforded the 1,2,4-triazine derivative of betulonic acid (21). From triazine (21), further substitution with different alkyl groups was conducted to get two S-alkylated triazine derivatives (22, 23). By heating the S-butynyl triazine derivative (23) in xylene under reflux, thienopyridine derivative (24) was obtained by intermolecular Diels–Alder reaction (Scheme 8). The cytostatic activity of five derivatives (20–24) were evaluated in murine leukemia L1210 and human cervix carcinoma HeLa and lymphoblast CEM tumor cell cultures (Table 3). Whereas 21 is

Table 3 Inhibitory effects of test compounds on the proliferation of murine leukemia cells (L1210) and human T-lymphoblast cells (CEM) and human cervix carcinoma cells (HeLa) IC50a (lM)

Compound

20 21 22 23 24 a

L1210

CEM

HeLa

12 ± 7 196 ± 12 7.3 ± 1.1 4.9 ± 0.2 7.8 ± 0.1

13 ± 4 204 ± 25 2.6 ± 1.5 1.2 ± 0.4 3.8 ± 0.5

19 ± 3 160 ± 6 7.3 ± 3.5 9.1 ± 5.7 10 ± 6

50% Inhibitory concentration.

devoid of significant cytostatic activity (IC50: 160–204 lM), 20, and in particular 22–24, showed pronounced cytostatic activity (lower lM-range) with some preference for the human CEM lymphoblast tumor cells (IC50 range: 1.2–5 lM). The molecular mechanism of cytostatic action of these new derivatives is currently unclear and should be subject of further study. Compounds 20–24 have also been evaluated for their inhibitory activity against a wide variety of DNA and RNA viruses (Table 4). Compound 20 showed antiviral activity against HSV-1, HSV-2 and VV at an EC50 of 10–20 lM, although it should be noticed that this antiviral activity is at concentrations that are in the cytostatic activity range. It may, therefore, be questioned whether the activity is due to an underlying cytostatic activity rather than a direct antiviral effect. Interestingly, several of the compounds 20–24 were invariably inhibitory against human cytomegalovirus in HEL cell cultures in the lower micromolar range that is at compound concentrations well below their toxicity threshold. In this respect, they may represent a potential lead for further synthesis of novel compounds with a more pronounced anti-CMV activity. 3. Conclusion A series of new triterpenoid triazine derivatives were obtained from allobetulin (2) and betulinic acid (4). The ketone functional

Table 4 Inhibitory activity of test compounds against cytomegalovirus in human embryonic lung (HEL) cells Antiviral activity EC50a (lM)

Compound

20 21 22 23 24 a b c

Cytotoxicity (lM)

AD-169 strain

Davis strain

Cell morphologyb (MCC)

Cell growthc (CC50)

3±1 P20 1.8 ± 0 >4 8.6 ± 6.6

2.9 ± 0.9 P20 2 ± 0.2 P4 11 ± 9

20 >100 20 20 0 ± 40

51 ± 3.5 >100 45 ± 7 55 ± 4 74 ± 4.8

Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. Cytostatic concentration required to reduce HEL cell growth by 50%.

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group of the A-ring of allobetulone (3) and betulonic acid (5) were efficiently used as the starting point for the synthesis of triazines. The intramolecular hetero Diels–Alder reaction of S-alkylated triazine derivatives (10 and 23) lead to interesting fused thienopyridine derivatives (17 and 24) or the benzofuropyrido-fused derivative 19. Several derivatives showed pronounced cytostatic activity in cell culture and were also endowed with anti-cytomegalovirus activity in the low micromolar range. 4. Experimental section General: Chemicals were purchased from Sigma Aldrich, Acros or ABCR, Betulin was purchased from Kaden Chemicals GmbH and used as received. All reactions were carried out in flame-dried glassware, but no special precautions were taken to exclude moisture. Solvents were mostly dried and in some cases were used as received. 1H NMR and 13C NMR spectra were recorded on a Bruker 300 (operating respectively at 300 MHz and 75 MHz) Bruker 400 Advance (operating respectively at 400 MHz and 100 MHz) and a Bruker 600 Advance (operating at respectively 600 MHz and 150 MHz). Infrared spectra were measured and processed on a Bruker Alpha-T FT-IR spectrometer with universal sampling module coupled to OPUS software. All samples were applied neat unless stated otherwise. Melting points were determined with a Reichert Thermovar with microscope, and are uncorrected. Mass spectra were recorded on a Hewlett–Packard 5989A mass spectrometer (EI or CI mode), coupled with an HP Apollo 900 series. High resolution mass data were recorded on a Kratos MS50TC, with electron impact ionizer energy of 70 eV, at temperature of 250 °C. 4.1. Procedures and experimental data of compounds 4.1.1. Thioxo-1,2,4-triazino (18a)-19b, 28-epoxyoleanane (Allobetulin triazine) (7) A solution of allobetulon-1,2-ene-2-ol (0.547 g, 1.20 mmol) and thiosemicarbazide (0.143 g, 1.56 mmol) in 50 ml ethanol was vigorously stirred at reflux for 30 min, after that 1.564 mmol of K2CO3 (0.216 g) was added and continuously stirred at reflux. TLC was checked with solvent heptane/EtOAc = 8:2 to show complete reaction and the reaction finished after 20 h. Then the reaction mixture was diluted with 50 ml of water and acidified with acetic acid till pH = 4.0. The resulting orange compound was immediately precipitated, filtered and washed with water. The crude mixture was purified in column chromatography on silica with eluent heptanes/EtOAc = 8:2. Yield: 0.512 g (83%); yellow crystals; mp 226–228 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.81 (d, J = 7.8 Hz, 1H, H-28), 3.57 (s, 1H, H-19), 3.49 (d, J = 7.5 Hz, 1H, H-28), 3.0 (d, J = 16.4 Hz, 1H, H-1), 2.24 (d, J = 16.3 Hz, 1H, H-1), 1.7–1.4 (21H-complex CH, CH2), 1.36 (s, 3H), 1.32 (s, 3H), 1.03 (s, 3H), 0.95 (s, 6H), 0.82(s, 3H), 0.81(s, 3H) (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 180.9 (C-31), 173.1 (C-3), 145.6 (C-2), 87.9 (C-19), 71.2 (C-28), 53.0, 48.7, 46.7, 44.8, 41.4, 41.1, 40.8, 40.4, 36.9, 36.6, 36.2, 34.2, 32.7, 32.6, 30.9, 28.7, 26.3, 26.2, 26.1, 24.5, 24.2, 21.6, 20.0, 16.3, 15.3, 13.4. HRMS: C31H47N3OS, calculated: 509.3440, found: 509.3436. FT-IR (neat, cm 1): 2947, 2928, 2903, 2864, 1542. 4.1.2. 3-Methylsulfanyl-1,2,4-triazino (18a)-19b,28-epoxyolean ane (Allobetulin methyltriazine) (8) A solution of triazine (100 mg, 0.196 mmol) and triethylamine (0.028 ml, 0.196 mmol) in 4 ml THF and methyl iodide (0.019 ml, 0.294 mmol) was stirred at room temperature. The reaction finished after 1 h. After the reaction had finished, the solvent was evaporated and the reaction mixture was diluted with 15 ml of

