6614
`
`J. Med. Chem 2006, 49, 6614—6620
`
`Synthesis and Pharmacokinetics of Valopicitabine (NM283), an Efficient Prodrug of the Potent
`
`Anti-HCV Agent 2’-C~Methylcytidine
`
`Claire Pierra,l Agnes Amador,i Samira Benzaria,’r Erika Cretton—Scott,I Marc D’Amours,I John Mao,t Steven Matthieu}
`Adel Moussa,I Edward G. Bridges} David N. Standring,I Jean-Pierre Sommadossi,1 Richard Storer,§'v and Gilles Gosselin‘hl
`Laboraloire Coopémtifldenix—CNRS— Université MontpeNiet‘ 11, Care Courrier 008, Université Mom‘pellier 11, Place Eugene Bataillon, 34095
`Montpellt'er Cedex 5, France, and 102er Pharmaceuticals Inc, 60 Hampshire Street, Cambridge, Massachusetts 02139, and Laboratoires
`Idem'x SARL, Labaratoire de Chimie Médicinale, Cap Gamma, 1682 Rue de la Valsiére, BP 50001, 34189 Montpellter Cedex 4, France
`
`Received March 28, 2006
`
`In our search for new therapeutic agents against chronic hepatitis C, a ribonucleoside analogue,
`2’-C—methylcytidine, was discovered to be a potent and selective inhibitor in cell culture of a number of
`RNA viruses, including the pestivirus bovine viral diarrhea virus, a surrogate mode] for hepatitis C virus
`(HCV), and three flaviviruses, namely, yellow fever virus, West Nile virus, and dengue-2 virus. However,
`pharmacokinetic studies revealed that 2’—C~methylcytidine suffers from a low oral bioavailability. To overcome
`this limitation, we have synthesized the 3’-O—L—valinyl ester derivative (dihydrochloride form, valopicitabinc,
`NM283) of 2’-C—methylcytidine. We detail herein for the first time the chemical synthesis and physicochcmical
`characteristics of this anti—HCV prodiug candidate, as well as a comparative study of its pharmacokinetic
`parameters with those of its parent nucleosidc analogue, 2’—C—methylcytidine.
`
`Introduction
`
`Hepatitis C virus (HCV) has infected an estimated 170 million
`individuals, 3% of the world‘s population] The virus establishes
`a persistent infection in the majority of cases, leading to chronic
`hepatitis that often develops into cirrhosis and, in many cases,
`causes hepatocellular carcinoma.1 There is no vaccine available
`against HCV, and current
`therapies, namely, pegylated or
`nonpegylated interferon—(1 (IFN-o.) monotherapy and combina~
`tion of IFN-o. with oral ribavirin, are expensive, often poorly
`tolerated, and effective only in half of the patient population}3
`Therefore, there is an urgent need to develop new and more
`effective therapies in response to this important unmet medical
`need.4
`
`In the course of our HCV program, we recently discovered
`that 2’-C—methylcytidine (1, Figure l) is a potent and selective
`inhibitor of Flauiuiridae virus replication in cell culture.5-6 More
`particularly, 1 inhibited the replication of the bovine viral
`diarrhea virus (BVDV, a pestivirus surrogate model for HCV),7
`eliminated persistent BVDV infection at nontoxic concentra-
`tions, and was synergistic in combination with interferon—omJ
`but not with ribavirin.8 Compound 1 has no activity against
`human immunodeficiency virus (HIV) or against DNA viruses.
`In contrast to ribavirin, which is not effective alone in reducing
`viral RNA levels but stimulates the immune boosting capacity
`of interferon—a when used in combination, 1 is the first example
`of an anti-RNA virus agent
`that
`is active via a nucleosidc
`analogue mode of action, Thus, in primary human hepatocyte
`cultures, in a human hepatoma cell line (HepGZ), and in a bovine
`kidney cell
`line (MDBK),
`1
`is converted into its major
`metabolite, 2’-C—methylcytidine—5’—triphosphate, along with
`smaller amounts of 2’—C~methyluridine-5'-tiiphosphate, resulting
`from deamination. The active metabolite 2’~C~methylcytidine
`
`‘ Corresponding author Tel: + 33-4-67143855. Fax: + 33-4—67549610.
`Email: gosselin@univ-montp2ifr.
`fUniversité Montpellicr ll.
`3 Idenix Pharmaceuticals Inc.
`§Laboratoires Idcnix SARL,
`V Present address: VASTox plc, 91 Milton Park, Abingdon, Oxfordshire,
`OX14 4RY, U.K,
`
`H0
`
`NH;
`\N
`l as
`"05
`N
`l" H,i:,v’
`OH
`HO
`2'-C-methyleylidine (1)
`
`0
`
`0
`[-
`
`HO
`
`'
`'NH
`l
`‘N’RO
`O
`l” chf
`OH
`HO
`'-C-mathyluridlno(2)
`
`@e
`NH,
`CI
`‘N
`(:K
`,0“ 54
`H3433
`H
`0
`
`0
`
`HO
`
`L;
`-
`o; 0
`OleGHSNJY'
`3'-O-vallnyl ester of
`2‘-C-methyleytidlna (3)
`(cf/hydrochloride salt)
`
`Figure 1. Structures of compounds 1-3.
