Synthesis and evaluation of novel podophyllotoxin analogs

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Bioorganic & Medicinal Chemistry Letters 22 (2012) 4293–4295

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Synthesis and evaluation of novel podophyllotoxin analogs

Jie Li a,b, Hui Ming Hua b, Yan Bo Tang a, Shipeng Zhang a, Emika Ohkoshi c, Kuo-Hsiung Lee c,d, Zhi yan Xiao a,*

a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China bKey Laboratory of Structure-Based Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China cNatural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA dChinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan

a r t i c l e i n f o

Article history: Received 23 February 2012 Revised 2 May 2012 Accepted 8 May 2012 Available online 15 May 2012 Keywords: Podophyllotoxin Structural modification Cytotoxicity

a b s t r a c t

Because prior studies have shown inconsistency between structure–activity relationships for podophyllotoxin derivatives as topoisomerase II inhibitors and cytotoxic agents, eight novel podophyllotoxin analogs were synthesized to further explore the effects of structural variations on both A and D rings on activity. The new compounds contain a 4,5-dimethoxy substituted A ring and opened D-ring variants and were prepared by appropriate functional and stereochemical operations at the methylenedioxy group, C7, C8, and C80. Four compounds (15, 18, 21 and 22) demonstrated noticeable inhibitory activity against less than A549, 10 lg/mL.

DU145, KB and KBvin tumor cells, and the most active compound 18 showed IC 50

values

The natural lignan podophyllotoxin (1) has been the focus of extensive chemical modification and biological investigation in re- cent decades. In particular, the discovery of the semi-synthetic anticancer drugs etoposide and teniposide has stimulated prolonged research interest in this structural phenotype.

Two alternative molecular mechanisms are generally involved in the antineoplastic activity of podophyllotoxin analogs:

prevent- ing the assembly of tubulin into microtubules and inhibiting the catalytic activity of DNA topoisomerase II (Topo II). As the primary mechanism for therapeutically useful podophyllotoxin analogs, Topo II inhibition has been the major focus for previous structure-activity relationship (SAR) studies, and intact A/D rings are

believed to be essential for Topo II inhibition.1 However, podophyllotoxin derivatives 2 and 3, which lack the trans-lactone D ring, showed significant cytotoxicity against various tumor cell lines.2,3 Furthermore, although the A-ring modified derivatives 4 and 5 were only weak inhibitors of Topo II catalytic activity, they

OH O R NO 2 NO 2 O

5 A O 4

8 7

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D O COOCH 3 HN

HN O HO

N 8' O

H 3 CO OCH 3 OCH 3 HO O O

O N

O H 3

CO 4' OCH

3 OCH 3

2 R = CHO H

3 1 3 R = N 4 5

inhibited KB cell growth at sub-micromolar concentrations.4 These results implied conflicting SAR for Topo II inhibition and cytotoxicity, and supported further SAR exploration on various molecular areas of the structural phenotype, particularly the A and D rings.

Accordingly, we synthesized a series of novel podophyllotoxin analogs with structural variations on both A and D rings (15–

22). These new analogs feature 4,5-dimethoxy substitution as well as structural alterations at C7, C8, and C80. To investigate the

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effects of C80 stereochemistry on cytotoxicity, D-ring variants with opposite chirality at C80 were deliberately incorporated. We report

OH OCH 3 H 3 CO OH OCH

3 CH 2

CH 3

*

Corresponding author. E-mail address: [email protected] (Z.y. Xiao).

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Bioorganic & Medicinal Chemistry Letters

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herein the synthesis,5 structural characterization, and preliminary biological evaluation of these novel podophyllotoxin analogs.

