Accepted Manuscript Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification Shreyans K. Jain, Samdarshi Meena, Ajai P. Gupta, Manoj Kushwaha, R. Uma Shaanker, Sundeep Jaglan, Sandip B. Bharate, Ram A. Vishwakarma PII: DOI: Reference:

S0960-894X(14)00488-0 http://dx.doi.org/10.1016/j.bmcl.2014.05.001 BMCL 21614

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

1 March 2014 26 April 2014 1 May 2014

Please cite this article as: Jain, S.K., Meena, S., Gupta, A.P., Kushwaha, M., Uma Shaanker, R., Jaglan, S., Bharate, S.B., Vishwakarma, R.A., Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.001

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Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification

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Shreyans K. Jain, Samdarshi Meena, Ajai P. Gupta, Manoj Kushwaha, R. Uma Shaanker, Sundeep Jaglan, Sandip B. Bharate and Ram A. Vishwakarma OH O OMe O

O

N

HO HO

N

N

N O

O

OH O Camptothecin

O

OH O 9-Methoxy camptothecin

Fonts or abstract dimensions should not be changed or altered.

N CH3 Rohitukine

CH3

1

Bioorganic Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification Shreyans K. Jain,a,b Samdarshi Meena,a,b Ajai P. Gupta,c Manoj Kushwaha,c R. Uma Shaanker,d Sundeep Jaglan,e Sandip B. Bharate,*,b,f and Ram A. Vishwakarma*,a,b,f a

Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India Academy of Scientific & Innovative Research (AcSIR), Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India c Quality Control and Quality Assurance Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India d Department of Crop Physiology and School of Ecology and Conservation, University of Agricultural Sciences, GKVK, Bangalore 560 065, India e Plant Biotechnology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India f Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed showed presence of CPT (1) and its 9methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. T his is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free procotol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D.binectariferum.

Keywords: Camptothecin, 9-methoxy camptothecin, Dysoxylum binectariferum, anticancer, rohitukine, quantification by LCMS

. 2014 Elsevier Ltd. All rights reserved.

Camptothecin (CPT, 1) is a quinoline alkaloid first isolated from Camptotheca acuminata (Nyssaceae)1 as a potent anticancer natural product. Later, it was isolated from several other plants belonging to different families such as Icacinaceae (Nothapodytes foetida,2 N. nimmoniana,3 Pyrenacantha klaineana4 and Merrilliodendron megacarpum),5 Rubiaceae (Ophiorrhiza pumila, O. pumila, O. filistipula), 6, 7 Apocynaceae (Ervatamia heyneana)8 and Gelsemiaceae (Mostuea brunonis).9 All these plants belongs to the unrelated order of angiosperms but they might have evolutionary relationship.10, 11 The two clinically used anticancer drugs topotecan and irinotecan are semi-synthetic analogs of CPT. More than million dollar worldwide market of topotecan and irinotecan has pushed the production of CPT as a raw material.12 In turn, this has influenced the development of CPT-containing plants as “Cash Crops” mainly in Southeastern Asia.13 Due to increased demand, there is an increased burden on plant sources and tissue culture methods for commercial production of CPT. Consequently, there has been an unprecedented pressure on the natural populations of these plants, which is leading to decline in their natural population. Thus, identification of an alternative plant source for production of CPT is highly demanding. -----*Corresponding author E-mail: [email protected] (RAV); [email protected] (SBB) Fax: +91-191-2569333; Tel: +91-191-2569111 IIIM Communication number. IIIM/1603/2013

