BIOLOGICALLY ACTIVE COMPOUNDS FROM

PLANTS: 40 YEARS OF SEARCHING FOR A

SCIENTIFIC POT OF GOLD

Second Professor Frank Fish

Memorial Award Lecture

School of Pharmacy

University College London

October 9, 2013

Prof. A. Douglas Kinghorn

Professor and Jack L. Beal Chair

College of Pharmacy

The Ohio State University

Professor Frank Fish,

OBE

(1924-2011)

B. Pharm (London), 1946

Ph.D. (Glasgow), 1955

Established the M.Sc.

Course in Forensic

Science at the University

of Strathclyde in 1966

Dean, School of

Pharmaceutical

Sciences, University of

Strathclyde, 1977-1978

Dean, School of

Pharmacy, University of

London, 1978-1988

OUTLINE OF PRESENTATION

Continued Importance of Natural Product-Derived Drugs in the 21st Century.

The Crucial Role of Mentorship in Career Development – a Personal Perspective.

Examples of Promising Lead Compounds from Tropical Plants:

Hernandulcin, a Highly Sweet Sesquiterpenoid

Silvestrol, a Potential Agent to Combat B-Cell Malignancies

“Active Constituents” of Botanical Dietary Supplements, as Exemplified by 2-Methoxy-2,4,6-trihydroxyanthraquinone from Noni and -Mangostin from Mangosteen

Conclusions.

CONTINUED IMPORTANCE OF

NATURAL PRODUCT-DERIVED

DRUGS IN THE 21ST CENTURY

NUMBERS OF ORGANISMS FOR DRUG

DISCOVERY

Eubacteria (bacteria), cyanobacteria (blue-green algae) 4,000a

Archaea (halobacteria, cyanogens)

Protoctista (e.g., protozoa, diatoms, “algae”, including “red

algae” and “green algae”)

Unknown

80,000

Plantae (mosses and liverworts, ferns, seed plants)b 270,000

Fungi (e.g., molds, lichens, yeasts, mushrooms)c 72,000

Animalia (e.g., mesozoa, sponges, jellyfish, corals, flatworms,

roundworms, sea urchins, mollusks, segmented worms,

arthropods, insects, fish, amphibians, birds, mammals)d-f

1,320,000

a Figures are species described taxonomically to date in each group. b Plants are the second largest group of classified organisms, representing 15% of the known biodiversity. c Only a relatively small proportion (5%) of the estimated 1.5 m fungi have been classified taxonomically to date. d The largest numbers of organisms are the arthropods, inclusive of insects (ca. 950,000 species). e Of the 28 major animal phyla, 26 are found in a marine environment. f Over 200,000 species of invertebrate animals and algal species occur in the sea.

(Tan et al., Curr. Drug Targets 7, 265, 2006)

NUMBER OF DRUGS APPROVED IN

THE UNITED STATES (1981 TO 2007)

(Li and Vederas, Science 325, 161, 2009)

NATURAL PRODUCT-DERIVED DRUGS

INTRODUCED IN THE U.S. (2000-2008)

[Chin et al., AAPS J., 8 (2), E239-E253, (Article 28), 2006; http://www.aapsj.org; Butler, in Natural Product Chemistry for Drug Discovery, eds. A.D. Buss and M.S. Butler, RSC:

Cambridge, U.K., 2010; p. 321]

Source Organism Type Number Terrestrial Plants

(apomorphine HCl, arteether, dronabinol/cannabidiol (mixture), galanthamine

HBr, lisdexamfetamine, methylnatrexone Br, nitisinone, tiotropium Br)

8

Terrestrial Microorganisms

(amrubicin HCl, anidulafungin, biapenem, caspofungin acetate, cefditoren

pivoxil, ceftobiprole medocaril, daptomycin, doripenem, ertapenem, everolimus,

fumagillin, gentumazumab ozogamicin, ixabepilone, micafungin Na, miglustat,

mycophenolate Na, pimecrolimus, pitavastatin, retapamulin, rosuvastatin Ca,

telithromycin, temsirolimus, tigecycline, zotarolimus)

24

Marine Organisms

(trabectidin, ziconotide) 2

Terrestrial Animals

(bivalirudin; exenatide; synthetic versions of natural forms) 2

http

://ww

w.n

ature.co

m/n

rc/journ

al/v2/n

2/slid

eshow

/nrc7

23_F

2.h

tml

IXABEPILONE (IXEMPRATM)

Lead compound was

epothilone B isolated

from Sorangium

cellulosum (a

myxobacterium).

Anticancer agent

(refractory breast

cancer) (B-MS, 2007).

