Bioorganic & Medicinal Chemistry Letters 25 (2015) 1799–1803

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Synthesis of novel pleuromutilin derivatives. Part 1: Preliminary studies of antituberculosis activity Ying-Jie Dong a, Zi-Hui Meng a, Yan-Qing Mi a, Chun Zhang a, Zhi-Hao Cui a, Peng Wang a, Zhi-Bin Xu a,b,⇑ a

Department of Applied Chemistry and Pharmaceuticals, Beijing Institute of Technology, Beijing 100081, PR China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China b

a r t i c l e

i n f o

Article history: Available online 17 February 2015 Keywords: Pleuromutilin derivatives Antitubercular activity N-Benzylamine side chain

a b s t r a c t The worldwide threat from tuberculosis (TB) has resulted in great demand for new drugs, particularly those that can treat multidrug-resistant TB. We synthesized novel pleuromutilin derivatives with N-benzylamine side chain substituted at the C14 position and evaluated their activity in vitro against a virulent strain of Mycobacterium tuberculosis (H37Rv). The primary assay results showed that five compounds inhibited the H37Rv at 20 lM, with a MIC of one of the analogues as low as 7.2 lM. Ó 2015 Elsevier Ltd. All rights reserved.

Infection by Mycobacterium tuberculosis, the causative bacterial agent of tuberculosis (TB), is amongst the biggest worldwide health threats, especially in countries lacking modern health care systems.1 Similar to other infectious diseases, tuberculosis is facing a serious drug-resistance problem: in some countries, resistant strains can account for up to 22% of infections.2 Treatment failure leads to increased morbidity, mortality and health care costs. Although implementation of ‘the directly observed therapy short course’ (DOTS), recommended by the World Health Organization (WHO), has achieved an 84% cure rate according to the WHO 2011 TB report, the development of new drugs therapies is still the most effective way to tackle the TB pandemic.3 Natural products isolated from plants have traditionally played an important role in the treatment of human diseases. In the field of cancer and infectious diseases, about 65% of the drugs on the market are of natural origin; thus serious attention has been paid to natural products with activities against multidrug-resistant tuberculosis.4 The first drug to treat TB was streptomycin (Fig. 1), isolated from the actinobacterium Streptomyces griseus in 1943.5 Another outstanding example is rifampin (Fig. 1), a semisynthetic compound derived from rifamycins, a member of the ansamycins family of antibiotics.6 Remarkably few of new medicines for the treatment of TB have been developed in the past 40 years. One of the major reasons for this is a lack of critical funding, R&D capacity and commercial interest. Inspired by ‘open source’ philosophy, The Global Alliance for Drug Development (TB Alliance) was established in 2000 with a ⇑ Corresponding author. E-mail address: [email protected] (Z.-B. Xu). http://dx.doi.org/10.1016/j.bmcl.2015.02.023 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

NH HN

H 2N

OHC

AcO AcO CH 3

OH

N H O

H3 C

CH 3 CH 3 CH 3

NH2 OH

HO NH

OH H 3C

NHCH3

HO

OH

OH H N

CH 3 O

O

OH O O

OH

OH

O OH CH3O

N

N

OH Streptomycin

N

CH 3

Rifampin

Figure 1. Structures of streptomycin and rifampin.

mission to accelerate early-stage drug discovery and harness the power of innovation across the world to develop new tuberculosis treatments. In recent decades, the interest in new anti-TB drugs from natural products, especially those active against multidrug-resistant TB, has continued to grow, resulting in discovery of drugs including erythromycin, an analogue of capuramycin RS-118641 (Fig. 2) and the pacidamycin family (Fig. 3).7–13 Pleuromutilin 1 (Fig. 4) is a diterpene with a fused 5-6-8 tricyclic skeleton, first isolated in 1951.14 Its two derivatives, tiamulin 2 (Fig. 4) and valnemulin 3 (Fig. 4), have been successfully developed as antibiotic agents for veterinary use.15,16 Their mechanism of action was identified as the selective inhibition of bacterial pro-

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O HO

HO

OH

OH O

O

OH

HO O

O

O

N

OCH 3

O

H N

HN

H 3CO

O

O

N

O

O

H N

O O

O

O

NH 2

OCO(CH 2 )8CH 3

H3 C

OH

Erythromycin

RS-118641

Figure 2. Structures of erythromycin and RS-118641.