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The key intermediate 8 was synthesized from 40-demethylepip- odophyllotoxin (DMEP, 6) in two steps with a protocol modified from the literature method (Scheme 1).6

As illustrated in Scheme 2, five analogs 15–19 with a-configuration at C80 were prepared from 8. Oxidization with pyridinium dichromate (PDC) and subsequent acid-catalyzed methanolysis gave the D-ring opened variant 10. Stereoselective reduction of the 7-carboxyl in 10 afforded compound 11. The stereoselectivity in the reduction of 10 should be attributed to the asymmetric environment of the Re and Si faces of the 7-carbonyl. The bulky aromatic group and carboxylate substitution preclude hydrogen addition from the rear face (i.e., the Re face), therefore, reduction from the front face (i.e., the Si face) would be dominant. Under Swern conditions, oxidation and dehydration of 11 occurred simultaneously to yield the unsaturated aldehyde 12.2,7 Com- pound 12 was reacted with the appropriate acetophenones in the presence of p-toluenesulfonic acid (p- TsOH) as catalyst to

O NO 2 18 R=

OH 19 R=

15-19

Scheme 2. Reagents and conditions: (a): PDC/CH 2

Cl 2

, rt, 2 h, 51%; (b): H 2

SO 4 /CH 3

OH, reflux, 2 h, 55%; (c): NaBH 4

/CH 3

OH, rt, 0.5 h, 90%; (d): (COCl) 2

, DMSO, Et 3

, À65 to À70 oC, 74%; (e): corresponding acetophenones, p-TsOH, DCM, reflux, 2–5 d.

O OH OH OH

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O O

20 R= O O

O O 21 R=

22 R= 8 13

20-22

R H 3

CO H 3 CO H 3 CO H 3 H 3 H 3 H H 3 CO O CO a CO N b CO N c H

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3 N O H 3 H 3 H 3

Scheme 3. Reagents and conditions: (a): pyrrolidine, reflux, 2 h; (b): (COCl) 2

CO CO OCH

3 OCH 3

CO OCH 3 CO OCH

3 OCH 3

OCH 3 14 H 3 CO OCH

3 OCH 3

, À65 to À70 oC, 66%; (c): corresponding acetophenones, p-TsOH, DCM, reflux, 2–5 d, 20–50%.

, DMSO, Et 3

N, CH 2 Cl 2

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4294 J. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4293–4295

provide analogs 15–19. The trans-D9,10 stereochemistry in compounds 15–19 was confirmed by the measured J 9,10

values (around 15.0 Hz).

Another three analogs 20–22 with b-configuration at C80 were also prepared from 8 (Scheme 3). Aminolysis of 8 under reflux with pyrrolidine as both reactant and solvent provided the dihydroxya- mide 13 in good yield. Swern oxidation of 13 afforded the aldehyde–amide 14, and subsequent aldol condensation of 14 with the corresponding acetophenones produced compounds 20–

22. The C80b-configuration in analogs 20–22 was achieved with a basic reaction milieu and supported by the 1H NMR data. It has been well recognized that C80 epimerization occurs readily under even mildly basic conditions. In fact, C80 epimerization was previously ob- served in the presence of 0.1M piperidine.8 Furthermore, the chemical shifts of H-70 in 20–22 were upfield from those in 15–19 ($4.34 vs $4.56), which is consistent with the previous observation for etoposide and its C80b-isomer.9

Analogs 15–22 were evaluated for their inhibitory activity against the growth of tumor cell lines with an SRB assay. Four OH

O O OH

OH O HO

H 3

CO O

O O a HO

O b H 3 CO O H 3 CO OCH 3 H 3 CO OCH 3 H

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3 CO OCH

3 OH OH

OCH

3 DMEP 6 7

8

Scheme 1. Reagents conditions: (a): (i) BCl 3

/CH 2 Cl 2

, 0 oC, 6 h; (ii) acetone–water–CaCO 3

, reflux, 3 h; (b): CH 3

I, K 2 CO 3 , Et 4

NF, acetone, rt, 24 h; (yield 82% for two steps).

OH O

O H

3 CO H 3

CO O

O H 3

O H

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3 O H 3 CO OCH

3 OCH 3

OCH 3 O H 3 12 H CO a

CO b c d 8 e

CO OCH

3 OCH 3

H 3 CO R 15 R=

H 3 H 3 OCH 3 O O OH OH

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OH H 3

CO H 3 CO H 3 CO H 3 CO OCH 3 H 3 CO OCH 3 H 3

CO O O

H 3 CO OCH 3 H 3 CO OCH 3 H 3 CO OCH

3 OCH

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3 OCH 3 OCH

3 9 10 11

CO 16 R=

CO OCH 3 17 R=

OCH 3 N, CH 2 Cl 2

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corresponding Table 1

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target molecules. Compound 15: Yield:

16.9%; mp: 100À102 °C; Inhibitory activity of selected analogs against A549, DU145, KB and KBvin tumor cell 1⁄2a lines

Compound IC 50

1H 20 D

NMR +322.4 (300 (c, MHz, 0.55, CDCl CHCl

3

3 ): ); d HR-ESI-MS: 8.31 (d, 2H, m/z 590.2011 [M+H]+ (calcd 590.2021); J = 8.4 Hz, –ArH), 8.08 (d, 2H, J = 8.4 Hz, (lg/mL)

–ArH), 7.68 (d, 1H, J = 15.6 Hz, 9-H), 7.10 (d, 1H, J = 15.6 Hz, 10-H), 7.07 (s, 1H, 7- H), 6.85 (s, 1H, 6-H), 6.62 (s, 1H, 3-H), 6.59 (s, 2H, 20, 60-H), 4.57 (d, 1H, J = 6.9 Hz,

A549 DU145 KB KBvin 70-H), 3.73À3.93 (16H, 5ÂOCH 3

15 16.8 ± 1.32 20.3 ± 0.61 16.0 ± 1.66 23.0 ± 0.35 18 6.79 ± 0.76 5.84 ± 0.91 5.90 ± 1.13 5.17 ± 0.96 21 12.0 ± 0.92 12.8 ± 2.15 12.28 ± 1.84 12.29 ± 2.36 22 24.6 ± 2.02 15.46 ± 1.19 16.6 ± 3.87 16.9 ± 0.71 GL-331* 0.113 ± 0.014 0.800 ± 0.056 0.819 ± 0.162 2.10 ± 0.378

* GL-331 is a podophyllotoxin analog that previously reached clinical trials.1

, 80-H), 3.50 Yield: 627.2928 55.1%; [M+H]+ mp: (calcd 107À109 627.2952); °C; 1⁄2a

20 D

1H +265.2 NMR (s, 3H, –COOCH

3 ). Compound (c, 0.7, CHCl 3 ); HR-ESI-MS: (300 MHz, CDCl 3

): d 7.90 (d, 16: m/z 2H, J = 8.1 Hz, ÀArH), 7.62 (d, 1H, J = 15.6 Hz, 9-H), 7.29 (d, 2H, J = 8.1 Hz, ÀArH), 7.17 (d, 1H, J = 15.3 Hz, 10-H), 6.99 (s, 1H, 7-H), 6.83 (s, 1H, 6-H), 6.60 (s, 3H, 20, 60-H, 3-H), 4.56 (d, 1H, J = 7.5 Hz, 70-H), 3.72À3.93 (16H, 5ÂOCH 3

, 80-H), 3.49 (s, 3H, –COOCH 3 ), Yield: 9.2%; mp: 95À97 oC; 1⁄2a

) 5

–). Compound 17: [M+H]+ (calcd 2.56 601.2796); (m, 1H, –CH–), 1H 20 D

NMR +260 1.26À1.86 (c, (300MHz, 0.5, CHCl (m, 10H, acetone-d

3

); HR-ESI-MS: –(CH

(16)

2 6

m/z 601.2780 ): d 7.91 (d, 2H, J = 8.1 Hz, –ArH), 7.60 (d, 1H, J = 15.3 Hz, 9-H), 7.33 (d, 2H, J = 8.1 Hz, –ArH), 7.24 (d, 1H, J = 15.3 Hz, 10-H), 7.16 (s, 1H, 7-H), 7.02 (s, 1H, 6-H), 6.76 (s, 2H, 20, 60-H), 6.67 (s, 1H, 3-H), 4.56 (d, 1H, J = 7.1 Hz, 70-H), 3.99 (d, 1H, J = 7.1 Hz, 80-H), 3.66À3.85 (15H, 5ÂOCH 3

), 3.32 (s, 3H, –COOCH 3

), 2.56 (d, 1H, J = 7.1 Hz, –CH–), 1.91 (m, 1H, –CH–), 0.99 (s, 41.7%; [M+H]+ mp:

(calcd. 126À129 561.2119); °C; 1⁄2a 1H 20 D 3H, CH

3

), 0.96 (s, 3H, CH 3

). Compound 18: Yield: +260.9 (c, 0.7, CHCl 3

); HR-ESI-MS: m/z 561.2132 NMR (300 MHz, CDCl 3

): d 7.93 (d, 2H, J = 8.4 Hz, – ArH), 7.62 (d, 1H, J = 15.3 Hz, 9-H), 7.16 (d, 1H, J = 15.6 Hz, 10-H), 6.99 (s, 1H, 7- H), 6.88 (d, 2H, J = 9.0 Hz, –ArH), 6.83 (s, 1H, 6-H), 6.60 (s, 3H, 20, 60-H, 3-H), 4.56 (d, COOCH CHCl

J=7.2 3 ); 3

HR-ESI-MS: ). Hz, Compound 1H, 70-H), m/z 19: 3.87 621.2471 Yield: (s, 1H, 13.7%; 80-H), [M+H]+ 3.72À3.92 mp: (calcd 124À126 621.2483); (15H, °C; 5ÂOCH

1⁄2a

1H 20 D

NMR 3 +311.1 ), 3.49 (300 (s, (c, MHz, 3H, 0.3, - CDCl

3

): d 8.05 (d, 2H, J = 8.4 Hz, –ArH), 7.45À7.69 (m, 5H, –ArH), 7.63 (d, 2H, J = 8.7 Hz, –ArH), 7.47 (d, 1H, J = 14.4 Hz, 9- H), 7.22 (d, 1H, J = 15.0 Hz, 10-H), 7.03 (s, 1H, 7-H), 6.85 (s, 1H, 6-H), 6.61 (s, 3H, 20, 60-H, 3-H), 4.57 (d, 1H, J = 6.9 Hz, Compound HR-ESI-MS: 70-H), 3.73À3.93 (16H, 5ÂOCH

3 , 20: Yield: 10.4%; mp: 110À113 °C m/z 628.2564 [M+H]+ (calcd. 649.2541); 80-H), ; [a]

1H D

20 3.51 NMR -244.4 (s, (300 3H, (c, MHz, 0.6, -COOCH CHCl CDCl 3

3 3 ); ): ).

d 7.55 (d, 1H, J = 15.6 Hz, 9-H), 7.43 (dd, 1H, J = 8.4; 1.5 Hz, –ArH), 7.36 (d, 1H, J = 1.5 Hz, –ArH), 7.08 (s, 1H, 7-H), 6.82 (d, 1H, J = 9.0 Hz, –ArH), 6.81 (s, 1H, 6-H), 6.74 (d, 1H, J = 15.6 Hz, 10-H), 6.55 (s, 1H, 3-H), 6.46 (s, 2H, 20, 60-H), 6.04 (s, 2H, -OCH

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2

O-), 4.34 (d, 1H, J = 6.3 Hz, 70-H), 3.96 (d, 1H, J = 6.3 Hz, 80-H), 3.78À3.91 (15H, 5ÂOCH 3

–). Compound 21: ), 3.32À3.56 Yield: 10.0%; (m, mp: 4H, 113À115 –N(CH

°C; 2 ) 2

–); 1⁄2a

1.87À2.04 (m, 4H, –(CH 2

)

2 ESI-MS: m/z 666.3406 [M+H]+ (calcd 666.3425); 20 D 1H À488.9 NMR (300 (c, 0.3, MHz, CHCl CDCl 3

); 3

HR- ): d 7.77 (d, 2H, J = 8.4 Hz, –ArH), 7.56 (d, 1H, J = 15.9 Hz, 9-H), 7.27 (d, 2H, J = 8.1 Hz, –ArH), 7.08 (s, 1H, 7-H), 6.81 (s, 1H, 6-H), 6.78 (d, 1H, J = 16.2 Hz, 10-H), 6.56 (s, 1H, 3-H), 6.46 (s, 2H, 20, 60-H), 4.33 (d, 1H, J = 6.0 Hz, 70-H), 3.97 (d, 1H, J = 6.0 Hz, 80-H), 3.78À3.91 (15H, 5ÂOCH 3