`triphosphate is a competitive inhibitor of purified BVDV RNA
`polymerase in vitro (K, = 160 nM).g
`Preliminary pharmacokinetic studies in animals revealed that
`further development of 1 would be hampered by its low oral
`bioavailability. To overcome this limitation, we devoted our
`efforts to the design of 2’-C—rnethylcytidine prodrugs with more
`favorable oral absorption profiles. From a literature survey, it
`was found that a broad variety of amino acid ester derivatives
`have been studied and successfully employed as nucleosidc
`prodrug forms. More particularly, efficacy of such derivatives
`has been proved in the case of valacyclovir, the L—valinyl ester
`of acyclovir (Figure 2).'°'“ After active absorption via peptide
`transport mechanism, valacyclovir is rapidly and almost com—
`pletely converted into acyclovir by enzymatic hydrolysis,
`increasing considerably the oral bioavailability and cellular
`uptake of the parent drug.'°'” The efficacy of L-valinyl
`derivatives has been also demonstrated in the case of ganciclovir
`(GCV), since valganciclovir (L-valinyl ester of GCV, Figure 2)
`has an oral bioavailability 10—fold higher than the parent
`nucleosidcfllu Recently, as part of our hepatitis B program,
`we have synthesized and studied several L—valinyl ester prodrugs
`of 2’—deoxy-,B-L-cytidine (L—dC)
`in order to improve the oral
`bioavailability of L—dC, Among them, the 3’—0—L—valinyl ester
`of L-dC (Val-L—dC, valtorcitabine, Figure 2) emerged as the most
`attractive L<dC prodrug14 and is currently in phase II clinical
`studies.”
`On the basis of these considerations, we decided to synthesize
`and study the 3’—O~L-valinyl ester derivative (NM283, valop—
`
`10.1021/jm0603623 CCC: $33.50 © 2006 American Chemical Society”
`Published on Web 10/06/2006
`‘
`
`CLARK EXHIBIT 2125
`Sommadossi v. Clark
`Contested Case 105,871
`
`1
`
`G|L2018
`I-MAK, INC. V GILEAD PHARMASSET LLC
`|PR2018—00122
`
`1
`
`GIL2018
`I-MAK, INC. V GILEAD PHARMASSET LLC
`IPR2018-00122
`
`

`

`Synthesis and Pharmacokinetics of Valopicitabine
`o
`
`ee
`
`0
`y
`H3N .JL.O
`
`c!
`
`I
`
`l”
`M” NH
`
`2
`
`0
`
`</N
`N
`\/
`VEIZCIMOVII'
`
`Cl
`
`as)
`
`‘
`
`o
`
`N
`/
`0
`<
`l
`_:L_
`HaNl" O‘KOVN N
`
`NH
`/L
`/
`
`””2
`
`(a e
`3
`c:
`NH
`N/J
`N-___.0.
`t.
`
`o
`
`_
`
`OH
`
`.
`
`.
`
`Or
`
`o
`
`.
`
`-[
`one ©H;N""|
`
`Val-L-dc
`
`Valgancicluvlr
`
`‘
`
`Figure 2. Structures of valacyclovir, valganciclovir, and val-L—dC.
`
`in order to improve the oral
`icitabine, 3; Figure l) of 1
`bioavailability of the parent nucleoside.
`Results and Discussion
`
`Synthesis. The 3’—O-valiny1 ester prodrug of 2’—C—methyl—
`cytidine (3) was first prepared by the route described in Scheme
`1. Our multistep sequence involved successive protections of
`the exocyclic amino function and of the 5'—hydroxyl group of
`1, followed by condensation with N-tert~butyloxycarbonyl-L—
`valine (L—Boc~valine) and,
`finally,
`total deprotection. N,N—
`Dialkylformamidines and especially N,N-dimethylformamidine
`have been widely used as protective groups for exocyclic amino
`function of nucleosidesfléi17 In the present work, the synthesis
`ofN4—[(dimethylamino)methylene]—2’-C-methylcytidine (4) was
`carried out following a procedure described by Kerr et al for
`the protection of 1 using N,N—dimethylformamide dimethylac—
`eta].18 Selective silylation of the 5’ primary hydroxyl group using
`a reported method19 led to the protected key intermediate 5.
`Condensation of 5 with L—Boc-valine using the coupling agent
`N'-(3~dimethylaminopropyl)—N—ethylcarbodiimide hydrochloride
`(EDC),20 with 4-(dimethylamino)pyridine (DMAP) as a cata-
`lyst,“ provided the N-Boc-protected ester derivative 6. Finally,
`desilylation of 6 with excess ammonium fluoride (NH4F) in
`methanol,21 an economical alternative to tetrabutylammonimn
`
`Scheme 1. A Conventional Route for the Synthesis pf 3"
`
`Journal of Medicinal Chemistry, 2006, V01. 49, No. 22 6615
`
`fluoride in tetrahydrofuran (TBAF/TIIF), followed by acidic
`hydrolysis using a saturated solution of hydrogen chloride
`in ethyl acetate,22 led to the target prodrug 3 as a dihydro—
`chloride salt.
