Antiviral Compounds

This invention relates to novel compounds, processes for their preparation, pharmaceutical compositions containing them and methods of treatment involving their 5 use.

Ribavirin(l-, β-D-ribofuranosyl-l,2,4-triazole-3-carboxamide) is a nucleoside analogue that has demonstrated efficacy in treating viral diseases both as monotherapy (respiratory syncytial virus, Hall, C. B. ; McBride, J. T. ; Walsh, E. E. ; Bell, D. M. ;

10 Gala, C. L. ; Hildreth, S. ; Ten Eyck, L. G. ; W. J. Hall. Aerosolized ribavirin treatment of infants with respiratory syncytial viral infection. N. Engl. J. Med. 1983, 308, 1443- 1447), and in combination therapy with interferon-alpha (hepatitis C virus, Reichard, O.; Norkrans, G.; Fryden, A.; Braconier, J H.; Sonnerborg, A.; Weiland, O: Randomized, double blind, placebo controlled trial of interferon alpha2B with and

15 without ribavirin for chronic hepatitis C, Lancet 1998, 351, 8387). Combinations of ribavirin with pegylated interferon cc2a and with pegylated interferon cc2b have also been reported.

Recently reported studies indicate that the in vivo utility of ribavirin can result not only 20 from direct inhibition of viral replication, but also from its ability to enhance T cell- mediated immunity (Hultgren, C; Milich, D.R.; Weiland, O.; Sallberg, M. The antiviral compound ribavirin modulates the T helper Typel/Type2 subset balance in hepatitis B and C virusspecific immune responses, J. Gen. Virol. 1998, 79, 2381-2391 ; Ning, Q. ; Brown, D. ; Parodo, J. ; Cattral, M. ; Fung, L. ; Gorczynski, R. ; Cole, E., 25 Fung, L.; Ding, J. W. ; Liu, M. F.; Rotstein,O. ; Phillips, M. J.; Levy, G. Ribavirin inhibits viral- induced macrophage production of tumor necrosis factor, interleukin-1, procoagulant activity fgl2 prothrombinase and preserves ThI cytokine production but inhibits Th2 cytokine response. J. Immunol. 1998, 160, 3487-3493; Martin, M. J. ; Navas, S.; Quiroga, J. A. ; Pardo, M.; Carreno, V. Effects of the ribavirin interferon 30 alpha combination on cultured peripheral blood mononuclear cells from chronic hepatitis C patients.; Cytokine 1998, 79, 2381-2391). This immunomodulatory effect of ribavirin is demonstrable in vitro by measuring the levels of Type 1 cytokines produced by activated T cells from both humans and mice (Tarn, R. C.Pai, B.; Bard, J.; Lim, C;

2

Averett, D. R.; Phan, U. T. ; Milovanovic, T. Ribavirin polarizes human T cell responses towards a Type 1 cytokine profile; J. Hepatol. 1999, 30, 376-382), and by other measures. The induction of a Type 1 cytokine bias by ribavirin is functionally significant in vivo in murine systems (Tarn, R. C. ; Lim, C; Bard, J.; Pai, B.: Contact 5 hypersensitivity responses following ribavirin treatment in vivo are influenced by Type 1 cytokine polarization, regulation of ILlO expression and co-stimulatory signaling. J. Immunol. 1999, 163, 3709-3717).

Mammalian immune systems contain two major classes of lymphocytes: B 10 lymphocytes (B cells), which originate in the bone marrow; and T lymphocytes (T cells) that originate in the thymus. B cells are largely responsible for humoral immunity (i.e. antibody production), while T cells are largely responsible for cell-mediated immunity.

15 T cells are generally considered to fall into two subclasses, helper T cells and cytotoxic T cells. Helper T cells activate other lymphocytes, including B cells and cytotoxic T cells, and macrophages, by releasing soluble protein mediators called cytokines that are involved in cell-mediated immunity. As used herein, lymphokines are a subset of cytokines.

20

Helper T cells are also generally considered to fall into two subclasses, Type 1 and Type 2. Type 1 cells produce interleukin 2 (IL-2), tumor necrosis factor (TNFa) and interferon gamma (IFNγ), and are responsible primarily for cell-mediated immunity such as delayed type hypersensitivity and antiviral immunity. In contrast, Type 2 cells

25 produce interleukins, IL4, IL-5, IL-6, IL-9, IL-10 and IL-13, and are primarily involved in assisting humoral immune responses such as those seen in response to allergens, e. g. IgE andIgG4 antibody isotype switching (Mosmann, 1989,Annu Rev Immunol, 7: 145- 173).

30 As used herein, the terms Type 1 and Type 2 "responses" are meant to include the entire range of effects resulting from induction of Type 1 and Type 2 lymphocytes, respectively. Among other things, such responses include variation in production of the corresponding cytokines through transcription, translation, secretion and possibly other

3

mechanisms, increased proliferation of the corresponding lymphocytes, and other effects associated with increased production of cytokines, including motility effects.

According to the invention there are provided compounds of formula (I),

5

CONH ₂

R ¹ X

R ² Cf OR ³ (I)

wherein, at least one of R ¹ , R ² and R ³ represents a group S-L- and the remainder represent H, S represents a binding group, L represents a linker chain, X represents O 10 or NH, and pharmaceutically acceptable derivatives thereof.

The present invention will now be described with reference to the following drawings, in which:

Fig. 1 shows an NMR spectrum of compound 1; 15 Fig. 2 shows an NMR spectrum of compound 2;

Fig. 3 shows an NMR spectrum of compound 3;

Fig. 4 shows an NMR spectrum of compound 4;

Fig. 5 shows plasma concentrations of certain compounds according to the invention;

Fig. 6 shows red blood cell (RBC) concentrations of certain compounds according to 20 the invention.

SL represents one of the following groups:

O ORK ⁴ V. OR ^*4

R ⁴ O^^^λ

D. V \o,-

25 (II) (III)

Wherein in formula (II), either: one of A and A' represents L and the other represents H, or A is L and -D-A' represents -OH, -OAcyl or -NHAcyl, or 5 A represents H and D represents NH and A' = L, and in each case R ⁴ = Acyl or H and L is as defined above.

Preferably, group S represents an oligosaccharide terminating in a galactosamine or galactose residue. Suitable sugars that S may represent include galactosamine, 10 galactose or lactose.

L may be linked at any of the hydroxy substituents on the ribavirin moiety (Ia), i.e. the 2- , 3- or 5- OH groups, to give O-L-S. Alternatively, L may be linked to the ribavirin moiety (Ia), represented by the following structure, 15

O

N f NH ₂

5 ^s JX _/ N-N

~X

^i

(Ia)

by substituting any one of the 2-, 3- or 5- OH substituents, e.g. by NH-L-S. Preferably, group X represents O when either or both of R ² and R ³ represent S-L-, and X 20 represents O or NH when R ¹ represents S-L-.

Preferably, L includes COCH ₂ -, CO- or benzylene which is linked to -O- or -X- of the ribavirin moiety (Ia).

25 Preferably, R ¹ represents L and R ² and R ³ both represent H, or

X represents O, R ¹ represents H, R ² represents L and R ³ represents H, or X represents O and R ¹ represents H, R ² represents H and R ³ = L, or

5

X represents O and R ¹ represents H, R ² represents L and R ³ represents L.

L may be further substituted by S', where S' has the same definition as S defined above. Preferably, S binds to an asialoglycoprotein receptor. 5

Preferably, L represents one of the following groups:

O

S-(CH ₂ ) Y " £ ⁿ (IV) wherein, 10 n is an integer from 0 to 6 inclusive;

Y represents O; -OCH ₂ -; -(CR ⁶ R ⁷ )d-, where R ⁶ and R ⁷ independently represent H or alkyl; cycloalkylene; arylene; or single bond; and d represents an integer from 0 to 6 inclusive;

15 or

S-(CH ₂ )JLN-Q- (CH ₂ J ₅

I J

/I ^" — U— fcH ₂ ]— N — (-CH ₂ ) — π — fCH ₂ -} £

O

O

S-(CH ₂ ) — " — N— Q ¹ — (cH ₂ ) _t

(V) wherein, 20 p and z independently represent 1 or 2; s represents an integer from 1 to 4; b, c, r and t independently integers from 0 to 3 inclusive;

Q and Q' independently represent -(CR ⁶ R ⁷ ) _q CONH-, where R ⁶ and R ⁷ are as defined above, and q is 1 or 2; or a single bond;

T is CH or N; 25 U is a single bond, -CONH- or -CO-;

or

S — CH ₂ - ¹ I ¹ l — H N-E Z—

(VI) wherein,

E represents -(CR ⁶ R ⁷ ) _q -, where R ⁶ and R ⁷ are as defined above, q is as defined above; 5 or benzylene;

Z represents a single bond, -CO-, -NHCO(CH ₂ ) _r -, wherein r is as defined above;

or

Z O

H

S-f \CH ₂ ^ /—w U'-C H— f \CH ₂ /)—q N-LcH ₂

(VII) 10 w represents 0 or 1 ; q is as defined above; U' represents -CO- or -CONH-;

Z' represents -ONHCO-CH ₂ -'' or H; and

15 S is as defined above.

Although it is not necessary for the functioning of the invention, we prefer that L is cleavably connected to the ribavirin moiety (Ia), such that in vivo, particularly in hepatocytes, the compound of formula (I) releases ribavirin or an antiviral derivative 20 thereof. Where L is linked to a ribavirin 5 -hydroxy substituent, it is preferably cleavably linked. However, linkages at the 2- or 3- hydroxyl position may be metabolically stable, as the compounds of formula (I) may be active as such.

According to the invention we also provide a process for the production of a compound 25 of formula (I), or a pharmaceutically acceptable derivative thereof, which comprises a) reacting a compound of formula (VIII),

Si-L-Zi (VIII)

7

in which Zi represents a leaving group,

Si is a group S as defined above or a protected S, and L is as defined above, with a compound of Formula (IX),

5 Ra (IX)

in which R _a is a ribavirin or protected ribavirin, or b) reacting a compound of Formula (X),

10 Z ₂ -L-R _a (X)

in which Z ₂ is a leaving group and L and R _a are as defined above, with a compound of Formula (XI),

15 Sa (XI)

in which S _a is a group S as defined above, or a protected derivative thereof, and where desired or necessary, converting the resulting compound into a compound of formula (I) by removing protecting groups.