O R 1HN CH 3

CH 3 N CH 3 O

R2

CH 3 O

H N

N H

O NH

N H

Liabilities of the pleuromutilin class of compounds include low solubilities and oral bioactivities. Chemical modifications resulted in the preparation of a series of pleuromutilin derivatives containing a purine ring and a piperazine ring spacer 5. These analogues showed good increased solubility in water and good pharmacokinetics, as well as excellent in vitro and in vivo antibacterial activities.20 Inspired by the N-benzoyl carbamate derivatives 6 and 7 (Fig. 4) that show increased affinity for the ribosome,21 we synthesized some novel pleuromutilin derivatives (8a–g) (Fig. 4) possessing N-benzylamine substitution at C-14 acyloxy group and evaluated their anti-TB ability with virulent strain of Mycobacterium tuberculosis (H37Rv) in liquid medium. The synthetic route to our target compounds is outlined in Scheme 1. The starting material 1 was reacted with p-toluene sulfonyl chloride (TsCl) in the presence of triethylamine at room temperature for 24 h in dichloromethane to yield the 14-O-(p-toluene sulfonyloxyacetyl) mutilin 9.22 The substituted benzoic acids were reacted with oxalyl chloride and ethylamine solution and converted into amides 11a–e in a one-pot process.23 The resulting amides were then reduced with LiAlH4 in THF, resulting in the formation of the di-substituted amines 12a–e which can be used in next step without purification. Compounds 12f and 12g were synthesized from 4-phenoxybenzoic acid through reduction and substitution reactions.24 It should be noted that 1-(chloromethyl)-4-phenoxybenzene, the precursor of 12f and 12g, was obtained from reduc-

COOH

O

O

N NH OH O

Pacidamycine f amily R 1=H, alanyl and glycyl; R 2=3-indoyl, benzyl and 3-hydroxybenzyl Figure 3. General structure of the Pacidamycine family.

tein synthesis through interaction with the 50S subunits of prokaryotic ribosomes, and consequently, has rarely exhibited cross-resistance with marketed antibacterial classes.17 Hundreds of pleuromutilin derivatives have been tested in vitro against Mycobacterium tuberculosis by GlaxoSmithKline and a number of lead compounds with high potency and most promising pharmacokinetics have been identified.18 Analogues of this class of antibiotics 4 (Fig. 4) have been patented for their antimycobacterial properties, and we were interested in synthesizing better derivatives of pleuromutilin and evaluating their anti-TB bioactivities.19

OH

S

3 R= 1

R= OH

2

R=

NH2

H N O

O 14

R

NH

O S

N

4

R=

N H

S

O

OH OH

O N

N

N N N

H2 N

N

O

O

N

O N H

O

O

O

O

O

5

6 OH OH

O

O N H

N

O

R''

R' N

O O

14

O O 7 (SB-264128)

Target compounds 8a-h

Figure 4. Important pleuromutilin derivatives and target compounds.

NH 2

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OH

OH

OH R2 N

R1

12a-g

TsO

O

55%

O

R2

O

(i)

O HO

N H

R1

O O

(ii) O

O

8a 44% 8c 48% 8e 23% 8g 79%

9

1 O

O

R1

(iv)

Cl

R1

(v)

N H

R1

12a R1 =H 12b R 1 =3-Cl 12c R1 =3-CH 3 12d R 1 =4-N(Et) 2

82% 50% 78% 29%

12e R1 =4-Phenoxyl

11e R1 =4-Phenoxyl 84% O

(vi) OH

N H

R1

11a R1 =H 11b R 1 =3-Cl 11c R1 =3-CH 3 11d R 1 =4-N(Et) 2

O

O

O

(iii)

OH

8b 78% 8d 33% 8f 52%

O

(vii)

H N

Cl

72%

R2

O 12f R2 = PhCH2 51% 12g R 2 =Cyclohexyl 35% Scheme 1. Reagents and conditions: (i) p-toluene sulfonyl chloride, Et3N, CH2Cl2, 24 h at room temperature; (ii) cat. KI, K2CO3, CH3CN, reflux; (iii) oxalyl chloride, DMF, DMAP, CH2Cl2, 5 to 0 °C; (iv) ethylamine solution, CH2Cl2 for 12a–e; (v) LiAlH4, THF, reflux; (vi) LiAlH4, THF, reflux; then 4-toluene sulfonyl chloride, Et3N, DMAP, CH2Cl2, 10 h at room temperature; (vii) benzylamine, K2CO3, DMF, 24 h at room temperature for 12f; cyclohexylamine, K2CO3, CH3CN, 4 h reflux for 12g.

