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DirectactingHCVantiviralagent,Sofosbuvir,makesonepillthatfitsall
ZahraRashidi
01/25/2017
1
CONTENT
§ IntroductionI. hepatitisCII. treatment1990-2013III. currenttreatmentIV. HCVlifecycle§ Developingofsofosbuvir§Mechanismofinhibitoryeffectofsofosbuvir§ synthesisofsofosbuvir
2
• A majorpublichealthproblemworldwide• 180millionindividualsinfectedworldwide4timesmorethanHIV• Themostcommonindicationforlivertransplantation,accountingfor40–50%oflivertransplants
HepatitisC:
3http://www.educatingwomen.org/hepatitis-c/
Nat.Rev.DrugDiscov.2013 ,12,595-610
hepatitisCvirus(HCV)exhibitsenormousgeneticvariability duetohighmutationrates,TherearesixmajorHCVgenotypesthatdifferfromeachotherby30%-35%,andalsoover100subtypes.
Thereisnoapproved vaccine.
4
Genotypediversity:
pegylated interferon
Ribavirin(RBV)
Generalstandardofcare:
5
J.Hepatol. 2015,62,s87-s99
Potentialsideeffects:• influenza-likesymptoms• fatigue• hemolyticanemia• depression
currentproblems:
6
IsitpossibletodesignanewefficientdrugagainsthepatitisC?
efficientdrugwithfollowingfeatures:• Highpotency• Pangenotypiccoverage• Highbarriertoresistance• Oral–singledosetherapy• INF-free• Minimaltoxicityandsideeffect
developmentofdirectactingantivirals(DAAs)
targetingessentialviralproteins ??
7
Featuresofnewdrug:
O
HO F
NH
N
O
O
CH3
OPO
HN
OO
O
CH3
Sofosbuvir( Sovaldi)
• discoveredin2007andapprovedformedicaluseintheUnitedStatesin2013• $10.3billionsoldin2014,thebest-sellingdrugintheworldinonlyitsfirstyearonthemarket• thecostofa12-weekregimenofSovaldi isaround$84,000• upto97%curerate
J.Hepatol.2015,62,s87-s99 8
Sofosbuvir treatment:
9
HepatitisCvirus:
http://www.medicalnewstoday.com/articles/294705.php
Nat.Rev.Gastroenterol.Hepatol. 2009,6,403-411
HCVlifecycle:
10
ERmembrane
targetingRNApolymerase:
11NatureRev.Microbiol. 2013,11,482-496
conservedactivesite
CONTENT
§ IntroductionI. hepatitisCII. treatment1990-2013III. currenttreatmentIV. HCVlifecycle§ Developingofsofosbuvir§Mechanismofinhibitoryeffectofsofosbuvir§ synthesisofsofosbuvir
12
N
NNH
NNH2
NH
NNH
NO
NH2
N
NH
NH2
O
NH
NH
O
O
Adenine Guanine
Uracil Cytosine
O
Base
OHHO
2ʹ3ʹ
O
PO
OO
P
O
O
OP
O
OO
5ʹ
nucleosidenucleoside monophosphate
nucleoside diphosphatenucleoside triphosphate
pentose
2ʹ OH = Ribose
2ʹ H = Deoxyribose
RNA
DNA13
PreviousBase IncomingBaseNEWRNACHAIN
RNATEMPLATE
OOOH
OH
OHO O
O
PO
O
OOH
OHO
P
OOP
O
O O
P
OO
OO
DesignaRNApolymeraseinhibitor:
14
PreviousBaseNEWRNACHAIN
RNATEMPLATE
IncomingBase
OOH
OHO
P
OOP
O
O O
P
OO
OO
DesignaRNApolymeraseinhibitor:
OOH
OHO
OO
P
OO
Z
X Y
15
Isolatedenzymeassay
RNApolymerase O
OHHO
HOBase
HCVRNAreplicationinacell-basedassay
TheIC50istheconcentrationofaninhibitorwheretheresponse(orbinding)isreducedbyhalf.TheEC50istheconcentrationofadrugthatgiveshalf-maximalresponse.