Interacts with tubulin.

http

s://ww

w.g

enetik

.uni-b

ielefeld.d

e/Gen

oM

ik/clu

ster6.h

tml

Ixabepilone R = NH

Epothilone B R = O

ECTEINASCIDIN 743: AN ANTICANCER

AGENT FROM A MARINE TUNICATE

Ecteinascidia turbinata

Ecteinascidin 743; ET-743;

trabectedin, Yondelis® [Soft

tissue sarcoma (Europe)]

www.pharmamar.com

htt

p:/

/ww

w.t

erid

anie

lsb

ook

s.co

m/S

tate

s/re

pti

le_

liza

rd_gil

a_m

on

ster

.jp

g

GILA MONSTER (HELODERMA SUSPECTUM)

SOURCE OF EXENATIDE – ANTIDIABETIC AGENT

PLANT NATURAL PRODUCTS AND DERIVATIVES APPROVED BY THE

U.S. FDA FROM JANUARY 2001-FEBRUARY 2013a

Year

Approved

Generic Name Natural Lead Compound Trade

Name

Indication

2001 Galantamineb Galanthamine Razadyne Dementia associated with Alzheimer's disease

2002 Nitisinoneb Leptospermone Orfadin Hereditary tyrosinemia type 1

2003 Miglustatb 1-Deoxynojirimycin Zavesca Type 1 Gaucher disease

2004 Tiotropiumb bromide Atropine Spiriva COPD and exacerbation of COPD

2004 Trospiumb chloride Atropine Sanctura Overactive bladder

2004 Solifenacinb Quinine VESIcare Overactive bladder

2005 Paclitaxel (protein-bound) Paclitaxel (taxol) Abraxane Breast cancer (also approved for lung cancer in

2012 )

2006 Sinecatechinsb,c Green tea phenols Veregen Genital and perianal warts

2006d Nabiloneb Δ9-Tetrahydrocannabinol Cesamet Chemotherapy-induced nausea

2008 Methylnaltrexoneb bromide Morphine Relistor Opioid-induced constipation

2008 Tetrabenazineb Emetine Xenazine Huntington's-associated chorea

2009 Artemetherb and lumefantrine Artemesinin Coartem Malaria

2009 Colchicined Colchicine Colcrys Gout

2009 Capsaicin Capsaicin Qutenza Postherpetic neuralgia

2010 Cabazitaxelb Paclitaxel (taxol) Jevtana Hormone-refractory metastatic prostate cancer

2010 Dextromethorphan and

quinidine

Morphine and quinidine Nuedexta Pseudobulbar Affect

2011 Ioflupane I-123b Cocaine Datscan Brain imaging with suspected Parkinsonian

syndromes

2011 Gabapentin enacarbilb -Aminobutyric acid (GABA) Horizant Restless leg syndrome

2012 Ingenol mebutateb Ingenol-3-angelate Picato Actinic keratosis

2012 Lorcaserinb hydrochloride Ephedrine Belviq Obesity

2012 Icosapent ethyl Eicosapentaenoic acid (EPA) Vascepa Hypertriglyceridemia

2012 Omacetaxine mepesuccinateb Homoharringtonine Synribo Chronic myeloid leukemia

2012 Crofelemerb,c Croton lechleri oligomeric

proanthocyanidin

Fulyzaq HIV/AIDS anti-retroviral-associated diarrhea

2013 Trastuzumab emtansine

(antibody-conjugate)

Maytansine Kadcyla Breast cancer

2013 Ospemifeneb Phytoestrogens Osphena Menopause-associated dyspareunia

a Information taken from Drugs@FDA (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm) and Kinghorn, et al. J.

Nat. Prod. 74, 1539, 2011 b New molecular entity. c Approved as a mixture of compounds as a “botanical drug”. d Originally

approved in combination therapy in 1985; newly approved as a monotherapy.

INGENOL MEBUTATE FOR THE TREATMENT

OF ACTINIC KERATOSIS

Approved by U.S. FDA in 2012 for the treatment of actinic keratosis, a precursor

to sun-related squamous cell carcinoma (used as a topical gel) (Lebwohl et al., N.

Engl. J. Med. 366, 1010, 2012; Mason, Pharm. J. 290, 141, 2013). This compound was isolated from Euphorbia peplus (Hohmann et al., Planta Med. 66, 291, 2000). New 14-step synthesis: Jorgensen et al., Science 341, 878, 2013.