20

Reactions (a) and (b) may be carried out using conventional conditions well known to the person skilled in the art of synthetic organic chemistry. Conditions are described, for example, in standard text books of practical organic chemistry, e.g. Fieser, Organic Syntheses, and the like.

25

Specific compounds of formula (I) may be synthesised as follows:

Scheme 1:

AcO -- OAc AcO --OAc

^i-O TBDPSCI

Acθλ^--^<^-O^|^,OR Acθλ-«^*>-^--O _^ >-i ,OTBD OAc ⁿ pyridine OAc ^ ' "

I 44 RK == B Bnn,, aa nn == 11 ( (8899%%)) 7a n = 1 (96%)

Pd(OH) ₂ /C, H ₂ , EtOH ^ 5 R = H, a n = 1 (74%)

1 Na, MeOH 2 BnBr, NaH, DMF

BnO ,--OBn BnO --OBn

TBAF

B n O \«-""^"«-X- O _^ Jς\ ^ O H Bnθ\---^λ---O^ >J ,OTBDPS

TBABr, OBn " NaHCO ₃ , DCM OBn n

10a n = 1(84%) 9a n = 1(89%)

8a n = 1(88%)

BnO ,-OBn _HO ^>yN-N NH ₂ BnO --OBn

Ul-O EDCI U-O -O _N v ϊr BnO -^CK _x / -OH ⁺ ° _{\ /} ° ^N-N NH;

DMAP Bnθλ««^>V-O^,,^ HO ^''

OBn DCM OBn ¥

OMe 65% O O. _/ O 10 11 ¹² OMe

TFA

DCM/MeOH/H ₂ O

82%

HO --OH BnO --OBn

U-O _o /^/ .O ^u .x^N-N NH ₂ Pd/C, H, -A--O .N-N NH; Hθ\-->^-O _^ BnO-X-^-^-O^ _/ ^^O^*^ V* OH MeOH OBn T ^N — O HO OH 1eq HCI O HO OH

21%

13

Compound 1

5 Figure 1 shows an NMR spectrum of compound 1.

Scheme 2:

N _x O I ) O ₂ N _N

BnO -OBn

BnO -OBn

λ--o Ho^rV ^N - ^N O "Cl L\_ _^ 0 pyridine ^N-N NH ₂

BπOJ OH O

OBn 2)DMAP, 7 38% O _{N ^} - O ₇ OMe

10 OMe

TFA

DCM/MeOH

74%

-OH N BnO -OBn N /P _X -- 0 O _x f ϊr

NH ₂ Pd-C, H ₂ X t

O _j ^N-N BnθN-^ O^Bn- ^O' --^ ^Oλ -o'^V ^{N"N NH2}

HO OH

12

Compound 2

10

Figure 2 shows an NMR spectrum of compound 2. Scheme 3:

N^ _/ P

^ ^O x^.N-N NH ₂

BnO OBn OH

BnOr-OBn _ Bnθ\*^^-O ^{'^} ~-^ ^< *o

N I _j l-o 9

V // \ TBDPSCI BnOV-T^r 0 ^" ~~-^0H ° ^Bn 35% / ^u x^N-N NH ₂ pyridine O .N-N NH ₂

TBDPSO

HO OH 98% TBDPSO-^T V ^{N"N NH2}

BnO OBn ₀

Ui^-o i ^O

Bn0V*^~-^-0 ^{'^} ~- ^{^X} 0 OBn ,

O

BnO O^

-OBn BnO _x / I

BnO

17%

N £> N .0

N _{x /} P

^°x^.N-N NH ₂ HO- ^" X^-N-N NH ^°\^.N-N NH ₂ J

BnO OBn HO ^O H

O O H O O

BnO ^OBn ₀ O λ— O

TBAF O ³ k^T-X-O' ^" ^ , H ₂ »■ Bn ^N ΎoI γ OBn X O Y ⁰ Pd-C

OBn ' 81% (

O r BnO P- HO p

BnO PV ^c OBn OH

BnO. HO _x

OBn

BnO, BnO HO

BnO

Compound 3

5 Figure 3 shows an NMR spectrum of compound 3.

Scheme 4:

HO _^ -^,OH

25

I TBDPSCI, pyr

HO^^^-OTBDPS

AcO ^. OAc AcO ^- OAc

26 AcO ^- OAc BnO ^OBn

L\__,0 TMSOTf L J\ _^ ,0 1 Na, MeOH \—-o

AcθX*^--^-OAc ' AcO- V.**^^ TMSOTf AcO X- _X^ ,OTBDPS - BnOJ ^OTBDPS 70-74% N _x \ DCE NHAc 2 BnBr, BaO, NHAc V" 57-62% 23 24 [ 27 Ba(OH) ₂ , 28 43-74%

TBAF THF 75-92%

BnO ^-OBn BnO ^OBn BnO ^-OBn

CICOCOCI V-O

Bnoλ^^^-O^^ _" - _γ OH ^ NaCIO ₂

BnOJ NaH ₂ PO ₄ - ^" -^ ⁰ DMSO BnOJ ^OH NHAc NHAc O 2-methyl- Et ₃ N NHAc 30 81% 29

82%

N. +P

HO^ °γ.N N NH ₂

DMAP 80% o _v o

OMe

BnO _r -0Bn N^^O HO ^-OH

N -O ^OyiN N NH ₂ 1 TFA, 60%

Bnθλ^^T--i-O^ HOJ NHAc Y 2 Pd-C, H ₂ , HCI

V 21% O HO OH

OM ⁰ e

Compound 4

10

10

Figure 4 shows an NMR spectrum of compound 4.

Scheme 5:

HO OH 0..

V/ HO OMe Ph. „

I ⁰ / I ⁰ T ⁰ ^ OMe

HO OH o o O O

O

N— / ~OMe ^" OMe

HO. Ph. _^ O PlK _^ O.

T ¥ ^{^0} %°xT

O n. ^< ψ ^r yi OAc

AcO OAc AcO OAc AcO OAc

Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C

10 iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

OAc OAc OAc

AcO AcO / AcO

Vλ o ° ,θ o

AcO-V^-V^OAc AcOA^-V^ ⁰ λ AcO λ, Jl OAc OAc -^OBn OAc OH

II , IV

O

OAc

AcO / \1 OMe

Wo o

AcOλ^λ-O _x H OAc

AcO / OAc "NT H

O H^ _/ - _^ AcOX--vV-O U NH

(' if OAc

AcO OAc

⁰ \J

AcO OAc

o

HO ₍ ^OH _\ \

O Nγ^NH ₂

H

Hθλ^--\ ^V ' ⁰ . OH - a "i -\ _° r

HO OH

15 Compound 5

Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT

11

ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP then NH ₂ CH ₂ CH ₂ NHZ, DMF, NEt ₃ iv Pd/C, EtOH, hydrogen v PFP, DCC, DCM, DMAP, Ribavirin derivative vi 7N NH ₃ , MeOH

Scheme 6:

O o

NH, NH, , 0 NH,

_/ S

HOλ θ| HO ^{^} λ O^

^ ⁰ / HO OH o o o o

o O O

NH.

// ^N T ^HHl NH,

M ^N

λ°f J

TES O OTES HO OH ^" X OJ O

O

W

N-^ NH ₂

H ₂ NI o F

TESO OTES

10 Reaction conditions: i Diol protection ii Pyridine, MsCl, DMAP iii NaN ₃ , DMF, 60°C iv 30% aqueous TFA

15 v TES-Cl, pyridine vi Pd/C, EtOH, hydrogen

12

AcO _/ ^0AC : AcO / ^0AC ■■ AcO / ^0AC

AcOλ^-^OAc AcOλ^V^O Jl Acθλ--\^ ^o _N J. _nH

OAc OAc ^ ^0Bn OAc ^{N/ OH}

N^λNH ₂

,,-N

H ₂ N-^O ! ^>

TESO OTES

O O

OH OAc

AcO / NH,

HO OH ^TES ° ^0TES

Compound 6

5 Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP, Ribavirin derivative, DMF, NEt ₃ iv TFA, THF, MeOH 10 v 7N NH ₃ , MeOH

Scheme 7:

HO-I o , ^0H i HO-λ o , ^0Me ii ^Ph — ⁰ Y^O-X o . ^0Me

V7 — - v7 — - ° v7

HO OH O O O O

III

O O

N-γ^ ^0Me / _/ ^N lf ° ^Me

Y oγ| ^ ^^ ^Ph -Yoγi ^N Y^ "-Voγ ^OAc

AcO OAc ^{Ac0 0Ac} AcO OAc

15 Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN 20 v Pd/C, MeOH, hydrogen

13

HJM^, BocHN- BocHN^. HJM _^ .

^OH V-OH -N, KN,

BocHN- BocHN-

Reaction conditions: i BoC ₂ O, K ₂ CO ₃ , aq. Dioxane ii DCM, MsCl, NEt ₃ , 0°C iii DMF, NaN ₃ , 85°C iv Pd/C, EtOH, hydrogen

OAc OAc

AcO / ^0AC AcO / AcO

VV-o °

AcOλ _^ -V^OAc Vq O

AcO- OAc OAc N/ OBn Acθλ _^ --\ ^λ -'O _{χ /} OAc OH

H,N-

^N,

H,N-

OAc OAc

AcO / AcO /

\ \ O VA O

AcO \ \ V _^ °Vo _x ^NH ₂ AcO λ, -N ₃

IV

OAc K H- _r f OAc vλ H _n OAc ^

Acθ ₍ ^{OAC H} ^ o ro \ \ ,o r U

AcO V λ _^ --- Vv-O AcO V^ -γV-0

OAc OAc

0

DMe

OH O

V HO

V O ^o % \ \

V / A _/ 0 p N-

HO-

AcO OAc Vv \ I

N /--.. N

V _l -O ϋ

O _M H ^^ ^{^} N _H \

O °λ

Vl OH ^H V \/ \ ^C

J /

HO / L HO OH

HO VλV^-v°^O ' °

10 OH Compound 7

Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT

15 ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP, then amine, DMF, NEt ₃ iv Pd/C, EtOH, hydrogen v PFP, DCC, DCM, DMAP, Ribavirin derivative vi 7N NH ₃ , MeOH

20

Scheme 8:

14

HOλ O . ^OH HO OMe Ph. _^ ,0 _^

T OM

\ / λ°i O n°) e

HO OH O O O O

O

N _^ "OMe N- ~0Me

HO _^ PtK „0 PtK „0 OAc

ϊ ^' °λ° F ^■ °λ ° /

AcO OAc AcO OAc AcO OAc

Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

10

BocHN^ BocHN— _\

KOH VOH

H ₂ N- KNH,

BocHN- BocHN -^

Reaction conditions: i BoC ₂ O, K ₂ CO ₃ , aq. Dioxane

15 ii Amination of alcohol

15

^ OAc OAc OAc

AcO / AcO / AcO /

VL-o VV-O Q \ \

AcOλ _^ -V^OAc _A cOX-^V^-O AcO V O OAc OAc OBn OAc / OH

BocHN^

~OMe

⁰ Yl/ OAc

AcO OAc AcO /

\λ _^ o , — NH,

Acoλ _^ -V--- ⁰ _^ OAc N- H ^x — NH,

NH,

O

OH HN ^'

HO

\\ _/ 0

HO OH

OH O

/ N _^ / NH ₂

""if ^O - ^λ o} " o \ /

HO OH Compound 8

Reaction conditions:

5 i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP, then amine, DMF, NEt ₃ iv Pd/C, EtOH, hydrogen v PFP, DCC, DCM, DMAP, Ribavirin derivative.