DesignexperimenttomeasureIC50 andEC50:
BaseO
OHHO
OPOO
OPOO
OPO
OO
16
• bindingaffinityofNTPtoRNApolymerase• inhibitoryeffectofNTP
• cellularuptakeofnucleosides• conversiontoNTP• metabolismanddeactivation• andotherfactors
O B
OHHO
HOHH 2′
3ʹ• modificationofribosering• substitutiononC1ʹandC4ʹ
• substitutiononC2ʹandC3ʹ
O B
OHHO
HOB
OHHO
HOO
O B
OH
HO
O B
OHHO
HOO B
OHHO
HO
X O B
OHHO
HOO B
OHHO
HO
X
J.Med.Chem.2012,55,2481-2531 17
Modificationofnucleosidestogetactiveinhibitors:
O B
OHHO
HO
Nucleoside EC50(µM)Cytidine 200
3ʹ-Deoxycytidine 45
3ʹ-Deoxyuridine 200
3ʹ-Deoxyguanosine 500
3ʹ-Deoxyadenosine 150
2ʹ,3ʹ-DideoxycytidineZalcitabine
200
3ʹ-fluoro-3ʹ-deoxyguanosin 1.2
AntiviralRes.2003,58,243–251
Nucleotide Km(µM) Ki(µM)CTP 0.06± 0.001
GTP 0.23± 0.05
UTP 0.24± 0.05
ATP 10± 2
3ʹ-dCTP 0.72± 0.2
3ʹ-dGTP 0.93± 0.08
3ʹ-dUTP 5.9± 0.5
3ʹ-dATP 23± 5
2ʹ,3ʹ-Dideoxycytidine
9100
3ʹ-fluoro-3ʹ-dGTP 1.8
removalof3ʹ-hydroxylgroupreducesanucleotide’saffinitytotheenzymecomplex
Roleof3’-hydroxylgroup:
18
Isolatedenzymeassay
O
OHHO
HOBase
Kinase1 Kinase2
Kinase3
NucleosidetriphosphateisthesubstrateforRNApolymeras
BaseO
OHHO
OPOO
O
BaseO
OHHO
OPOO
OPOO
O
BaseO
OHHO
OPOO
OPOO
OPO
OO
19
Phosphorylationisnecessary:
Kinase1
NatureStruct Biol,2003,10,513-519
O
OH
HOBase O
OH
OBase
PO
OO
20
Phosphorylationisnecessary:
ComparisonbetweensubstrateefficiencyofUTPanalogs
UTPanalogue relativesubstrate affinity commentUTP 1 naturalsubstrate
2ʹ-F-uridine 0.16 substrate butnotinhibitor
2ʹ-NH2 -uridine 0.03 substrate butnotinhibitor
2ʹ-arabinouridine 0.0015 poorsubstrate
2ʹ-N3 -uridine 0.0005 poorsubstrate
unabletoinhibitHCVNS5B
Antimicrob.AgentsChemother. 2004,48,651–654
O B
HOH
HO
HO
2ʹ-arabinouridine
nucleosideanalogueshouldhavehydrogenbonddonor/acceptor in2ʹposition
Roleof2’-hydroxylgroup:
21
O B
OHHO
HO 2ʹ
O B
ba
HO
HO
a b B Inhibitorypotency(IC50),µM
RepliconEC50µM
H OCH3 adenine 47 >50
H OCH3 guanine 1.6 >50
H OCH3 cytosine 3.8 21.2
CH3 OH adenine 1.9 0.3
CH3 OH guanine 0.13 3.5
CH3 OH cytosine 0.09-0.18 1.23
CH3 OCH3 adenine >50 >50
Et OH adenine >50 >50
J.Med.Chem.2004,47,2283-2295
InhibitoryPotencyof2ʹ-ModifiedNucleosides/NucleosideTriphosphates
Modificationofaandb onC2ʹ:
22
O B
OCH3
HHO
HOO B
OH
CH3HO
HO
O
ba
HO
HO c
N
NN
NNH2
a b c Inhibitorypotency(IC50),µM
RepliconEC50µM
H OH CH3 >50 >50
EffectofsubstitutiononC3:
J.Med.Chem.2004,47,2283-2295
nosubstitutionatc
23
OHO
HO OCH3
N
N
NH2
O
Pharmacokineticsstudy:
2ʹ-O-methylcytidine
OHO
HO OH
N
N
NH2
O
CH3
2ʹ-C-methylcytidine
OHO
HO OCH3
N
N
NH2
OO
O
HO OCH3
N
N
NH2
OPOPOPO
OOO
OOOO
O
HO OH
N
N
NH2
OPOPOPO
OOO
OOO+
A B
24
HPLCofextractfromcellsincubatedwith2ʹ-O-methylcytidine
AB
J.Biol.Chem.2003,278,11979-11984
OHO
O OH
N
N
NH2
O
CH3O
H2NNM283
discontinuationofhumanclinicaltrialduetosignificantgastrointestinaltoxicity
OHO
HO OH
N
N
NH2
O
CH3
2ʹ-C-methylcytidine
Humanclinicaltrialof2ʹ-C-methylcytidine:
25
OHO
HO F
N
N
NH2
O
potentialtoxicity lackofinhibitoryeffect goodselectivity&inhibitoryeffect
_ InhibitorypotencyIC50 =>1000µM
IC50 =0.21± 0.05
OHO
HO OH
N
N
NH2
O
CH3
OHO
HO F
N
N
NH2
O
CH3
Efficacyof2ʹ-F-2ʹ-C-methylclassofnucleosides:
26
HPLCprofileofanextractofprimaryhumanhepatocytesincubatedwith2ʹ-F-2ʹ-C-methylcytidine(PSI-6130)
referencecompounds
extractfromhepatocytesincubatedwithPSI-6130
Pharmacokineticsstudy2ʹ-F-2ʹ-C-methylcytidine:
OHO
HO F
N
N
NH2
O
CH3
PSI-6130
a
OO
HO F
N
N
NH2
O
CH3O3P2
PSI-6130-MP
d
OO
HO F
N
N
NH2
O
CH3(O3P)22
PSI-6130-DP
f
OO
HO F
N
N
NH2
O
CH3(O3P)32
PSI-6130-TP
h
27J.Biol.Chem.2007,282,29812-29820
OHO
HO F
N
N
NH2
O
CH3
HPLCprofileofanextractofprimaryhumanhepatocytesincubatedwith2ʹ-F-2ʹ-C-methylcytidine(PSI-6130)
referencecompounds
extractfromhepatocytesincubatedwithPSI-6130
OHO
HO F
NH
N
O
O
CH3
OHO
HO F
N
N
NH2
O
CH3O
O
HO F
NH
N
O
O
CH3(O3P)32O
O
HO F
NH
N
O
O
CH3(O3P)22O
O
HO F
NH
N
O
O
CH3O3P2
c eg
b
Pharmacokineticsstudy2ʹ-F-2ʹ-C-methylcytidine:
28J.Biol.Chem.2007,282,29812-29820
29
OHO
HO F
N
N
NH2
O
CH3
OO
HO F
N
N
NH2
O
CH3O3P2 O
O
HO F
N
N
NH2
O
CH3(O3P)22
OO
HO F
N
N
NH2
O
CH3(O3P)32
Kinase1 Kinase2 Kinase3
OO
HO F
NH
N
O
O
CH3(O3P)32
OO
HO F
NH
N
O
O
CH3(O3P)22O
O
HO F
NH
N
O
O
CH3O3P2O
HO
HO F
NH
N
O
O
CH3
deamination
Kinase2 Kinase3
Secondmetabolicpathwayfor2ʹ-F-2ʹ-C-methylcytidine:
process EnzymeEfficiency(Kcat/Km)µM-1S-1 foreachsubstrate2ʹ-F-2ʹ-C-methyl cytidine 2ʹ-F,2ʹ-methyluridine