–), 2.56 (m, 1H, – CH(CH 2

), 3.31À3.59 (m, 4H, –N(CH 2

)

2 –), 1.33À1.41 (m, 6H, – (CH 2

) 2

–), 1.79À1.99 (m, 8H, –(CH 2

) 2 –; )

2 ) 3 –). Compound 22: Yield: CHCl

3 ); HR-ESI-MS: m/z 660.2937 21.2%; mp:

[M+H]+ compounds (15, 18, 21, and 22) demonstrated noticeable inhibitory activity against A549, DU145, KB and KBvin tumor cells, and the 10 lg/mL most (Table active 1).

compound 18 exhibited IC 50

values less than

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In summary, a series of novel podophyllotoxin analogs featuring 4,5-dimethoxy substitution and an opened D ring were synthesized and evaluated for cytotoxic activity. In contrast to previous SAR deduced from Topo II inhibition, which requires intact A and D rings for retention of activity, analogs with modified A and D rings, such as 18, exhibited evident in vitro anticancer activity.

Acknowledgments

This investigation was supported by the National S&T Major Project (No. 2009ZX09301-003) and also in part by the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH100-TD-C-111-005).

References and notes

1. Cragg, G. M.; Kingston, D. G. I.; Newman, D. J. Anticancer Agents from Natural

Products; CRC Press Taylor & Francis Group: London UK, 2005. 1st ed., pp. 71. 2. Gordaliza, M.; Castro, M. A.; Miguel del Corral, J. M.; López-Vázquez, M. L.;

–CH(CH

2 115À117 (calcd 660.2956); °C; 1⁄2a

1H 20 D

À358.8 (c, 0.55, NMR (300 MHz, García, P. A.; Feliciano, A. S.; García-Grávalos, M. D.; Broughton, H. Tetrahedron CDCl

3

): d 7.92 (d, 2H, J = 8.4 Hz, –ArH), 7.66 (d, 2H, J

= 8.1 Hz, –ArH), 7.42À7.64 1997, 53, 15743.

(m, 5H, –ArH), 7.47 (d, 1H, J = 14.4 Hz, 9-H), 7.11 (s, 1H, 7-H), 6.82 (d, 1H, 3. Castro, M. A.; MigueldelCorral, J. M.; Gordaliza, M.; García, P.; Gómez-Zurita, M.

J = 15.9 Hz, 10-H), 6.82 (s, 1H, 6-H), 6.56 (s, 1H, 3- H), 6.47 (s, 2H, 20, 60-H), 4.35 A.; García-Grávalos, M. D.; de la Iglesia-Vincente, J.; Gajate, C.; An, F.; Mollinedo,

(d, 1H, J = 6.0 Hz, 70-H), 3.99 (d, 1H, J = 6.3 Hz, 80-H), 3.79À3.91 (15H, 5ÂOCH 3

) , F.; Feliciano, A. S. J. Med. Chem. 2004, 47, 1214.

3.34À3.60 (m, 4H, –N(CH 2

) 2

–); 1.84À2.00 (m, 4H, –(CH 2

) 2

–).

4. Cho, S. J.; Kashiwada, Y.; Bastow, K. F.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1996,

6. Wang, Z. Q.; Hu, H.; Chen, H. X.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1992, 35, 39, 1396.

871. 5. General Preparation of Compounds 15À22:

To a solution of 12 or 14 (0.25 mmol)

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7. Mancuso, A. J.; Swern, D. Synthesis 1981, 165. in 15mL of anhydrous CH

2 Cl 2

were added p-TsOH (0.15mmol) and the

8. Gensler, W. J.; Gatsons, C. D. J. Org. Chem. 1966, 31, 3224. corresponding acetophenones (0.5 mmol). The reaction mixture was stirred at

9. Aso, Y.; Hayashi, Y.; Yoshioka, S.; Takeda, Y.; Kita, Y.; Nishimura, Y.; Arata, Y. room temperature for 2 to 5days and then washed with 5% NaHCO

3 and

Chem. Pharm. Bull. 1989, 37, 422. saturated aqueous NaCl. The organic layer was dried over Na

2 SO 4

and concentrated. The residue was chromatographed on silica gel and afforded the

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