10 vi 7N NH ₃ , MeOH

Scheme 9:

HO OH

υi HO OMe PtK _^ O _^ O UMl e

λ

HO OH ⁰ Yf o o o o

O O

\\

N^Y ^ OUe ^/ "OMe

HO. N ^N Ph^, fir

V

λ -^ ϊ ^Ph - °lf o- _V o t OAc

V ⁰ / I o 1 o \ 7

A cO OAc AcO OAc AcO OAc

16

Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

NH, NH,

NHBoc NHBoc

X H,N } H

BocHN X CO ₂ H BocHN 1 ^* ^^ ^H ^" NHZ ϊ NHZ O

10 O

Reaction conditions: i BoC ₂ O, NaOH, dioxane, water ii DCC, DMAP, DCM, H ₂ N(CH ₂ ) ₂ NHZ 15 iii HCl, MeOH, THF

OAc OAc OAc

AcO / AcO / AcO /

VV-O VV-O O \ \

Vλ ,O O

Acθλ^--V^ ^OAc AcO \^χ-'i~~O AcO λ, -γV-θ OAc OAc OBn OAc OH

NH,

0

W

J ^H

HC> N

OAc

^{^0} Y/ AcO ,

O

AcO A^-V^ ⁰ . OAc NH

OAc

OH AcO

HO

-O

,θ o

-Tλ-O 1 AcO^

HO^ O I 1? ₁₁ I I ₇ N ^H . .

OAc ^{^} l_j Il "NH,

OH -^NH

L

OH 0

HO /

N Y^ NH ₂ HO

Viu -/ NT if -^ N, /\ N

OH H Il Y^ ⁰ Yf

HO OH

Compound 9

20 Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP, then amine, DMF, NEt ₃ iv Pd/C, EtOH, hydrogen

25 v PFP, DCC, DCM, DMAP, Ribavirin derivative vi 7N NH ₃ , MeOH

17

Scheme 10:

HO-i oj OH

HO OMe PtK .,O.

\ 7 V l Y ⁰ V / OMe

HO OH O O O O

N _^ / ^{^} OMe N~^/^OMe

Hθ _{^ /} ~. \, N _v Ph^O^. ^ _{„ N} N Ph^O^^ OAc

O ^/ O \ / O \ J

AcO OAc AcO OAc AcO OAc

Reaction conditions:

5 i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen 10

NHBoc o H _; O

I N^ Il N-

VN H \ ^- H \

3 * ^"

H / -N ₃ — H

\ N^ \ N- J -N ₃

NHBoc O H. ₂ N O

Reaction conditions: i TBTU, DMF, NEt ₃

15 ii HCl, MeOH, THF

18

OAc OAc OAc

AcO / AcO / AcO

\λ -o p ,θ O

AcOA^-V^-OAc AcO VV- ^O AcOA^-V^- ⁰ OAc OAc OBn OAc OH

H ₂ N o w

OAc

AcO / hN ₃

H /

λ°f N

ACOVλ^O JlH

OAc ^V ^ ^N χ P

N

H

V OAc -NH,

AcO / H

Vl o \ N

_A c _O VλV _^ -Vo _^ O

OAc / N O

OH H

HO /

VVo O

HθV-λ^-θ OH O O

\\

N-V^NH ₂

H \ , H </ I

HO ₍ ^OH H /

_* ^r %

VVo o \_ O Y N/ O °r I

OH ^X / J ₁ H HO OH

Compound 10

Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP then amine, DMF, NEt ₃ iv Pd/C, EtOH, hydrogen

10 v PFP, DCC, DCM, DMAP, Ribavirin derivative vi 7N NH ₃ , MeOH

Scheme 11:

OH

HO OMe Ph. ^O _{^ /} •

λ °λ ,o OMe

"λ

HO OH o o o o

o

"OMe N~~/ ~OMe

C Il

HO. N

^~ \°r Ph, _,0 _{^ /} • Ph _^ .,O.

λ >f ^{0^} X ⁰ J OAc o N /

AcO OAc AcO OAc AcO OAc

15

Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine

19 iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

CN

HJvL

"NH, NHBoc _^ N.

^" NHBoc - ^" ^"-""^NHBoc

NC ^'

Reaction conditions: i Boc ₂ O, CHCl ₃ , 0°C ii Acrylonitrile, AcOH iii Hydrogen, Raney-Ni, 1.4N NaOH, EtOH

10

OAc OAc OAc

AcO / AcO AcO /

W-O ,θ o VV-o

AcO A^-YV- ^OAc AcO λ _^ -V^-O 11 Acθλ---\ ^λ -- ^o _N OAc OAc -^OBn OAc OH

o

OAc

AcO / , N ^/-

^" NHBoc

λ°r ,θ O H,N . J

AcOA^-V^ ^O _^ OAc ^N ¹

OAc

AcO

,θ O NH,

AcO-\--V^ ^o _x OAc

HO / ^OH

O

HO-V- O AH

OH N _^

O

HO / ^OH N / "NH,

VV-o O -N_^H

HO-V-V^ O OH ° Jt! O

HO OH

Compound 11

Reaction conditions:

15 i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii PFP, DCC, DCM, DMAP then amine, DMF, NEt ₃ iv 5% TFA, DCM v PFP, DCC, DCM, DMAP, Ribavirin derivative

20 vi 7N NH ₃ , MeOH

Scheme 12:

20

OAc OAc OH

AcO / AcO HO

VVo \λ-o o \ \

Acθλ^--V^ ^OAc _Ac oV-Vv-O Ji HO- ^O OAc OAc OMe OH -^OMe

P rh

Vo oi iλ _^ o o ^o 11 OMe

OH

Reaction conditions: i DCM, HBr/AcOH, 0°C then HOCH ₂ COOMe , DCM, AgOTf, 0°C to RT ii Na, MeOH, CHCl ₃ then DOWEX (H+) iii CSA, PhCH(OMe) ₂ , DMF 60°C, 260mBar o O

N- NH ₀

NH ₀ NH ₀

HO ZHN.

HO Vf V N _t / / H λ° ^: f

HO OH o o o o

OMe OMe

Ph

VO

Ol N- NH ₀

NH Il Vo

HOV ¹ A-f ⁰ , -O i

OH ^N 0-λ o I*

¹ ^N o-λ _O P HOλ _^ -Vv- ⁰ OH ^x

O

O O \ O I _/ γ OMe

OMe

OH o

HO

N Y^ NH ₂

OH ^ ^N ^/oi ol N

HO OH

Compound 12

10 Reaction conditions: i HC(OMe) ₃ ,PPTS, THF ii CDI, NEt ₃ , DMF then ZNH(CH ₂ ) ₂ NH ₂ iii Pd/C, MeOH, hydrogen iv MeOH, 80°C 15 v 2% TFA in MeOH

Scheme 13:

21

HO OMe PK _^ O-

λ°γ HO W O- λλ o OMe

HO OH O O O O

O O

~OMe OMe

PtK _^ O _^ Ph. ,0..

λ^ ^* ° ^~ W Oλ λ o OAc

AcO OAc AcO OAc AcO OAc

Reaction conditions: i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

OH OH

HO OAc OAc

AcO /

VV-o o — A-- ^O _{x nH} ,θ _^ o — ^ ^LOAC

AcO λ,

OH OH AcO- OAc OAc

ACO / ^OAC < ^OAC II, IV ACO / ^OAC < ^OAC

\Vo o— V—°\ o λ \Vo o— A-P,

AcO V-V^ Acθ ^λ - ^{^} ^ ^i/ OH ACOλ^- ^T λ^ ACO -^-V

OAc OAc OAc AcO Br

N-/ ~OMe ^ N

T ⁰ Y/

AcO OAc

OAc OAc

AcO / / O

\\/O _/ O r^ ^O \ .O^ A /\ _/ NH,

AcOV-V^ AcO^-^V^ ^ N ^ ² OAc ^{OAc H}

O

OH OH

HO O N- -/ "NH v L πJ Il

V-O _O λ _^

HO-: HO- ¹ -^ V °\^- o _^ ^/i N /^ ^ NI N

OH OH H Y O V

10 HO OH Compound 13

Reaction conditions: i Ac ₂ O, iodine

15 ii HBr/AcOH iii Benzyl glycolate, AgOTf, 0°C to RT iv Pd/C, EtOH, hydrogen

22

v PFP, DCC, DCM, then H ₂ NCH ₂ CH ₂ NHZ, Et ₃ N, DMF vi Pd/C, EtOH, hydrogen vii Ribavirin derivative, PFP, DCC, DCM, then amine, Et ₃ N, DMF viii 7N NH ₃ , MeOH

Scheme 14: ₎ o=

^H0 X°y ^0H Z. ' Ho-i o ? ^Me „ ^Phχ/ Yoλ o ? ^Me

HO OH O O O O

III

O Y

\\

^" OMe N~ _^ /~~OMθ

// T

HO, PtK _^ O, of " _J__ PtK^O^^

- Y °% OAc s ⁰ Yf V ⁰ V

AcO OAc A cO OAc AcO OAc

Reaction conditions:

10 i MeOH, acetone, 2,2-dimethoxy propane, HCl^g) ii NaH, THF, 70°C then alkyl iodide, 0°C iii 70% aqueous AcOH, 90°C then acetic anhydride, pyridine iv Triazole, DBU, TMSOTf, MeCN v Pd/C, MeOH, hydrogen