`
`Although this conventional synthetic route afforded the
`desired prodrug in a satisfactory overall yield of 52%, several
`chemical difficulties (instability of the formamidine intermediate
`4 and partial raceinization of the L—valine during the coupling
`step), as well as cost issues (use of expensive tert-butyldiphe-
`nylsilyl group), made scale-up using this initial route unattrac-
`tive. In our search for a more direct and scalable synthetic
`process, we then developed a new synthetic strategy based on
`the direct condensation of 1 with L-Boc-valine, followed by
`removal of the N—tert—butyloxycarbonyl group, thus reducing
`the number of the synthetic steps from 5 to 2 (Scheme 2). We
`decided to explore this direct coupling approach after examining
`a report by McCormick et al, who described the room—
`temperature reaction of unprotected guanosine with Boc—
`anhydride in the presence of triethylamine, DMAl’, and DMSO.
`This reaction selectively gave 3’—Boc—guanosine in 55% yield.23
`In our first attempt, we reacted compound 1 in DMSO with
`carbonyldiimidazole(CDI)-activated L-Boc-valine in the presence
`of triethylamine and DMAP. The reaction did not progress at
`all when kept at room temperature. The reaction produced the
`desired product and several byproducts when heated at 50 °C
`using 4 equiv of CDI-activated L—Boc-valine. Optimum selectiv-
`ity was achieved by using 1.1 equiv of CDI-activated L-Boc-
`valine and running die reaction for only 1 h at 80 °C. We used
`only 0.] equiv of DMAP in order to avoid the racemization of
`the L—valine. Other coupling reagents such as EDC and MN-
`dicyclohexylcarbodiimide (DCC) did not produce desired
`product, and DMF was difficult to change due to the insolubility
`of compound 1 in most organic solvents. HPLC analysis showed
`68% of the desired compound, 11% of starting nucleoside l,
`and the two 3’,5’/3’,N‘-divalinyl ester byproducts. Pure product
`3 was obtained in 54% yield and 99% purity by using simple
`acid—base extraction, eliminating the extensive chromatographic
`step. Thus, this time- and labor-saving alternative process could
`be accomplished without
`involvement of chromatographic
`
`.
`
`H0
`
`NH2
`.
`N
`,1,
`
`l
`0
`N
`[K'Hfig'
`HO
`OH
`1
`
`0
`
`_',_
`
`Ho
`
`O
`
`7
`(78% for 3 steps)
`(i) MexNCl-I(0Me)2, DMF, 11, 1.5 11; (ii) TBDPSCl, imidazolc, pyridine, it, 6 h; (iii) N—Boc-L-valinc, EDC, DMAP, CH3CN/
`" Reagents and conditions:
`DMF, rt, 2 d; (iv) NHqF, McOH, reflux, 3 11; (v) HCl/EtOAc, EtOAc, rt.
`
`5
`
`NflN’
`J:N
`i
`.. A
`N
`..o..
`" H3?
`HO
`OH
`4
`(60% first crop
`crystallization)
`NH2
`‘N
`' t
`,N
`
`,
`
`N4\N:
`.t
`7]
`N
`II
`t A
`"
`-
`N 0'
`.0.
`-TSi-Ct'
`" Hit?“
`rial
`|
`"H“ HO
`OH
`5
`
`_
`___II _
`
`J iii
`
`0
`
`.
`
`IV
`
`E
`
`MN:
`“N
`'P/KO
`
`o.
`—|~SI-O
`'ng'C
`I”
`'3
`‘5 DVD
`OH
`0* "'f'
`NH
`
`><
`
`HO-
`
`_o__
`
`’ ch
`v
`0 0:.0
`0H
`XOJKNH '[J
`
`(96
`NHa
`c1
`‘N
`(a
`N
`
`0
`
`HO-
`
`.0.
`' Hat:
`i—’
`cc 0
`OH
`CIGGDNH't'l/
`s
`3'-O-valinyl ester of 1
`(hydrochloride form, 3, 81%)
`
`2
`
`

`

`Pierm et a].
`
`Table 1. Pharmacokinctic Parameters of Compounds 1 and 3,
`Following a Single Oral Administration of 3 to Sprague—Dawlcy Rats“
`prong 3
`oompd 1
`dose
`equiv dose
`AUC
`Ti/2
`Cmax
`Tum
`F
`
`(mg/kg)
`(mykg)
`diug
`(ug li/mL)
`(h)
`(fig/mL)
`(h)
`(%)
`100
`72
`3
`8.95
`0.64
`3.62
`1.0
`-
`1
`30.0
`7.10
`6.12
`2.0
`33.6
`
`
`" AUClH: area under the plasma concentration—time curve from time
`0 to r. Tm:
`terminal elimination half-life. Cm“: maximum plasma
`concentration. Tm“:
`time to maximum plasma concentration. F: apparent
`oral bioavailability calculated on the basis of a dose—normalized AUC value
`of 1.24 (fig li/mL) from a single iv dose of] to the rat.
