DRUG DISCOVERY AND DEVELOPMENT“Synthesis analog compounds and

Its Biological Activity”

MUHAMMAD HANAFIResearch Centre

forChemistry (RC Chem) - LIPI

INTRODUCTION

Isolation Salicin from bark Salix alba (bitterness) for analgesic drug (Rev Edward Stone 1760), hydrolisis & oxidation (Raffaele Piria, 1838), acetylation of Salisylic acid (Charles Frederich Gerhardt, 1853), and finally pill form as 500 mg tablets in 1990) . Smith and Willis (1971) to prove that the blood-thinning properties (antiplatelets)

OH

O

OH

O

OH 1. Hydrolysis

2. OxidationGlu

Acetylation

Salisylic acid- more effective, - no bitter taste- gastric bleeding

salicin

Hystory of Drug Discovery :

O

O

OH

Ac

Acetylsalicylic acid (aspirin) - less irritating - ester hydrolyzes to active drug

Target IdentificationAnd Validation

Search of Lead Structure

Optimization of Lead Structure

PreclinicalDevelopment

Research Phases in Drug Development

Idea

Lead Structure

Candidate for Development Product

Development Product

FOUR MAIN APPROACHES TO DISCOVERING NEW DRUGS

1. From Natural Products : Screening to find biologically active component

2. From the drugs in use : Modification to improve activity or to find different

3. From synthetic chemicals and animal modelsScreening of chemical library by disease animal models

4. From the modern approach to drug designDesigning drugs based on physiological mechanism

DISCOVERY of NOVEL DRUGSfrom NATURAL PRODUCT

1. Screening of Natural Compounds for Biological Activity :

plants, microbes, marine, etc

2. Isolation and Purification of Active Principle

3. Determination of Structure : NMR, IR, MS

4. Structure-Activity relationships(SAR) :

Identification of Pharmacophore

5. Synthesis of Analogues :

Increase activity, reduce side effects

6. Receptor Theories : binding site information

7. Design and Synthesis of Novel Drug Structure

Lead Compouns from Natural Products

H

O

O

HO

H

O

O

Lovastatin Aspergillus tereus Anticholesterol -

O

O

O

OOR

HN

N

OOH

O

UK-3A

Streptomycesp sp. 517-02Cytotoxic to P338, KB

N

N

HO O

Phenazine carbioxylatePseudomonas pycocyaneae

O

O O

O

HO

CalanoneCallophyllum tesmanii

O

O

Methyl cinnamte

Time & Cost for A New Drug Development

Research

Preclinical Phase

Development/Clinical Phase

Phase I

Phase II

Phase III

Authority”sAssesment/NDA Phase

Duration Costs (Mio US$)

3-6 yr 140

1.5 yr 30

2 yr 80

3.5 yr 330

1.5 yr 60

11 – 15 yr ca. 750 Mio US$

Compounds

5000-10000

250

5

1 drug

Drugs Fail Because of two Major Reason

39 % fail due to deficiencies in Absorption, Distribution, Metabolism & Elimination (ADME)

30% fail due to lack of efficacy

11% fail due to animal toxicity

10% fail due to adverse effects in man

5% fail due to commercial reason

5% miscellaneous

Lipinski’s “Rule of Five”

Christopher Lipinski proposed four parameters that define the "drug- likeness" of potential drug candidates based on analysis of existing drug molecules. "The Rule of Five" got its name from the cut-off values for each of these parameters of which all have values of five or a multiple of five.

The “rule” states that poor absorption or permeation is more likely when :

–A compound has > 5 H-bond donors (sum of OHs and NHs); –There are > 10 H-bond acceptors (sum of Ns and Os); –The MW is > 500; –TheLogP is > 5 (or MLogP is over 4.15).

The “rule” is used by many as a useful guide in drug design.

The rule of five - formulation

Poor absorption or permeation are more likely when:

There are more than There are more than 55 H-bond donors.H-bond donors. The molecular weight is over The molecular weight is over 500500.. The LogP is over The LogP is over 55.. There are more than There are more than 1010 H-bond H-bond

acceptors.acceptors.