5

DCM and washed 2 times with saturated NaHCO3 (2  15 ml) and water (1  15 ml) and dried over MgSO4. The resulting slightly yellow compound was purified in column chromatography on silica with eluent EtOAc/heptane = 1:9. Yield: 86 mg (84%); yellow crystals; mp 276–277 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.79 (d, J = 7.7 Hz, 1H, H-28), 3.57 (s, 1H, H-19), 3.47 (d, J = 7.8 Hz, 1H, H-28), 3.32 (d, J = 16.4 Hz, 1H, H-1), 2.63 (s, 3H, H-32), 2.22 (d, J = 15.9 Hz, 1H, H-1), 1.7–1.4 (20H-complex CH, CH2), 1.31 (s, 3H), 1.28 (s, 3H), 1.05 (s, 3H), 0.95 (s, 6H), 0.82 (s, 3H), 0.81 (s, 3H) (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 171.5 (C-31), 165.4 (C3), 145.6 (C-2), 87.9 (C-19), 71.2 (C-28), 52.8, 49.1, 46.7, 45.5, 41.4, 40.8, 40.4, 39.5, 36.8, 36.7, 36.2, 34.2, 32.8, 32.7, 31.0, 28.8, 26.4, 26.3, 26.2, 24.5. HRMS: C31H47N3OS, calculated: 523.3596, found: 523.8159. FT-IR (neat, cm 1): 2928, 1505, 1452. 4.1.3. 3-(3-Cyanopropyl)sulfanyl-1,2,4-triazino (18a)-19b,28epoxyoleanane (Allobetulino[3,2-e]-(31-propanenitrilo)-1,2,4triazine) (9) A mixture of allobetuline-1,2,4-triazine 7 (0.150 g, 0.294 mmol), Et3N (0.041 ml, 0.294 mmol) and 3-bromopropanenitrile (0.32 ml, 0.383 mmol) in 4 ml THF was stirred for 48 h with monitoring by TLC. After completion of the reaction the solvent was removed under reduced pressure, and the crude product was redissolved in DCM. The organic layer was washed 2 times with a saturated NaHCO3 solution and one time with water. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified in column chromatography on silica with eluent: heptanes/EtOAc 4:1. Yield: 85 mg (51%); white crystals; mp 249–251 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.80 (d, 1H, J = 7.7 Hz, H-28), 3.57 (s, 1H, H-19), 3.51–3.41 (m, 3H, H-28, H-33), 3.46 (t, 2H, J = 7.2 Hz, H33), 3.34 (d, 1H, J = 16.5 Hz, H-1), 2.94 (t, 1H, J = 7.0 Hz, H-32), 2.50 (d, 1H, J = 16.7 Hz, H-1), 1.81–0.71 (m, 39-H), 1.32 (s, 3H), 1.29 (s, 3H), 1.05 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.82 (s, 6H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 169.5 (C-31), 166.4 (C-3), 153.3 (C-2), 118.1 (C-34), 87.9 (C-28), 71.3 (C-19), 52.8, 49.1, 46.8, 45.6, 41.5, 40.8, 40.5, 39.6, 36.9, 36.7, 36.3, 34.3, 32.8, 32.7, 31.1, 28.8, 26.4, 26.4, 26.2, 24.6, 23.9, 21.6, 19.9, 18.4, 16.3, 15.4, 13.5. HRMS: calculated: C34H50N4OS: 562.37053; found: 562.37013. FT-IR (neat, cm 1): 2926 (br, CH), 1505 (CN), 1504, 1452. 4.1.4. 3-(But-1-ynesulfanyl)-1,2,4-triazino (18a)-19b,28-epoxyoleanane (3-(but-1-ynesulfanyl)-1,2,4-triazinoallobetulin) (10) A mixture of allobetuline-1,2,4-triazine 7 (120 mg, 0.235 mmol), Et3N (0.033 ml, 0.235 mmol) and 4-bromobut-1-yne (0.029 ml, 0.306 mmol) in THF (3 ml) was stirred for 18 h with monitoring by TLC. After completion of the reaction the solvent was removed under reduced pressure, and the crude product was redissolved in DCM. The organic layer was washed 2 times with a saturated NaHCO3 solution and one time with water. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified in column chromatography on silica with eluent heptanes/EtOAc = 9:1. Yield: 124 mg (94%); White crystals; mp 226–228 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.80 (d, J = 7.4 Hz, 1H, H-28), 3.56 (s, 1H, H-19), 3.47 (d, J = 7.4 Hz, 1H, H-28), 3.41–3.26 (m, 3H, H-1, H-32), 2.71 (t, J = 7.0 Hz, H-33), 2.49 (d, J = 16.5 Hz, 1H, H-1), 2.05 (s, 1H, H-35), 1.7–1.1 (20H-complex CH, CH2), 1.31 (s, 3H), 1.28 (s, 3H), 1.05 (s, 3H), 0.97 (s, 6H), 0.95(s, 3H), 0.81(s, 3H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 170.6 (C31), 165.8 (C3), 152.1 (C2), 87.9 (C-19), 82.3 (C-34), 71.3 (C-28), 69.7 (C-35), 52.8, 49.1, 46.7, 45.5, 41.5, 40.8, 40.5, 39.5, 36.9, 36.7, 36.3, 34.3, 32.8, 32.7, 31.1, 29.6, 28.8,