`
`+ 3 in = 3)
`
`«O~
`
`'1 (n '-" 3)
`
`
`
`Ti
`“a.E
`
`E iE
`
`(J
`
`.i
`
`1
`
`0
`
`
`.
`.
`__.
`.
`x
`lo
`M
`.32
`to
`Timeth}
`
`__.
`as
`
`I:
`1
`.9
`::
`g Q3.
`
`g 0,6-
`.
`8 0.4
`2 0,2
`g 0
`a:
`
`D
`
`500 1000 1500 2000
`Incubation tlme (min)
`G @
`NHa
`Cl
`‘N
`6
`’0
`NAG
`H3?
`OH
`
`H
`
`Ho
`
`6616
`
`Journal of Medicinal Chemistry, 2006, Val. 49, No. 22
`
`Scheme 2. Alternative Route for the Synthesis of 3“
`
`NH2
`b.
`..n.. N
`H0:
`
`“0'
`
`O
`
`.
`_I,
`
`NH;
`er
`“0- .u.
`l“
`l" Hp;
`
`O
`
`HO'
`
`ii
`
`696
`NH;
`Cl
`0“.
`0.
`N
`, Hat
`
`O
`
`HO
`
`oH
`
`1
`
`OH
`
`..
`
`00__o
`Jk __
`X0 NH Ii
`7
`(54%)
`
`0.
`
`OH
`
`to
`,_ .-
`GE)
`'l
`Cl
`NH,
`3'-O-va|iny| ester of 1
`(dihydrochloride salt, 3,
`96.5%l
`
`" Reagents and conditions:
`THF; (ii) HCl/EtOH.
`
`(i) N—Boc-L-valine, CDI, DMAP, TEA, DMF/
`
`At H45
`
`Atpl-l 7.2
`
`=
`l
`g
`E
`E 0.9
`
`§ 0.6
`m
`3 0.4
`E 0.2
`%
`0
`m
`
`0
`
`
`
`_.I
`—--i
`5000 10000 15000 20000
`Incubation time (min)
`6) G
`NH3
`CI
`\N
`(x
`N’KO
`'
`0H
`
`5
`H'
`
`_
`
`
`Tm=6.1daysatpH4.5
`Tm: 3.9 hours at pH 7.2
`
`H5.
`0- .0
`eo 'l
`CI
`H,N’ ‘1’
`O = 3‘-0vallnyl ester 0! 2'»C—melhylcytidlne (3)
`1 ,m = 273.5 nm
`
`A = 2'-C-meihylcytidlne (1)
`A w = 273.5 nm
`
`Figure 4. Mean plasma concentration of compounds 1 and 3, following
`a single oral administration of 3 to Sprague-Dawley rats.
`
`Figure 3. Kinetic curves ofhydrolysis of prodrug 3 at pH 4.5 and 7.2.
`
`purifications, and compound 3 was obtained in high purity using
`simple crystallization.
`Aqueous Solubility, Lipophilicity, and Chemical Stability
`of Prodrug 3. To be considered as a suitable prodrug for oral
`administration, the 3'—0—valinyl ester of 2’-C-methylcytidine (3)
`should possess adequate solubility in aqueous media in order
`to dissolve in the small
`intestine,
`thus being available for
`absorption. The aqueous solubility of 3 has been determined in
`comparison with the solubility of the parent nucleoside 1. The
`concentration of saturated solutions of 1 and 3 in water were
`32 and 423 g/L, respectively. Both compounds were found to
`be highly soluble in aqueous media, which has been cor-
`roborated by their low distribution coefficient (log P) values:
`#0965 and —1.34 for 1 and 3, respectively.
`The main aim of stability studies was to determine whether
`the prodrug of 2’-C—methylcytidine would be sufficiently
`chemically stable in the gastrointestinal tract before its absorp—
`tion. Prodrug 3 appeared to be fully stable at pH 1.2 but was
`hydrolyzed at pH 4.5 and 7.2 into the parent drug 1 following
`first-order kinetics. The half-lives of 3 at pH 4.5 and 7 .2 were
`6.1 days and 3.9 h, respectively (Figure 3). It is noteworthy
`that 1 was fully stable at all the acidic and neutral studied pHs.
`Stability in Human Blood, Plasma, and Liver Cytosol.
`Prodrug 3 was rapidly converted into 1 in both human plasma
`and whole blood, exhibiting in vitro half-life values of 130 and
`40 min, respectively. This rapid conversion is primarily due to
`the presence of esterases in blood and plasma. Conversion of 3
`into 1 was also observed in human liver cytosol and S9 fractions,
`with approximately 30% of 3 converted into 1 within 1 h.
