Transcript Slide 1

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DIRECT FACTOR Xa INHIBITOR
AS ANTICOAGULANT
PREET PAL SINGH SIDHU
DEPT OF MEDICINAL CHEMISTRY
SCHOOL OF PHARMACY, VCU.
1
OVERVIEW
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1.
2.
3.
4.
5.
6.
Introduction
Comparison with current therapy.
Natural inhibitors of Factor Xa.
Structural details of active site.
Trend in Factor Xa inhibitors.
Rivaroxaban
- SAR
- Synthesis
- Crystal structure
- pharmacokinetic profile
7. Apixaban
- Synthesis
- Crystal structure
- Pharmacokinetic profile
8. Conclusion.
2
INTRODUCTION
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Factor Xa is K-dependent serine protease.
Molecular Wt is 45 K.
Factor Xa occupies the pivotial position in coagulation cascade.
Factor X is zymogen of Factor Xa.
Circulates in plasma as a light- and heavy-chain held together by a disulfide bond.
N-terminal light chain contain GLA and two epidermal growth factor-like domain.
C-terminal heavy chain contain the trypsin-like catalytic domain.
First Factor Xa deficiency was described in 1950s.
3
INTRODUCTION
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Two physiological inhibitor:
1)Tissue factor pathway inhibitor:
Regulate inhibition by Fxa dependent inhibition of FVIIa-Tissue factor complex
during initial stage.
Function by binding the active site of Fxa and FVIIa-Tissue factor Via second and
first kunitz domain.
No co-factor requirement.
2)Antithrombin:
Serine protease type inhibitor.
Function by binding site of Fxa through exposed reactive center loop and
induces the conformational change which trap enzyme in inactive, stable complex.
Require co-factor ( Glycosaminoglycans).
4
Rezaie et al, biochemistry, 2002, 41, 6780
MECHANISM
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EXTRINSIC PATHWAY
Prothrombinase
complex
FACTOR Xa
INTRINSIC PATHWAY
Direct
Factor Xa
inhibitor
PHOSPHOLIPID
FVa-Fxa
Ca+
Prothrombin
thrombin
fibrinogen
fibrin
platelet
aggregation
clot
FXa
5
DISADVANTAGES OF CURRENT THERAPY
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General disadvantage of current therapy:
Slow onset of action
parenteral mode of administration
excessive bleeding
Stringent monitoring requirement
HEPARIN:
Parenteral administration.
Monitoring requirement.
Excessive bleeding.
WARFARIN:
Excessive bleeding.
Monitoring requirement.
Severe enzyme induction.
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PREFERRED PROFILE
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A wide therapeutic window of antithrombotic effect versus bleeding.
A rapid onset and offset of action.
Oral bioavailability
No coagulation monitoring required.
Inhibition of free and clot- bound thrombin.
Reproducible PK and PD profile.
Minimal interaction with food and other drugs.
Minimal dose frequency.
7
Weitz et al blood 2005 105 453
Hirish et al thromb res 2003 9 suppl 1:S1-8
COMPARISON OF ANTICOAGULANT
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Agent
Inh. of clot
bound enzyme
monitoring
?
Heparin induced
thrombocytopenia
Route of
admin.
Risk/
Benefit
NO
I.V, oral
++++
Direct
FXa
YES
Direct IIa
YES
YES
NO
S.C., I.V.
+
Heparin
NO
YES
YES
I.V., S.C.
+
LMWH
NO
NO
YES
S.C., I.V.
++
Pentasac
caride
NO
?
?
S.C., I.V.
+++
Warfarin
NO
NO
Oral, I.V.
+8
YES
NATURAL INHIBITORS
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Antistasis•Isolated from extracts of mexican leech in 1987.
•It is slow, tight binding, selective Factor Xa inhibitor.
•Ki=0.3-0.6 nM.
TAP•A single 60 amino acid peptide isolated from extract of tick.
•Reversible, slow, tight binding inhibitor of Factor Xa.
•Ki=0.5 nM.
Ghilanten•Isolated from south American leech.
Simpson et al, j. biol. Chem, 1989, 264, 16694 9
Waxman et al, science,1990, 248, 593
S1 POCKET
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Most important pocket for binding for substrate or inhibitor.
Formed by 214-220 and 189-195 loops tied together by CYS 220- CYS 191
Disulfide bond.