1st phosphorylation 1.9 ⤬ 10-4 _
2nd phosphorylation 0.012 0.009
3rd phosphorylation 0.015 0.046Antimicrob.AgentsChemother.2008,52,458–464
compound Inhibitorypotency(IC50),µM RepliconEC50µM
2ʹ-F-2ʹ-C-methyl cytidine ND 0.6± 0.04
2ʹ-F,2ʹ-methyluridine ND >100
2ʹ-F-2ʹ-methyl cytidine-TP 0.023± 0.002 ND
2ʹ-F,2ʹ-methyluridine-TP 0.141± 0.03 ND
OHO
HO F
N
N
NH2
O
CH3
PSI-6130
OHO
HO F
NH
N
O
O
CH3
2ʹ-F,2ʹ-methyluridine
meanhalf-lives:4.7h38h
Inhibitoryeffectof2ʹ-F-2ʹ-C-methyluridine:
bettercandidate
30J.Biol.Chem.2007,282,29812-29820
OHO
HO F
NH
N
O
O
CH3
OO
HO F
NH
N
O
OPO
O
O CH3
OO
HO F
NH
N
O
OPOPOPO
OOO
OOO CH3
firstphosphorylation
OO
HO F
NH
N
O
OPO
O
O CH3
OO
HO F
NH
N
O
OPOPOPO
OOO
OOO CH3
Skipthefirstphosphorylation:
31
O
POAr
O
O NucleosideO
POAr
O
O NucleosideOH
O
PHO
O
O Nucleoside
O
Ar
OH
P O Nucleoside
O
Ar
O
HO
cytochrome 450 spontaneous
spontaneous
Antivir.Chem.Chemother. 2011,22,23–49
Designmonophosphateprodrug1:
32
P O Nucleoside
OHP O NucleosideO
HO
EsteraseO
OO
S
SRO
RO
P O Nucleoside
OOO
S
HS
RO
S
P O Nucleoside
OOHO
SRO
Esterase
S
33
Designmonophosphateprodrug2:
Antivir.Chem.Chemother. 2011,22,23–49
P O Nucleoside
OHP O NucleosideO
HO
O
Nucleoside
OAr
HNO
O
R2
R1
Carboxyesterase
orcathepsin A
P O NucleosideO
OAr
HNHO
O R1
Spontaneous
HNO
P
O
O
OR1
H2OP O NucleosideO
OHHNHO
O R1
Phosphoramidase
J.Med.Chem.2010,53,7202–7218Antivir.Chem.Chemother.2011,22,23–49 34
Designmonophosphateprodrug3:
O
HO F
NH
N
O
O
CH3
OPO
HN
OO
O
CH3
Rp (PSI-7976) Sp (PSI-7977)PSI-7851
J.Med.Chem.2010,53,7202–7218J.Biol.Chem. 2010,285,34337–34347
Differentactivityoftwodiastereoisomer ofPSI-7851:
Sofosbuvir
35
O
HO F
NH
N
O
O
CH3
OPO
HN
OO
O
CH3
O
HO F
NH
N
O
O
CH3
OPO
HN
OO
O
CH3
EC50µM=0.149± 0.001 EC50µM=1.07± 0.04 EC50µM=0.092± 0.005
P O NucleosideO
OAr
HNO
O
R2
R1
Carboxyesterase
orcathepsin A
OO
O F
N
N
NH2
O
CH3O
O
RG7128
OHO
O OH
N
N
NH2
O
CH3O
H2N
NM283
O
HO F
NH
N
O
O
CH3
OPO
HN
OO
O
CH3
PSI-7977
firstgeneration secondgeneration
800mgtwicedaily 1000mgtwicedailydiversemetabolizationnotoxicity
400mgoncedailylongerhalflifenotoxicity
discontinued CompletedPhaseII FDAapprovedstatus
36
Comparisonofdifferentagents:
Antivir.Chem.Chemother. 2011,22,23–49
CONTENT