`Differences in half-life for prodrug 3 in plasma, whole blood,
`
`human liver cytosol, and S9 fractions are not surprising and
`depend on the activity/quantity of enzyme(s) present in these
`samples. In all cases, no further metabolism of 1, such as
`deamination to its corresponding uridine metabolite 2, has been
`observed.
`Protein Binding of Compounds 1 and 3 to Rat Plasma.
`The in vitro binding of compounds 1 and 3 to rat plasma proteins
`was investigated by the ultrafiltration method using radiolabeled
`test articles ([3H]-2’-C—methylcytidine and [3H]-3’—0-valinyl
`ester of 2’-C—methylcytidine) at a concentration of 20 #M for
`each compound. As expected for nucleosides, the protein binding
`for the two compounds was low, with 7% and 5% bound for 3
`and 1, respectively.
`Oral Bioavailability and Pharmacokinetic Studies of
`Compounds 1 and 3. The pharmacokinetics (PK) of prodt‘ug
`3 were evaluated in Sprague—Dawley rats in a mass balance
`using radiclabeled 3. Both intact and bile duct cannulated (BDC)
`rats were used in this study. PK parameters of 1 and its 3’—O-
`valinyl ester 3 are presented in Table 1, Plasma concentration-
`time profiles of 1 and 3 are shown in Figure 4.
`Following a single oral dose of ['4C]-3’-O-valinyl ester of
`2’-C—methylcytidine (3) at 100 mg/kg (as free base), Lu‘ine, feces,
`and cage rinses were collected for a period of 72—168 h
`postdose to evaluate the excretion of radioactivity and the overall
`mass balance. Plasma samples were collected over a 48-h period
`postdose to study the PK of 1 and 3. 1n intact rats, fecal excretion
`was also the predominant route of elimination of total radio—
`activity, accounting for 63.5% of the administered dose.
`Excretion of radioactivity occurred largely within the first 24 h
`(averaging 60% of the dose). Urinary excretion accounted for
`31.9% of the administered dose. The mean total recovery of
`radioactivity, in urine, feces, and cage rinses, was 96.6%. Biliary
`excretion of total radioactivity in BDC rats accounted for a mean
`
`3
`
`

`

`Synthesis and Pharmacokinetics of Valopicitabine
`
`Journal of Medicinal Chemistry, 2006, V01. 49, No. 22 6617
`
`of 0.3% of the dose over the 72—h collection period. In this
`group, fecal excretion was the predominant route of elimination,
`accounting for a mean 65.9% of the dose. A mean 29.8% of
`the dose was recovered in urine. Excretion of radioactivity was
`largely completed by 24 h in BDC rats. On the basis of the
`combined mean recovery of radioactivity in bile and urine, it
`was apparent that at least 30.0% of the [14C]-3’-0-valinyl ester
`of 2’—C-methylcytidiue (3) oral dose was absorbed. The mean
`recovery of total radioactivity in this group was 97.0% of the
`administered dose. In the group following a single dose of M03
`at 100 mg/kg (as free base), maximum plasma concentrations
`of total
`radioactivity (averaging 11.9 ng equiv/mL) were
`achieved 1—2 h after the oral dose. Plasma concentrations of
`3, determined by LC/MS/MS, were greatest 0.5~l h after
`dosing, and Cmax averaged 3.62 yg/mL. Plasma 3 concentrations
`decayed with a half—life of 0.64 h, and no prodrug 3 was detected
`in plasma by 12 h. Plasma concentrations of 1, determined by
`LC/MS/MS, were greatest 1—2 h after dosing, and Cmax
`averaged 6.12 rig/mL. Plasma concentrations of1 decayed with
`a half-life of 7.1 h. The AUC of 1 was more than 3—fold greater
`than the AUC of 3. 2’—C—Methyluridine (2) was not detected in
`rat plasma (<0.1 ,ug/rnL).
`The pharmacokinetics of 1 were also studied in Sprague—
`Dawley rats in a toxicokinetic (TK) study in which compound
`1 was administered intravenously daily for 15 days. There were
`three dose groups (60, 120, and 300 mg/kg/day) in the study,
`and TK sampling was conducted on day 1 following the first
`dose and on day 15 after the last dose. For the scope of this
`paper, only the TK parameters of the 60 mg/kg/day dose group
`on day 1 are presented. Following a single iv dose of compound
`1 to rats at 60 mg/kg, the mean plasma Cmax of l was 75.6
`,ug/mL and the mean AUC was 74.4 pg h/mL. The mean AUC
`value of 1 from this dose group was compared to the AUC value
`of 1 from the prodrug 3 rat study above, and an apparent oral
`bioavailability was calculated for 1. Thus, the apparent oral
`bioavailability of 1 following oral administration of the valinyl
`ester derivative 3 at 100 mg/kg (as free base) was calculated to
`be 33.6%.
