DRUG DISCOVERY AND DEVELOPMENT
“Synthesis analog compounds and
Its Biological Activity”
MUHAMMAD HANAFI
Research Centre forChemistry
(RC Chem) - LIPI
INTRODUCTION
Hystory of Drug Discovery :
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 bloodthinning properties (antiplatelets)
O
OH
1. Hydrolysis
Glu
2. Oxidation
O
OH Acetylation
OH
salicin
Salisylic acid
- more effective, - no bitter taste
- gastric bleeding
O
OH
O
Ac
Acetylsalicylic acid (aspirin)
- less irritating
- ester hydrolyzes to active drug
Research Phases in Drug Development
Target Identification
And Validation
Search of Lead
Structure
Optimization of
Lead Structure
Preclinical
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 models
Screening of chemical library by disease animal
models
4. From the modern approach to drug design
Designing drugs based on physiological mechanism
DISCOVERY of NOVEL DRUGS
from 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
HO
O
H
O
O
N
H
N
O
O
Lovastatin
O
O
O
OH
O
H
O
R
O
Aspergillus tereus
Anticholesterol -
UK-3A
Streptomycesp sp. 517-02
Cytotoxic to P338, KB
O
N
O
O
N
HO
HO
O
Phenazine carbioxylate
Pseudomonas pycocyaneae
O
O
Methyl cinnamte
O
Calanone
Callophyllum tesmanii
Time & Cost for A New Drug Development
Compounds
Duration
5000-10000
Research
250
Preclinical Phase
Costs (Mio US$)
3-6 yr
140
Phase I
1.5 yr
30
Phase II
2 yr
80
Phase III
3.5 yr
330
Authority”s
Assesment/NDA Phase
1.5 yr
60
Development/
Clinical Phase
5
1 drug
11 – 15 yr
ca. 750 Mio US$
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 5 H-bond donors.
The molecular weight is over 500.
The LogP is over 5.
There are more than 10 H-bond acceptors.
OPTIMAZATION ACTIVITY:
SYNTHESIS OF DERIVATIVES/
ANALOGOUS
SYNTHESIS DERIVATIVE OF
LEAD COMPOUNDS
R-OH
Alcohol
Ether
CH3-I
R-OMe
CH3COCl
R
O
or Ac2 O
CH3SO2C;l R
Ester
O
O
O
R-NH2
Amine
S
LiALH4
O
CH3COCl
R
or Ac2O
R-H Alkane
H
N
Amide
O
OPTIMIZE LEAD COMPOUND
O
OH
Reduction
O
O
R
R'
Ketone
NaBH4/LiALH4
R
R'
Alcohol
O
O
O
O
O
Strong base :
R
O
Ester
NaOH/KOH
LiALH4
R
OH
Acid
R
OH
Alcohol 1o
O
HO
O
O
O
ESTERS AS PRODRUGS
Prodrug
Drug
Fatty
barrier
esterase
O
C
R
O
C
Fatty
barrier
Prodrug
O
C
R
O
O
Drug
O
C
O
OH
O
esterase
C
R
O
R
Ester masking polar groups
allowing passage through
fatty cell membranes
OH
R
C
H
N
R'
NaOH
R
C
OH
+
H2N R'
O
O
Carboxylic acid
Amide:
LiAlH4
R
C
H2
Amine
NH2
1o Amine
NaH / MeI
CH3
N
R
C
R'
O
3o Amide
R
C
OH
H+ / R'OH
R
C
OR'
O
O
Ester
Carboxylic acid:
LiAlH4
R
C
H2
OH
1o Alcohol
OPTIMIZE LEAD COMPOUND
Analogs of pharmacophore (remember morphine)
Goals?
1.
Variation of alkyl substituents
2.