Ingenol mebutate (Ingenol-3-angelate)

( Picato )

http://www.discoverlife.org/mp/20q?search=Euphorbia+peplus&mobile=iPhone

Euphorbia peplus (Euphorbiaceae)

http://luirig.altervista.org/flora/taxa/index1.php?scientific-name=euphorbia+peplus

DEFINITION OF A “BOTANICAL

DRUG PRODUCT”

In June 2004, the U.S. FDA issued the publication

“Guidance for Industry. Botanical Drug Products”

(http://www.fda.gov/cder/guidance/index.htm).

A “botanical drug product” is defined as containing as

ingredients vegetable materials, which may include plant

materials, algae, macroscopic fungi, or combinations

thereof, for use as a drug. It may be prepared, for example,

as a solution (e.g., tea), powder, tablet, capsule, elixir,

topical agent, or injectable.

Excluded are: fermentation products, highly purified or

chemically modified botanical substances, genetically

modified plants, allergenic extracts, and vaccines that

contain botanical ingredients.

(J. Dou, U.S. FDA, CDER)

OVERVIEW OF NDA PROCESS OF

FDA FOR “BOTANICAL DRUGS”

Purification to single active molecules is not required, although strict documentation of “chemistry, manufacturing, and controls” (CMC) must be carried out.

Identification of “active principles” is not essential.

The same requirements for safety assurance and clinical efficacy as for synthetic drugs are needed for NDA approval.

Purification to single active molecules is not required.

The same levels of safety requirements and clinical efficacy as non-botanical drugs are needed for NDA approval.

Nearly 500 IND/pre-IND applications for botanical drugs have been submitted to CDER, FDA between 1999-2012.

Most of these are in Phase 2, with a few in Phase 3.

(J. Dou, U.S. FDA, CDER)

SINECATECHINS FROM GREEN TEA (CAMELLIA

SINENSIS) AS THE FIRST “BOTANICAL DRUG”

Green tea contains mixtures of a sub-type of flavonoids (flavans)

known as “catechins”, which are esterified with a small phenolic

acid, gallic acid.

Epigallocatechin gallate (EGCG), the major flavonoid ester in

green tea, is one of the most highly studied natural product

molecules in the biomedical literature.

A mixture of green tea catechins (“sinecatechins”; Veregen)

was the first “botanical drug” approved by the U.S. FDA in 2006

(used topically to treat venereal and perianal warts) (Chen et al.,

Nature Biotechnol. 26, 1077, 2008).

CROFELEMER, THE FIRST FDA-APPROVED

“BOTANICAL DRUG” TO BE TAKEN ORALLY J. D

ou

, CD

ER

, FD

A

Crofelemer is a complex

mixture of proanthocyanidins

(literally thousands of

compounds co-occur).

The plant of origin is called

“Dragon’s Blood”.

Crofelemer (Fulyzaq) was

approved as a “botanical drug”

by the USA FDA in 2012 as an

oral antidiarrheal agent.

THE CRUCIAL ROLE OF MENTORSHIP

IN CAREER DEVELOPMENT – A

PERSONAL PERSPECTIVE

B.PHARM. (SPECIAL IN

PHARMACOGNOSY) AT BRADFORD

(1966-1969)

Dr. Peter

A. Linley

Dr. Keith J.

Harkiss

served as

tutor and

supervised

a thee-part

under-

graduate

thesis

MASTER’S IN FORENSIC SCIENCE AT

STRATHCLYDE (1969-1970)

Prof. Frank Fish, Coordinator of the M.Sc. Course in Forensic

Science

(Thesis performed under the supervision of Mr. Peter F.

Nelson)

TEACHING FELLOWSHIP AND PH.D.

STUDENTSHIP AT THE SQUARE (1971-1975)

Late Prof. Fred

Evans, Ph.D.

Supervisor (Dissertation carried

out on the skin-irritant

diterpene esters of

Euphorbia species)

SENIOR PHARMACOGNOSY ACADEMIC STAFF

AT “BRUNSWICK SQUARE” (1970s-1990s)

LATE PROF. JAMES W. FAIRBAIRN

(ANTHRAQUINONES; OPIUM POPPY;

CANNABIS SATIVA)

EMER. PROF. J. DAVID

PHILLIPSON (ALKALOIDS; ANTIPROTO-

ZOALS; TISSUE CULTURE)

LATE PROF. FRED J. EVANS

(PHORBOL ESTERS; CANNABIS SATIVA;

PH.D. SUPERVISOR)

DR. MARGARET F. ROBERTS

(ALKALOID BIOSYNTHESIS;

PAPAVER ALKALOIDS)

U.K. PHARMACOGNOSISTS AT A CELEBRATION IN THE LATE 1970S

Photograph Provided by Prof. J. David Phillipson

PRESENT PHARMACOGNOSY LEADERS AT

“BRUNSWICK SQUARE”