Residues 225-228 form the base and lower strand of pocket.
Important residues are ASP 189, ALA 190 and GLN 192.
10
Rai et al current med chem, 2001, 8,101
S2 POCKET
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A small pocket merged with S4 pocket.
Likely this pocket defines the selectivity of selective Factor Xa inhibitor over thrombin.
11
Rai et al current med chem, 2001, 8,101
S3 POCKET
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Located on the rim of S1 pocket.
Most of Factor Xa inhibitor do not interact with this region.
12
Rai et al current med chem, 2001, 8,101
S4 POCKET
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S4 and S1 pocket represent the important binding region.
It is large and well defined pocket.
S4 pocket the 3 sub-regions:
Hydrophobic box
Cationic hole
water site
Hydrophobic box composed of Phe 174, Tyr 99, Trp 215.
Cationic hole is formed by Glu97 and Lys96.
Water is trapped underneath the Thr98, Ile175, thr175.
13
Rai et al current med chem, 2001, 8,101
TREND IN Fxa INHIBITORS
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Factor Xa inhibitors can be divided into three fragments:
P1 Group: Binds in the S1 sub-pocket.
Central Scaffold: project the substituent's into sub-pocket.
P4 Group: Binds in the S4 sub-pocket.
P1 group is further divided into 3 types:
- Highly Basic Group.
- Moderately Basic Group.
- Neutral Group.
Scaffold is further divided into 2 types:
- Flexible Scaffold.
- Rigid Scaffold.
14
Maignan et al. Curr. Top. Med. Chem. 2001, 1, 161
HIGHLY BASIC P1 GROUPS
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-salt bridge with Asp189
-H-bond with Gly219
-Poor bioavailability
HN
-10 fold increased potency
--Enhanced oral absorption
NH2
Benzamidine
H 2N
NH
Napthyl amidine
NH
S
HN
NH2
Amidino thiophene
-Similar inhibition constant
-Enhanced oral absorption
-4-fold more active than
benzamide series
HN
NH2
Amidino indole
MODERATELY BASIC P1 GROUPS
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Form water mediated interaction with Asp189
Form H- bond with Gly219
10 times better at crossing Caco-2 monolayer
than benzamidine
H 2N
N
Amino isoquinolines
HN
Similar pharmacokinetic activity and binding
mode as amino isoquinolines
N
Aza indole
NEUTRAL P1 GROUP
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O
-Introduced by Lilly pharm.
-17-fold lower inhibition
-Improved Pk profile
Cl
O
-6-fold increase in potency
-Efficacy same as benzamidine
Chloro phenyl
Methoxy benzoyl
Cl
Cl
S
Chlorothiophene
S
Chloro thiophene
LINKER SCAFFOLDS (FLEXIBLE)
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COOCH3
O
H3COOC
NH
NH
Aliphatic chain
- By Daiichi Pharm.
O
NH
Allyl linker
Amide bond
- To remove chiral center
- Gain H-bond to Gly219
Amide ester
-By Aventis Pharm.
-Increases potency by 9 fold
-Increases selectivity by 5 fold
-Ester group located in small
hydrophobic pocket
O
NH
O
NH
S
Sulfonamide
O
COOH
Pentanoic acid
-Designed to circumvent
chiral center
LINKER SCAFFOLDS (RIGID)
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H O
N
S
O
O
N
Sulfonamido pyrrolidinone
N
N
O
Cyclo heptanonediene
By Aventis pharm.
Potential for
additional interaction.
By Berlex.
Photochemical liable
O
O
Ether link
Cyclic urea
By Berlex
Optimal linker
By DuPont
H- bond with Gly216
N
N
O
N
N
Benzimidazole
O
Carbazole
By Berlex
To add hydrophilicity
Benzoxazinone
By Berlex
To add hydrophilicity
Introduced by Pfizer
P4 GROUPS
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O
N
O
Methoxy benzene
By Lilly Pharm.
Phenyl lies parallel to
Trp215
mM potency
O
H
N S
O
Amido diphenyl
Phenyl sulfonamide
N
Pyridyl piperidine
To increase hydrophilicity
Pyridine stack with Trp215
H-bond with structural
water
Additional H-bond
with Gly219
By DuPont Pharm.