`
`Conclusion
`
`3’-0-Valinyl ester of 2’-C—methy1cytidinc (dihydrochloride
`salt, NM283‘, valopicitabine‘, 3) has been synthesized in order
`to improve the oral bioavailability of the parent compound 2’—
`C-methylcytidine (1). For that purpose, two different strategies
`have been developed, both starting from 1. The first one is a
`conventional route that involves successive protection steps, and
`the second one is more appropriate for large-scale synthesis and
`is based on selective 3’-0-esterification. Physicochemical,
`pharmacokinetic, and toxicokinetic studies have shown that
`compound 3 is an acid—stable prodrug of 1 with excellent
`pharmacokinetic and toxicokinetic profiles. Prodrug 3 is rapidly
`converted into compound 1 in both human plasma and whole
`blood, probably due to the presence of esterases. The apparent
`oral bioavailability of the parent drug 1 following oral admin-
`istration of the prodrug 3 is 34%.
`Thus, on the basis of its ease of synthesis, its physicochernical
`properties, and phamracokinetic profile, 3’-0-valinyl ester 3 has
`emerged as a promising prodrug of 2’—C—methylcytidine (1). The
`. 3’—0~va1inyl ester of 2’-C—methylcytidine (3, NM283, valopic—
`itabine) is currently undergoing phase Ilb clinical trials for the
`treatment of chronic HCV infection.24
`
`Experimental Section
`General Methods for Chemistry. 1H NMR spectra were
`recorded at ambient temperature on a Bruker AC 200 MHz, 250,
`
`300, or 400 MHz spectrometer. 1H NMR chemical shifts (d) are
`quoted in parts per million (ppm) referenced to the residual solvent
`peak [dimethyl sulfoxide (DMSO-dg] set at 2.49 ppm. The accepted
`abbreviations are as follows: 5, singlet; d, doublet; t, triplet; m,
`multiplet. FAB mass spectra were recorded in the positive—ion or
`negative-ion mode on a JEOL DX 300 mass spectrometer operating
`with a JMA—DA 5000 mass data system and using a mixture of
`glycerol and thioglycerol (l/l, v/v, G—T) as the matrix. Melting
`points were determined in open capillary tubes with a Bijchi 13-545
`apparatus and are uncorrected. UV spectra were recorded on an
`Uvikon XS spectrophotometer. Elemental analyses were carried out
`by the Service de Microanalyscs du CNRS, Division de Vernaison
`(France). Thin-layer chromatography (TLC) was performed on
`precoated aluminum sheets of silica gel 60 F254 (Merck, Art. 5554),
`visualization of products being accomplished by UV absorbance
`and by charring with 10% ethanolic sulfirric acid with heating or
`with a 0.2% ethanolic ninhydrin solution for compounds bearing
`an amide function.
`_
`Column chromatography was carried out on silica gel 60 (Merck,
`Art. 9385). Evaporation of solvents was carried out in a rotary
`evaporator under reduced pressure. All moisture-sensitive reactions
`were carried out under rigorous anhydrous conditions under an
`argon atmosphere using oven-dried glassware. Solvents were dried
`and distilled prior to use and solids were dried over P105 under
`reduced pressure. Analytical high—performance liquid chromatog-
`raphy (HPLC) studies were carried out on a Waters Associates unit
`(multisolvent delivery system, 717 autosampler injector, 996
`photodiode array detector and a Millenium data workstation) using
`a reverse-phase analytical column '(Nova-Pak Silica 60 A, 4 am,
`C13, 150 x. 3.9 mm). The compound to be analyzed was eluted
`using a linear gradient of 0—50% acetonitrile in 20 mM trierhy—
`lammonium acetate buffer (TEAC, pH 7) programmed over a 30—
`min period with a flow rate of 1 mL/min.
`General Procedure for Aqueous Solubility Studies. Aqueous
`solubilin'es were determined in distilled water at room temperature.”
`An excess of studied compounds was added to aqueous solution,
`and the suspension was shaken and centrifugated. Samples of
`supernatant were analyzed by HPLC (same conditions as described
`in General Methods for Chemistry). and the concentration of
`saturated solution was determined according to a calibration curve.
`The experiment was repeated three times.
`General Procedure for Distribution Coefficient Studies.
`Distribution coefficients between l-octanol and an aqueous phase
`(phosphate buffer solution 0.02 M) were determined at room
`temperature using a shake-flask procedure.25 An aliquot of a 10‘2
`M aqueous solution of studied compounds was diluted to 1 mL
`with the aqueous phase previously saturated with octanol. An equal
`volume of octanol, previously saturated with the aqueous phase,
`was added to give a total volume of 2 mL. The mixture was shaken
`vigorously. The two phases were centrifugated and separated.
`Samples of each phase were collected and analyzed by HPLC (same
`conditions as deScribed in General Methods for Chemistry). UV
`absorbances for both phases were measured at the respective
`maximum wavelength. The distribution coefficient was calculated
`from the ratio of the area of the signal detected in the oetanol and
`aqueous phases. Each experiment was repeated twice.