PROF. SIMON GIBBONS (ANTI-

INFECTIVE AGENTS FROM PLANTS;

DRUGS OF ABUSE)

PROF. MICHAEL HEINRICH

(ETHNOBOTANY, PHYTOTHERAPY;

PHARMACEUTICAL BIOLOGY)

M.SC. DEGREE IN PHARMACOGNOSY

AT UCL SCHOOL OF PHARMACY

Dr. Jose Prieto-Garcia

Programme Director

Prof. Simon Gibbons

Deputy Programme Director

Invited Guest Lecturers from outside UCL include Dr. Colin Wright (University

of Bradford), Prof. Peter Houghton (King’s College London), Prof. Monique

Simmonds (RBC, Kew), and Prof. Elizabeth Williamson (University of Reading)

SABBATICAL OF PROFESSOR DAVE PHILLIPSON AT

THE OHIO STATE UNIVERSITY IN THE LATE 1960s

Late Professor Jack L.

Beal

The Ohio State

University

Emeritus Professor

Raymond W. Doskotch

The Ohio State

University

A NIGHT ON THE TOWN WITH PROFESSOR

NORMAN R. FARNSWORTH AT “TRADER VICS”

Photograph Provided by Prof. Norman R. Farnsworth

DEPARTMENT OF PHARMAGNOSY, UNIVERSITY OF

MISSISSIPPI, 1975 (HEAD: NORMAN J. DOORENBOS)

THE ARROWS SHOW PROF. DOORENBOS AND

THE FUTURE MRS. KINGHORN

Late Dr. Norman R. Farnsworth University of Illinois at Chicago

1930-2011

Slide Provided by Professor J. David Phillipson

(Photograph by Jimmy W. Crawford, RTI)

DRS. MONROE WALL AND MANSUKH WANI – DISCOVERERS OF TAXOL AND CAMPOTHECIN

EXAMPLES OF PROMISING

LEAD COMPOUNDS FROM

TROPICAL PLANTS

EXAMPLES OF LEAD COMPOUNDS OF

INTEREST FROM TROPICAL PLANTS

Pervilleine A (Cancer Res., 2001)

α-Mangostin

(Nutr. Res., 2012)

Abrusoside A (Chem. Commun., 1989)

trans-Resveratrol (Science, 1997)

2-Methoxy-1,3,6- anthraquinone

(J. Nat. Prod., 2005)

Betulinic acid (Nature Med., 1995)

Silvestrol

(J. Org. Chem., 2004)

Pentalinonsterol (Phytochemistry, 2012)

Hernandulcin (Science, 1985)

TASTE AND THE TONGUE Humans have ca. 10,000 taste buds with each taste bud

containing 50-100 taste receptor cells.

The popular taste map of the tongue taste is based on faulty

interpretation of a 1901 paper, in fact taste buds on many

areas of the tongue can detect different tastes.

Bitterness has a much lower taste threshold than sweetness

due to the biological imperative to select for nutritious foods

while avoiding toxins.

Lindeman, B. Nature Med., 1999, 5, 381-382. Scott, K. Neuron, 2005, 48, 455-464

Image from www.rickbakas.com Image from Lindeman

“Low-Caloric” (“High Potency Sweeteners”) “Reduced-Calorie” (“Bulk Sweeteners”)

Compound Sweetness Intensity1 Compound Sweetness Intensity1

Glycyrrhizin 50-100 Lactitol 0.4

Stevioside 150-250 Isomalt 0.45-0.65

Perillartine2 370 Sorbitol 0.6

Phyllodulcin 400 Mannitol 0.7

Rebaudioside A 200-300 Maltitol 0.9

Mogroside V 250-450 Xylitol 1.0

MGGR2 941 High-Fructose

Corn Syrup

(90%)

1.0

Hernandulcin 1500

Monellin 1500-2000

Neohesperidin

dihydrochalcone2

1800

Thaumatin 2000-3000

COMPARISON OF THE SWEETNESS INTENSITIES OF “LOW-

CALORIC” AND “REDUCED-CALORIE” NATURAL PRODUCT

SWEETENERS (ALL OF HIGHER PLANT ORIGIN)

1 Relative to sucrose (=1). Sweetness intensity varies with concentration being tasted.

2Semi-synthetic compound.

HERNANDULCIN, A HIGHLY SWEET COMPOUND

FROM LIPPIA DULCIS (VERBENACEAE) In Mexico, L. dulcis is used as an

emmenagogue, to induce menses, as an abortifacient, and to treat coughs and stomachache.

The plant was purchased in bulk at a marketplace in Mexico City, and collected in the field at Tlayacapan, Morelos, Mexico in May-June, 1982 by then graduate student Cesar M. Compadre.