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26.4, 26.2, 24.6, 23.8, 21.6, 19.9, 19.4, 16.3, 15.4, 13.5. HRMS: C35H51N3OS, calculated: 561.37528, found: 561.37824. FT-IR (neat, cm 1): 2945, 2866, 2360, 2341, 1504, 1453. 4.1.5. 3-Phenyl-1,2,4-triazino (18a)-19b,28-epoxyoleanane (allobetulino[3,2-e]-(31-phenyl)-1,2,4-triazine) (11) A mixture of allobetulin S-methylated triazine 8 (0.100 g, 0.191 mmol), CuTC (0.127 g, 0.668 mmol), Pd(PPh3)4 (0.011 g, 9.55 lmol) and phenylboronic acid (0.070 g, 0.573 mmol) in 3 ml of THF was refluxed for 22 h. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was dissolved in DCM. The solution was filtered over a Celite pad and washed with ample amounts of water. The organic layer was dried using MgSO4, filtered and concentrated under reduced pressure. The crude mixture was purified using column chromatography with eluent heptane/EtOAc = 1:1. Yield: 52 mg (78%); Yellow crystals; mp 306–308 °C; 1H NMR (CDCl3, 300 MHz, ppm): 8.54 (m, 2H, H-33), 7.52 (m, 3H, H-34, H-35), 3.81 (d, 1H, J = 7.6 Hz, H-28), 3.58 (s, 1H, H-19), 3.48 (d, 1H, J = 6.5 Hz, H-28), 3.44 (d, 1H, J = 15.7 Hz, H-1), 2.59 (d, 1H, J = 16.7 Hz, H-1), 1.81–1.13 (m, 20 H), 1.40 (s, 3H), 1.38 (s, 3H), 1.07 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.85 (s, 3H), 0.83 (s, 3H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 165.4 (C31), 162.5 (C2), 154.5 (C3), 135.6 (C32), 131.0 (C35), 128.7 (C34), 127.9 (C33), 87.9 (C19), 71.3 (C28), 53.0, 49.2, 46.8, 45.9, 41.5, 40.8, 40.5, 39.6, 37.0, 36.7, 36.3, 34.3, 32.8, 32.7, 31.2, 28.8, 26.4, 26.4, 26.2, 24.6, 24.0, 21.6, 19.9, 16.4, 15.4, 13.5. HRMS: C37H51N3O, calculated: 553.40321, found: 553.40344. FT-IR (neat, cm 1): 2912 (br, CH), 2870, 1515, 1452. 4.1.6. Allobetulino[3,2-e]-(31-(35-tBu-phenyl))-1,2,4-triazine (12) A mixture of allobetulin S-methylated triazine 8 (0.100 g, 0.191 mmol), CuTC (0.127 g, 0.668 mmol), Pd(PPh3)4 (0.011 g, 9.55 lmol) and t-butylphenylboronic acid (0.102 g, 0.573 mmol) in 3 ml of THF was refluxed for 20 h. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was dissolved in DCM. The solution was filtered over a Celite pad and washed with ample amounts of water. The organic layer was dried using MgSO4, filtered and concentrated under reduced pressure. The crude mixture was purified in column chromatography with eluent heptanes/EtOAc = 4:1. Yield: 89 mg (76%); Yellow crystals; mp 306–308 °C; 1H NMR (CDCl3, 300 MHz, ppm): 8.46 (d, 2H, J = 8.2 Hz, H-33), 7.54 (d, 2H, J = 8.3 Hz, H-34), 3.81 (d, 1H, J = 7.7 Hz, H-28), 3.58 (s, 1H, H-19), 3.48 (d, 1H, J = 8.5 Hz, H-28), 3.43 (d, 1H, J = 17.7 Hz, H-1), 2.58 (d, 1H, J = 16.3 Hz, H-1), 1.81–1.12 (m, 20H), 1.40–1.36 (s, 18H), 1.61 (s, 3H), 1.07 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H), (all s, 103H, 23-, 24-, 25-, 26-, 27-, 29-, 30-, 337Me). 13C NMR (CDCl3, 75 MHz, ppm): 165.2 (C-31), 162.5 (C-2), 154.4 (C-3), 154.2 (C-32), 132.8 (C-35), 127.7 (C-34), 125.7 (C33), 87.9 (C-19), 71.3 (C-28), 53.0, 49.2, 46.8, 45.9, 41.5, 40.8, 40.5, 39.5, 36.7, 36.3, 34.9, 34.3, 32.8, 32.7, 31.2 (C-37), 28.8, 26.4, 26.4, 26.2, 24.6, 24.0, 21.6, 19.9, 16.4, 15.4, 13.5. HRMS: C41H59N3O, calculated: 609.46581, found: 609.46551. FT-IR (neat, cm 1): 2963 (br, CH), 1612, 1511. 4.1.7. Allobetulino[3,2-e]-(31-methylsulfonyl)-1,2,4-triazine (13) A mixture of mCPBA (527 mg, 2.291 mmol) and MgSO4 (551 mg, 4.58 mmol) was stirred for 1 h in 10 ml dry DCM under an argon atmosphere. 400 mg (0.764 mmol) of allobetulin methyl triazine 8 was added, and the mixture was stirred overnight. After completion of the reaction, the mixture was diluted with 20 ml of DCM and washed 2 times with a saturated NaHCO3-solution