15

-(

OH OAc

OAc

HO AcO

AcO / Il

\ λ _/ O V V--O HO- ,OH AcO λ ^OH

I V

AcO A ^OAc

NH ₂ HCI N ^ N H ₂ TFA

N ^,NHR H ^" ~X\

H Y O

O

N~/ ^~ OMe

( Il

HO ^" I

O N~^/ "NH,

HOXW^OH H () Il ' iv ' ^HO r° AvcO / OAc ^N πγ o^V o n XoJ J

HO OH Compound 14

20 Reaction conditions: (R = Z, Fmoc, Boc): i a) HATU, DIPEA, DMF, b) Ac ₂ O, pyridine ii R=Z: Pd/C, hydrogen, TFA (1 eqv), EtOH ; R=Fmoc: piperidne; R=Boc: TFA/DCM, 1/1, v/v iii Ribavirin derivative, PFP, DCC, DCM, then amine, NEt ₃ , DMF 25 iv 7N NH ₃ , MeOH

23

Scheme 15:

OAc OAc OH

AcO / AcO / HO

,θ ,θ

AcOλ^-V^OA ⁰ Acθλ^--v ^v ' ^o _N HO- OAc OAc OMe OH OMe

Ph Ph to

O ,θ O

HOA^V^° _X HO V o OH OH i-

OH -^OMe

Reaction conditions: i DCM, HBr/AcOH, 0°C then HOCH ₂ COOMe , DCM, AgOTf, 0°C to RT ii Na, MeOH, CHCl ₃ then DOWEX (H ⁺ ) iii CSA, PhCH(OMe) ₂ , DMF 60°C, 260mBar iv NaOH (leq), MeOH:H ₂ O (v:v 3:1) then IM HCl (0.95 eq).

o O w

NH,

~NH, o NH,

HO ZHN. N

HO \°? o-) o

OH

HO OH o o y O Y O OMe OMe

Ph

P o rh

VO o

?l _^ o O ^Y^- NH ₂ ^H0 ^ OH ⁰ ^ TPA . H ₂ N _^ λ a

HOV- ,-γ^-o o N

OH o

Me V f O Y O o Y o OMe

OMe

OH O

HO / \\

N Y^NH _{2 HO &^YO JlH 9 N}

OH — 0 ^{^} λ ₀ ;

Me \

HO OH

10 Compound 15

Reaction conditions: i HC(OMe) ₃ , PPTS, THF ii ECDI ₅ DCM, DMAP 15 iii Pd/C, EtOH, hydrogen, TFA iv HATU, DIPEA, DMF

24

4% TFA, MeOH/H ₂ O

Scheme 16:

OAc OAc OH

AcO AcO / HO

.0

AcO^ AcO-Y^A-O HO^ -τλ-0

OAc OAc ^ OMe OH ^ OMe

Ph Ph

V :.o

Ol Ol

O o

HO^ λ _^ O HO^ -γV-0 11

OH ^ OH OH OMe

Reaction conditions: i DCM, HBr/AcOH, 0°C then HOCH ₂ COOMe , DCM, AgOTf, 0°C to RT ii Na, MeOH, CHCl ₃ then DOWEX (H ⁺ ) iii CSA, PhCH(OMe) ₂ , DMF 60°C, 260mBar iv NaOH (leq), MeOH:H ₂ O (v:v 3:1) then IM HCl (0.95 eq).

10 o U

N-^NH ₂ NH, λ-

„ N ⁰ // Y NH,

HO Yf HO Yf ZHN ^' λ ⁽ N ^N

O O ^{ZHN 0H}

HO OH Y o o

OMe OMe

Ph

O Ol o

VO U Uv,o o

N- _^ /^NH, HO-V-V-o^i N^NH _{2 ^ O o O \-0 X X. N M N Hθ\, -χ}

OH N H ⁰ Yf ⁰ Yf

O O O O γ Y _>

OMe OMe

H0 / ^0H N-Y ~NH,

VV-o P

HOA^-V^- ⁰ OH ^■Jl B " ° ^" W

HO OH Compound 16

Reaction conditions: 15 i HC(OMe) ₃ , PPTS, THF ii ECDI ₅ DCM, DMAP iii Pd/C, EtOH, hydrogen, TFA

25 iv HATU, DIPEA, DMF v 4% TFA, MeOH/H ₂ O

Scheme 17:

OAc OAc OH

AcO AcO , ii HO ,

VV-o V V-o O * ^■ V V-O O

AcO-V-'V -OAc AcO λ ^V-O Hθλ _-λ^-O Jl

OAc OAc / OMe OH v/ OMe

Ph Ph

O VO ol 91

[ \λ -O o o Il HO^

OH -" OH ° OH ^{^} o v> S0Me

Reaction conditions: i DCM, HBr/AcOH, 0°C then HOCH ₂ COOMe , DCM, AgOTf, 0°C to RT ii Na, MeOH, CHCl ₃ then DOWEX (H+) iii CSA, PhCH(OMe) ₂ , DMF 60°C, 260mBar

10 iv NaOH (leq), MeOH:H ₂ O (v:v 3:1) then IM HCl (0.95 eq).

Ph

({ Il \, N OH ^ ^0H

HOλ o) ^N ' . ^H °-X°J

HO OH °γ°

OMe O

OH W

H ^H _θ ^O vλL>Vo^-O _^ J o _^ _ P\Y N ^ OH -H ^o J

HO OH Compound 17

Reaction conditions: 15 i HC(OMe) ₃ , PPTS, THF ii ECDI ₅ DCM, DMAP iii 4% TFA, MeOH/H ₂ O

20 Scheme 18:

26 o O

W

N-^ NH ₂

Y NH,

N

^H °-W ^{N j} TIPSO V/

HO OH HO OH

O

NH, NH,

¹ Y NH ₀

TIPSO ^{^}

Tl PSO -γf TIPSO To/ V/

O OH HO O o o

O ₂ N ^' O ₂ N

O ₂ N ^ NO,

i O

♦ N-^NH ₂

M ^N

TIPSO-λ o ^ ¹

HO O

Reaction conditions: i TIPS-Cl, imidazole, DMF ii Ag ₂ O, TBAI, DCM, 4-nitrobenzyl bromide iii Pd/C, EtOH/DCM, hydrogen

OAc OAc OAc AcO / AcO / AcO / o O

Acθλ---V^- ^OAc AcO AcO \to OAc OAc OBn OAc OH

O

O \ M

TIPSO-I O . ^N

NH,

< \ N HO O

HO O IV, V

OH

HO / H H O

OHr°λ Compound 18

10 Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen

27 iii HATU, DIPEA, DMF, Ribavirin derivative iv TBAF, THF, DIPEA, DMF v NH ₃ , MeOH

Scheme 19: o

Nγ~NH ₂ NH, N

Ho- Vλ o/ r I TIPSO \°r

HO OH HO OH

O

NH, NH, NH,

TIPSCλ o TIPSO λ°f TIPSO V/

O OH HO O o o

O ₂ N O ₂ N ^' O ₂ N NO,

O

NH,

TIPSO V/

O OH

H ₂ N

Reaction conditions: i TIPS-Cl, imidazole, DMF

10 ii Ag ₂ O, TBAI, DCM, 4-nitrobenzyl bromide iii Pd/C, EtOH/DCM, hydrogen

28

^ OAc

Aco / ^0Ac AcO / AcO / ^0AC

VV-O vvo O VVo o

Acθλ _^ -V^- ^OAc AcO-VV^ ⁰ OAc OAc OBn Acθλ^-V^-° OAc OH

MH,

O \,-N

^" NH,

V 'Y HO I O

HO W ( j

IV, V

O OH

OH

HO

_HO W-Oj OH

Compound 19

Reaction conditions: i DCM, HBr/AcOH, 0°C then benzyl glycolate, DCM, AgOTf, 0°C to RT ii Pd/C, EtOH, hydrogen iii HATU, DIPEA, DMF, Ribavirin derivative iv TBAF, THF, DIPEA, DMF v NH ₃ , MeOH

10

Scheme 20:

AcO -- OAc AcO --OAc AcO --OAc

TBDPSCI \~^°\

AcO λ»—^--L-- OAc HO^ i-J -OBn AcO-X >--V-O^-(pj _^ -OR Ac0-V-- ¹ >^-0 _^ pyridine _0Ac AcO OAc ⁿ /r ^" b: n=3 4 R = Bn, b n=3, (77%)

Pd(OH) ₂ /C, H ₂ , EtOH _| 7b n=3 (98%) 5 R = H, b n=3 (70%)

1. Na, MeOH 2. BnBr, NaH, DMF

BnO ^OBn BnO ,--OBn BnO-' OH ^^O _^ i-4 -OH

OBn * M TBABr, OBn ⁿ OBn NaHCO ₃₁ DCM

10b n=3 (93%) 9b n=3 (98%) 8b n=3 (70%) TBAF

BnO ^OBn ^ ⁰ V BnO ---OBn

HO ^' ^T .N.

EDCl .0.