§ IntroductionI. hepatitisCII. treatment1990-2013III. currenttreatmentIV. HCVlifecycle§ Developingofsofosbuvir§Mechanismofinhibitoryeffectofsofosbuvir§ synthesisofsofosbuvir
37
Science,2015,347,771-775
structuralstudyofRNAreplicationbyRNApolymerase:
RNAtemplate
newRNA
incomingnucleotide
38
D319 D318 D220
R158
N291
D225
S282
Complementarynetworksforrecognizingribonucleotidesubstrates:
O
O
H2N NH
NH2
HO
H2N
O39Science,2015,347,771-775
O
B
4-(O3P)3O
OH
HN
O
O
H
O
O
N291
S282
D225
2.1 Å
2.7 Å
2.2 Å
H
OH
O
B
4-(O3P)3O
F
HN
O
O
H
O
ON291
S282
D225
7.0 Å
5.1 Å
H2.1 Å
Me
OH
Hydrogenbonddonor/acceptorin2ʹposition:
40
Chainterminationofsofosbuvir:
OHO
HO F
NH
N
O
O
CH3
41Science,2015,347,771-775InfectDisord DrugTargets2006,6,17-29J.Biol.Chem.2003,278,49164-49170
CONTENT
§ IntroductionI. hepatitisCII. treatment1990-2013III. currenttreatmentIV. HCVlifecycle§ Developingofsofosbuvir§Mechanismofinhibitoryeffectofsofosbuvir§ synthesisofsofosbuvir
42
XPO
OPhNH
O
O+O N
CH3FHO
NHO
O
O
PO
OPhNH
O
O O N
CH3FHO
NHHO
O
OSofosbuvir
9
Synthesisofsofosbuvir:
43
O N
OHHO
NHO
NH2
O
O N NNHBz
O
HO
HOHO
O N
OO
NNHBz
O
OTIPDS
O N
OHO
NNHBz
O
O
Si
SiO
i_Pri_Pr
i_Pr
i_Pr
O N NNHBz
O
BzO
HOBzO
1. Bz2O, DMF, rt
2. TIPDSCl2, DMF
DMSO, TFAATEA, -15 ℃ 1. MeLi, -78℃
2.TBAF, conc HOAc
BzClpyridine, rt
(58%, 3 steps)
(61%, 2 steps) (67%)
1 2 3
4 5
1.80% HOAc, reflux, 87%
2. MeOH/NH3, rt, 76%
O N NHO
O
HO
FHO
9
J.Med.Chem.2005,48,5504-5508
1.Late-StageFluorinationApproachtoprovideNucleosideCore
44
O N NNHBz
O
BzO
FBzO
O N NNHBz
O
BzO
OHBzO
O N NNHBz
O
BzO
BzO
DASTtoluene, -20℃
+
(19%)
(15%) (14%)6 7
8
SF
FF
NEt
Et
XPO
OPhNH
O
O+O N
CH3FHO
NHO
O
O
PO
OPhNH
O
O O N
CH3FHO
NHHO
O
OSofosbuvir
9
Synthesisofsofosbuvir:
45
OHO
HO F
N
N
NH2
O OHO
HO F
ONH
N
NH2
O
46
2.Early-StageFluorinationApproachtoprovideNucleosideCore O
O
CHOO
O
CO2Et
MeOEt
OMe
PPH3
101191%
E/Z: 97:3
O
O
CO2EtMe
OHHO12
AD-mix βt-BuOH, H2O, rt
O OHO
OHHO
HCl, H2O,EtOH,rt
16
BzClpyridine,rtO OBzO
OHBzO 55%
DAST or deoxyfluorTHF, 0℃
O OBzO
BzO OHDIPEA
DAST or deoxyfluorTHF, 0℃
34
35
50%
CH2 (OMe)2,TfOH O OMOMO
OHMOMO
DAST or deoxyfluor-78℃, amine
87%
O OMOMO
MOMO F
36 37
17
O OBzO
BzO F
U.S.Patent8,481,713B2,Jul.9,2013.