OPTIMAZATION ACTIVITY: SYNTHESIS OF DERIVATIVES/

ANALOGOUS

SYNTHESIS DERIVATIVE OF LEAD COMPOUNDS

R-OHCH3-I R-OMe

CH3COClR

O

O

CH3SO2C;l RO

S

OO

Alcohol

Ether

Ester

AlkaneLiALH4 R-H

or Ac2O

R-NH2 CH3COClR

HN

OAmine Amide

or Ac2O

OPTIMIZE LEAD COMPOUND

R

O

R' R

OH

R'

Reduction

NaBH4/LiALH4

Ketone Alcohol

R O

LiALH4

OStrong base :

NaOH/KOHR OH

Ester Acid

R OHAlcohol 1o

O

O

O O

O

HO

O

O

O

O

O

ESTERS AS PRODRUGS

Fatty barrier

RC

HN

O

R'

LiAlH4

NaOH RC

OH

O

R'H2N

RCH2

NH2

+

NaH / MeIR

C

CH3N

O

R'

Carboxylic acid Amine

1o Amine

3o Amide

Amide:

RC

OH

O

LiAlH4

H+ / R'OH RC

OR'

O

RCH2

OH

Ester

1o Alcohol

Carboxylic acid:

OPTIMIZE LEAD COMPOUND

Analogs of pharmacophore (remember morphine)

Goals?

1. Variation of alkyl substituents

2. Variation of chain length

ANALOGUE

C

CH3

CH3H3C

van der Waals interactions

LEAD COMPOUND

CH3

Hydrophobicpocket

Salbutamol (Ventolin) (Anti-asthmatic)

Adrenaline

Propranolol(-Blocker)

OH

O NH

CH3

CH3H

HOCH2

HO

HN

CCH3

OH

CH3

H

CH3

HO

HO

HN

CH3

OHH

EXAMPLE:

OH

NHMe

OMe

HOOC

Ph

Cl

Drug

OH

NHMePh Drug

Excess ring

Simplification

DRUGS /LEAD COMPOUNDSDEVELOPMENTS

PRODRUG- EUQUININE

Euquinine is the esterification product of quinine with

chloro-formic acid ethyl ester

N

NO

O

O

O

HH

N

NHO

O

H H

+ O

O

Cl

ÓÅ¿üÄþEnquinine

SYNTHESIS OF ARTEMISINI DERIVATIVES

ReductionNaBH4/EtOH

Methyllation (MeI),

Ethylation

Artesunate

DihydroartemisininArtemisinin

CALANONE DERIVATIVES AND ITS CYTOTOXIC ACTIVITY*

O

O O

HO

HO

O

O O

O

HO

CalanoneEster Calanol

Log P 2.32Against colon cancer cells HCT116: IC50 = 1.29 µg/mL P388 : IC50 = 7,5 µg/mL

Log P 0.43Against colon cancer cells HCT116: IC50 > 20 µg/mL L1210 : 59.4 µg/mL P388 : IC50 = 15

Cisplatin IC50 = 1.02 µg/ml

O

O OHO

R

O

Calanol

Log P -0.42Against colon cancer cells HCT116: IC50 > 20 µg/mL L1210 : 70.0 µg/mL P388 : IC50 = 15

*atent: M. Hanai, 2006

N

OH

NH

O

OO

O

A

B C

O

PSMOE

UK-3A Analog Development

N

OH

NH

O

O

O

O

O

O

OUK-3A

UK-3A Ring opening (Analog UK-3A)

DEVELOPMENT OF ANALOG UK-3A POTENTIAL FOR BREAST CANCER

TREATMENT

DEVELOPMENT OF ANALOG UK-3A POTENTIAL FOR BREAST CANCER

TREATMENT

PSMOEPSMOE

HN

O

O O

N

OOH

O

BcL-xL Protein

QSAR PARAMETER & CYTOTOXIC

TEST RESULTS N

OH

HN

O

OH

O OCH3

O

O

O

OOR

HN

N

OOH

O

UK-3A

Log P -1.18Ebinding = -7.1 kcal/molIC50 = >100 g/ml

Log P 1.61Ebinding = -11.65 kcal/molP388 : IC50 = 38 g/ml

O

O

O OH

OO

O

OOHO

O

O

OH

NH

OTaxol

Log P 1.67Ebinding = -10.39 kcal/mol

O

OHN

OOH

HN

O

O

OH O

OAntimycin A3

Log P 1.30, Ebinding = -10.24 kcal/mol

KB :IC50 = 0.23 mg/mlYMB-1:IC50 = 0.015 mg/ml

CYTOTOXIC TEST RESULTS TO P388, KB AND YMB-1

NHN

O

O

OH

O

OO

PDBGE : R = Butyl

N

HN

O

O

O

OO

OH

NDBGE : R = Butyl

IC50 34 g/ml (P388)IC50 2.28 g/ml (KB)IC50 1.83 g/ml (YMB-1)