B nO λ^-^>~^- O ./ > -OH .N-N NH ₂

Ck ,0 DMAP BnO-V- ¹¹¹¹ ^V- OBn ^Kη s T DCM OBn

OMe 60% tϊTϊ 0~ ^' O

10b 11 12b OMe

TFA

DCM/MeOH/H ₂ O

66%

BnO --OBn ,N _% ,p

Pd/C, H ₂ N-N NH ₂ -O γ°γ.N-N NH ₂ BnO-^ -O

OH π , t MeOH OBn H ₃ T ^U

O H0~ OH 1eq HCI O HO ^S OH

51 %

13b

15 Compound 20

29

Scheme 21:

Hσ^M^OH

TBDPSCl pyridine

62% AcO OAc AcO OAc

AcOJ .O^

^~V ^, -SPh -

BF ₃ OEt ₂ OAc NIS TMSOTf DCM AcOλ ^-O-^f^OTBDPS ⁺ OAcO-V" ⁰ OAc

AcO S^ AoAc 18(36%)

12(26%)

1 Na MeOH

2 BnBr NaH DMF 70%

BnO ,OBn

>_-J ^~ ^QLJ -^l ^« ^-_-0 ^' (~1 ~0TBDPS

NaOCI TBABr OBn ⁵

KRr NaHCD., 19

M:

'yN N NH ₂

EDCI

DMAP 45% V OM ⁰ e

BnO OBn

TFA DCM/MeOH OH_OH ,N. 0

Lj^o M

^ ⁰ V-N N H ₂ O 81% \ ϊr

BnθX-- ¹ T-^- ^θ' TH' ^H0 ^^CT ^ ⁰ X--N-N NH ₂ 0^ ,0

⁴ OMe

Compound 21

Scheme 22:

O^ ,OEt Ph

AcO OAc ^^ O

O OEt ₂ OEt O

1)Na, MeOH, qu

AcO - -^OAc (S . BF ₃

DCM i(

AcOJ O' _^ O

2) Bz(OMe) ₂

OAc 61%

OH OAc pTsOH HOJ

14 24 58% CJ OEt

23

NaOH, MeOH 60%

HO-vO NM _N H ₂

Ph O

N _/ P 1

OMe OH N-N NH ₂

EDCI, DMAP HO^ DMF, 24%

HO^ O, ,O

OH

26 Y OMe

TFA, DCM/MeOH

^ ^υ x^.N-N NH ₂

HOJ

Compound 22

10 Scheme 23:

30

OBn OBn

OBn ₀ Bn l-V-0 O

BnoV-'^-O^ ,OH UV-0

BnOJ OBn -o A NH

AIlBr Cs ₂ CO ₃ k OH DMF -.....^k. _/ OAII PFP, DCC, DMAP OBnOBn

RHN ^' Y 78% ^RHN Y DMF ₁ Et ₃ N

O O 36% .,OR

BnO -Ck HN'

BoC ₂ O, NaOH I 50R=H 52 R = Boc 1 _{TFA nrM} dioxane, quant I— 51 R = Boc 53 _{R = H} TFA^J quant

43R= hi" Zm ^K ° ^tBu _' ^Me0H . ⁸⁴ %

O O ^N _x

Jl ^" _N NH ₂ j≡ ^P q _RHN ^λ ₀ ^yNN NH ₂

ZHN'^- ^' ^OH EDCl,

0 0 ^81% 0 0 DMAP,

51%

OMe OMe

Pd-C ₁ H ₂ i 52R=Boc

TFA, 31% I— 53R=HTFA

OBn _06n

O O

1 UV-o

NH BnθX-^u-0' ^" ^ 1

NH OBn

OBn ₀ Bn A _0^ «γ , ^υ 0y..N N NH ₂ HCI, MeOH U\_0 ^N N NH ₂

HOJ k NH ^' -x _^

\ 7 ₀ ^ ^υ k NH ^' Nλ,

Bnθ\--?*V-O^ HN' 0 π HO OH OBn Il o O _x ,0

Y

OMe

Compound 23

Scheme 24:

AcO ^- OAc

L AcO _% ,, OAc

■λ^o

Y-O «— "^V-OAc + HO _{x /} γOMe BF ₃ OEt ₂

AcO- V Na, MeOH _^ /^ _x _^0Me

OAc DCM ' AcOλ. -""- _X "--^ O quant O 90% OAc T 10 O 10

Ph Ph

/

D \ ⁽ D

HO ^OH O

Bz(OMe) ₂ O NaOH \—-o

HO^ OMe pTsOH HO Iλ -^-O _x/ ^0Me dioxane HO Iλ

OH MeCN OH quant OH O 82% O 10 11

Chemical Formula C ₁₈ H ₂₈ N ₄ O ₁₂ Exact Mass 49217

HO N

TFA ^π V r-° ^H

12 +

DMAP ^► V^- ⁰ _X ^•^/Oχ^.N-N NH ₂ DCM DCM/MeOH Hθ\*--^--V_-O _x/ ~ _x ^,O ^' 38% H ₂ O ^X _OH Y

30% ₀ HO OH

Compound 24

Scheme 25:

31

AcO ,-OAc

BF ₃ OEt ₂

HO _x ^" V ,OMe Na, MeOH π DCM ¹ AcO OMe BnBr, NaH, DMF O 95%

BnO ,-OBn

BnO ,,0Bn BnO- -0, V ,OMe EDCI BnO ^- OBn ^N U jV ,0H — V OBn T Bn0λ----?<^ ^, 0^ „-0

O ,0 f iT OBn ¥ DMAP yφ N-N NH ₂

10 (40%) DCM BnOλ-

10 (24%) 60% OBn Il o & Jo

OMe

TFA DCM/MeOH H ₂ O 71%

,,OH N

BnO , ^, OBn N

M 0 f ⁰

^.N-N NH, Pd-C, H ₂

(

— 0,. X ,0^Y .N-N NH ₂

HCI, MeOH BnO ° _v \ // ^'

OH HO OH OBn T — '

O HO OH

Compound 25

Scheme 26:

OAc, OAc Y I) NaOMe, MeOH I

AcO OAc 2) PhCH(OMe) ₂ \\ DCM ~~OMe MeCN, pTSOH 0

AcθX-*?^-y,OAc ⁺ I BFo.OEt,,

OR

OAc 54% AcO 59% over 2 steps

14 OH HO- e ¹ ^°\ ,,O XJ 15

Vo S ^{" Me} I ^Na0H . ^Me0H . ⁹⁶ %

^°x _> N-N NH ₂

,O _{x N} OM© ₂

TFA, DCM/MeOH J \ (I \

X ⁰ X^N-N NH ₂

HO OH

Compound 26

Scheme 27:

32

CK ,OR

I) NaOMe ₁ MeOH

AcO ,OAc

BF ₃ OEt ₂ , DCM 2) PhCH(OMe) ₂ OMe MeCN, pTSOH

OR

70%

OAc 14% over 2 steps HO-J .0^-

14 22

^ NaOH, MeOH, 65%

2J1 R R = = H Me ~3 DIC, DMAP, DCM, MeOH ²³ R ^{" Me "} ^]

81%

,N. J3

NH ₂

HO ^" «-γ°γ.N N

EDCI DMAP

DMF, 54%

⁰ V ⁰ ₂

Ph OMe '

N 0 L

\ N .0

0

NH, TFA, DCM/MeOH 0 /

21% X 0 \

~~-\---~- 0 0' / I HO HO^ ^-V-0- _^ - O^ OH ø ⁰ V N- N NH ₂ v" ⁰ 25 OMe

Compound 27

Scheme 28:

-OH AcO ^- OAc

1 HATU, DlPEA

HO DMF

Y ^-^ NHBoc + L\ ^o

W-Oh AcO- -OAc

0 HoX.

NH

44 2 HN.

-" ^""" NHBoc °l ^uant V NH ₂

1 BoC ₂ O 45 47 O

I NaOH dioxane quant

HO

Y ^-^ ^{^} NH ₂ N N NH ₂ DlPEA

HO _^ o-c°r DMF

0 43 AcO OAc 48%

48

OH ,- -OH AcO ^ OAc

_^ - q ^N O NH ₃ N x.0

HOX- 4jj« 0H O //

.0. AcoX"* ^ OAc \

HN. JJ N-N NH ₂ MeOH γ«N-N NH,

O HN ,0 T ^■ N ^' 19% J H

O HO OH AcO OAc

Compound 28

O

BnO -^Br

NaI,

Acetone quant

O

HO ^ ^O ^OH Acetone ^/ Me ^O H _H0 ^(V ^0Me ^ ' o B-nJO LxrVV° ^{Me 1 Ac0H} ' "^ n B.n^O O^ ⁰ )- -OAc

HCI O O NaH AcO OAc

HO OH 0-^0 2 Ac ₂ O, pyr 72% 51 %

-^^ 60% 2

TMSOTf i M O DBU ( W

MeCN HN N NH ₂

50% T

P _d -C H ₂ 5_ ₀ ^° _r N N NH ₂ j^ ₀ ^°^N N NH ₂ BnO \ — /

HO MeOH AcO OAc

AcO OAc 77% 48

10 Scheme 29:

33 tsnu ^ubn

1 NaN ₃ Tf ₂ O S ₃ N Lλ^-0 quan ^~ t~ Bnθ\

NH, ^H OH ^{2 CuS} ° ^{4 K2C} ° ³ NH ₂ OBn ^{2 UM} 3 BnBr NaH DMF

BnO ,-OBn Pd C H ₂ L\ _-0

^N. MeOH BnO- OBn 9

₀ ^°yN N NH ₂ HCI ^u y.N N NH ₂ iN N NH ₂ ^H 2°

43%

Compound 29

Scheme 30:

AcO ^ OAc

AcO ,,OAc

TBDPSCI \

AcO ^- OAc DMAP -λ _^ -o

BF ₃ OEt ₂ ^Ac0 * -CX / ,OH

AcO-

HO^. \*^-\,0 pyridine 49% 43% OAc

N^O _H0 ^^NN NH ₂ o _v o

OMe

EDCl

DMAP 29R = TBDPS-H _{TBAF T} H _F

56% 30R=H ^■ * — I Goo _/

Compound 30

Scheme 31 :

AcO _^-OAc AcO _r ^OAc TEMPO AcO ^- OAc

BF ₃ OEt ₂ I \ NaOCI KBr

HO^^, -J _^ -OBn UC-o

AcOλ ^-^-^-OAc ⁺ DCM AcoX ^-0 TBABr' Acθ\*^«V,O^ >v^ _^ , OH

AcO OAc ^JrV' ^0R NaHCO ₃ DCM OAc ^V 'nfl a n= 1 O

3 b π=3 4 R = Bn a π = 1(89%) 6a π = 87%

Pd(OH) ₂ /C H ₂ EtOH _i

5 R = H a π = 1 (74%) O

EDCI --, ⁰ N-NN NH

HO ^"

DMAP \ — /

DCM O _γ O

67% OMe

2

AcO ^ OAc N _%

I )) — - AcO ^OAc N

\ _^ -0 Il f° N NH ₂ TFA

AcO \ N

AcO-X--^

OAc DCM/MeOH/H

O HO OH ₂ 0 OAc T 60% O O. O

10 Compound 31

Scheme 32:

34

MW

V — V-OH 14Q ⁰ C , 2, EDCI O

DMAP —O A _^* - _^ / ^O γ.N-N NH ₂ 36 15% - ⁰ ^ X ⁾ H DCM

37 38 80%

39

TFA ° ^Me DCM/MeOH H ₂ O 28%

.O _N x Ir

V- — \^-O "O y N-N NH ₂

HO OH

Compound 32

In the above processes it may be necessary for any functional groups, e.g. hydroxy or amino groups, present in the starting materials to be protected. Suitable protecting groups and methods for their removal are, for example, those described in "Protective Groups in Organic Synthesis" by T.W. Greene and P.G.M. Wuts, John Wiley and Sons

10 Inc., 1991.

Hydroxy groups may, for example, be protected by arylmethyl groups such as phenylmethyl, diphenylmethyl or triphenylmethyl, or as tetrahydropyranyl derivatives.