Worldwide, there exists around eighty species in the genus Dysoxylum (Family: Meliaceae), growing widely across the regions of South and South-East Asia, the western Pacific ocean, Australia, and tropics between the Pacific and Indian oceans. In India, it is distributed mainly in South India and western Ghats (Elamalai, Anaimalai in South Sahyadri).14 Maximum species are tree in habit and have commercial values, many of them are widely used in timber industries for building construction, boxes, turnery and ply-board. The D. binectariferum (Roxb.) Hook is being cultivated in South India as a sacred tree for use as an alternative for ‘Sandal’. The decoction of wood has been reported to be useful in arthritis, anorexia, cardiac debility, expelling intestinal worms, inflammation, leprosy and rheumatism.15 Rohitukine (3) is a chromone alkaloid, which was first isolated from the leaves and stem of Amoora rohituka (Roxb.) and then from the stem barks of D. binectariferum,16 both plants belonging to Meliaceae family.17 Recently, rohitukine has also been isolated from endophytic fungi of these plants.18 Rohitukine (3) led to the discovery of two clinical candidates flavopiridol (Alvocidib) and P-276-00 for treatment of cancer; the former has also received ‘orphan-drug’ status for treatment of chronic myeloid leukemia.19, 20 As a part of our phytochemical,

2

Bioorganic Medicinal Chemistry Letters

synthetic and medicinal chemistry efforts21-24 on D. binectariferum, we have isolated CPT (1) and 9-methoxy CPT (2) along with chromone alkaloid rohitukine (3) through bioassay-guided isolation from stem bark of this plant.16, 17 This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient protocol for enrichment of CPT in crude extract and isolation of CPT in large amount has also been established. Additionally, the LCMS (MRM)-based method was established for quantification of CPT in the crude extract. The chemical structures of alkaloids 1-3 are shown in Figure 1. OH O

12

13

N 1

4

O

N

16a

14 15 20 19 18

1

O

HO HO

N N O

21

OH O 2

O

8 2' 3'

OH O

3

7

O

16 17

3

4

5

OMe

6

8

1'

1

CH3

5'

N 4' CH3 3

Figure 1. Chemical structures of isolated alkaloids 1-3

CPT content. For this, the obvious aim was to remove rohitukine from the extract as much as possible. The preliminary phytochemical analysis of EtOH extract using TLC and HPLC showed rohitukine as single major component, whereas other components were not detected because of higher amount of rohitukine. The LCMS analysis in SIM mode (specific-ion mode) detected CPT [m/z 348 (M-H)-] but not in TIC mode (total-ionic current) because of high content of rohitukine. The EtOH extract was then suspended in minimum cold MeOH and subjected to repeated precipitation of rohitukine by adding cold EtOAc which resulted in isolation of rohitukine (3, 2.1 g, approx 5.2% in dry plant material). The filtrate was concentrated, resuspended in water and extracted with EtOAc. The LCMS (MRM)-based quantification of EtOAc fraction indicated 209 µg of CPT per mg of this extract (21% enrichment). x10 4

+ES I TIC MRM (**-> **) C1000 0 TMS.d x10 4 +ESITIC MRM(** ->**) C10000TMS.d

7 6

* 6 .6 0

R ohitukine

0.9

(a)