(2  20 ml), and 1 time with water (20 ml). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified using column chromatography on silica with eluent DCM (1% MeOH). Yield: 345 mg (82%); White crystals; mp 314–316 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.81 (d, 1H, J = 7.6 Hz, H-28), 3.57 (s, 1H, H-19), 3.53–3.41 (m, 5H, H-1, H-28, H-32), 2.64 (d, 1H, J = 17.2 Hz, H-1), 2.81–1.13 (m, 20 H), 1.40 (s, 3H), 1.38 (s, 3H), 1.07 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.83 (s, 6H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 169.1 (C-31), 165.9 (C-2), 160.0 (C-3), 87.9 (C-28), 71.2 (C19), 53.4, 49.1, 46.7, 46.2, 41.5, 40.9, 40.5, 40.3, 39.7, 36.9, 36.7, 36.3, 34.2, 32.7, 32.6, 31.3, 28.8, 26.4, 26.3, 26.2, 24.6, 24.2, 21.6, 19.8, 16.5, 15.4, 13.5. HRMS: C32H49N3O3S, calculated: 555.34946, found: 555.34999. FT-IR (neat, cm 1): 2927 (br, CH), 2866, 2358, 1773. 4.1.8. Allobetulino[3,2-e]-(31-phenoxy)-1,2,4-triazine (14) A mixture of phenol (0.017 mg, 0.180 mmol), allobetulin methylsulfonyl triazine 13 (0.050 g, 0.09 mmol), K2CO3 (0.025 g, 0.180 mmol) and 18-crown-6 (0.012 g, 0.045 mmol) in 4 ml of DMF was refluxed for 24 h. After completion of the reaction the solution was diluted with 15 ml of Et2O and washed 3 times with water (3  20 ml). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified in column chromatography on silica with eluent heptanes/EtOAc 9:1. Yield: 30 mg (59%); White crystals; mp 275–277 °C; 1H NMR (CDCl3, 300 MHz, ppm): 7.46–7.34 (m, 2H, H-34), 7.32–7.15 (m, 3H, H-33, H-35), 3.80 (d, 1H, J = 6.6 Hz, H-28), 3.57 (s, 1H, H-19), 3.52–3.30 (m, 2H, H-1, H-28), 2.6–2.4 (m, 1H, H-1), 1.81–1.12 (m, 20 H), 1.31 (s, 3H) 1.26 (s, 3H), 1.05 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.81 (s, 6H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13 C NMR (CDCl3, 75 MHz, ppm): 182.7 (C-31), 152.8 (C-3, C-33), 141.9 (C-2), 129.5 (C-35), 125.4 (C-36), 121.4 (C-34), 87.9 (C-28), 71.3 (C-19), 52.8, 49.1, 46.8, 41.5, 40.8, 40.5, 37.0, 36.7, 36.3, 34.3, 32.8, 32.7, 31.0, 28.8, 26.4, 26.3, 26.2, 24.6, 23.8, 21.6, 19.9, 16.3, 15.4, 13.5. HRMS: C37H51N3O2, calculated: 569.39813, found: 569.39942. FT-IR (neat, cm 1): 2925 (br, CH), 2861, 2356, 1593, 1523. 4.1.9. Diethylollobetulino[3,2-e]-1,2,4-triazin-31-ylmalonate (15) A mixture of allobetulin methylsulfonyl triazine 13 (0.050 g, 0.090 mmol) and NaH (6.5 mg, 0.270 mmol) in diethyl malonate (0.027 ml, 0.180 mmol) was refluxed for 18 h. After that, the mixture was cooled down to 0 °C, 15 ml of EtOAc was added, and the solution was carefully quenched with water. The organic layer was washed 3 times with water (3  20 ml), dried over MgSO4 and concentrated under reduced pressure. The crude product was purified in column chromatography on silica with eluent heptanes/EtOAc 7:3. Yield: 35 mg (61%); White crystals; mp 186–188 °C; 1H NMR (CDCl3, 300 MHz, ppm): 5.23 (s, 1H, H-32), 4.28 (q, 4H, J = 7.0 Hz, H-34), 3.80 (d, 1H, J = 7.6 Hz, H-28), 3.57 (s, 1H, H-19), 3.47 (d, 1H, J = 7.7 Hz, H-28), 3.42 (d, 1H, J = 17.1 Hz, H-1), 2.55 (d, 1H, J = 16.7 Hz, H-1), 1.82–1.12 (m, 26H), 1.31 (s, 3H), 1.27 (s, 3H), 1.05 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 166.3 (C-33), 166.2 (C-2), 161.3 (C-31), 155.5 (C-3), 87.9 (C-28), 71.3 (C-19), 62.1 (C-34), 59.8 (C-32), 52.8, 49.2, 46.8, 45.8, 41.5, 40.8, 40.5, 39.5, 36.9, 36.7, 36.3, 34.3, 32.8, 32.7, 31.0, 28.8, 26.4 (3C), 26.2, 24.6, 23.8, 21.6, 19.8, 16.4, 15.4, 14.0, 13.5. HRMS: C38H57N3O5, calculated: 635.42982, found: 635.43184. FT-IR (neat, cm 1): 2980 (br, CH), 1741 (CO).