Variation of chain length
ANALOGUE
LEAD COMPOUND
CH3
H3C
C
CH3
CH3
Hydrophobic
pocket
van der Waals
interactions
EXAMPLE:
Adrenaline
H
HO
OH
H
N
CH3
HO
Salbutamol
(Ventolin)
HOCH2
(Anti-asthmatic)
HO
H
OH
H
N
CH3
CH3
CH3
H
Propranolol
(b-Blocker)
C
CH3
O
N
H
OH
CH3
Simplification
OH
HOOC
Ph
Drug
OH
NHMe
Cl
Ph
Drug
NHMe
OMe
HO
HO
HO
O
N
N
CH3
H
H
H
H
CH3
N
Me
Me
H
H
HO
Morphine
Excess functional groups
Levorphanol
Excess ring
Metazocine
CH3
DRUGS /LEAD COMPOUNDS
DEVELOPMENTS
PRODRUG- EUQUININE
Euquinine is the esterification
product of quinine with
chloro-formic acid ethyl ester
H
H
O
N
O
HO
+
O
N
O
Cl
O
H
H
N
O
O
N
ÓÅ¿üÄþ
Enquinine
SYNTHESIS OF ARTEMISINI DERIVATIVES
Reduction
NaBH4/EtOH
Artemisinin
Dihydroartemisinin
Methyllation (MeI),
Ethylation
Artesunate
CALANONE DERIVATIVES AND ITS CYTOTOXIC
ACTIVITY*
O
O
O
HO
HO
O
O
O
HO
O
O
HO
O
O
O
R
Calanone
Calanol
Log P 0.43
Against colon cancer cells
HCT116: IC50 > 20 µg/mL
L1210 : 59.4 µg/mL
P388 : IC50 = 15
Log P -0.42
Against colon cancer cells
HCT116: IC50 > 20 µg/mL
L1210 : 70.0 µg/mL
P388 : IC50 = 15
Cisplatin IC50 = 1.02 µg/ml
Ester Calanol
Log P 2.32
Against colon cancer cells
HCT116:
IC50 = 1.29 µg/mL
P388 : IC50 = 7,5 µg/mL
*atent: M. Hanai, 2006
DEVELOPMENT OF ANALOG UK-3A
POTENTIAL FOR BREAST CANCER TREATMENT
UK-3A Analog Development
OH
O
O
PSMOE
PSMOE
O
N
N
H
O
O
O
BcL-xL Protein
O
UK-3A
OH
N
A
N
O
O
O
O
N
H
B
O
C
UK-3A Ring opening (Analog UK-3A)
OH
O
H
N
O
O
O
O
PSMOE
QSAR PARAMETER & CYTOTOXIC
TEST RESULTS
N
O
N
H
N
O
H
N
O
OH
O
OH
O
O
R
O
OH
CH3
O
O
UK-3A
Log P -1.18
Ebinding = -7.1 kcal/mol
IC50 = >100 mg/ml
Log P 1.61
Ebinding = -11.65 kcal/mol
P388 : IC50 = 38 mg/ml
O
O
O
O
OH
H
N
HN
O
O
OHHO
O
NH
O
O
O
O
O
O
Taxol
Log P 1.67
Ebinding = -10.39 kcal/mol
OH
H
O
O
O
O
Antimycin A3
O
O
O
Log P 1.30, Ebinding = -10.24 kcal/mol
KB
:IC50 =
YMB-1:IC50 =
0.23 mg/ml
0.015 mg/ml
CYTOTOXIC TEST RESULTS TO P388, KB AND YMB-1
N
O
Ebinding=-9.66 kcal/mol),
H
N
O
OH
O
PDBGE : R = Butyl
O
N
O
OH
O
O
NDBGE : R = Butyl
O
IC50 34 mg/ml (P388)
IC50 2.28 mg/ml (KB)
IC50 1.83 mg/ml (YMB-1)
Ebinding=-10.29 kcal/mol);
O
H
N
Log P 1.5
Log P 1.62
IC50 38 mg/ml (P388)
IC50 1.92 mg/ml (KB)
IC50 5.46 mg/ml (YMB-1)
O
Log P 2.09
P388 :IC50 = 40,0 mg/ml
KB :IC50 = 0,82 mg/ml
YMB-1:IC50 = 2,69 mg/ml
Metabolite Secundar from Microbial Soil
Pseudomonas pycocyanea
8,53(dd)
8,35(dd)
H
H
8,02(dd)
130,15
H
133,22
N
125,08
130,29
143,46
139,91
137,45
128,03
7,98(dd)
H
8,28(dd)
H
144,16
131,73
H
135.14
N
140,12
H
8,04(dd)
8,98(dd)
165,88
HO
dH15,5 ppm
O
p-Carboxyl-phenazine
MIC 4,8 mg/ml (E. coli); 0,07 mg/ml ( S. aureus)
IC50 : 5,20 mg/ml (L1210)
Erythromycin : MIC 5,08 (E.coli), 4,06 (S. aureus) and 3,36 mg/ml
SYNTHESIS SALYCIL ANILIDE (SA)
NH2
OH
OH
+
H
N
DCC, DMAP,
O
CHCl3, RT, 1 d
OH
SA
O
SALYCIL ANILIDE DERIVATIVES
(PHENAZINES ANALOGS)
Log P 3.29
Ebinding = -10.21 kcal/mol
OH
L1210: IC50 = 4.8 mg/ml
N
H
P388 :IC50 = 7.75 mg/ml
KB :IC50 = 0.6 mg/ml
YMB-1:IC50 =2.97 mg/ml
O
OCH3
OH
N
OH
N
H
O
L1210 IC50 7,0 mg/ml
N
H
O
L1210 IC50 5,5 mg/ml
M. Hanafi, Paten P00200200449, 2002
CYTOTOXIC ACTIVITY RESULTS
Log P 3.29
H
N
OH
N
Salycil octyl amide (SOA)
O
P388 :IC50 = 7,55 mg/ml
KB :IC50 = 0,78 mg/ml
OH
H
N
O
NOA : Log P 3,02
IC50 (T47D) : 4,67 mg/mL
EFFICACY & TOXICITY TEST OF SALYCIL ANILIDE (SA)
OH
N
H
P388 :IC50 = 7.75 mg/ml
KB :IC50 = 0.6 mg/ml
YMB-1:IC50 =2.97 mg/ml
O
1. Acute Toxycity (LD50) : 365.83 mg/kg bw
and 429.46 mg/kg bw
1. Effective dose : 30 mg/kg bw
a
SYNTHESIS METHYL CINNAMTE DERIVATIVES
OH
O
CH3
OH
p-TSOH
+
kalor
o-Cresol
Cinnamic acid
O
O
CH3
8-Methyl-4-phenylchroman-2-one
OH
O
OH
p-TSOH
+
calor
Cinnamic acid
phenol
O
O
4-phenylchroman-2-one
CYTOTOXIC TEST TO LEUKEMIA CELL LINE P388
No.