R ohitu kine

6.51 * 6.51

1

8

0.8 0.7

* 7 .9 4

0.6

Camptothecin

0.5

5 4

0.4 0.3 0.2

3 2 1

The ethanolic extract of Dysoxylum binectariferum bark was fractionated on HP20 resin using methanol: water (10:90 to 100: 0). All collected individual fractions were pooled into five fractions. The well-known major component (rohitukine) of this plant was found in fraction 2 (eluted at 30-40% methanol in water). All five fractions were screened for cytotoxicity in human leukemia HL-60 cell line at 10 µg/mL, wherein 50, 55, 70, 95 and 10% growth inhibition was observed, respectively. The IC50 value of most potent fraction (fraction 4) was found to be 1.5 µg/mL (The flow chart of isolation is depicted in supporting information). This most active fraction 4 showed Dragendorffpositive characteristic fluorescent bands at higher wavelength (366 nm). After repeated purifications on alumina column, two additional alkaloids 1 and 2 were isolated. These compounds were characterized as CPT and 9-methoxy CPT by spectroscopic data including UV, 1H, 13C, 2D NMR, MS and IR data and further their comparison with literature values.1, 25 The CPT (1) was further confirmed via comparison of TLC and HPLC profile with commercially available reference standard.26 Since the D. binectariferum is a rich source of rohitukine alkaloid (5-7%),16 the HPLC chromatogram of the EtOH extract showed presence of only rohitukine (inset of Figure 2a). Therefore, the isolation of CPT from this extract was found to be difficult. Several approaches were investigated to isolate pure CPT (in large amount) including routine column chromatography on silica gel and basic alumina. The silica gel protocol was not suitable however the basic alumina protocol was somewhat successful. Although, the alumina protocol was successful, but it was time-consuming and also required large amount of organic solvent. Next, the utility of HP-20 resin was investigated for removal of rohitukine from the extract. Dianion HP-20 resin was used and the volume of resin used was 15% v/v of the material to be loaded. The extract was suspended in deionised water and was loaded on HP-20 column. The column was eluted with increasing amounts of MeOH in water. Rohitukine was eluted at 35% MeOH in water whereas the CPT was eluted at 65% MeOH in water. Highly pure CPT was obtained (~ 1 g) by repeating the HP-20 protocol. This protocol has several advantages such as it is environmentally-friendly, work process involves semi-aqueous medium, it is faster and reproducible at large scale; and it do not involve any heat or acid-base treatment. Next, our aim was to devise a practical and economical chromatography-free protocol for enrichment of crude extract for

0.1

9-Meth oxy camptothecin

0

* 8 .3 6

5.6 5.7 5.8 5.9 6 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 Counts vs. AcquisitionTime (min)

6

7

9

8

Counts vs. Acquis ition Time (min)

1

2

3

4

5 6 7 8 9 C o u n t s v s . A c q u is it io n Tim e ( m in )

Scan:1 Scans) Frag=135.0V.d +ESI Sca n:1 (6.5-6(6.5-6.7 .7 min, 10 min, Scans)10 Frag =135.0V C AMPTOTHICIN25PPM0001.d x10 5 +ESI 2.25

[M+H] Rohitukine mw = 305

1.25 1 0.75

(c)

[M+H]

0.8

1.5

12

349.0

1

2 1.75

11

min, Scans) Frag=135.0V.d +ESI ScScan:1 an:1(7.9-8.(7.9-8.1 1 min, 13Sc ans) F13 rag=1 35.0V CAM PTOTHICIN25PPM0001.d x10 5 +ESI

(b)

306.0

10

Camptothecin mw = 348

0.6 0.4

0.5 0.25 0

+E S can:1 (8.3-8. 6 Scan min,s)12 Scans) Frag= 135.0V .d SI SI Scan:1 (8.3-8.6 min, 12 Frag=1 35.0V CAM PTO THICIN25 PPM0001.d x10 3 +E 2.5

379.0

[M+H]

(d)

9-Methoxy camptothecin mw = 378

1.5 1.25

50 100 100 150 200 200 250 300 300 350 400 400 450 500 500 550 6 600 00 650 700 700 750800 800 850 Countsvs. vs.M ass-to-Charge (m/z) (m /z ) Counts Mass-to-Charge x10 3

(e)

Rohi tukine

4 Camptothecin

3 2

1 0.75 0.5 0.25

719.0

305.0

0

50 100 100 150 200 200 250 300 300 350 40 400 0 450 5 500 00 550600 600 650 700 700 750 800 800 850 Co unts Counts vs. vs.Ma Mass-to-C ss-to-Charge harge(m/z)(m/z)

2.25 2 1.75

[Dimer + Na]

0.2

245.0

1 187.0

0

0 50 100 100 150200 200 250 300 300 350 400 400 450 500 500 550 600 600 650 700 700 750 800 800 850 Count vs. M ass-ts-to-Charg o-Charge(m/z) Cou ntssvs. Mas e (m/ z)