OH

OH

O TsO

(i)

O

HN N

O

O

90% O

O

9

(ii) 93%

10

OH

O O S N

O N H

(iii)

O N

O

79% O

13

OH

O O S N H 2N

O N

O O 8h

Scheme 2. Reagents and conditions: (i) piperazine, cat. KI, K2CO3, CH3CN, reflux; (ii) p-acetamidobenzene sulfonyl chloride, Et3N, CH2Cl2, 12 h at room temperature; (iii) HCl, CH3CN, reflux for 4 h.

tion of 4-phenoxybenzoic acid followed by addition of 4-toluene sulfonyl chloride in the same pot.25 Displacement of the tosyl group of 9 with a series of N-benzylamines 12a–g gave the target compounds 8a–g after reflux for 4 h in acetone.26 Since sulfonamides are a common pharmacophore in antibiotics, we also designed the target compound 8h. It was synthesized using the same route as described in Scheme 2. The activitated compound 9 was reacted with piperazine to yield the amine derivative 10,27 followed by treatment of p-acetamidobenzene sulfonyl chloride for 12 h at room temperature to give the sulfonamide 13.28 The target compound 8h was obtained after deprotection of the acetyl group by refluxing in hydrochloric acid.29 The antituberculosis activity of the novel pleuromutilin derivatives 8a–h were assessed in vitro using the tuberculosis screening module provided by TB Alliance. In the primary assay, all compounds were tested for their ability to prevent growth of a

genetically-modified, fluorescent reporter strain of Mycobacterium tuberculosis (H37Rv) at a single concentration. Table 1 summarizes the biological data for our eight new compounds. Interestingly, four of the compounds 8a, 8d, 8e and 8f, containing electron-donating groups substituted at the 4-position of benzene, exhibited good inhibition against M. tuberculosis at 20 lM. Interestingly, compound 8c containing a methyl group substituted at the meta-position of the phenyl ring, had greatly reduced activity compared to 8b. Replacing the ethyl group on the N atom with either a benzyl or cyclohexyl group retains the potent activity. However, introduction of piperidine as the linker group (compound 8h), causes the activity to decline sharply, due to the rigidity of the six-membered ring. Compound 8d was further tested to determine the minimum inhibitory concentration (MIC) in a second assay, showing that 8d completely inhibited proliferation of M. tuberculosis at 7.2 lM (MIC).

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Table 1 Antituberculosis activity (% inhibition) of pleuromutilin derivatives (8a–h) in whole cell activity H37Rv cells

OH R'' R'

O

N O

14

O

Compd

R0

R00

H37Rv (% inhib @20 lM)

8a 8b 8c 8d 8e 8f 8g 8h

–CH2Ph –CH2Ph (30 –Cl) –CH2Ph (30 –CH3) –CH2Ph (40 –NEt2) –CH2Ph (40 –phenoxyl) –CH2Ph (40 –phenoxyl) –CH2Ph (40 –phenoxyl) –SO2Ph (40 –NH2)