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T. Dinh Ngoc et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

4.1.10. Allobetulino[3,2-e]1,2,4-triazinyl-3-ol (16) Allobetulin methylsulfonyl triazine 13 (0.10 g, 0.18 mmol) was dissolved in 1 ml of THF at room temperature. NaOH (0.014 g, 0.36 mmol) and 1 ml of water were added, and the resulting mixture was refluxed for 18 h. After completion of the reaction, the THF was removed under reduced pressure, and 30 ml of EtOAc was added. The organic layer was washed 3 times with water (3  20 ml), dried over MgSO4 and concentrated under reduced pressure. The crude product was purified using column chromatography (SiO2, eluent DCM 5% MeOH). 0.081 mg of white crystals were obtained. Yield: 81 mg (91%); White crystals; mp >350 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.80 (d, 1H, J = 7.7 Hz, H-28), 3.57 (s, 1H, H-19), 3.48 (d, 1H, J = 7.8 Hz, H-28), 2.98 (d, 1H, J = 15.7 Hz, H-1), 2.19 (d, 1H, J = 15.9 Hz, H-1), 1.79–1.12 (m, 21 H), 1.35 (s, 3H), 1.31 (s, 3H), 1.04 (s, 3H), 0.95 (s, 6H), 0.82 (s, 6H) (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 179.4 (C-31), 156.9 (C-2), 141.4 (C-3), 87.9 (C-28), 71.2 (C-19), 52.9, 48.7, 46.7, 44.9, 41.5, 41.3, 40.8, 40.4, 37.1, 36.7, 36.3, 34.2, 32.7, 32.7, 31.1, 28.8, 26.4, 26.3, 26.2, 24.5, 24.3, 21.6, 20.1, 16.2, 15.4, 13.4. HRMS: C31H47N3O2, calculated: 493.36683, found: 493.36557. FT-IR (neat, cm 1): 2945 (br, CH), 2929, 2903, 2857, 1598, 1458. 4.1.11. Thieno[2,3b]quinolone allobetulin (17) 3-(But-1-ynethio)-1,2,4-triazinoallobetulin (75 mg, 0.13 mmol) was dissolved in xylene (5 ml). The mixture was refluxed for 20 h. After the reaction had finished, the solvent was evaporated. The crude product was purified in column chromatography on silica with eluent heptanes/EtOAc = 9:1. Yield: 35 mg (50%); White crystals; mp 276–278 °C; 1H NMR (CDCl3, 300 MHz, ppm): 6.98 (s, 1H, H-35), 3.80 (d, J = 7.6 Hz, 1H, H-28), 3.56 (s, 1H, H-19), 3.46 (d, J = 7.6 Hz, 1H, H-28), 3.36 (t, J = 7.3 Hz, 2H, H-32), 3.21 (t, J = 7.3 Hz, 2H, H-33), 2.66 (d, J = 15.4 Hz, 1H, H-1), 2.26 (d, J = 15.4 Hz, 1H, H-1), 1.7–1.1 (20Hcomplex CH, CH2), 1.29 (s, 3H), 1.23 (s, 3H), 1.04 (s, 3H), 0.95 (s, 6H), 0.81(s, 3H), 0.80 (s, 3H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 162.6 (C-3, C-31), 133.0 (C-35), 124.4 (C-2, C-34), 87.9 (C-19), 71.3 (C-28), 53.7, 49.4, 46.8, 45.8, 41.5, 40.8, 40.5, 39.4, 36.7, 36.3, 34.3, 33.2, 33.1, 32.7, 31.6, 31.2, 28.8, 26.5, 26.4, 26.3, 24.6, 23.9, 21.5, 20.1, 16.0, 15.4, 13.5. HRMS: C35H51NOS, calculated: 533.36914, found: 533.36834. FT-IR (neat, cm 1): 2923, 2865, 2356, 1734, 1557. 4.1.12. Allobetulino[3,2-e]benzofuropyridine (19) Allobetulin methylsulfonyl triazine 15 (0.1 g, 0.180 mmol), K2CO3 (0.055 g, 0.396 mmol), 18-crown-6 (0.024 g, 0.090 mmol) and 2-[(trimethylsilyl)ethynyl]phenol 18 (0.068 g, 0.360 mmol) were dissolved in 1 ml of dry DMF under argon atmosphere. The resulting mixture was stirred at 70 °C for 18 h. After completion of the reaction the solvent was removed under reduced pressure, and the residue was redissolved in 20 ml of DCM. The organic layer was washed 3 times with brine (3  20 ml), dried over MgSO4 and concentrated under reduced pressure. The crude product was purified in column chromatography on silica with eluent heptanes/EtOAc 95:5. Yield: 63 mg (62%); white powder; mp: 297–298 °C; 1H NMR (CDCl3, 300 MHz, ppm): 7.88–7.80 (m, 2H, H-33, H-39), 7.56 (d, 1H, J = 8.1 Hz, H-36), 7.45 (t, 1H, J = 7.5 Hz, H-34), 7.32 (t, 1H, J = 7.5 Hz, H-35), 3.81 (d, 1H, J = 7.8 Hz, H-28), 3.58 (s, 1H, H-19), 3.47 (d, 1H, J = 7.8 Hz, H-28), 2.98 (d, 1H, J = 15.3 Hz, H-1), 2.51 (d, 1H, J = 15.3 Hz, H-1), 1.80–1.14 (m, 20 H), 1.41 (s, 3H), 1.36 (s, 3H), 1.07 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.85 (s, 3H), 0.83 (s, 3H), (all s, 73H, 23-, 24-, 25-, 26-, 27-, 29-, 30-Me). 13C NMR (CDCl3, 300 MHz, ppm): 162.1 (C31), 161.8 (C-3), 154.6 (C-32), 130.9 (C-39), 127.5, 125.2, 122.9, 122.7, 120.8, 114.1, 111.9, 87.9