IC50 38 g/ml (P388)

IC50 1.92 g/ml (KB)IC50 5.46 g/ml (YMB-1)

Ebinding=-9.66 kcal/mol), Log P 1.5

Ebinding=-10.29 kcal/mol);

Log P 1.62

P388 :IC50 = 40,0 mg/mlKB :IC50 = 0,82 mg/mlYMB-1:IC50 = 2,69 mg/ml

Log P 2.09

Metabolite Secundar from Microbial SoilPseudomonas pycocyanea

MIC 4,8 g/ml (E. coli); 0,07 g/ml ( S. aureus) IC50 : 5,20 g/ml (L1210)

Erythromycin : MIC 5,08 (E.coli), 4,06 (S. aureus) and 3,36 g/ml (B. subtillis)

N

N

HO O

H

H

H

H

H

H

H 8,98(dd)

8,53(dd)

8,04(dd)

8,35(dd)

8,02(dd)

7,98(dd)

165,88

139,91

140,12

137,45

135.14

130,29125,08

143,46

144,16

131,73

133,22

130,15

8,28(dd)

128,03

H15,5 ppm

p-Carboxyl-phenazine

SYNTHESIS SALYCIL ANILIDE (SA)

OH

OOH

+

HN

OOH

NH2

DCC, DMAP,

CHCl3, RT, 1 d SA

SALYCIL ANILIDE DERIVATIVES (PHENAZINES ANALOGS)

L1210 IC50 5,5 g/ml

L1210 IC50 7,0 g/ml

NH

N

O

OH

NH

O

OH

NH

O

OH

OCH3

L1210: IC50 = 4.8 mg/ml

M. Hanafi, Paten P00200200449, 2002

Log P 3.29Ebinding = -10.21 kcal/mol

P388 :IC50 = 7.75 g/mlKB :IC50 = 0.6 g/mlYMB-1:IC50 =2.97 g/ml

CYTOTOXIC ACTIVITY RESULTS

P388 :IC50 = 7,55 mg/mlKB :IC50 = 0,78 mg/ml

Log P 3.29 HN

OOH Salycil octyl amide (SOA)

N

HN

O

OHNOA : Log P 3,02 IC50 (T47D) : 4,67 g/mL

EFFICACY & TOXICITY TEST OF SALYCIL ANILIDE (SA)

1. Acute Toxycity (LD50) : 365.83 mg/kg bw and 429.46 mg/kg bw 1. Effective dose : 30 mg/kg bw

a

NH

O

OH

P388 :IC50 = 7.75 g/mlKB :IC50 = 0.6 g/mlYMB-1:IC50 =2.97 g/ml

SYNTHESIS METHYL CINNAMTE DERIVATIVES

OH

O

+

OH

O OCinnamic acid

p-TSOH

kalor

CH3

CH3

o-Cresol

8-Methyl-4-phenylchroman-2-one

OH

O

+

OH

O OCinnamic acid

p-TSOH

calor

phenol

4-phenylchroman-2-one

CYTOTOXIC TEST TO LEUKEMIA CELL LINE P388

No.

Compound IC50 (ppm)

1 Metthyl cinnamate (1) 20.35

2 Cinnamic acid (2) 48.85

3 4-phenylchroman-2-one (3) 209.20

4 8-Methyl-4-phenylchroman-2-one

68.42

5 Phenyl cinnamte (5) 0.5

32O O

CH3

O

O

O

O

O O

OH

O

Log P 2.2

Log P 1.93

Log P 3.85 Log P 3.36Log P 3.86

FIND AND OPTIMIZED A LEAD COMPOUND: LOVASTATIN

» Minimise energy of structure : Chem3D, Gaussian, Mopac,

» Structure Activy Correlationship : HyperChemPro

» Direct Ligand Design (HMG-CoA rductase): Arguslab 4.0

» Synthesis» Bioaactivity Test

O

OO

OHO

O

OO

OR1O

O

O

R1O O

O

O

O

HO O

O

12, 3, 4

5

Lovastatin

Simvastatin

Siynthesis Simvastatin from Lovastatin (1)*

1. Protection :t-Bu(Me)2SiCl or (MeO)2CH2/P2O5

2. Hydrolysis (KOHaq or LiOH)3. Cyclization, Heat/cyclohexane/pTsOH4. Esterification : RCOCl, DMAP 5. Deprotection, TBAF/THF or PhSH