`General’Procedure for Chemical Stability Studies. Chemical
`hydrolysis rates were determined in KCl—HCl buffer 0.135 M
`solution (pH 1.2, 37 °C), acetate buffer 0.02 M solution (pl-l 4.5,
`37 °C), and phosphate buffer 0.02 M solution (pH 7.2, 37 °C).26
`Solutions (10‘4 M) of 3 have been incubated at 37 °C in buffer
`solutions. Aliquot samples were collected at different time intervals
`and analyzed by HPLC (the same conditions as described in General
`Methods for Chemistry). Rates of decomposition have been easily
`dctcrmincd using a method developed in our laboratory and based
`on pseudo—first—order kinetic models.27
`General Procedure for in Vitro Blood and Plasma Stability
`Studies. A stock solution of 3 was aliquoted (1 mg/mL stored in
`MeOH at —20 ”C), dried down, and resuspended to 20 [Ag/mL with
`either human plasma or whole blood (Bioreclamation lnc., Hicks»
`ville, NY) preheated to 37 °C. Aliquot samples were collected at
`
`4
`
`

`

`6618
`
`Journal QfMedieinal Chemistry, 2006, V01. 49, Na. 22
`
`different time intervals 0, 2, 5, 10, 20, 40, and 60 min for blood
`studies and 0, 30, 70, 100, and 140 min for plasma studies. Samples
`were quenched in 5 volumes of ice-cold solvent consisting of 50%
`acetonitiile/50% methanol and centrifuged at 16 000 rcf for 3 min.
`Supernatant was removed, dried down in a centrifugal concentrator,
`resuspended in mobile phase (5% MeOH—95% potassium phos-
`phate, monobasic, pH 5.0), filtered (0.2 ,um nylon Spin X tubes),
`and analyzed by reverse-phase HPLC using an Agilent model 1100
`instrument with automatic injection and a diode~array spectropho-
`tometric detector. The mobile phase consisted of buffer A (25 mM
`potassium phosphate, pH 5) and buffer B (methanol). A linear
`gradient from 5% to 100% buffer B was run over 15 min. The
`HPLC system used a Cu Columbus column (5 pm, 250 x 4.6 mm,
`Phenomenex, Torrance, CA). Authentic standards were used to
`identify the peaks in HPLC. This assay was done one time (blood
`and plasma from one donor) in duplicate.
`General Procedure for Metabolic Stability Studies. The ability
`of derivative 3 to function as substrate for relevant metabolic
`enzymes was examined using human liver subcellular fractions
`obtained from BD_ Gentest (Woburn, MA). The prong was
`incubated for 1 h at 50 ug/mL with 1 mg/mL liver S9 or liver
`cytosol. Prior to HPLC analysis, samples were quenched in 5
`volumes of ice-cold solvent (50% acetonitrile/50% methanol) and
`centrifuged (16 000 ref for 3 min). Supernatant was removed, dried
`down in a centrifugal concentrator, resuspended in mobile phase
`(5% MeOH—95% potassium phosphate, monebasic, pH 5.0),
`filtered (0.2 ,um nylon Spin X tubes), and analyzed via HPLC (same
`conditions as described in General Procedure for in vitro Blood
`and Plasma Stability Studies).
`General Procedure for Protein Binding Studies. The protein
`binding of prodrug 3 and parent nucleoside 1 in rat plasma was
`investigated using the ultrafiltration centrifugation method at a
`concentration of 20 [2M for each. The studies used both nomadic—
`labeled and radiolabeled test articles. The [3H]-3’-0-valinyl ester
`of 2’-C—methylcytidine and [3H]-2’-C-methylcytidine, provided by
`Moravek Biochemicals, Inc. (Brea, CA), were prepared by tritium
`exchange (catalyst and labile tritium were removed during purifica—
`tion). To achieve the desired concentrations, concentrated stock
`solutions for each test article containing the appropriate amounts
`of the radiola‘belcd and nonradiolabeled articles were prepared in.
`HPLC grade water. Rat plasma (1 mL) was then added to each
`tube and vortexed. The sample tubes were incubated at 37 0'C for
`1 h.
`Centrifee micropartition units (l-mL capacity, Amicon Inc.) with
`a molecular weight cutoff of 30 000 Da were used to separate the
`unbound from the bound test article by membrane filtration.
`Fortified plasma (0.6 mL) was added to the sample reservoir portion
`of the ultratiltration device. Plasma samples were centrifuge for
`10 min at 37 °C and 1800g so that the amount of sample filtered
`ranged between 20 and 50% of the total volume. After centrifuga-
`tion, the ultrafiltrate of each sample was collected, and 10 pL
`aliquots were analyzed for radioactivity using liquid scintillation
`counting (LSC) on a Beckman Coulter LSéSOO. Protein binding
`determinations were done in duplicate for each test article.
`General Procedure for Pharmacokinetic Studies. The phar-
`macokinetic and mass balance study of the [14C]—3’-O-valinyl ester
`of 2’-C—methylcytidinc (14C label at C-2, Moravek Biochemicals,
`Inc.) was conducted in three groups of male Sprague—Dawley rats.