1

2

3

4

5 6 7 8 9 10 Counts Cou nts vs.vsAcqu . Ac qu isitisioition n Tim Tim e(m e (in m) in )

11

12

Figure 2. LCMS analysis of D. binectariferum crude extract for identification of CPT (1) and rohitukine (3). (a) LCMS chromatogram of CPT-enriched EtOAc fraction obtained after repeated precipitation of rohitukine from EtOH extract (Inset: LCMS chromatogram of initial EtOH extract of D. binectariferum bark); (b) MS spectrum of peak eluted at t R 6.6 min; (c) MS spectrum of peak eluted at t R 7.9 min; (d) MS spectrum of peak eluted at tR 8.36 min; (e) LCMS chromatogram showing spiking of rohitukine and CPT in crude extract.

For quantification, 2.1 mg of EtOAc fraction was used. The LCMS analysis of EtOAc fraction in TIC mode (Figure 2a) showed three major peaks with mass m/z 305 (at tR 6.6 min), m/z 349 (at tR 7.9 min) and m/z 379.6 (at tR 8.4 min) as depicted in Figure 2b-d. After running a LCMS chromatogram with pure reference standards and performing a spiking experiment (Figure 2e), these peaks were identified as rohitukine (3), CPT (1) and 9methoxy CPT (2), respectively. The protonated molecular ions [M+H]+ of rohitukine (3) and CPT (1) which were predominantly generated were chosen for multiple reaction monitoring (MRM) analysis. The fragmentor and CE (collision energy, eV) were optimized in order to obtain the maximum sensitivity of analysis. Both negative and positive ion modes were conducted, which showed that the positive ion mode to be more sensitive for both compounds. Based on these results, the precursor/product ion pair of transition mass m/z 306/245 and 306/70 were taken as quantifier for rohitukine and pairs m/z 349/305.1 and 349/249 were taken as qualifier for CPT for the MRM scan (Section S14 of Supporting information). The peak areas obtained from the MRM of both the standards were utilized for quantification. The

3 sample solution of processed extract was injected directly, separated and detected under the optimized conditions. During MRM method development, fragmentor voltage for maximum abundance of rohitukine (3) and CPT (1) and collision energy for the generation of product ion were optimized. Maximum resolution was obtained at fragmentor voltage of 115 and 155 V and collision energy 20 and 25 V for rohitukine and CPT, respectively. The identification of 1 and 3 was done on the basis of retention time and comparing the presence of peak in the MRM in sample and standards. The calibration equation of rohitukine and CPT were obtained by plotting LC-MS peak area (y) versus the concentration (x, mg/ml) of calibrators as y = 14.079943x + 253.704408 (r 2 = 0.997) and y = 14.552128x + 142.943875 (r2 = 0.996), respectively. The equation showed very good linearity over the range 25-2500 ng/ml. The CPT content of EtOAc fraction was found to be 209 µg/mg of the EtOAc extract. The total CPT content in the dry plant material was found to be 0.105% (41.8 mg in 40 g dry plant material). Next, we also investigated leaves, seed, and seed coats of D. binectariferum for the presence of CPT alkaloids. The EtOH extracts of these parts were processed in similar fashion and were analyzed by LCMS (MRM) method. The LCMS results indicated that, rohitukine was the major component present in these parts; whereas they do not contain CPT alkaloids. The content of CPT in different sources is summarized in Table 1. The highest concentration of CPT was reported in C. acuminata young leaves (0.4 to 0.5 % of dry powder)3 and N. nimmoniana bark (0.3 to 0.7 % of dry powder).3 The CPT content in D. binectariferum bark (0.11% ) was found to be similar to Ervatamia heyneana bark (0.13%), 8 and Ophiorrhiza pumila roots (0.10%).5 In general, the young leaves comprises the higher contents of secondary metabolites, thus bioprospecting of various parts of D. binectariferum at different life stages and their clonal multiplication via developing appropriate in vitro production systems will further lead to identification of optimized highyielding CPT source.