Edge to face with Trp215
OH interact with Tyr99
O
O
N S
O
O
Methoxy naphthalene sulfonamide
Sulfonamide has
conformational role
HN
H2N S O
O
Benzyl sulfonyl
piperidine
Edge to face interaction
with Trp215
Piperidine play no role
H
N
HN
N
NH2
Amido benzofuran
Phenyl imidazoline
By Berlex
Both ring are parallel to
each other and
perpendicular to Trp215
H-bond with Asn97
Parallel to Trp215
DRUGS AND THEIR CURRENT STATUS:
DRUG
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MANUFACTURER
STAGE AND INDICATION
Rivaroxaban
Bayer healthcare
and scious.Inc
Phase III for VTE, acute DVT, strokes in patient
with AF
LY517717
Lilly
Phase II for VTE prevention
YM150
Astellas
Phase II for VTE prevention
DU-176b
Daiichi Sankyo
Phase II for VTE prevention, phase IIb for strokes
in patient with AF
Apixaban
Bristol myers
squibb
Phase III for VTE prevention , acute DVT
813893
GSK
Phase I
PRT-054021
Portola
Phase II for VTE prevention
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RIVAROXABAN(BAY-59-7939)
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Oral , direct Fxa inhibitor under development.
It is oxazolidinone derivative.
IC50=21nM.
Currently in Phase III of clinical trail.
If approved , it will be marked by name Xarelto.
It is joined product of Bayer and ortho-McNiel pharmaceuticals.
22
SAR OF RIVAROXABAN
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O
N
N
1
IC50= 120nM
H2N
F
N
O
O
NH
S
O
HN
HN
Cl
S
O
N
O
N
3
IC50= 20μM
O
F
2
IC50= 8nM
HN
S
O
R
N
O
Cl
S
O
O
O
O
HN
N
4
IC50= 90nM
O
HN
S
O
Cl
SAR OF RIVAROXABAN
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O
F
S
O
N
N
Cl
HN
S
O
I.
SAR around thiomormpholine
ring and the enatiomeric
position.
R2
R3
R1
II.
Modifications on the amide
linker and thiophene moiety
O
O
O
N
O
*
Cl
HN
O
N
N
R2
N
R1
X
S
O
O
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Roehrig S, Straub A, Pohlmann J, et al. J. Med. Chem. 48 (19): 5900
SAR around the aryl moiety
For
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R2
R1
O
N
R3
R1
*
N
Cl
HN
O
S
O
R1
R2
R3
N
O
N
F,H
s
90
F,H
N
s
H,H
N
N
F,H
N
HN
N
s
s
N
N
32
43
N
s
74
F,H
s
140
H,H
s
0.7
F,H
s
1.4
CF3, H
s
1.O
NH2, H
s
2.5
O
O
N
H , CH3
s
1260
O
N
H,H
R
2300
40
H,H
4.0
O
O
O
s
O
O
O
H,H
O
ent IC50 [nM]
O
S
R2 R3 ent IC50 [nM]
O
25
Modifications on the amide linker and thiophene moiety
R1
X R2 IC50 [Nm]
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Cl
S
CO H
2000
CO H
8.5
CO H
290
CO H
29
CO H
94
CO CH3
197
Cl
NH2
R1
X
R2
IC50 [Nm]
S
S
S
Cl
Br
S
CH3
O
Br
CO H
0.7
CO H
0.4
Cl
N
N
Cl
CO H
9.2
N
Cl
CO H
26
CO H
20
CO H
1170
Cl
S
Cl
S
Cl
Cl
SO2 H
1200
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SYNTHESIS OF RIVAROXABAN (SCHEME I)
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O
NO2
NH
O
NaH, NMP
+
O
F
NH2
O
H2, Pd/C, 700C
N
O
1
NO2
N
O
3
4
2
O
O
N
O
EtOH, H20
O
O
5
O
N
H
N
OH
CDI, DMAP, THF
N
toluene
aminoalcohol adduct O
6
27
Bayer healthcare Drug Fut 2006, 31, 484
SYNTHESIS OF RIVAROXABAN (SCHEME I)
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O
O
O
N
O
N
oxazolidinone
7
O
O
S
9
Cl
O
O
O
Cl
CH3NH2, H2O
EtOH, reflux
O
N
O
O
N
N
N
O
NH2
8
O
N
Cl
O
Rivaroxaban
S
H
N
O
28
Bayer healthcare Drug Fut 2006, 31, 484
SYNTHESIS OF RIVAROXABAN (SCHEME II)
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OH
HO
Cl
S
O
Cl
NH2. HCL
2
NaHCO3, H2O, MTHF
HBr, AcOH
N
N
OH
Br
O
H
N
S
3
1
4
Cl
O
Bromohydrin
Cl
O
O
NH2
O
H
N
5
OH
S
H
N
O
O
CDI, NMP
toluene,
O
N
O
N
Cl
O
Rivaroxaban
S
H
N
O
Bayer healthcare Drug Fut 2006, 31, 484
CRYSTAL STRUCTURE
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MAJOR BINDING COMPONENT
1. Hydrogen with Gly219
2. L-Shape provided by oxazolidine
3. Carbonyl doesn’t interact
4. Chlorine-Tyr228 interaction
Bayer healthcare, J. Med. Chem, 2005, 48, 5900
PHARMACOKINETIC PROFILE
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ABSORPTION AND DISTRIBUTION:
Peak plasma conc. is reached in 2 to 4 hours after oral administration.