The compound was determined as a novel bisabolane sesquiterpene by spectroscopic methods.

The structure was checked by synthesis in the (±)-form by directed-aldol condensation of the ketones 3-methyl-2-cyclohexen-1-one (I) and 6-methyl-5-hepten-2-one (II).

(Compadre et al., Science 227, 417, 1985)

Prof. Cesar M. Compadre

IDEAL PROPERTIES OF A NONCALORIC AND NONCARIOGENIC SWEETENER FOR POTENTIAL

USE AS A SUCROSE SUBSTITUTE

Highly sweet, odorless, colorless, with taste

characteristics similar to sucrose.

Water soluble, stable.

No toxicity nor cariogenicity, from either the

parent substance or its metabolites.

Easy to synthesize or to obtain by cultivation.

Should fit existing techniques for the application

of sweeteners.

Economically competitive with currently

approved sweeteners.

(Hough et al., Eds. In Developments in Sweeteners – 1; Applied Science Publishers, London, 1979; p. v)

STRUCTURAL CHARACTERIZATION OF

SILVESTROL FROM AGLAIA FOVEOLATA

Drs. Bang Yeon Hwang and Baoning Su

Silvestrol

X-ray Structure by Drs. Bernard Santarsiero and Andrew Mesecar (UIC)

(Hwang et al., J. Org. Chem. 69, 3350, 2004;

ibid., 69, 6156)

Collaboration between:

The Ohio State University, Columbus, OH;

University of Illinois at Chicago, Chicago, IL;

University of North Carolina at Greensboro, NC;

Mycosynthetix Inc., NC;

Bristol-Myers Squibb, Princeton, NJ.

Funded by the United States National Cancer Institute, NIH (1990-2006; 2007-2013)

COLLABORATIVE PROJECTS ON NATURAL

PRODUCT ANTICANCER DRUG DISCOVERY

National Cooperative Drug Discovery Group (NCDDG) Grant

(U19 CA52956)

Program Project Grant

(P01 CA125066)

[Most recent review: Kinghorn et al., Pure Appl. Chem. 81, 1051, 2009]

NCDDG GROUP AT THE MEDICINAL PLANT GARDEN,

UNIVERSITY OF ILLINOIS AT CHICAGO IN 2003

SHOWS LEFT TO RIGHT (UIC UNLESS STATED) MARCY BALUNAS, GEOFF

CORDELL, STEVE SWANSON, DOEL SOEJARTO, YALI FU (NCI), NORM

FARNSWORTH, GORDON CRAGG (NCI), MANSUKH WANI (RTI), DOUG

KINGHORN, WILL JONES, NICK OBERLIES (RTI), TATIANA LOBO, AIKO ITO,

YINGMEI TAN. GHEE TAN, BANG YEON HWANG, AND FAUSTO RIVERO-CRUZ

IN VITRO CYTOTOXICITY OF SILVESTROL

Compound Cell linea

Lu1 LNCaP MCF-7 HUVEC

Silvestrol 1.2 1.5 1.5 4.6

Methyl rocaglateb

163 325 Not determined

203

Paclitaxelc 2.3 4.7 0.7 105.5

Camptothecinc 28.7 28.7 28.7 258.6

a ED50 values (nM) - Lu1 = human lung cancer; LNCaP

= hormone-dependent human prostate cancer; MCF-7

= human breast cancer; HUVEC = human umbilical

vein endothelial cells. b Isolated in our previous work (Rivero-Cruz et al., J.

Nat. Prod., 67, 343, 2004). c Used as positive control.

(Hwang et al., J. Org. Chem. 69, 3350, 2004)

EFFECT OF SILVESTROL IN THE IN VIVO HOLLOW FIBER TEST AND IN THE MURINE P-388 LEUKEMIA

MODEL

Active at maximum tolerated dose of

2.5 mg/kg/inj, given by intraperitoneal

injection daily for five consecutive

days (qd5) in ip P388 model.

Achieved maximum lifespan increase

corresponding to T/C of 150%.

Hollow fiber: Murine P-388 leukemia:

Inactive (T/C = 100%) in iv P388 leukemia model when administered by either the iv or ip route using a daily times five schedule (qd5).

Active (T/C = 129%) in same tumor model when injected iv on a twice-daily schedule (2qd5) using the same cumulative dose (2 mg/kg/day).