1
Compound
IC50 (ppm)
Metthyl cinnamate (1)
O
O
20.35
Log P 2.2
2
Cinnamic acid (2)
48.85
O
3
4-phenylchroman-2-one (3)
209.20
OH
4
8-Methyl-4-phenylchroman-2-one
5
Phenyl cinnamte (5)
Log P 1.93
68.42
0.5
O
O
O
Log P 3.85
O
CH3
O
Log P 3.86
O
Log P 3.3632
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
Siynthesis Simvastatin from Lovastatin (1)*
HO
O
R1O
O
O
O
O
O
O
2, 3, 4
1
Lovastatin
R1O
O
HO
O
R1 = TBDMSi or OCH2OMe
O
O
O
O
O
5
O
O
Simvastatin
1. Protection :t-Bu(Me)2SiCl or (MeO)2CH2/P2O5
2. Hydrolysis (KOHaq or LiOH)
3. Cyclization, Heat/cyclohexane/pTsOH
4. Esterification : RCOCl, DMAP
5. Deprotection, TBAF/THF or PhSH
*US Patent, 6,506,929 B1, Jan. 14, 2003
SYNTHESIS DEHYDROLOVASTATIN
(LIPISTATIN)
HO
O
O
O
O
O
O
H +, Cyclohehane
H3
C
O
H 3C
O
CH 3
H3C
CH3
H3C
Lovastatin
Dehydrolovastaton
88,3 % (EtOH)
H:EtOAc (4:1)
INTERACTION ENERGY
WITH HMG CoA REDUCTASE AND LOG P
NO
Compounds
1 Substrat (HMG-CoA)
Interaction Energy (kcal /
mol)
Log P
- 10,5055
2 Dehydrolovastin
- 9.95
4.80
3 Lovastatin
- 9,48
3.77
4 Simvastatin
5 Buthyl ester (Lovastatin)
- 8,86
- 9,91
Interaction Dehydrolovastatin
and the active site of HMG-CoA reductase
5.73
4,92
Lipistatin Spectrum : 1H and
0.92 (t) O
1.12 (d, )
13C
6.02
H (d, 7.3 Hz)
6.86 (dt)
H
O
O
O
H
0.91 (d)
H 7.95 Hz)
5.99 (d,
H
H
5.39 (d, 2.6.Hz)
5.78 (dd, ...Hz)
1.08 (d, )
6.02
H (d, 7.3 Hz)
0.92 (t)
11.91
1.12 (d, )
16.47
O
O
164,53
O
27.02
41.64
176,81
O
67.92
36.80
27.65
121.68
H
145,.02
29.75
77.46
32.86
H
24.44
36.79
37.46
131,74
14.06
0.91 (d)
32.60
133,20
??
H
128,51 129,88 5.99 (d,
7.95 Hz)
1.08 (d, )
H
H
5.39 (d, 2.6.Hz)5.78 (dd, ...Hz)
NMR
Evaluation Results of Antihiperlipidemic Activity on Mice
for Lipistatin and Simvastatin
Parameter
Total
cholesterol
(mg/dl)
(%)
Trigliseride
(mg/dl)/(%)
LDLcholesterol
(mg/dl)/(%)
HDLcholesterol
(mg/dl)/(%)
Simvastatin
Normal Hiperlipi(7,2 mg/
control
demic
200 g bw)
Lipistatin
(7,2 mg/
200 g bw)
Lipistatin
(14,4 mg/
200 g bw)
111,79
156,66
112,03
(28,49%)
106,64
(31,93 %)
105,54
(32,55 %)
106,29
172,53
102,28
(40,72%)
103,85
(40,0%)
94,79
(45,06%)
32,34
72,99
30,23
(58,58%)
25,00
(65,75%)
28,77
(60,58%)
58,20
49,16
61,34
(24,77%)
60,87
(23,82%)
57,81
(17,60%)
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:
CONCLUSION
1. 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