R1 = TBDMSi or OCH2OMe

*US Patent, 6,506,929 B1, Jan. 14, 2003

SYNTHESIS SYNTHESIS DEHYDROLOVASTATIN DEHYDROLOVASTATIN

(LIPISTATIN)(LIPISTATIN)

CH3

O

H3C

H3C

O

O

O

CH3

O

H3C

H3C

O

O

O

Lovastatin

HO

H+, Cyclohehane

Dehydrolovastaton

88,3 % (EtOH)

H:EtOAc (4:1)

Interaction Dehydrolovastatin and the active site of HMG-CoA

reductase

NONO CompoundsCompounds Interaction Energy (kcal / Interaction Energy (kcal / mol)mol)

Log PLog P

11 Substrat (HMG-CoA)Substrat (HMG-CoA) - 10,5055- 10,5055

22 DehydrolovastinDehydrolovastin - 9.95- 9.95 4.804.80

33 Lovastatin Lovastatin - 9,48- 9,48 3.773.77

44 SimvastatinSimvastatin - 8,86- 8,86 5.735.73

55 Buthyl ester (Lovastatin)Buthyl ester (Lovastatin) - 9,91- 9,91 4,924,92

INTERACTION ENERGY WITH HMG CoA REDUCTASE AND LOG P

O

O

O

HO

H

H

H

HH

5.99 (d, 7.95 Hz)

5.78 (dd, ...Hz)

6.02 (d, 7.3 Hz)

5.39 (d, 2.6.Hz)

1.12 (d, )

1.08 (d, )

0.91 (d)

0.92 (t)6.86 (dt)

Lipistatin Spectrum : 1H and 13C NMR

O

O

O

HO

176,81

164,53

133,20131,74

129,88128,51

121.68

145,.02

67.92

29.75

32.86

24.44

36.79

32.6037.46

77.46

27.65

36.80

41.6427.02

16.47

11.91

14.06

??

H

H

H

HH

5.99 (d, 7.95 Hz)

5.78 (dd, ...Hz)

6.02 (d, 7.3 Hz)

5.39 (d, 2.6.Hz)

1.12 (d, )

1.08 (d, )

0.91 (d)

0.92 (t)

Parameter Normalcontrol

Hiperlipi-demic

Simvastatin (7,2 mg/

200 g bw)

Lipistatin(7,2 mg/

200 g bw)

Lipistatin(14,4 mg/200 g bw)

Total cholesterol

(mg/dl)(%)

111,79 156,66 112,03 (28,49%)

106,64 (31,93 %)

105,54 (32,55 %)

Trigliseride (mg/dl)/(%)

106,29 172,53 102,28 (40,72%)

103,85 (40,0%)

94,79 (45,06%)

LDL-cholesterol (mg/dl)/(%)

32,34 72,99 30,23 (58,58%)

25,00 (65,75%)

28,77 (60,58%)

HDL-cholesterol (mg/dl)/(%)

58,20 49,16 61,34 (24,77%)

60,87 (23,82%)

57,81 (17,60%)

Evaluation Results of Antihiperlipidemic Activity on Mice for Lipistatin and Simvastatin

Comparative study on HDL-cholesterol raising

effects of atorvastatin and dehydrolovastatin*

* Marissa A Indah D. D, T. Yuliani, YAnita, L Meilawati, MJP, Andrianopsyah, and Hanafi, M. Journal of Applied Pharmaceutical Science 02 (03); 2012:

CONCLUSION1. To get a new drug is very complex, take time, and costly

2. Starting material (lead comp) could be isolated from the major comps.

3. The Lipinski’s “Rule of Five is used by many as a useful guide in drug design.

4. To optimized acrtivity of lead compouunds can be make derivatives, by simple methods: methylation. reduction, esterification, hydrolisis, and simplification

5. Lipofilicity FG is important for biological activity

6. Analog UK-3A were potential candidate anticancer

7. Dehydrolovastatin is potential a new candidate drug for anticholesterol

ACKNOLEDMENTS

Indonesian Institute of Science (LIPI) & Ministry of Sci & Tech (KNRT) and JSPS for fund

RC Chem LIPI for support facilities

Osaka City Univeristy Japan for cytotoxic test

TERIMAKASIH