(Hwang et al., J. Org. Chem. 69,

3350, 2004)

Incubation Time (hours)

B Cells

0

20

40

60

80

100

120

0 24 48 72

Incubation Time (hours)

T Cells

0

20

40

60

80

100

120

0 24 48 72

EFFECTS OF SILVESTROL ON B AND T CELLS

IN WHOLE BLOOD FROM CHRONIC

LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS

80 nM Silvestrol 1 µM 2-F-ara A

% L

ive C

ell

s R

ela

tive t

o U

ntr

eate

d

(Lucas et al., Blood 113, 4656, 2009)

The remaining three mice appeared normal 12+ weeks post-

engraftment, 6 weeks after the last treatment.

SILVESTROL SIGNIFICANTLY IMPROVED

SURVIVAL IN AN ACUTE LYMPHOBLASTIC

LEUKEMIA (ALL) XENOGRAFT MOUSE MODEL

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45

Days Post Engraftment

% S

urv

ival

Control (N=13)

Silvestrol (N=14)

1.5 mg/kg i.p. M, W, F; started 1

wk post-engraftment

Treatment stopped

85

Median survival difference P = 0.002

(Lucas et al., Blood 113, 4656, 2009)

Silvestrol was totally synthesized by two groups in 2007, the

groups of Porco (Boston University) (Gerard et al., Angew.

Chem. Int. Ed., 46, 7831, 2007) and Rizzacasa (University of

Melbourne) (El Sous et al., ibid., 46, 7835, 2007).

Pelletier and co-workers have shown that silvestrol is a

translation inhibitor by targeting eukaryotic initiation factor

(eIF) 4A (Cencic et al., PLoS ONE 4(4), e5223, 2009).

SILVESTROL: WORK PERFORMED BY OTHER GROUPS

The figure shows the translation

initiation complex and known

inhibitors (Lucas et al., Curr. Drug

Targets 11, 811, 2010); based in part

on Cencic et al., 2009, as cited

above).

In very recent work, using

biotinylated 5-epi-silvestrol, a

specific interaction was shown with

eIF4AI and eIF4AII (Chambers et al.,

Org. Lett. 15, 1406, 2013).

SILVESTROL: A POTENTIAL NEW THERAPEUTIC

AGENT FOR B-CELL MALIGNANCIES

At The Ohio State University, silvestrol has shown promising in vivo

activity in models of acute lymphoblastic leukemia, acute myeloid

leukemia, EBV-driven lymphoma, and mantle cell lymphoma (Lucas et

al., Blood 113, 4656, 2009; Alinari et al., Clin. Cancer Res. 18, 4600, 2012;

Alahkar et al., J. Hematol. Oncol. 6, 21, 2013).

Silvestrol was selected for preclinical development by NCI through the

DDG IIA mechanism in 2007, and is now in the NCI NExT program having

been subjected to additional peer and program review in August 2013.

The mechanism of antileukemic action is being further investigated

through Project 5 of a NCI/NIH SPORE (P50) award (J. C. Byrd, PI) (M.R.

Grever, D.M. Lucas) (2009-2014).

EFFECTS OF SILVESTROL ANALOGUES ON GROWTH

INHIBITORY ACTIVITY OF HUMAN CANCER CELLS

Methyl ester group is

required for potency

Absence of OCH3 at C-4' diminishes

resultant cytotoxicity slightly

Esterification or change

in configuration of the

hydroxy group at C-1

diminishes cytotoxicity

(Hwang et al., J. Org. Chem. 69, 3350, 2004; Adams et al., J. Am. Chem. Soc. 131, 1607, 2009; Pan et

al., J. Nat. Prod. 73, 1873, 2010; Woodard IV et al., Unpublished results)

The presence of a 1,4-dioxanyloxy moiety at C-6

enhances activity dramatically

The cyclopenta[b]benzofuran moiety

and other aromatic units confer basic

cytotoxicity to this compound class

Inversion of configuration at C-2'''

reduces cytotoxicity much more

than when inverted at C-5'''

Esterification of the hydroxy

groups at C-5''' and C-6'''

decreases resultant cytotoxicity

The natural product silvestrol is the most potent of ca. 15 close analogues investigated

AGREEMENT FOR DEVELOPMENT OF SILVESTROL

FOR POTENTIAL TREATMENT OF B-CELL MALIGNANCIES

In June 2012, after protracted

negotiations, an agreement was

signed to jointly develop silvestrol

between The Ohio State University

and the Sarawak Biodiversity Center,

with the immediate goal of

conducting preclinical toxicology.

The U.S. NCI will be involved

through the NExT program.

This slide shows the two other

faculty participants in this work at

Ohio State, Drs. Michael Grever (top

left) and David Lucas (top right), and

Dr. Rita Manurung, Chief Operating

Officer, Sarawak Biodiversity Center,

Kuching, Sarawak, Malaysia

(bottom). Silvestrol will be sourced

from Aglaia stellatopilosa grown in

Sarawak.