–CH2CH3 –CH2CH3 –CH2CH3 –CH2CH3 –CH2CH3 –CH2Ph Cyclohexyl Piperidino

100 99 16 99 100 101 88 60

We have synthesized eight novel pleuromutilin derivatives and investigated their antibacterial activity in vitro against a Mycobacterium H37Rv cell line. The results revealed that most derivatives showed moderate to good inhibitory characteristics. The structure–activity relationship showed that changes in the para-position of phenyl ring retain the potent activity, while rigidifying the linker group results in a dramatic decrease in the activity. Acknowledgments We gratefully acknowledge financial support by the State Key Laboratory of Bioactive Substance and Function of Natural Medicines; and also thank for the Open Innovation Drug Discovery (OIDD) program providing the biological screen. We also wish to express gratitude for Professor Jennifer M. Schomaker in University of Wisconsin-Madison help with our manuscript. References and notes 1. World Health Organization. Multidrug and Extensive Drug Resistant Tuberculosis: 2010 Global Report on Surveillance and Response. http:// whqlibdoc.who.int/publications/2010/9789241599191_eng.pdf. 2. Wright, A.; Zignol, M.; Van Deun, A.; Falzon, D. Lancet 1861, 2009, 373. 3. World Health Organization. Global Tuberculosis Control: WHO Report 2014. http://www.who.int/tb/publications/global_report/en/. 4. Newman, D. J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66, 1022. 5. Kingston, W. J. Hist. Med. Allied Sci. 2004, 59, 441. 6. Floss, H. G.; Yu, T. W. Chem. Rev. 2005, 105, 621. 7. Negi, A. S.; Kumar, J. K.; Luqman, S. Med. Res. Rev. 2010, 30, 603. 8. Krist, H. A. Expert Opin. Drug Disc. 2013, 8, 479. 9. Copp, B. R.; Pearce, A. N. Nat. Prod. Rep. 2007, 24, 278. 10. Guido, F.; Pauli, G. F.; Case, R. J.; Inui, T. Life Sci. 2005, 78, 485. 11. Mims, C.; Dockrell, H. M.; Goering, R. V.; Roitt, I.; Wakelin, D. Medical Microbiology, 3rd ed., London, 2004, Chapter 33. 12. Koga, T.; Fukuoka, T.; Doi, N.; Harasaki, T.; Inoue, H.; Hotoda, H. J. Antimicrob. Chemother. 2004, 54, 755. 13. Boojamra, C. G.; Lemoine, R. C.; Blais, J. V.; Vernier, N. G.; Stein, K. A.; Magon, A. Bioorg. Med. Chem. Lett. 2003, 13, 3305. 14. Kavanagh, F.; Hervey, A. W. J.; Robbins, W. J. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 570. 15. Drews, J.; Georgopoulos, A.; Laber, G.; Schutze, E.; Unger, J. Antimicrob. Agents Chemother. 1975, 7, 507. 16. Hannan, P. C. T.; Windsor, H. M.; Ripley, P. H. Res. Vet. Sci. 1997, 63, 157. 17. Hogenauer, G. Eur. J. Biochem. 1975, 52, 93. 18. Global TB Alliance Annual report 2004-05; Available at: http://www.tballiance. org/downloads/2005-2006 annual report.pdf. 19. Ascher, G.; Stauffer, F.; Berner, H.; Mang, R.WO Patent 2003082260, 2003. 20. Hirokawa, Y.; Kinoshita, H.; Tanaka, T.; Nakamura, T.; Fujimoto, K.; Kashimoto, S. Bioorg. Med. Chem. Lett. 2009, 19, 175. 21. (a) Long, K. S.; Hansen, L. H.; Jakobsen, L.; Vester, B. Antimicrob. Agents Chemother. 2006, 50, 1458; (b) Brooks, G.; Burgess, W.; Colthurst, D.; Hinks, J. D.; Hunt, E.; Pearson, M. J. Bioorg. Med. Chem. 2001, 9, 1221. 22. Xu, P.; Zhang, Y.; Sun, Y. Chem. Biol. Drug Res. 2009, 73, 655.