1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

Reference

15.

0.4-0.5 0.18-0.2 0.39-0.55 0.2-0.40 0.13

27 27 28 28 8

16.

0.05 0.10 0.0012 0.08 0.004 0.04 0.3-0.7 0.08 0.11

HPLC HPLC HPLC HPLC HPLC LCMS HPLC HPLC LCMS

5 5 7 9 4 6 3 3 This paper

Plant Part

% of CPT

C. acuminata

Leaves Bark Young leaves Leaves Wood and Bark Leaves Hairy root Whole Plant Whole Plant Stem Wood Bark Leaves Bark

D. binectariferum

References and notes

Method of analysis HPLC HPLC HPLC HPLC HPLC

Plant source

M. megacarpum O. pumila O. mungos M. brunonis P. klaineana N. foetida N. nimmoniana

Acknowledgements. The authors gratefully acknowledge D. Singh and S. Aravinda for analytical support. S.K.J. is a Senior Research Fellow receiving financial support from CSIR, New Delhi. This study was supported by grant BT/PR/10546/NDB/172/2008 (awarded to R.A.V. and R.U.S.) from Deparment of Biotechnology, Govt. of India.

14.

Table 1. The content of CPT in different sources

C. lowreyana C. yunnanensis E. heyneana

1

Supplementary data. Supporting information available. H, C, DEPT 135, COSY, HSQC, HMBC NMR and MS data of compound 1. This material is available free of charge via the internet at http://sciencedirect.com. 13

In summary, through bioassay-guided isolation, we have identified D. binectariferum bark as a new alternative plant source for isolation of CPT. The LCMS-based quantification method was established, which indicated the 0.11% of CPT content in the dry barks. The discovery of CPT from D. binectariferum bark will certainly create global interest in phytochemical investigations of other Dysoxylum species and also other plants from Meliaceae family. The cultivation of this plant as a new crop for commercial production of CPT will relieve the pressure on natural populations of other CPT sources.

17. 18. 19. 20. 21. 22.

23.

24.

25. 26.

Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888. Aiyama, R.; Nagai, H.; Nokata, K.; Shinohara, C.; Sawada, S. Phytochemistry 1988, 27, 3663. Ramesha, B. T.; Amna, T.; Ravikanth, G.; Gunaga, R. P.; Vasudeva; Ganeshaiah, K. N.; Shaanker, R. U.; Khajuria, R. K.; Puri, S. C.; Qazi, G. N. J. Chromat. Sci 2008, 46, 362. Zhou, B.; Hoch, J.; Johnson, R.; Mattern, M.; Eng, W.; Ma, J.; Hecht, S.; Newman, D.; Kingston, D. J. Nat. Prod. 2000, 63, 1273. Arisawa, M.; Gunasekera, S. P.; Cordell, G. A.; Farnsworth, N. R. Planta Med. 1981, 43, 404. Yamazaki, Y.; Urano, A.; Sudo, H.; Kitajima, M.; Takayama, H.; Yamazaki, M.; Aimi, N.; Saito, K. Phytochemistry 2003, 62, 461. Tafur, S.; Nelson, J. D.; DeLong, D. C.; Svoboda, G. H. Lloydia 1976, 39, 261. Gunasekera, S. P.; Badawi, M. M.; Cordell, G. A.; Farnsworth, N. R.; Chitnis, M. J. Nat. Prod 1979, 42, 475. Dai, J. R.; Hallock, Y. F.; Cardellina II, J. H.; Boyd, M. R. J. Nat. Prod. 1999, 62, 1427. Lorence, A.; Nessler, C. Phytochemistry 2004, 65, 2735. Hsiao, H.-Y.; Cheng, T.-J.; Yang, G.-M.; Huang, I. J.; Chen, R. Phytochem. Anal. 2008, 19, 136. Raskin, I.; Ribnicky, D. M.; Komarnytsky, S.; Ilic, N.; Poulev, A.; Borisjuk, N.; Brinker, A.; Moreno, D. A.; Ripoll, C.; Yakoby, N.; O'Neal, J. M.; Cornwell, T.; Pastor, I.; Fridlender, B. Trends Biotechnol. 2002, 20, 522. Wu, S. F.; Hsieh, P. W.; Wu, C. C.; Lee, C. L.; Chen, S. L.; Lu, C. Y.; Wu, T. S.; Chang, F. R.; Wu, Y. C. Molecules 2008, 13, 1361. Sasidharan, N., Ed. Biodiversity documentation for Kerala- Flowering Plants; Kerala Forest Research Inst, Peechi, Kerala, India, 2004. Bodare, S.; Tsuda, Y.; Ravikanth, G.; Uma Shaanker, R.; Lascoux, M. Ecol. Evol. 2013, 3, 3233. Mohanakumara, P.; Sreejayan, N.; Priti, V.; Ramesha, B. T.; Ravikanth, G.; Ganeshaiah, K. N.; Vasudeva, R.; Mohan, J.; Santhoshkumar, T. R.; Mishra, P. D.; Ram, V.; Shaanker, R. U. Fitoterapia 2009, 81, 145. Yang, D. H.; Cai, S. Q.; Zhao, Y. Y.; Liang, H. J. Asian Nat. Prod. Res. 2004, 6, 233. Kumara, P. M.; Soujanya, K. N.; Ravikanth, G.; Vasudeva, R.; Ganeshaiah, K. N.; Shaanker, R. U. Phytomedicine 2013, 21, 541. Jain, S. K.; Bharate, S. B.; Vishwakarma, R. A. Mini-Rev. Med. Chem. 2012, 12, 632. Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009, 109, 3012. Jain, S. K.; Meena, S.; Singh, B.; Bharate, J. B.; Joshi, P.; Singh, V. P.; Vishwakarma, R. A.; Bharate, S. B. RSC Adv. 2012, 2, 8929. Vishwakarma, R. A.; Bharate, S. B.; Bhushan, S.; Jain, S. K.; Meena, S.; Guru, S. K.; Pathania, A. S.; Kumar, S. Indian Patent Application. 1142DEL2013 (Prov. Date: 17th April 2013), 2013. Vishwakarma, R. A.; Jain, S. K.; Bharate, S. B.; Dar, A. H.; Khajuria, A.; Meena, S.; Bhola, S. K.; Qazi, A. K.; Hussain, A.; Sidiq, T.; Uma Shaanker, R.; Ravikanth, G.; Vasudeva, R.; Patel, M. K.; Ganeshaiah, K. N. Indian Patent Application. 1077DEL2013 (Prov. Date: 10th April 2013). 2013. Jain, S. K.; Meena, S.; Qazi, A. K.; Hussain, A.; Bhola, S. K.; Kshirsagar, R.; Pari, K.; Khajuria, A.; Hamid, A.; Uma Shaanker, R.; Bharate, S. B.; Vishwakarma, R. A. Tetrahedron Lett. 2013, 54, 7140. Ezell, E.; Smith, L. J. Nat. Prod. 1991, 54, 1645. Procedure for bioassay-guided isolation. The 100 g of powdered D. binectariferum bark was vigorously extracted with ethanol (50 mL x 5 times, 50 °C, each cycle for 5 h sonication) which produced 32 g crude extract. Extract was suspended in deionized water (500 mL) and loaded over HP20 resin (30 mL) to adsorb organic components on