DIETARY HEALTH SUPPLEMENT AND

EDUCATION ACT (DSHEA) (1994)

Defined terms “dietary supplement” (including

herbal remedies) and “dietary ingredient”.

These are taken orally and may be used as in forms

such as tablets, capsules, soft gels, gel liquids, and

powders.

Exempted dietary supplements marketed before

October 15, 1994 from new dietary ingredient (NDI)

review.

Established requirements for new dietary

ingredients.

A dietary supplement may be declared as a hazard

to public health or safety.

“Structure and function” claims may be made, but

most labels have a disclaimer about health benefits.

NONI AS A DIETARY SUPPLEMENT

Marketed in the U.S. by several companies. Noni juice is available in many retail outlets, including supermarkets, health-food stores, and pharmacies.

Tahitian and Hawaiian noni juice are both available.

Marketing toward layman primarily based on the likely fictitious alkaloid, “xeronine”.

(Heinecke, Pac. Trop. Bot. Gard.

Bull. 10, 15, 1985)

NONI (MORINDA CITRIFOLIA)

(RUBIACEAE) Occurs in tropical and subtropical regions.

All parts (fruit, leaf, bark, flower, and seed) have been utilized medicinally.

Roots and bark are used as a dye.

In initial isolation work performed at The Ohio State University, a lignan (americanin A) and a flavonoid glycoside (narcissoside) were isolated as antioxidant constituents of noni fruits.

No trace of xeronine was found!

(Su et al., J. Nat. Prod. 68, 592, 2005).

htt

p://w

ww

.nps.g

ov/k

aho

/KA

HO

ckL

s/K

AH

Op

lnt/n

on

i.h

tm

(Review: Pawlus and Kinghorn, J. Pharm. Pharmacol. 59, 1587, 2007)

Dr. Alison Pawlus

QUINONE REDUCTASE-INDUCING ACTIVITY OF

ISOLATED CONSTITUENTS OF NONI FRUITS

1 (new, 2-methoxy-1,3,6,- trihydroxyanthraquinone)

aCD = Concentration required to double quinone reductase induction. bIC50 = Concentration

for 50% inhibition of cell viability. cCI = Chemopreventive Index. d Only 2 mg of 1,3,6-

trihydroxy-2-methoxyanthraquinone (1) isolated from ca. 9 kg dried noni fruits. * = Control.

(Pawlus et al., J. Nat. Prod. 68, 1720, 2005)

2

Compound CDa, M (g/mL) IC50b, M (g/mL) CIc

1d 0.009 (0.0027) >69.9 (>20) >7770

2 1.67 (0.52) >66.6 (>20) >39.9

L-sulforaphane* 0.34 (0.061) 9.77 (1.73) 28.7

INHIBITORY EFFECT OF NONI FRUITS

ON RAT ESOPHAGEAL TUMORIGENESIS

Male F344 rats were fed a diet of 5% w/w dried

powdered fruits of M. citrifolia (noni), and six other

dried fruits, including black raspberry (Rubus

occidentalis), in a comparison study.

The standard carcinogen, NMBA (N-

nitrosomethylbenzylamine was used to treat rats for

five weeks, and the experiment was terminated after

35 weeks.

All seven fruits had similar effects on reducing

esophageal tumor incidence, size, and multiplicity,

and also reduced the levels of two serum cytokines.

Noni fruits have lower levels of anthocyanins and

ellagitannins than black raspberries.

(Stoner et al., Pharm. Res. 27, 1138, 2010)

GARCINIA MANGOSTANA (MANGOSTEEN) (CLUSIACEAE)

The tropical fruit Garcinia

mangostana L. (Clusiaceae;

Mangosteen), has become a

major botanical dietary

supplement in the U.S.

Mangosteen extracts and/or their

purified xanthone constituents,

such as -mangostin, have

antioxidant and e.g., putative

anti-inflammatory and

antimicrobial activities.

In initial collaborative work with

Dr. Bill Keller, several

mangosteen xanthones were

found to be potent antioxidants

(Jung et al., J. Agric. Food

Chem., 54, 2077, 2006).

DR. WILLIAM J.

KELLER

NATURE’S SUNSHINE

PRODUCTS, INC.

XANTHONES ISOLATED FROM THE PERICARP OF MANGOSTEEN

PROFILE OF XANTHONE CONTENT IN THE MANGOSTEEN JUICE USED IN THE STUDY

(Chitchumroonchokchai et al., J. Nutr. 142, 675, 2012)

When analyzed by

HPLC, the 100%

mangosteen juice

used in the study

provided 5.3 + 0.1 mM

total xanthones, with

-mangostin being the

most abundant

(59.9%), as indicated

in the table opposite.