23. (a) Du, Y.; Hyster, T. K.; Rovis, T. Chem. Commun. 2011, 12074; (b) Xu, Z.; Meng, Z.; Mi, Y.; Dong, Y. CN Patent 103265442, 2013. 24. (a) The synthesis of compounds 12f: A solution of 1-(chloromethyl)-4phenoxybenzene (0.22 g, 1.0 mmol), phenyl- methanamine (0.22 mL, 2.0 mmol), anhydrous K2CO3 (0.69 g, 5.0 mmol) in 5.0 mL DMF was stirred at room temperature for 24 h. The solution was washed with water and extracted by ethyl acetate for four times. The organic layer was separated and dried over anhydrous MgSO4, then the solvent was evaporated in vacuum and the residue was purified by column chromatography (petroleum ether/ethyl acetate/ triethylamine = 40:8:1, v/v/v) to obtain 12f as a yellow oil (0.16 g, 51%). 1H NMR (600 MHz, CDCl3) d ppm: 7.37–7.31 (m, 9H), 7.11–7.08 (m, 1H), 7.02–6.99 (m, 4H), 3.84 (s, 2H), 3.80 (s, 2H), 1.76 (s, br, 1H). 13C NMR (150 MHz, CDCl3) d ppm: 157.4, 156.1, 140.2, 135.2, 129.7, 129.5, 128.4, 128.1, 126.9, 123.0, 118.9, 118.6, 53.1, 52.5. (b) The synthesis of compounds 12g: A solution of 1-(chloromethyl)-4phenoxybenzene (0.22 g, 1.0 mmol), cyclohexanamine (0.23 mL, 2.0 mmol), anhydrous K2CO3 (0.69 g, 5.0 mmol) in 15 mL acetonitrile was refluxed for 24 h. The solution was washed with water and extracted by ethyl acetate for four times. The organic layer was separated and dried over anhydrous MgSO4, then the solvent was evaporated in vacuum and the residue was purified by column chromatography (petroleum ether/ethyl acetate/triethylamine = 90:30:4, v/v/v) to obtain 12g as a yellow oil (0.098 g, 35%). 1H NMR (600 MHz, CDCl3) d ppm: 7.33–7.28 (m, 4H), 7.09–7.07 (m, 1H), 7.01–6.97 (m, 4H), 3.79 (s, 2H), 2.53–2.48 (m, 1H), 1.94–1.92 (m, 2H), 1.77–1.74 (m, 2H), 1.64–1.62 (m, 1H), 1.31–1.11 (m, 5H). 13C NMR (150 MHz, CDCl3) d ppm: 157.4, 155.9, 135.9, 129.6, 129.4, 122.9, 118.9, 118.6, 56.2, 50.4, 33.5, 26.1, 24.9. 25. Ding, R.; He, Y.; Wang, X.; Xu, J.; Chen, Y.; Feng, M.; Qi, C. Molecules 2011, 16, 5665. 26. General procedure for the synthesis of target compounds 8a–g: A mixture of amine 12a–g (1.0 mmol), compound 9 (0.64 g, 1.2 mmol), anhydrous K2CO3 (0.35 g, 2.5 mmol), KI (0.20 mmol) was dissolved in 20 mL acetonitrile and refluxed for 3 h. The solution was washed with water and extracted by ethyl acetate for 4 times. The organic layer was separated and dried over anhydrous MgSO4, then the solvent was evaporated in vacuum and the residue was purified by column chromatography (PE/EtOAc = 10:1–3:1) to afford the target compounds 8a–g. Analytical data for selected compounds 8g: Yield 79%, yellow oil, 1H NMR (600 MHz, CDCl3) d ppm: 7.31 (m, 4H), 7.07 (m, 1H), 6.97 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.49 (dd, J1 = 11.4 Hz, J2 = 17.4 Hz, 1H), 5.78 (d, J = 8.4 Hz, 1H), 5.33 (d, J = 10.8 Hz, 1H), 5.17 (d, J = 17.4 Hz, 1H), 3.74 (m, 2H), 3.46 (s, 2H), 3.34 (m, 1H), 3.19 (s, 2H), 2.57 (s, br, 1H), 2.38 (m, 1H), 2.21 (m, 2H), 2.04 (m, 2H), 1.86–1.76 (m, 6H), 1.66–1.56 (m, 4H), 1.55–1.41 (m, 4H), 1.26–1.13 (m, 6H), 1.12–0.88 (m, 4H), 0.86 (d, 3H), 0.63 (d, 3H); 13C NMR (150 MHz, CDCl3) d ppm: 217.3, 171.4, 157.4, 155.9, 139.2, 135.0, 129.8, 129.6, 122.9, 118.7, 118.6, 117.0, 74.7, 67.7, 59.9, 58.3, 53.9, 51.8, 45.4, 45.2, 43.8, 41.7, 36.8, 36.0, 34.4, 30.4, 30.2, 29.5, 26.8, 26.3, 26.1, 25.9, 24.8, 16.5, 14.9, 11.5; HR MS(ESI): m/z Calcd for C41H56NO5: 642.41530. Found: 642.41441(M+H+). 