7

(C-28), 71.3 (C-19), 53.6, 49.3, 46.8, 46.2, 41.5, 40.8, 40.5, 40.2, 36.7, 36.5, 36.3, 34.3, 33.1, 32.7, 31.9, 28.8, 26.5, 26.4, 26.3, 24.6, 24.3, 21.6, 20.2, 15.9, 15.5, 13.5. HRMS: C39H51NO2, calculated: 565.39198, found: 565.39294. FT-IR (neat, cm 1): 2945 (CH), 2929, 2903, 2857, 1598, 1458. 4.1.13. (3-Thioxo-1,2,4-triazino)betulonic acid (21) 2-Oxobetulonic acid (340 mg, 0.725 mmol) and thiosemicarbazide (134 mg, 1.451 mmol) were dissolved in ethanol (10 ml). The mixture was refluxed for 30 min, after that potassium carbonate (201 mg, 1.451 mmol) was added and the mixture was continuously refluxed for 9 h. After the reaction had finished, the mixture was diluted with 20 ml water and acidified by acetic acid until pH = 4. The resulting orange compound was immediately precipitated, filtered and washed with water. The crude product was purified by column chromatography on silica with eluent heptanes/ EtOAc = 8:2. Yield: 302 mg (79%); Yellow crystals; mp: 250–251 °C; 1H NMR (CDCl3, 300 MHz, ppm): 4.75 (s, 1H, H29-Z), 4.63 (s, 1H, H29-E), 3.0 (m, 1H, H-19), 2.9–1.0 (24H-complex CH, CH2), 1.70 (s, 3H), 1.48 (s, 3H), 1.35 (s, 3H), 1.30 (s, 6H), 1.0 (s, 3H), 0.79 (s, 3H), (all s, 63H, 23-, 24-, 25-, 26-, 27-, 30-Me). 13C NMR (CDCl3, 300 MHz, ppm): 181.0 (C31), 180.4 (C28), 173.1 (C3), 150.2 (C2), 145.7 (C20), 109.8 (C29), 56.3, 52.8, 49.0, 48.2, 46.8, 44.6, 42.5, 41.0, 40.5, 38.3, 37.0, 36.9, 33.0, 32.0, 30.9, 30.5, 29.6, 25.3, 24.2, 21.4, 20.0, 19.3, 15.9, 15.7, 14.6. HRMS: C31H45N3O2S, calculated: 523.32325, found: 523.32576. FT-IR (neat, cm 1): 2939, 2868, 1692, 1643, 1521, 1452, 1354. 4.1.14. 3-(Methylthio)-1,2,4-triazinobetulonic acid (22) (3-Thioxo-1,2,4-triazino)betulonic acid (192 mg, 0.37 mmol), methyl iodide (0.046 ml, 0.74 mmol), and triethylamine (0.104 ml, 0.74 mmol) were dissolved in THF (20 ml). The mixture was stirred at room temperature for 1 h. After the reaction had finished, the solvent was evaporated, the mixture was diluted in DCM (40 ml), washed with water (2  40 ml), dry over MgSO4. Column chromatography with eluent heptane/EtOAc = 8:2. Yield: 135 mg (68%); Yellow crystals; mp 239–240 °C; 1H NMR (CDCl3, 300 MHz, ppm): 4.76 (s, 1H, H-29Z), 4.64 (s, 1H, H-29E), 3.28 (d, J = 16.47 Hz, 1H, H1a), 3.02 (m, 1H, H-19), 2.63 (s, 3H, H-32), 2.35 (d, J = 16.44 Hz, 1H, H-1b), 2.3–1.0 (21H-complex CH, CH2), 1.71 (s, 3H), 1.29 (s, 3H), 1.25 (s, 3H), 1.02 (s, 6H), 1.01 (s, 3H), 0.77 (s, 3H), (all s, 63H, 23-, 24-, 25-, 26-, 27-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 180.8 (C28), 171.5 (C31), 165.5 (C3), 152.2 (C2), 150.2 (C20), 109.9 (C29), 56.3, 52.7, 49.1, 48.6, 46.8, 45.3, 42.5, 40.5, 39.5, 38.4, 37.0, 36.8, 33.1, 32.0, 31.0, 30.5, 29.7, 25.4, 23.8, 21.4, 19.9, 19.4, 15.9, 15.6, 14.6, 13.7. HRMS: C32H47N3O2S, calculated: 537.33890, found: 537.33937. FT-IR (neat, cm 1): 2927, 2868, 1685, 1640, 1506, 1451, 1351. 4.1.15. 3-(But-1-ynesulfanyl)-1,2,4-triazinobetulonic acid (23) (3-Thio-1,2,4-triazino)betulonic acid (200 mg, 0.382 mmol), 4-bromobut-1-yne (0.071 ml, 0.764 mmol), and triethylamine (0.108 ml, 0.764 mmol) were dissolved in THF (10 ml). The mixture was stirred at room temperature for 22 h. After the reaction had finished, the solvent was evaporated, the mixture was diluted in DCM (40 ml) washed water (2  40 ml) and dried over MgSO4. The crude product was purified by column chromatography on silica with eluent heptanes/EtOAc = 8:2. Yield: 120 mg (55%); White crystals; mp 231–232 °C; 1H NMR (CDCl3, 300 MHz, ppm): 4.76 (s, 1H, H-29Z), 4.64 (s, 1H, H-29E), 3.36 (m, 2H, H-32), 3.28 (d, J = 16.47 Hz, 1H, H-1a), 3.01 (m, 1H, H-19), 2.69 (m, 2H, H-33), 2.43 (d, J = 16.53 Hz, 1H, H-1b), 2.3–1.0 (22H-complex CH, CH2), 1.71 (s, 3H), 1.30 (s, 3H), 1.25 (s,3H), 1.03 (s, 6H), 1.01 (s, 3H), 0.78 (s, 3H), (all s, 63H, 23-, 24-, 25-, 26-, 27-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm):

