Designing Anti-Tumor Drugs Using Natural and Synthetic Agents

Herman L. Holt, Jr.

University of North Carolina, Asheville

Asheville, NC 28804

Medicinal Chemistry Folklore• Medicinal chemistry is the science

that deals with the discovery of therapeutic chemicals and their development into useful medicines

• Medicinal chemistry has been practiced for thousands of years

• The earliest written records of the African, Chinese, Indian, South American, and Mediterranean cultures and biblical languages describe the therapeutic effects of various plant concoctions

• Man has search for cures by chewing on bark, roots, leaves and berries

Dr. Milton Brown, University of Virginia

...Milton Brown, a native of Baltimore, MD, received his bachelor’s degree from Oakwood College in 1987, a Ph.D. in organic chemistry from the University of Alabama, and a M.D. from the University of Virginia, where he currently serves as an associate professor of Chemistry and principal investigator for various NIH funded research projects. He has published innumerous prestigious academic journals as well as receiving awards in research and teaching.

A Father of Modern Medicinal Chemistry

• Edward E. Smissman, University of Kansas

– Smissman Award of the ACS Division of Medicinal Chemistry named in his honor

Tsung Ying (T.Y.) Shen University of Virginia

Professor Emeritus

Famous for Synthesis and Discovery of the Mechanism of Action of:IndomethacinSulindacDiflunisalOther anti-inflammatory-analgesic (NSAIDS) and immunoregulatorsMore than 210 U.S. Patents and scientific publications

• Medicinal Chemistry is defined as an interdisciplinary science situated at the interface of organic chemistry and life sciences (such as biochemistry, pharmacology, molecular biology, immunology, pharmacokinetics and toxicology) on one side and chemistry-based disciplines (such as physical chemistry, crystallography, spectroscopy and computer-based information technologies) on the other.

Chemistry based Chemistry based disciplinesdisciplines

Organic ChemistryOrganic ChemistryLife SciencesLife Sciences

Medicinal ChemistryMedicinal Chemistry

• Terms more or less synonymous with medicinal chemistry– Pharmacochemistry

– Molecular pharmacochemistry

– Drug design

– Selective toxicity

Definition and Objectives• Medicinal chemistry relates to the design and production of compounds that can be used in medicine for the

prevention, treatment or cure of human and animal diseases

• Medicinal chemistry covers three critical steps:

• A discovery step consisting of the identification and production of new active substances usually called lead compounds. Leads can originate from synthetic organic chemistry, from natural sources or from biotechnological processes.

• An optimization step that deals mainly with the synthetic modification of the lead structure in order to improve potency, selectivity and lessen toxicity. Its characteristics are the establishment and analysis of structure-activity relationships (SAR).

• A development step consisting of:– the optimization of the synthetic route for bulk production – modification of the pharmacokinetic and pharmaceutical properties of the active substance to

render it suitable for clinical use. This may cover optimization of properties associated with:• Chemical formulation• Solubility• Elimination of unpleasant taste or irritation• Reduction of pain at site of injection

Challenges for Medicinal Chemistry

• Medicinal chemist must excel in organic synthesis and understanding modern approaches to structure-activity analysis. Medicinal chemist are involved in the design, synthesis, optimization and selection of new lead compounds

• A survey of a range of company hiring representatives suggested these desired qualities:– Ability to fit a multidisciplinary team– Ability to search for novel molecules– Increased knowledge of how to synthesize molecules for focused

biological testing– Understanding of the reasons for making compounds– Understanding drug design– Insight in SAR with insufficient data– Knowledge in collateral fields (pathophysiology, cell biology, genetics)– Familiarity with new techniques

Designing Anti-Tumor Drugs Using Natural and Synthetic Agents

Herman L. Holt, Jr.

University of North Carolina, Asheville

Asheville, NC 28804

TUBULIN

•Globular Protein

•Taxoid Site

•Vanca Alkaloid

Domain

•Colchicine Site

MICROTUBULES

•Tubulin Polymers

MITOTIC SPINDLE

•Composed of Microtubules and associated proteins

•Needed for cellular division

MICROTUBULES

•Tubulin Polymers

What is the MTT assay?What is the MTT assay?

•A dose response curve is plotted and the concentrationA dose response curve is plotted and the concentration at 50% cell growth is obtainedat 50% cell growth is obtained

•Crystal is dissolved in a suitable solvent (e.g. DMSO, Crystal is dissolved in a suitable solvent (e.g. DMSO, acid-IPA) and the absorbance at 570 nm is obtainedacid-IPA) and the absorbance at 570 nm is obtained

•Living cells convert the yellow water soluble-tetrazolium Living cells convert the yellow water soluble-tetrazolium

salt into an insoluble purple formazan crystalsalt into an insoluble purple formazan crystal

•The amount of viable cells remaining can be determined spectrophotometrically The amount of viable cells remaining can be determined spectrophotometrically

••Cultured cancer cells are grown in the presence of potentialCultured cancer cells are grown in the presence of potential drug for a specific time period (e.g. 72, 96 h)drug for a specific time period (e.g. 72, 96 h)