15 When S, L or the ribavirin moity includes an amino protecting group, suitable amino protecting groups include arylmethyl groups such as benzyl, (R,S)-a-phenylethyl, diphenylmethyl or triphenylmethyl, and acyl groups such as acetyl, trichloroacetyl or trifluoroacetyl. Conventional methods of deprotection may be used.

20 Arylmethyl groups may, for example, be removed by hydrogeno lysis in the presence of a metal catalyst e.g. palladium on charcoal. Tetrahydropyranyl groups may be cleaved by hydrolysis under acidic conditions. Acyl groups may be removed by hydrolysis with a base such as sodium hydroxide or potassium carbonate, or a group such as trichloroacetyl may be removed by reduction with, for example, zinc and acetic acid.

25

Other compounds of formula (I) may be made from commercially available starting materials using analogous processes.

35

Pharmaceutically acceptable derivatives of the compound of formula (I) include pharmaceutically acceptable salts, esters and amides thereof.

5 Suitable pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts derived from inorganic and organic acids, such as hydrochlorides, hydrobromides, hydroiodides, sulphates, phosphates, maleates, tartrates, citrates, benzoates, 4-methoxybenzoates, 2- or 4-hydroxybenzoates, 4-chlorobenzoates, benzenesulphonates, p-toluenesulphonates, naphthalenesulphonates,

10 methanesulphonates, sulphamates, ascorbates, salicylates, acetates, diphenylacetates, triphenylacetates, adipates, fumarates, succinates, lactates, glutarates, gluconates, hydroxy-naphthalenecarboxylates, e.g. 1 -hydroxy or 3-hydroxy-2- naphthalenecarboxylates, or oleates.

15 The compounds may also form salts with suitable bases. Examples of such salts include alkali metal, e.g. sodium and potassium, and alkaline earth metal, e.g. calcium and magnesium, and ammonium, salts.

The compound of formula (I) may be obtained in the form of a salt, conveniently a 20 pharmaceutically acceptable salt. Where desired, such salts may be converted to the free bases using conventional methods. Pharmaceutically acceptable salts may be prepared by reacting the compound of formula (I) with an appropriate acid or base in the presence of a suitable solvent.

25 Suitable pharmaceutically acceptable esters of the compounds of formula (I) include alkyl Ci _-4 esters, e.g. ethyl ester. The esters may be made by conventional techniques, e.g. esterification or transesterification.

Suitable amides include unsubstituted or mono- or di-substituted alkyl Ci _-4 or phenyl 30 amides, and may be made by conventional techniques, e.g. reaction of an ester of the corresponding acid with ammonia or an appropriate amine.

36

The compounds of formula (I) may exhibit tautomerism, they may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various optical 5 isomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation.

10 By the term MW we mean microwave radiation.

By the term alkyl we mean straight, branched or cyclic saturated or unsaturated alkyl groups. Preferably alkyl is C _1-6 , more preferably Ci _-4 , for example methyl. Cycloalkylene includes C ₃ -Cs cycloalkylene, including cyclohexylene and preferably 15 includes the following group:

Arylene includes phenylene and preferably includes the following group:

We prefer compounds of formula (I) in which R ¹ represents S-L-. We further prefer 20 compounds of formula (I) in which X represents O.

Suitable sugars that may be mentioned include galactosamine, galactose and lactose. The sugar may be cleavably linked to -L- by any residue which may be cleaved in vivo, to release the parent sugar. The sugar may linked to -L- by an oxygen or a 25 nitrogen; for example the sugar may be a 6-galactosyl residue or a 5- galactosamine residue.

Specific examples of L include:

37

O O

~O

O O

O O

^{^} o

O

O O

N NH ^O H O

HN HN

O

\ H HrN

O

⁵ JN O

H O

-NH

O -N,

~NH

NH ¹ OI^

O

NH

O O

O

O O

³ ^ NH H ^S

38

O

H

O

O O O O

N ^' NH H

N ^H o

O O

S

. H HN

I l S- ⁷

H

HN O

wherein r, n and S are as defined above.

When R ⁴ represents acyl, acyl includes alkanoyl C ₁ -C ₆ , preferably acetyl. We 5 particularly prefer combinations of S-L that are likely to bind and to be cleaved by asialoglycoprotein within hepatocytes, to release ribavirin.

Most preferably, compounds of formula (I) include: Compound 1

HO / ^0H M ?

2

^Hθ ^ ^£ o^A,^y ^N - ^N

HO OH Compound 2

39

O

HO / ^OH _o N

O f NH,

O. N-N

O ^'

OH

HO OH

Compound 3

O

N

O. ^ f NH, N-N

HO ^'

OH ;λ c?T

HO -OH

HO-^-V

HO

Compound 4

O

H0/ ^0H N

O f NH, HO ^ S _0. O. .N-N

O NHAc

HO OH

Preferably, compounds of formula (I) additionally include: Compound 5

O

H0/ ^0H N-/ NH,

^HOH ° ^V fo ^{^ N}

HO OH

Compound 6

40

O

OH

HO ^" NH,

<' HO ⁰ -N

O OHH ⁰ ^ °N _H O.

HO OH

Compound 7

OH O HO /

O O N NH ₀

'/ O N <' I

N OH on o o

OH \ I,

HO

,o r o HO OH HO -O OH

Compound 8

O

N--/ NH,

O < ^/ I

OH HN HO /

O

HO OH

^HO λ-- ^{^} V^ ^{^0}

OH O N-/ NH,

^H T °λ HO! ^/ OH ^'N

Compound 9

41

OH

HO /

O

HOλ^^A-

OH NH

OH O

HO /

N NH _n

HO ⁰ ?ι -N

OH H O O.

O

O

HO OH

Compound 10

OH

HO /

O O

HO o IH

OH ^N O O

N NH,

<' M

OH

HO / Y ^{^} o

Ho^k ow I o O -o 1

OH ^NH HO OH

Compound 11

OH

HO /

HO^^OJH

O

H0/ ^0H N _^ / NH,

N. /^H

_HO ^{^} o ^K ^N JH^

HO OH

Compound 12

O H0/ ^0H

N NH,

H ₀ ^{^} LOJL _N O '/ I _^ N

^{^} N^O H

HO OH

Compound 13

42

O

N NH.

HO/ ^OH

< '/' I

r N

O n \of /

HO OH

Compound 14

W-O N _^ /^NH,

HO OH

Compound 15

HO / ^0H 1

Me \ /

HO OH

Compound 16

O

OH \\

HO / ^ _n N^/^NH ₂

HO OH

Compound 17

O

^HO vC _{° ?} f/- ^NH -

^HO ^ ^O ^on.oV ^N

HO OH Compound 18

43

O

N NH ₀

-N

HO

HO O

OH

HO / ^{v :}

HO °

OH ° J ^ N H

Compound 19

O

N NH,

<' N

HO-I n

O OH

OH

HO /

O O

HO

OH ⁰ ^ N H

Compound 20

OH

HO /

OH^O

HO OH

Compound 21

H0 / ^0H

Hθλ _^ --\\

OhTo

HO OH

Compound 22

O

O f // NH ₂

H0/ ^0H O. N-N

O

HO-V^V O ^'

OH HO OH

44

Compound 23

OH

HO /

O

HO

OH O NH

O

N

^HO vLo O O

/X _/ f NH, N-N

O

OH

O

HO OH

Compound 24

O H0/ ^0H

O NH,

HOλ _^ -λλ. O. .N-N OHO O

HO OH

Compound 25

O

HO / ^OH N

O f // NH, HO-\^V^ O. N-N

OH O" X \>-

HO OH

Compound 26

O

N

O

O. _/ f NH, N-N

H0/ ^0H O

HO-S^V^- ^0'

HO OH OH

Compound 27

H0/ ^0H ϊ-\^\^-' HO OH

Compound 28

45

OH

HO / O

N

U f NH,

N-N )

HN _^ ^\ . 0 ^'

Il ^^ N A ,

\ \ / I

H O

HO OH

Compound 29

OH

HO / O

J

Hθλ^^-»\OH r U\ NH,

,0^ fN-N >

HN ,0"

Il

O HO OH

Compound 30

O

H0 / ^0H N

HOλ _^ -\V f NH,

Ov N-N

OhT O

^{^} O

O

HO OH

Compound 31

OAc O AcO / N

O NH,

ACO \λL^^ToV _χ n _^ fN-N

\ O O OAc

HO OH

Compound 32

O

O f ^N v^

Ov

^ o^ o

HO OH

The compounds of formula (I) are useful in that they exhibit pharmacological activity in animals. In particular the compounds are prodrugs of ribavirin.

46

The compounds of the invention may be used to treat an infection, an infestation, a cancer or tumor or an autoimmune disease. It is further contemplated that the compounds of the invention may be used to target conditions or diseases in specific organs of a patient, such as the liver or heart. 5

Infections contemplated to be treated with the compounds of the present invention include respiratory syncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, 10 hantann virus (hemorrhagic fever), human papilloma virus (HPV), measles, and fungus.

Infestations contemplated to be treated with the compounds of the present invention include protozoan infestations, as well as helminth and other parasitic infestations.

15 Cancers or tumors contemplated to be treated include those caused by a virus, and the effect may involve inhibiting the transformation of virus-infected cells to a neoplastic state, inhibiting the spread of viruses from transformed cells to other normal cells and/or arresting the growth of virus-transformed cells.

20 Autoimmune and other diseases contemplated to be treated include arthritis, psoriasis, bowel disease, juvenile diabetes, lupus, multiple sclerosis, gout and gouty arthritis, rheumatoid arthritis, rejection of transplantation, giant cell arteritis, allergy and asthma.

Still other contemplated uses of the compounds according to the present invention 25 include use as intermediates in the chemical synthesis of other nucleoside or nucleotide analogs that are, in turn, useful as therapeutic agents or for other purposes.

In yet another aspect, a method of treating a mammal comprises administering a therapeutically and/or prophylactically effective amount of a pharmaceutical containing 30 a compound of the present invention. In this aspect the effect may relate to modulation of some portion of the mammal's immune system, especially modulation of lymphokines profiles of Typel and Type 2 with respect to one another. Where modulation of Type 1 and Type 2 lymphokines occurs, it is particularly contemplated

47 that the modulation may include suppression of both Type 1 and Type 2, and more preferably stimulation of Type llymphokines, or a relative increase of a type 1 response to a type 2 response.

