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
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