4

Bioorganic Medicinal Chemistry Letters resin. HP20 column was eluted with increasing concentrations of methanol in water. Fractions were pooled on the basis of TLC. Five pooled fractions were obtained, which were submitted to cytotoxicity on HL-60 cell line (Schematic shown in SI). Fraction 4 was found to be most potent (IC50 = 1.5 µg/mL). Fractions were then loaded over the basic alumina column and eluted by chloroform with increasing the proportion of MeOH. Approximately, 4 g of pure rohitukine (3) was obtained from fractions 1 and 2. Fraction 4 showed characteristic fluorescent band at 366 nm which was Dragendorff-positive. The repeated purification of this fraction on alumina column led to isolation of CPT (1, 70 mg) and 9-methoxy-CPT (2, 15 mg). The isolated compounds were characterized by comparison of spectroscopic data with literature values. Camptothecin (1). light yellow solid; mp 265-269 °C; [α]25D +29.8 (CHCl 3/EtOH, 3:2) (lit. value +31.8 in CHCl 3/MeOH, 4:1); IR (KBr) υmax 3448, 2929, 2072, 1741, 1653, 1463, 1351, 1198, 1157, 1042 cm-1; 1 H NMR (500 MHz, pyridine-d5): δ 8.46 (1H, d, J = 8.4, H-12), 8.33 (1H, s, H-7), 8.07 (1H, s, H-14), 7.97 (1H, dd, J = 4.0, 8.0 Hz, H-9), 7.83 (1H, dd, J = 4.0, 8.4 Hz, H-11), 7.63 (1H, d, J = 8.0 Hz, H-10), 5.92 (1H, d, J = 16 Hz, H-17b), 5.60 (1H, d, J = 16 Hz, H17a), 5.35 (1H, d, J = 6.0 Hz, H-5), 2.13 (2H, q, J = 7.4 Hz, H-19), 0.90 (3H, t, J = 7.4 Hz, H-18); 13 C NMR (100 MHz, Pyridine-d5): δ 173.8, 157.7, 153.4, 151.3, 146,5, 136.0, 135.0, 131.5, 130.6, 130.0, 128.8,

27. 28.

127.9, 124.0, 119.9, 97.9, 73.6, 66.3, 50.5, 31.9, 8.2; HRESIMS: m/z 349.1188 [M+H]+ calcd for 349.1183 (C20H16N2O4+H+). 9-Methoxy camptothecin (2). light yellow solid; mp 255-260 °C; IR (KBr) νmax 3449, 2925, 2075, 1637, 1465, 1416, 1115 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.83 (1H, m, H-7), 7.73-7.64 (3H, m, H-14, 10, 11), 6.96 (1H, d, J = 8.4 Hz, H-12), 5.72 (2H, d, J = 16 Hz, H-17), 5.30 (2H, d, J = 6.0 Hz, H-5), 4.06 (3H, s, CH3, H-9), 1.97-1.84 (2H, m, H-19), 0.88 (3H, t, J = 7.4 Hz, H-18); ESIMS m/z 379.1218 [M+H]+ . Rohitukine (3). light yellow solid; mp 218-220 o C; IR (KBr): vmax 3399, 2924, 2350, 1659, 1556, 1417, 1271, 1186 cm-1 ; 1H NMR (CD3 OD, 400 MHz): δ 6.08 (1H, s, H-6), 5.98 (1H, s, H-3), 4.19 (1H, brs, H-3´), 3.623.13 (6H, m, 2H of C-2´, H-4´, 1H of C-5´, 2H of C-6´), 2.83 (3H, s), 2.36 (3H, s), 1.67 (1H, m, H-5´a); 13C NMR (CD3OD+Pyridine-d5, 100 MHz) δ 182.9, 178.2, 166.7, 161.4, 156.9, 108.5, 108.0, 102.7, 69.3, 62.2, 56.6, 44.8, 37.3, 24.0, 23.6, 19.9; ESIMS m/z 306.0957 [M+H]+. Lopez-Meyer, M.; Nessler, C. L.; McKnight, T. D. Planta Med. 1994, 60, 558. Li, S.; Yi, Y.; Wang, Y.; Zhang, Z.; Beasley, R. S. Planta Med. 2002, 68, 1010.