5 Combination therapies according to the present invention comprise the administration of at least one compound of the present invention or a functional derivative thereof and at least one other pharmaceutically active ingredient. The active ingredient (s) and pharmaceutically active agents may be administered separately or together and when administered separately this may occur simultaneously or separately in any order. The

10 amounts of the active ingredient (s) and pharmaceutically active agent (s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. Preferably, the combination therapy involves the administration of one compound of the present invention or a physiologically functional derivative thereof and one of the agents mentioned herein below.

15

Examples of other drugs or active ingredients contemplated to be effective in combination with a modulator according to Formula (I) are anti-viral agents such as interferon, including but not limited to interferon α- and γ-, Ribavirin, acyclovir, and AZT™ ; anti-fungal agents such as tolnaftate, Fungizone™, Lotrimin™, Mycelex™,

20 Nystatin and Amphoteracin ; anti-parasitics such as Mintezol™, Niclocide™, Vermox™, andFlagyl™, bowel agents such as Immodium™, Lomotil™ and Phazyme™ ; anti-tumor agents such as interferon α- and γ-, Adriamycin™, Cytoxan™, Itnuran ™, Methotrexate, Mithracin, TiazofurinT-Nl. Taxon; dermatologic agents such as Aclovate™, Cyclocort™, Denorex, Florone™, Oxsoralen™, coal tar and salicylic

25 acid; migraine preparations such as ergotamine compounds; steroids and immunosuppresants not listed above, including cyclosporins, Diprosone™, hydrocortisone; Floron, Lidex, Topicort and Valisone ; and metabolic agents such as insulin, and other drugs which may not nicely fit into the above categories, including cytokines such as IL2, IL4, IL6, IL8, ILlO and IL12.

30

Especially preferred primary drugs are AZT, 3TC, 8-substituted guanosine analogs, 2,3-dideoxynucleosides, interleukin II, interferons such as α- and γ-interferons, tucaresol, levamisole, isoprinosine and cyclolignans. Other drugs that may be

48

mentioned are Toll-like receptor (TLR) agonists, such as Actilon, other immunomodulators and protease/polymerase inhibitors.

Examples of such further therapeutic agents include agents that are effective for the

5 modulation of immune system or associated conditions such as AZT, 3TC, 8- substituted guanosine analogs, 2', 3'-dideoxynucleosides, interleukin II, interferons, such as α-interferon, tucaresol, levamisole, isoprinosile and cyclolignans. Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the

10 metabolism or inactivation of other compounds and as such, are co-administered for this intended effect.

With respect to dosage, one of ordinary skill in the art will recognize that a therapeutically effective amount will vary with the infection or condition to be treated, 15 its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient (animal or human) treated.

For the above mentioned uses the doses administered will, of course, vary with compound employed, the mode of administration and the treatment desired. However,

20 in general, satisfactory results are obtained when the compound of formula (I) is administered at a daily dosage of from about 1 μg to about 20 mg per kg of animal body weight, preferably given in divided doses 1 to 4 times a day, e.g. twice a day (BID) or in sustained release form. For man the total daily dose is in the range of from 70 μg to 1 ,400 mg and unit dosage forms suitable for administration comprise from 20

25 μg to 1,400 mg of the compound admixed with a solid or liquid pharmaceutical diluent or carrier.

While treatment success may be achieved with some viral infections at relatively low plasma concentrations of the compounds of formula (I), other viral infections may 30 require relatively high dosages. It is contemplated, however, that an appropriate regimen may be developed by administering a small amount, and then increasing the amount until the side effects become unduly adverse, or the intended effect is achieved.

49

The compounds of formula (I) may be used on their own or in the form of appropriate pharmaceutical compositions for topical, enteral or parenteral administration.

Compositions in a form suitable for topical administration to the lung include aerosols,

5 e.g. pressurised or non-pressurised powder compositions; compositions in a form suitable for oesophageal administration include tablets, capsules and dragees; compositions in a form suitable for administration to the skin include creams, e.g. oil- in-water emulsions or water-in-oil emulsions; compositions in a form suitable for administration intravenously include injections and infusions; and compositions in a

10 form suitable for administration to the eye include drops and ointments.

According to the invention there is also provided a pharmaceutical composition comprising, preferably less than 80% and more preferably less than 50% by weight of, a compound of formula (I), or a pharmaceutically acceptable derivative thereof, in

15 admixture with a pharmaceutically acceptable diluent or carrier. Examples of such diluents and carriers are: for tablets and dragees - lactose, starch, talc, stearic acid; for capsules - tartaric acid or lactose; and for injectable solutions - water, alcohols, glycerin, vegetable oils.

20

When the compound of formula (I) is to be administered to the lung it may be inhaled as a powder which may be pressurised or non-pressurised. Pressurised powder compositions of the compounds of formula (I) may contain a liquified gas propellant or a compressed gas. In non-pressurised powder compositions the active ingredient in

25 finely divided form may be used in admixture with a larger-sized pharmaceutically acceptable carrier comprising particles of up to, for example, lOOμm in diameter.

Suitable inert carriers include, e.g. crystalline lactose.

30 The compounds of formula (I) have the advantage that they are less toxic, more efficacious, are longer acting, have a broader range of activity, are more potent, produce fewer side effects, are more easily absorbed or have other useful pharmacological properties, than compounds of a similar structure.

50

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable 5 carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable

10 carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carrier, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders,

15 disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients including those that aid dispersion 20 may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

25 The compounds of formula (I) have the advantage that they are less toxic, more efficacious, are longer acting, have a broader range of activity, are more potent, produce fewer side effects, are more easily absorbed or have other useful pharmacological properties, than compounds of a similar structure, in particular ribavirin.

30

In particular, we have found that the compounds of formula (I) are excluded from red blood cells, unlike ribavirin which is rapidly taken up and leads to anaemia. Blood partitioning of compounds of the invention and of ribavirin has been determined using

51

whole human blood. Compounds, at a nominal concentration of 50μM were incubated at 37 ⁰ C for up to 2 hours and concentrations determined in both the plasma and red blood cell fractions. Red cell samples were also treated with phosphatise to release potential phosphorylated parent compound and/or ribavirin which may have been 5 formed.

Ribavirin was seen to enter red cells and accumulate as phophorylated ribavarin during the course of the 2 hour incubation. This accumulation was mirrored by a decrease in plasma ribavirin, such that the final ratio of total ribavirin in red cells to that in plasma

10 was approximately 7:1. For the glycosylated ribavirin derivatives tested, red cell penetration was poor and there was no evidence that parent compounds or phosphorylated forms accumulated in the cells during the incubation. Indeed, the compounds of the invention showed higher concentrations in plasma than in red cells throughout the incubation, with red celhplasma ratios between 0.1:1 and 0.3:1 at 2

15 hours. No ribavirin was detected in any of these incubates.

This suggests that the compounds of the invention will be less toxic than ribavirin, may be used at both lower and higher doses, and for a longer treatment regime, than corresponding therapies with ribavirin. 20

In separate experiments, we have established the uptake of compounds of the invention into the HepG2 cell line, using a similar methodology to that described above.

Compounds 1, 2, 4, 5 and 8-19 appear stable in plasma. 25

Compounds 5-19 appear stable in simulated gastric fluid.

Compounds 6 and 7 enter Hep-G2 cells over a period of 90 minutes.

30 Compounds 6, 7, 15 and 16 liberate free ribavirin in Hep-G2 and SK-Hepl cells.

In particular, compounds 1-4, 20-23, 29 and 30 show inhibition of bovine viral diarrhea virus (BVDV) immunofocus formation. The method for testing the level of inhibition

52

of BVDV immunofocus formation involves adding the compound and BVDV to a cover plate of bovine kidney cells. A control is set up in which ribavirin and BVDV are similarly added to a cover plate of bovine kidney cells. The cover plates are then left overnight. The percentage inhibition of BVDV immunofocus formation is 5 expressed by comparing the degree of inhibition shown in the cover plate containing the compound against the inhibition shown in the control.

A more detailed description of the assay is as follows:

10 Method for BVDV assay

The appropriate number of 19mm coverslips were dipped in 70% ethanol, blotted and air dried in 12 well tissue culture plates. Each inhibitor and control Ribavirin was diluted to 5 mM in PBS. MDBK NBL cells (ATCC) were used between passages pi 16 15 and pi 25. Culture medium was aspirated from flask(s) and cells washed with PBS. An appropriate volume (~0.5ml/25cm ² ) Trypsin versine was added to the cells and incubated at 37°C/5% CO ₂ until cells were detatched from the flask (3-5min). Cells were then resuspended in at least 5vol of cell culture medium. A viable cell count was performed by mixing 0.1 ml cell suspension with 0.1ml Trypan Blue. Viable and non- 20 viable cells were counted in the three corner squares of a haemocytometer. The cells were then diluted to a density of 3.5 x 10 ⁵ / ml in cell culture medium. Stock BVDV (lot 1, ~3 x 10 ⁶ TCIU / ml) was added to the cell suspension to give a titre of - 6.5 x 10 ³ TCIU / ml. The diluted inhibitors were mixed with the cell/virus suspension to give a final inhibitor concentration of 50 μM (1 in 1000 dilution). For each inhibitor 750 μl 25 volume of the cell/virus/inhibitor mix was added to 3 wells of the 12 well tissue culture plates containing ethanol washed coverslips. Plates were incubated at 5% CO ₂ ; 37°C overnight. Working quickly to avoid drying of coverslips, cells were fixed and stained as follows.

30 Plates removed from incubator. Supernatant was removed by aspiration. 1 ml PBS added to wells and aspirated (x3 times per well). 0.5 ml 4% Paraformaldehyde in PBS added to each well and incubated 20 min at ambient temperature. 4%

53

Paraformaldehyde was removed by aspiration. ImI PBS added to wells and aspirated (x3 times per well)

1 ml 0.1% Triton XlOO in PBS added to each well and incubated 7 min at ambient 5 temperature. 4% 0.1% Triton XlOO was removed by aspiration. 1 ml PBS added to wells and aspirated (x3 times per well). On the third wash, the PBS was not aspirated.

1° Antibody: Mouse anti-BVDV (Bovine Viral Diarrhoea Virus Type 1 E2 (gp53)) catalogue no. 157 VMRD. 10

Diluted 1:100 in PBS + 10% FCS.