N

N

NN

S

N

Br-

N

HN

N

N

SN

Living cancer cells

Mitocondrial dehydrogenases

MTT Formazan

MTT = 3-(4’,5’-dimethylthiazol-2’-yl)-2,5-diphenyltetrazolium bromide

CISPLATIN

PtCl

Cl NH3

NH3

•Anti-cancer agent

•Ovarian•Testicular•Lung•Breast

CISPLATIN

•Cleaves DNA

TAXOL

•Anti-cancer agent

•Pacific Yew Tree

•Ovarian•Testicular•Lung•Breast

TAXOL

•Stabilizes microtubules and prevents

disassembly and re-polymerization

VANCOMYCIN

•Tubulin polymerization inhibitor

• Meadow saffron or autumn Crocus

COLCHICINE

• poisonous

Copyright @ 1997 Alice B. Russell, James W. Hardin, Larry Grand

OCH3H3CO

H3CO

NHCOCH3

OCH3

O

COLCHICINE

•Tubulin binder

COLCHICINE

•Inhibits tubulin polymerization

•Derived from the African bush willow tree, Combretum caffrum

COMBRETASTATIN

OCH3H3CO

H3CO

cis-Combretastatin A4

OH

OCH3

(CA4)

COMBRETASTATIN

•Tubulin binder

•Inhibits tubulin polymerization

OCH3H3CO

H3CO

cis-Combretastatin A4

OH

OCH3

(CA4)

OCH3H3CO

H3CO

NHCOCH3

OCH3

O

COLCHICINE

OCH3H3CO

H3CO

cis-Combretastatin A4

OH

OCH3

(CA4)

OCH3H3CO

H3CO

CA4P

OPO32-

OCH3

OCH3H3CO

H3CO

R = NHCOCHNH2CH2OH, AVE--8062

R

OCH3 OCH3H3CO

H3CO

AC-7739

NH2

OCH3

•Combretastatin A-4 is the most developed and furthest along in clinical studies among the vascular targeting agents.

OCH3H3CO

H3CO

cis-Combretastatin A4

OH

OCH3

(CA4)

H3CO

H3CO

H3CO

trans-Combretastatin A4

OH

OCH3

OCH3H3CO

H3CO

cis-Combretastatin A4

OH

OCH3

(CA4)

http://www.oxigene.com/vascular/video1.asp

HETEROCYCLE ANALOGS OF COMBRETASTATIN

OCH3H3CO

H3CO R

OCH3

N

N

N

OCH3H3CO

H3CO R

OCH3

SN

OCH3H3CO

H3CO R

OCH3

N

N

N

N

Ohsumi et al. Bioorg. Med. Chem. Lett. 1998, 8, 3153.

OCH3H3CO

H3CO R

OCH3

NHN

OCH3H3CO

H3CO R

OCH3

ON

OCH3H3CO

H3CO R

OCH3

HN

N

Wang et al. J. Med. Chem. 2002, 45, 1697.

HETEROCYCLE ANALOGS OF COMBRETASTATIN

OCH3H3CO

H3CO R

OCH3

NH

N

N

OCH3H3CO

H3CO

NH2

NH

N

N

Pati, Wicks, Holt, LeBlanc, Weisbruch, Forrest, Lee Heterocyclic Communications 2005, in press.

Maintain structural rigidity

Increase water solubility

COMBRETASTATIN AND TRIAZOLE SYNTHESIS

NaH

DMF, r.t.16 hrs.

PPh3

O

H

Br

+O2N OCH3

OCH3

OCH3

H3CO

H3CO

H3CO

NO2

NO2

H3CO

H3CO

H3CO

Br2

CH2Cl2

Br

Br

NO2

H3CO

H3CO

H3CO

Br

Br

H3CO

H3CO

H3CO

NO2

KOtBuHOtBu

50oC5 hrs.

H3CO

H3CO

H3CO

NO2

COMBRETASTATIN AND TRIAZOLE SYNTHESIS

NO2H3CO

H3CO

H3CO

H2, Lindlar's Catalyst

Quonoline, THF, 1 hr.

OCH3H3CO

H3CO

NH2

NO2H3CO

H3CO

H3CO

1. BnN3, PhCH3, , overnight

2. H2, 10% Pd/C, THF, r.t, overnight

OCH3H3CO

H3CO

NH2

NH

N

N

AZIRIDINE ANALOGS OF COMBRETASTATINS

H3CO OCH3

H3CO

OCH3

RPhINNs

Cu(acac)

I N SO2 NO2PhINNs =

H3CO OCH3

H3CO

OCH3

R

NsN

AZIRIDINE TYPE ANALOGS OF COMBRETASTATINS

H3CO OCH3

H3CO

OCH3

R

N

H3CO OCH3

H3CO

OCH3

R

N

N

H3CO OCH3

H3CO

OCH3

R

N NH

O

O

O

H2N

O

H2N

Mitomycin C

OCH3

MITOMYCINS

•Anti-cancer agent

•Bladder

MITOMYCINS

•DNA Alkylating Agent

N

NH2

O

O

H2N

HN

N

HN

N

O

HN

NH

N

HN

N O

NH

Mitomycin C binding two DNA Bases

OCH3

N NH

O

OO

Target Mitomycin Skeleton

MITOMYCINS

•SYNTHESIS IS CURRENTLY IN PROGRESS•RESEARCH PRESENTED AT:

► Southeast Regional Meeting of the American

Chemical Society (joint meeting with Southwest region)

► North Carolina Undergraduate Research Symposium

► National Conference on Undergraduate Research

(Held at UNCA in celebration of its 20th Anniversary)

REFERENCEShttp://en.wikipedia.org/wiki/Main_Page

http://www.ces.ncsu.edu/depts/hort/consumer/poison/Colchau.htmChemical and Engineering News 2005, 83(5), several pages therein.

MUCH APPRECIATION

Jessica MaddoxDavid Mabe

Regan LeBlancJohn DicksonDr. Moses Lee

Dr. Toni BrownDr. Karen Buchmueller

Lee GroupUNC-Asheville

Furman UniversityNSF-REU

NSF-MRPG