Xanthone

Content

in juice

(µM)

Percentage of

total xanthones

identified (%)

garcinone C 291 + 11.2 5.5

garcinone D 520 + 10.9 10.2

garcinone E 239 + 18.5 5.1

-mangostin 3190 + 123 59.9

β-mangostin 121 + 9.3 2.3

-mangostin 356 + 4.3 6.5

8-deoxygartanin 176 + 4.5 3.1

gartanin 157 + 6.9 2.8

tovophillin B 50 + 2.9 1.1

9-hydroxycalabaxanthone 193 + 7.4 3.6

Total 5290 + 166 100

Values are means + SD; n = 5 independent replicates

-Mangostin

DETERMINATION OF BIOAVAILABILITY IN HUMANS

OF XANTHONES FROM MANGOSTEEN JUICE

The bioavailability of mangosteen xanthones using

human subjects is of interest, since milligram amounts

are ingested on a daily basis.

In a preliminary study, Kondo et al. administered ca. 60

mL of a supplement (mangosteen; aloe vera; green tea;

multivitamins) to 20 fasted healthy human volunteers,

and it was concluded that -mangostin is bioavailable

(observed Cmax at tmax of ca. 1 hour) (Kondo et al. J. Agric.

Food Chem., 57, 8788, 2009).

In a study carried out at The Ohio State University, the

bioavailability of xanthones was investigated in ten

healthy adults (five females; five males), who consumed

a single dose of 100% mangosteen juice along with a

typical fast-food (high-fat) breakfast, which was

supplemented with canola oil and soybean oil

(Chitchumroonchokchai et al. J. Nutr., 142, 675, 2012).

EFFECT OF DIETARY α-MANGOSTIN IN A MURINE

HT-29 COLON CANCER CELL XENOGRAFT MODEL

(Chitchumroonchokchai et al., Mol. Nutr. Food Res. 57, 203, 2013)

Balb/c nu/nu mice were fed either the control diet AIN-93G or the control diet with

-mangostin (-MG; 900 mg/kg). After one week of acclimation to diet, mice were

injected sc with HT-29 cells, and then fed the same diets. After two weeks (panel A) or four weeks (panel B) from the initial injection of HT-

29 cells, the tumor masses were 27% and 41% less, respectively, in mice fed the

diet with -mangostin compared to those fed the control diet.

Xanthones and their metabolites were found in the serum, liver, and feces.

This work was carried out in collaboration with Professors Mark Failla and Steve

Clinton at The Ohio State University.

Values are

means + SD; n =

12 and 6 mice

for panels A and

B, respectively

0

200

400

600

800

1000

1200

Tum

or

mas

s (m

g)

AIN-93G AIN-93G+ α-MG

a

b

0

10

20

30

40

50

60

70

80

90

100

Tum

or

mas

s (m

g)

AIN-93G AIN-93G+ α-MG

a

b

A B

CONCLUSIONS: COLLABORATIVE APPROACH

TO THE DISCOVERY OF ANTICANCER AGENTS

FROM TROPICAL PLANTS

Tropical plants are more biodiverse than temperate plants, and thus hold the potential of offering greater chemical diversity for anticancer drug discovery.

Plant collections for drug discovery must cover the source country in terms of intellectual property agreements.

Efforts to harness plant compounds as potential cancer chemotherapeutic agents require a multidisciplinary approach with open and frequent communications.

Photograph by Jon Gladden (April 2013)

Shown (left to right) are Tony Gromovsky, Ben Naman, Lynette Bueno, Dr. Heebyung

Chai, Dr. Li Pan, Ms. Anecie Benatrahina, Dr. Patrick Still, and Dr. Yulin Ren

Support was obtained from NIH grants N01-DE-02425, R03-07560; R01-DE-

08937, U19 CA52956, and P01 CA125066 and faculty start-up funding from the

Molecular Carcinogenesis and Chemoprevention Program of the OSUCCC.

Many faculty, visiting scholars, postdoctorals, and graduate students at the

University of Illinois at Chicago and The Ohio State University are thanked for

very their kind collaboration.

This is a long-running

and well-respected

book series that was

established in 1938 by

Laszlo Zechmeister.

The series has

featured contributions

by nine Nobel

laureates. The

chapters refer to the

origin, distribution,

chemistry, synthesis,

biochemistry,

functions, and uses of

naturally occurring

substances, ranging

from small molecules

to biopolymers.

College of Pharmacy and OSU Comprehensive Cancer Center