For each coverslip to be stained, 30μl diluted antibody was placed on the upturned lid of a 12-well plate and the coverslips containing cells were carefully removed from the 15 wells and placed face down over the diluted antibody droplet.

Coverslips were diluted with antibody 60 min at ambient temperature.

After incubation, coverslips were carefully lifted and returned face up to the 12 well 20 dish containing ImI PBS per well.

2 ml PBS added to wells and aspirated (x3 times per well). On the third wash, the PBS was not aspirated.

25 2° Antibody: Alexa Fluor 594 conjugate donkey anti-mouse IgG monoclonal. Catalogue Number - A21203 (Molecular Probes, Invitrogen).

Diluted 1:200 in PBS + 10% FCS.

30 For each coverslip to be stained, 30μl diluted antibody was placed on the upturned lid of a 12-well plate and the coverslips containing cells were carefully removed from the wells and placed face down over the diluted antibody droplet.

54

Coverslips were diluted with antibody 60 min at ambient temperature.

After incubation, coverslips were carefully lifted and returned face up to the 12 well dish containing ImI PBS per well. 5

2 ml PBS added to wells and aspirated (x4 times per well). On the fourth wash, the PBS was not aspirated.

Coverslips (in PBS) were viewed on a fluorescent microscope and cells displaying 10 cytoplasmic fluorescence were counted.

Compound detection by MS-MS

Materials

15

All general solvents and reagents used for analyses performed by BioFocus DPI were of analytical or appropriate equivalent grade and were stored according to suppliers' recommendations. Any details of reagent suppliers and the equipment used in these experiments which are not detailed in this report, are held on file at BioFocus DPI,

20 Cambridge, UK.

The glycosylated ribavirin derivatives were supplied as 5OmM solutions. These stock materials and any working solutions prepared from them were stored at approximately - 20 ⁰ C. 25

Human plasma (Lot# 17-133) used in the assay was supplied, frozen, by SCIPAC (SCIPAC, Broad Oak Enterprise Village, Broad Oak Road, Sittingbourne, Kent, ME9 8AQ) and was stored at approximately -20 ⁰ C prior to use.

30 Methods

Mass spectrometry

A Micromass Quattro Micro mass spectrometer (S/N: QAA028, Waters Ltd, 730-740

55

Centennial Court, Centennial Park, Hertfordshire) was used for this study. The settings of the electrospray ion source used for method development and subsequent data acquisition are detailed in Table 1 :

Table 1 : Instrument parameters

Parameter Setting

Capillary voltage (kV) 3.0

Extractor cone voltage

3 (V)

RF lens (V) 0.2

Source temp (oC) 120

Desolvation gas temp

250 (oC)

Desolvation gas flow

350 (L/h)

Cone gas flow (L/h) 100

Multiple reaction monitoring (MRM) methods were created using QuanOptimise 10 software

(Waters Ltd 730-740 Centennial Court, Centennial Park, Hertfordshire.). The parameters selected are shown in Table 2.

15

20

56

Table 2: Compound tune parameters

Compound Ionisation Transition Cone Collision mode voltage energy

(V) (eV)

Ribavirin ESP+ 245.15 > 112.78 18 10

Compound 28 ESP+ 548.80 > 26 28 96.70

Compound 29 ESP+ 506.05 > 18 22 96.81

Compound 1 ESP+ 479.01 > 112.78 26 16

Compound 20 ESP+ 507.10 > 113.10 18 16

Compound 2 ESP+ 509.08 > 112.91 26 16

Compound 30 ESP+ 509.10 > 112.97 18 22

Compound 4 ESP+ 505.21 > 287.92 60 28

399.09

Compound 32 ESP+ 96.74 26 28 >

Methylcytidine ESP+ 258.18 > 125.89 18 10

5 Method files were created which contained the transitions (listed above in table 2) for the analyte, ribavirin and the analytical internal standard methylcytidine.

Chromatography

Chromatographic gradient methods previously developed for analysis of ribavirin and 10 derivatives of ribavirin were used for these analyses.

The chromatographic conditions used are detailed in Table 3 and compound retention times in Table 4.

15

57

Table 3: Chromatographic conditions

Parameter Setting

Phenomenex Gemini C18 150 x 4.6mm

Column

5μm *

Flow rate 1.Oml/min

Injection volume 20μL

A 0.01% Formic acid in water B 0.01%

Mobile phase Formic acid in acetonitrile

Gradient used for: Cpd 32 & Ribavirin Cpd 24

O.Omin 8% B O.Omin 2% B

2.5min 8% B 2.5min 2% B

Gradient profile 5.0min 95% B 5.0min 95% B ό.Omin 95% B ό.Omin 95% B

6.5min 5% B 6.5min 2% B

* Phenomenex column (Phenomenex, Melville House, Queens avenue, Hurdsfield Ind 5 Est,

Macclesfield, SKlO 2BN)

10

15

58

Table 4: Chromatographic retention times

Compound \ Retention time i (min)

Ribavirin 1.97

Compound 28 2.09

Compound 29 ! 2.17

Compound 1 2.22

Compound 20 2.24

Compound 1 2.22

Compound 30 ! 2.20

Compound 4 I 2.22

Compound 32 5.82

M ethylcytidine I 2.13

Once methods were created which contained working detection parameters for ribavirin, the novel derivatives and the internal standard 3 -methylcytidine, fresh dilutions in 50:50 (v/v) acetonitrile:water were made from the 5OmM stocks and the ribavirin content of these solutions was quantified.

This analysis indicated measurable levels of ribavirin to be present in stock solutions of Compounds 29 and 30. However, repeat injections of the 50:50 solvent solutions from

10 the same vials indicated that the ribavirin content was increasing over a relatively short period (<3 hours) in this mixture. These results are shown in table 5.

15

20

59

Table 5: Ribavirin detected in 50μM dilutions prepared from stock supplies (dilutions in 50:50, v/v, acetontrile:water for MS)

Compound Ribavirin detected Ribavirin detected between 0 & 1 5 hrs between 1 .5 & 3 hrs

μM % μM %

Compound 28 0 0

Compound 29 1.0 2 8.7 17

Compound 1 0 0

Compound 20 0 0

Compound 2 0 0

Compound 30 3.1 6 4.7 9

Compound 4 0 0

Compound 32 0 0

5 Plasma Stability

Compound stock solutions, supplied at 5OmM, were diluted 1:500 in water to give lOOμM working solutions.

Ribavirin and the standard compound, bisacodyl, were also diluted to lOOμM in water. 10 The aqueous working solutions were added (n=2) to human plasma (SCIPAC, Lot# 17- 133) at a final concentration oflOμM; initiating the incubations. The incubates were then transferred to a shaking water bath, maintained at 37oC.

Aliquots of plasma (50μl) were removed at prescribed time-points (0, 5, 15, 30 and 120 15 minutes), mixed with acetonitrile (lOOμl) and centrifuged at 4000rpm for 15 minutes prior to MS analysis of supernatant.

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As Compounds 29 and 30 had shown some instability in solution when awaiting MS analysis, the time between sampling of plasma incubates and processing via MS was kept to a minimum for these two compounds.

5 Red blood cell exclusion and plasma stability Materials

All general solvents and reagents used for analyses performed by BioFocus DPI were of analytical or appropriate equivalent grade and were stored according to suppliers' recommendations. Any details of reagent suppliers and the equipment used in these 10 experiments which are not detailed in this report, are held on file at BioFocus DPI, Cambridge, UK.

The glycosylated ribavirin derivatives were supplied as 5OmM solutions. These stock materials and any working solutions prepared from them were stored at approximately - 15 20 ⁰ C.

Whole human blood (Batch# HHB4584) used in the assay was supplied, refrigerated, by First Link (First Link (UK) Ltd, 1 Vale Pits Road, Garretts Green, Birmingham, B33 OTD) and used immediately on receipt at BioFocus DPI (Thursday, May 17, 2007).

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Methods

Mass spectrometry

A Micromass Quattro Micro mass spectrometer (S/N: QAA028, Waters Ltd, 730-740

Centennial Court, Centennial Park, Hertfordshire) was used for this study. The settings

25 of the electrospray ion source and the individual parameters for acquisition of compound data are given above.

Chromatography

Chromatographic gradient methods previously developed for analysis of ribavirin and 30 derivatives of ribavirin were used for these analyses.

Blood Partitioning

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Compound stock solutions, supplied at 5OmM, were diluted 1:1000 (n=2) in whole blood (pre-warmed to 37oC) to give 50μM incubations.

Incubations were mixed by inversion several times, the T=O sample was removed and 5 the sample tubes were transferred to an orbital shaker in an incubator maintained at 37oC.

Aliquots of blood (ImI) were subsequently removed at prescribed time-points (30, 60 and 120 minutes), and centrifuged at 15000rpm for 5 minutes to separate plasma and 10 red cells.

Samples of plasma were analysed directly; samples from the red cell pellets were lysed using ice-cold, purified water.

15 lOOμl aliquots of both cell lysate and plasma were then mixed with 200μl of acetonitrile to precipitate protein and extract compounds.

Blood Partitioning Experimental Procedure

20 1. Whole human blood pre-incubated at 37oC.

2. Compounds added to blood to give a nominal compound concentration of 50μM

3. ImI of blood removed per time point (0, 30, 60 and 120minutes)

4. Blood transferred to Eppendorf tube and centrifuged at 150Og for 5 minutes at 4 ⁰ C (rotor pre-chilled)

25 5. 400μl of plasma transferred immediately to 96 deepwell plate

6. 300μl of red blood cells transferred with a positive displacement pipette to fresh Eppendorf tube

7. ImI ice-cold water added

8. Samples vortexed for 30 seconds followed by brief centrifugation 30

Plasma Analysis lOOμl of plasma removed

200μl of acetonitrile added

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Samples centrifuged and supernatant analysed

Red Blood Cell Analysis lOOμl lysate removed 5 200μl of acetonitrile added

Samples centrifuged and supernatant analysed

Plasma concentrations are illustrated in Figure 5. These show that plasma concentrations of both ribavirin and Compound 32 rapidly decrease, suggesting uptake. 10

Red blood cell concentrations are illustrated in Figure 6. Both ribavirin and Compound 32 rapidly increase, suggesting uptake. The levels then decrease, suggesting phosphorylation.

15 Compound 32 is freely permeable into Hep-G2 and SK-Hepl cells and releases ribavirin within the cells.

In particular, compounds 1, 2, 4, 20 and 28 show plasma stability over a period of 120 minutes. 20