Please cite this article in press as: Dinh Ngoc, T.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.061

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T. Dinh Ngoc et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

180.8 (C28), 170.6 (C31), 165.8 (C3), 152.6 (C2), 150.2 (C20), 109.9 (C29), 82.3 (C34), 69.7 (C35), 58.3 (C17), 52.6, 49.2, 48.6, 46.8, 45.3, 42.5, 40.6, 39.5, 38.4, 37.0, 36.8, 33.2, 32.1, 31.0, 30.6, 29.7, 29.6, 25.5, 23.8, 21.5, 19.9, 19.4, 15.9, 15.6, 14.7. HRMS: C35H49N3O2S, calculated: 575.35455, found: 575.35774. FT-IR (neat, cm 1): 2939, 2868, 2017, 1685, 1641, 1519, 1453, 1426. 4.1.16. Thieno[2,3b]quinolobetulonic acid (24) 3-(But-1-ynesulfanyl)-1,2,4-triazinobetulonic acid (58 mg, 0.101 mmol) was dissolved in xylene (10 ml). The mixture was refluxed for 20 h. After the reaction had finished, the solvent was evaporated. The crude product was purified by column chromatography on silica with eluent heptanes/EtOAc = 8:2. Yield: 28 mg (51%); White crystals; mp 168–169 °C; 1H NMR (CDCl3, 300 MHz, ppm): 6.94 (s, 1H, H-35), 4.76 (s, 1H, H-29Z), 4.62 (s, 1H, H-29E), 3.35 (t, J = 7.2 Hz, 2H, H-32), 3.20 (t, J = 7.4 Hz, 2H, H-33), 3.01 (m, 1H, H-19), 2.60 (d, J = 15.6 Hz, 1H, H-1a), 2.23 (d, J = 17.1 Hz, 1H, H-1b), 2.3–1.0 (21H-complex CH, CH2), 1.71 (s, 3H), 1.27 (s, 3H), 1.21 (s,3H), 1.01 (s, 6H), 1.00 (s, 3H), 0.78 (s, 3H), (all s, 63H, 23-, 24-, 25-, 26-, 27-, 30-Me). 13C NMR (CDCl3, 75 MHz, ppm): 180.9 (C28), 162.6 (C3), 162.5 C31), 150.6 (C20), 133.1 (C34), 130.9 (C35), 124.5 (C2), 109.6 (C29), 56.3, 53.5, 49.2, 48.8, 46.8, 45.7, 42.4, 40.6, 39.4, 38.4, 37.0, 36.2, 33.5, 33.1, 31.9, 31.5, 31.2, 29.7, 25.6, 23.8, 22.7, 21.4, 20.1, 19.4, 15.7, 15.6, 14.7, 14.1. HRMS: C35H49NO2S, calculated: 547.34840, found: 547.34905. FT-IR (neat, cm 1): 2928, 2867, 1691, 1638, 1595, 1559, 1450, 1399, 1374. 4.2. Cytostatic activity assays All assays were performed in 96-well microtiter plates. To each well were added (5–7.5)  104 tumor cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37 °C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited tumor cell proliferation by 50%. 4.3. Antiviral activity assays The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK ) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK VZV strain 07-1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, parainfluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, Sindbis, reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain IIIB and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 or 100 plaque forming units (PFU) (VZV or HCMV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or

compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%. 4.4. Anti-HIV activity assays Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing 3  105 CEM cells/ml infected with 100 CCID50 of HIV per milliliter and containing appropriate dilutions of the test compounds. After 4–5 days of incubation at 37 °C in a CO2controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC50 (50% effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50%. Acknowledgments We thank the FWO (Fund for Scientific Research—Flanders), the Vietnam International Education Development Project (VIED) and the University of Leuven (KU Leuven) for financial support. The technical assistance of Mrs. Lizette van Berckelaer, Mrs. Leen Ingels, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, Mr. Steven Carmans, Mrs. Lies Van den Heurck and Mrs. Anita Camps for the biological evaluations is greatly appreciated. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.061. References and notes 1. Newman, D. J. J. Med. Chem. 2008, 51, 2589. 2. Breinbauer, R.; Vetter, I. R.; Waldmann, H. Angew. Chem., Int. Ed. 2002, 41, 2878. 3. (a) Shesadri, T. R.; Vedanthan, T. N. C. Phytochemistry 1971, 10, 897; (b) Green, B.; Bentley, M. D.; Chung, B. Y.; Lynch, N. G.; Jensen, B. L. J. Chem. Educ. 2007, 84, 1985; (c) Li, T. S.; Wang, J. X.; Zheng, X. J. J. Chem. Soc., Perkin Trans. 1998, 3957. 4. Urban, M.; Sarek, J.; Kvasnica, M.; Tislerova, I.; Hajduch, M. J. Nat. Prod. 2007, 70, 526. 5. Salin, O.; Alakurtti, S.; Pohjala, L.; Siiskonen, A.; Maass, V.; Maass, M.; YliKauhaluoma, J.; Vuorela, P. Biochem. Pharmacol. 2010, 80, 1141. 6. Krasutsky, P.A.; Carlson, R.M. PCT Int. Appl. WO 2002026761, 2002, [CA 2002, 136, 294955]. 7. Platanov, V. G.; Zorina, A. D.; Gordon, M. A.; Chizhov, N. P.; Balykina, L. V.; Mikhailov, Y. D.; Ivanen, D. R.; Kvi, T. K.; Shavva, A. G. Pharm. Chem. J. 1995, 29, 42. 8. (a) Ruzicka, L.; Govaert, F.; Goldberg, M. W.; Lamberton, A. H. Helv. Chim. Acta 1938, 21, 73; (b) Kazakova, O. B.; Tolstikov, G. A.; Suponitskii, K. Y. Russ. J. Bioorg. Chem. 2010, 36, 133; (c) Flekhter, O. B.; Medvedeva, N. I.; Karachurina, L. T.; Baltina, L. A.; Galin, F. Z.; Zarudii, F. S.; Tolstikov, G. A. Pharm. Chem. J. 2005, 39, 401. 9. Dehaen, W.; Mashentseva, A. A.; Seitembetov, T. S. Molecules 2011, 16, 2443. 10. Kuyper, L. F.; Garvey, J. M.; Baccanari, D. P.; Champness, J. N.; Stammers, D. K.; Beddel, C. R. Bioorg. Med. Chem. Lett. 1996, 4, 593. 11. Depecker, G.; Patino, N.; Giorgio, C. D.; Terreux, R.; Gobrol bass, D.; Bailly, C.; Aubertin, A.; Candom, R. Org. Biomol. Chem. 2004, 2, 74. 12. (a) Araguchi, K. H.; Kubota, Y.; Tanaka, H. J. Org. Chem. 2004, 69, 1831; (b) Nguyen, T. L. Anticancer Agents Med. Chem. 2008, 8, 710. 13. (a) Tori, M.; Maisuda, R.; Sono, M.; Kohama, Y.; Asakawa, Y. Bull. Chem. Soc. Jpn. 1988, 61, 2103; (b) Kim, D. S. H. L.; Chem, Z.; Nguyen Van, T.; Pezzuto, J. M.; Quit, S.; Lu, Z. Z. Synth. Commun. 1997, 27, 1607; (c) Flekhter, O. B.; Ashavina, O. Y.; Smirnova, I. E.; Baltina, L. A.; Galin, F. Z.; Kabal’nova, N. N.; Tolstikov, G. A. Chem. Nat. Comp. 2004, 40, 141; (d) Thibeault, B.; Legault, J.; Bouchard, J.; Pichette, A. Tetrahedron Lett. 2007, 48, 8416. 14. (a) Flekhter, O. B.; Ashavina, O. Y.; Boreko, E. I.; Karachurina, L. T.; Pavlova, N. I.; Kabalnova, N. N.; Savinova, O. V.; Galin, F. Z.; Nikolaeva, S. N.; Zarudii, F. S.; Baltina, L. A.; Tolstikov, G. A. Khim. Pharm. Zh. 2002, 36, 21; (b) Mukherjee, R.; Jaggi, M.; Rajendran, P.; Siddiqui, M. J. A.; Srivastava, S. K.; Vardhanb, A.; Burman, A. C. Bioorg. Med. Chem. Lett. 2004, 14, 2181; (c) Kumar, V.; Rani, N.; Aggarwal, P.; Sanna, V. K.; Singh, A. T.; Jaggi, M.; Joshi, N.; Sharma, P. K.; Irchhaiya, R.; Burman, A. C. Bioorg. Med. Chem. Lett. 2008, 18, 5058. 15. Dinh Ngoc, T.; Dehaen, W. Tetrahedron 2014, 70, 183. 16. Korovin, A. V.; Tkachev, A. V. Russ. Chem. Bull. 2001, 50, 304. 17. Nagai, S.; Ueda, T.; Takamura, M.; Nagatsu, A.; Murakami, N.; Sakakibara, J. J. Heterocycl.Chem. 1998, 35, 293.

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Synthesis of triterpenoid triazine derivatives from allobetulone and betulonic acid with biological activities.

The synthetic transformation and modification of natural products with the aim to improve the biological properties is an area of current interest. Th...
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