O OAcBzO
BzO F
1.Li(O-tBu)3AlHTHF, -20 0℃
2. Ac2O, DMAP, -20 0℃
2.NH3, MeOH, rt
N
N
OTMS
NHBz
1819
OHO
HO F
N
N
NH2
O OHO
HO F
N
NH
O
O
94:1 β/!29% β from 17
1.SnCl4, PhCl, 65℃
47
O
O
CHOO
O
CO2Et
Me
101191%
E/Z: 97:3
O
O
CO2EtMe
OHHO12
AD-mix βt-BuOH, H2O, rt
OEt
OMe
PPH32.Early-StageFluorinationApproachtoprovideNucleosideCore
17
O OBzO
BzO F
BzClpyridine,rt 71%
O
O
CO2EtMe
OHBzO
O
O
CO2EtMe
FBzO
O OHO
BzO F
DAST or deoxyfluorTHF, 0℃68%
TFA, MeCN, 80 0℃then PhCH3 ,Δ
58%
13
14
BzClpyridine,rt
EtOAc, 60 0℃91% 15
O
O
CO2Et
MeO
O
CO2EtMe
OHHO
O
O
CO2EtMe
OO SO O
O
O
MeFHO3SO
CO2Et
O
O
CHO
10 11 12 20
21
KMnO4acetone, 0℃
1.SOCl2,NEt3CH2Cl2, 0℃
2. TEMPO, NaOClNaHCO3, MeCNH2O, 0℃
J.Org.Chem.2009,74,6819–6824
O OBzO
BzO F
17
67%
3.Industrialearly-StageFluorinationApproachtoprovideNucleosideCore
48
Et4NF1,4 -dioxane,100 ℃
O OHO
HO F
67% from 12
O
OTBS
F
MeiPrO
OTMPOBn+ O
O
F
OH
OTMPMeOBniPr
TiCl2(O_iPr)2CH2Cl2, -20℃
79%22 23 24
J.Am.Chem.Soc.2014,136,5900–5903
25% overall yield
4.Developmentofearly-StageFluorinationApproach
O OBnO
HO F
1.Zn, AcOHEtOH, H2O,rt
2.TFA, toluene, rt80%
TBSOTf, 2,6-lutidine
CH2Cl2, 0℃95%25
99% ee
O OBnO
TBSO F
OBnO
TBSO F
OAc OBnO
TBSO F
N
N
NHBz
O OHO
HO F
N
N
NH2
O
N
N
NHBz
OTMS
1.DIBAL, CH2Cl2 -78℃
2.Ac2O, pyridineDMAP,CH2Cl2,-78℃92%
SnCl4, PhCl, 70℃ 1.BCl3,CH2Cl2,-78℃
2.HCl,MeOH,rt3.NH3,MeOH, -40℃94%
β: 62%!: 27%
26 27 28 19
20:1 dr isoated
49
Z:E ratio dose not given
O P ClO
ClPh +
O N NHO
O
HO
FHO
sofosbuvir (crude)
99.74%purity15.2%yieldfrom9
9N
N
NMI, THF, rtDCM, -78 ℃
O N
CH3FHO
NHO
O
OPO
OPhNHO
O
O N
CH3F
NH
O
O
OPO
OPhNHO
O
HOO N
CH3F
NH
O
O
OPO
OPhNH
OO
OPO
OPhNHO
O
O
ONH
PO
ClOPhO
ONH2 .HCl
J.Med.Chem.2010,53,7202–7218
29 30 31
50
Phosphoramidation:
O
ONH2 .HCl recryst.O P Cl
O
ClPh +
1.NEt3, -70℃2.C6F5OH, 0℃ O
ONH
PO
OC6F5OPh O
ONH
PO
OPhOC6F5
O
ONH
PO
OPhOC6F5 + O N NH
O
O
HO
FHO
t-BuMgCl,THF-5℃ to 5℃ , 17h recryst.
68% yield99.72% de99.79%purity
J.Org.Chem.2011,76,8311–8319
then aqueous workupcrystalization( PhMe/CH2Cl2)98.21% purity
32 33
51
Diastereoselective phosphoramidation approach:
(Sp:SR 1:1) 34%(Sp:SR = 99:1)
O N
CH3FHO
NHO
O
O
PO
OPhNH
O
O
Conclusion:
• HCVinfectionisacomplexdiseasewithlargepopulationofpatients,• TargetingtheactivesiteofvirusRNApolymeraseisaninterestingideatodevelopadrugformultiplegenotypes,• ModificationofriboseringofnucleosidebymethylandfluorinesubstitutiononC2’provideactiveinhibitorthatcanterminateRNAreplication,• Usingphosphoramidate prodrugstrategiestodelivernucleosidemonophosphatedrugtocells,• Phosphorusstereocenter isimportantinactivationofprodrug,• Improvementinsofosbuvir synthesis
52
Acknowledgment
Dr.Xuefei HuangMehdi,Berm,Zibin,Sherif,Kedar andmygroup
Saeedeh,Aliakbar
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