27. The synthesis of compounds 10: A mixture of piperazine (0.35 g, 4.0 mmol), compound 9 (1.1 g, 2.0 mmol), anhydrous K2CO3 (0.35 g, 2.5 mmol), KI (0.20 mmol) was dissolved in 50 mL acetonitrile and refluxed for 16 h. The solid was filtered and the filtrate was concentrated in vacuum. The residue was dissolved in ethyl acetate, washed with water and extracted by ethyl acetate for four times. The organic layer was separated and dried over anhydrous MgSO4, then the solvent was evaporated in vacuum and the residue was purified by column chromatography (silica gel, chloroform/methanol = 10:1, v/v) to obtain 10 as a white amorphous solid (0.81 g, 90%). 1H NMR (600 MHz, CDCl3) d ppm: 6.44 (m, 1H), 5.72 (dd, 1H), 5.26 (dd, 1H), 5.13 (dd, 1H), 3.30 (d, 1H), 3.10 (m, 1H), 2.98 (m, 1H), 2.91 (m, 4H), 2.56–2.46 (m, 4H), 2.32–2.28 (m, 1H), 2.19–2.12 (m, 2H), 2.05–1.99 (m, 2H), 1.73–1.70 (m, 1H), 1.63–1.57 (m, 2H), 1.52–1.49 (m, 1H), 1.42–1.31 (m, 4H), 1.30–1.29 (m, 1H), 1.25–1.22 (m, 2H), 1.10–1.05 (m, 4H), 0.83 (d, 3H), 0.66 (d, 3H). 13C NMR (150 MHz, CDCl3) d ppm: 216.9, 168.9, 139.0, 128.9, 117.0, 74.4, 68.2, 60.2, 58.0, 53.3, 45.3, 45.2, 44.9, 43.8, 41.6, 36.6, 34.3, 30.3, 26.7, 26.4, 24.7, 16.6, 14.8, 11.4. 28. The synthesis of compounds 13: A solution of 4-acetamidobenzene-1-sulfonyl chloride (70 mg, 0.03 mmol), compound 10 (90 mg, 0.02 mmol), and triethylamine (30 lL, 0.03 mmol) in 20 mL dichloromethane was stirred at room temperature for 12 h. Then the solvent was evaporated in vacuum and the residue was dissolved in ethyl acetate, washed with water and extracted by ethyl acetate for four times. The organic layer was separated and dried over anhydrous MgSO4, then the solvent was evaporated in vacuum and the crude product was purified by column chromatography (silica gel, chloroform/ methanol = 10:1, v/v) to obtain 13 as a white solid (0.13 g, 93%).1H NMR (600 MHz, CDCl3) d ppm: 8.16 (s, 1H), 7.68 (d, J = 9.0 Hz, 2H), 7.62 (d, J = 9.0 Hz, 2H), 6.44 (m, 1H), 5.72 (dd, 1H), 5.26 (dd, 1H), 5.13 (dd, 1H), 3.33 (m, 1H), 3.12 (m, 1H), 3.02 (m, 5H), 2.63–2.56 (m, 4H), 2.29–2.14 (m, 6H), 2.12–2.01 (m, 3H), 1.73 (m, 1H), 1.64–1.61 (m, 2H), 1.59 (m, 1H), 1.54–1.30 (m, 4H), 1.25–1.21 (m, 2H), 1.12–1.06 (m, 4H), 0.83 (m, 3H), 0.65 (m, 3H). 13C NMR (150 MHz, CDCl3) d ppm: 217.1, 168.9, 168.8, 142.4, 138.9, 129.4, 119.1, 117.1, 74.4, 68.5, 60.4, 59.3, 58.1, 51.9, 45.8, 45.3, 44.9, 43.8, 41.6, 36.6, 35.9, 34.4, 30.3, 26.7, 26.3, 24.7, 24.6, 16.6, 14.8, 14.1, 11.4. 29. The the synthesis of compounds 8h: A solution of compound 13 (0.13 g, 0.2 mmol) and 4.0 mL hydrochloric acid in 20 mL acetonitrile was refluxed for 4 h. The precipitation was separated by filtration and dried in vacuum to yield 8h as a white amorphous solid (98 mg, 79%).1H NMR (600 MHz, CDCl3) d ppm: 7.68 (d, J = 9.0 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 6.44 (m, 1H), 5.72 (dd, 1H), 5.26

Y.-J. Dong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1799–1803 (dd, 1H), 5.13 (dd, 1H), 3.33 (m, 1H), 3.15–3.00 (m, 6H), 2.65–2.58 (m, 4H), 2.30–2.02 (m, 8H), 1.74 (m, 1H), 1.64 (m, 2H), 1.53–1.39 (m, 5H), 1.32 (m, 1H), 1.23 (s, 1H), 1.13–1.07 (m, 4H), 0.83 (d, 3H), 0.66 (d, 3H). 13C NMR (150 MHz,

1803

CDCl3) d ppm: 216.9, 168.8, 138.9, 129.9, 129.4, 123.0, 117.2, 113.9, 74.5, 68.5, 59.4, 58.1, 51.9, 45.8, 45.4, 44.9, 43.9, 41.7, 36.6, 35. 9, 34.4, 30.4, 26.7, 26.4, 24.8, 16.6, 14.8, 11.5.