`A single oral dose of the [14C]-3’-0-valinyl ester of l was
`administered as a solution in 0.01 N HCl at a target dose of 100
`mg/kg (as free base) to the three groups of’male rats afier fasting
`overnight. Group 1 was used to characterize the excretion of the
`radioactivity derived from the [‘4C]—3’—0-valinyl ester of 2’-C—
`methylcytidine in urine and feces. Urine and feces were collected
`up to 168 h postdose from group 1 rats. Group 2 comprised bile
`duct cannulatcd (BDC) male rats and was used to characterize the
`excretion of the radioactivity derived from the [14C]-3’-0-valinyl
`ester of 2'-C-methylcytidine in bile, urine, and feces. Urine, bile,
`and feces were collected up to 72 h postdose in group 2 rats. Group
`3 consisted of jugular vein cannulated (JVC) male rats in which
`the pharmacokinetics of plasma radioactivity derived from the [14C]-
`
`Pierre er al.
`
`3'-0-valinyl ester of .2'-C-methylcytidinc, and 1 and 3 were
`evaluated. Blood samples were collected from group 3 male rats
`predose and 0.25, 0.5, 1, 2, 3, 4, 8, 12, 16, 24, and 48 h postdose
`(3 rats/time point), and the separated plasma was analyzed by LSC
`and by LC/MS/MS. The LC/MS/MS method was validated ac-
`cording to ICH guidelines for bioanalytical method validation prior
`to sample analysis. A daily cage rinse, a cage wash, and a cage
`Wipe at termination were also performed for groups 1 and 2, All
`postdose samples of bile, urine, feces, plasma, and cage residues
`(cage rinse, cage wash, and cage wipe) were analyzed for
`radioactivity by LSC. Mean plasma concentrations of 1 and its 3’-
`0—valinyl ester were used to calculate descriptive phannacokinctic
`parameters by noncompartmental analysis using WinNonlin 4.1
`(Pharsight).
`The 15-day toxicokinetic study of nucleoside analogue 1 was
`conducted to evaluate the pharmacokinetics of 1
`following iv
`administration to Sprague~Dawlcy rats. Compound 1 was admin»
`istered once daily to rats intravenously for 15 days at dose levels
`of 60, 120, and 300 mg/kg/day. Each dose group consisted of eight
`animals/gender. On days 1 and 15, blood samples were collected
`from the first set of four rats/sex/group at predose and approximately
`20 min and 2 h postdose and from the second set of four rats/sex/
`group at approximately 5 min and 1 and 4 h postdose. Plasma
`samples were analyzed for 2’-C—methylcytidine and 2'-C~methy-
`Iuridine by a validated bioanalytical method (LC/MS/MS), and
`composite PK parameters were calculated by noncompartmental
`analysis using WinNonlin 4.1 (Pharsight).
`For LC/MS/MS analysis, compound 1, its 3’-0-valiny1 ester, and
`added internal standards were extracted from plasma (50 ML) using
`protein precipitation with acetonitrile (0.5 mL). The supernatant
`was dried, reconstituted, and analyzed by LC/MS/MS using HPLC
`coupled to a PE Sciex API3000 tandem mass spectrometer with a
`Turbo IonSpray interface. The HPLC system used an Alltceh
`Platinum C13 column (3 ,um, 53 x 7 mm, Alltceh Associates,
`Deerfield, IL). HPLC elution was carried out using a gradient of
`ammonium acetate (10 mM, pH 4) and acetonitrile. The flow rate
`was 2.5, mL/min with a split of 0.5 mL/min into the mass
`spectrometer. Each, analyte was detected by multiple reaction
`monitoring (MRM) under the positive ion mode using the precursor
`to product ion pair of m/z 357 to 112 (collision energy 27 eV) and
`m/z 258 to 112 (collision energy 20 eV), for 3 and 1, respectively.
`Quantitation was achieved by constructing a calibration curve by
`weighted linear regression of the ratio of the analyte peak area to
`that of the added internal standard. Calibration standards were in
`blank rat plasma. The calibration range was 0.01~5 ,ug/mL in
`plasma for 3 and 0.05~25 ,ug/mL in plasma for the parent
`nucleoside I.
`.
`N“-[(Dimethylamino)methylene]—2’-C-methyI-fi-D-cytidine (4).
`A solution of 15,633 (1.65 g, 6.43 mmol) in NN—dimethylformamide
`(DMF, 32 mL) was treated with dimethylforrnamide dimethylacetal
`(8.2 mL, 61.73 mmol) and stirred for 1.5 h at room temperature.18
`The solution was evaporated under reduced pressure and coevapo-
`rated with ethanol. Crystallization from ethanol/ether yielded the
`hitherto unknown title compound 4 (first crop = 1.21 g, 60%;
`second crop slightly impure on TLC = 0.46 g, 23%) as crystals.
`All
`the following physicochemical characteristics have been
`determined on the