Bioavailability range from 60-80%.
Presence of food increases maximum conc., time to maximum conc.
and AUC.
Bound extensively to protein (90%).
No effect of increased gastric pH on PK-PD profile.
METABOLISM:
Hepatic metabolism Via cytochrome P-450 3A4.
EXCRETION:
Both renal and hepatic excretion.
36% of drug is excreted unchanged in urine.
31
PHARMACOKINETIC PROFILE
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EFFECT OF AGE:
AUC was higher in patient above 75 years.
EFFECT OF RENAL INSUFFICIENCY:
AUC was 44%, 52%, 64% higher in mild, moderate and severe
renal insufficiency.
Still no recommendation in dose adjustment for renal insufficiency.
EFFECT OF HEPATIC INSUFFICIENCY:
With mild hepatic disease, no difference in PK-PD profile.
With moderate hepatic insufficiency, decrease in total body
clearance.
Patient with severe renal failure are excluded from clinical trails.
EFFECT OF OBESITY:
Compared in different weight group (<50, 50-80, 80-120, >120).
In higher Wt. group, AUC and inhibition was lower.
In lower Wt. group, Cmax was higher and half life was 2 hour longer.
EFFECT OF GENDER AND RACE:
No difference in PK-PD was found between the gender.
APIXABAN
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Apixaban is follow-up compound of razaxaban, a direct Fxa inhibitor.
Highly selective and potent (Ki=0.8nM) inhibitor of both free and bound Fxa.
It is product of Bristol-Myers Squibb (BMS).
CURRENT STATUS:
1). Phase III for VTE prevention study.
2). Phase III for prevention of stroke.
3). Phase III for other thromboembolic events in patient
with atrial fibrillation.
33
SYNTHESIS OF APIXABAN
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4
2
1
3
6
5
CRYSTAL STRUCTURE
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35
PHARMACOKINETIC PROFILE
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ABSORPTION AND DISTRIBUTION:
Apixaban is rapidly absorbed, Cmax in 1 hour.
Oral bioavailability is 80%.
Average half life is 12 hours.
METABOLISM:
70% of drug excreted unchanged.
CYP3A4 may be involved in metabolism.
EXCRETION:
60% excreted by fecal route.
25% excreted in urine.
Minor route of excretion is biliary.
36
Zhang et al, Drug. Met. Disp. 2008 (in press)
DO FACTOR Xa RELATE WITH CANCER??
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Cancer patient have elevated level of coagulation abnormalities.
Thrombosis is most frequent complication and 2nd leading cause of death in cancer.
Factor Xa, thrombin and tissue factor contribute to tumor growth.
Factor Xa promote tumor growth in 2 ways:
Fibrin provide network for growth and block access of immune system.
Recognition and binding to tumor cell to initiate cellular event.
Heparin suppress small cell lung cancer and B16 melanoma cell growth in animals.
Antistasin suppress T241 sarcoma cell growth in mice.
Ghilanten also suppress B16 melanoma cell growth in mice.
DX-9065a shown to inhibit A549 lung adenocarcinoma cell proliferation.
37