Accepted Manuscript

Syntheses of Nucleosides with a 1’,2’-β-Lactam Moiety as Potential Inhibitorsof Hepatitis C Virus NS5B Polymerase

Qun Dang, Zhibo Zhang, Yunfeng Bai, Ruijun Sun, Jie Yin, Tongqian Chen,Stephane Bogen, Vinay Girijavallabhan, David B. Olsen, Peter T. Meinke

PII: S0040-4039(14)01415-4DOI: http://dx.doi.org/10.1016/j.tetlet.2014.08.072Reference: TETL 45039

To appear in: Tetrahedron Letters

Received Date: 29 July 2014Revised Date: 14 August 2014Accepted Date: 18 August 2014

Please cite this article as: Dang, Q., Zhang, Z., Bai, Y., Sun, R., Yin, J., Chen, T., Bogen, S., Girijavallabhan, V.,Olsen, D.B., Meinke, P.T., Syntheses of Nucleosides with a 1’,2’-β-Lactam Moiety as Potential Inhibitors ofHepatitis C Virus NS5B Polymerase, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.08.072

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Syntheses of Nucleosides with a 1’,2’-ββββ-

Lactam Moiety as Potential Inhibitors of Hepatitis C Virus NS5B Polymerase

Qun Dang, Zhibo Zhang, Yunfeng Bai, Ruijun Sun, Jie Yin, Tongqian Chen, Stephane Bogen, Vinay

Girijavallabhan, David B. Olsen, Peter T. Meinke

OHO

HONH X: CH, N

ON N

N

HO

HO

NH2

MK-608

OH

OO

HO

OH

PHN

OO

O

O

CN

NN

N

NH2

GS-6620

O

N

X

N

N

NH2

+

Leave this area blank for abstract info.

Syntheses of Nucleosides with a 1’,2’-ββββ-Lactam Moiety as Potential

Inhibitors of Hepatitis C Virus NS5B Polymerase

Qun Dang,a,*

Zhibo Zhang,b Yunfeng Bai

b, Ruijun Sun

b, Jie Yin

b, Tongqian Chen,

b Stephane

Bogen,a Vinay Girijavallabhan,

a David B. Olsen,

c Peter T. Meinke

a

a Discovery Chemistry, Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA

b Pharmaron Beijing Co., Ltd., 6 Tai-He Road, BDA, Beijing, 100176, China

c Discovery Biology, Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA

Abstract—To discover novel nucleosides as potential anti-HCV agents, nucleosides with a 1’,2’-β-lactam moiety was designed as a

hybrid scaffold of MK-608 and GS-6620. Synthetic strategies were successfully developed to prepare two series of C-nucleosides with a

1’,2’-β-lactam moiety: a 7-deaza-purine C-nucleoside analog 11 was prepared in 10 steps with an overall yield of 3.7%; a purine C-

nucleoside analog 22 was prepared in 9 steps with an overall yield of 9.7%.

Key words— nucleoside, HCV NS5B, C-nucleoside, prodrugs, β-lactam.

©2013 Elsevier Science Ltd. All rights reserved.

* Corresponding author. Tel.: +1 908 740 3789.

E-mail address: qun_dang@merck.com (Q. Dang).

Decades of active research to discover novel nucleosides as hepatitis C virus (HCV) NS5B polymerase inhibitors established extensive SAR in the field.

1-3 The majority of nucleosides with proven anti-

HCV activity have modifications at the 2’-position,4-11

while some active nucleosides were reported with modification at the 4’ and 1’ positions,

12, 13 Figure 1.

ON N

N

HO

HO

NH2

ON

HO

HO

N

NH2

O

OH

MK-608

OH

ON

O

HO

NH

O

O

F

NM-107 sofosbuvir

OO

HO OH

PHN

OO

O

O

CN

NN

N

NH2

GS-6620

ON

HO

HO

N

NH2

O

OH

R-1479

N3

PHN

OO

O

O

Figure 1. Representative Nucleoside HCV NS5B inhibitors

To discover novel nucleosides as potential

inhibitors of HCV NS5B polymerase, we designed novel nucleosides with a 1’, 2’-β-lactam moiety as hybrids of known inhibitors MK-608

14 and GS-6620

13

(Figure 2).

Figure 2. Design of 1’,2’-β-lactam-nucleosides as potential anti-HCV

agents Discovery of GS-6620 suggests that a small

hydrogen-bond acceptor is tolerated at the 1’-position, while MK-608 and many other nucleosides with proven anti-HCV activity indicate that a small hydrogen-bond donor is acceptable at the α-face of the 2’-position. Thus, we envisioned a 1’, 2’-β-lactam nucleoside which places a hydrogen-bond acceptor at the 1’ and a hydrogen-bond donor at the α-face of the 2’ (Figure 2). Our hope is that this hybridization of MK-608 and GS-6620 will lead to novel nucleosides with anti-HCV activity. Herein, syntheses of these novel nucleosides are reported.

Retrosynthetically, introduction of a β-lactam

moiety at the 1’,2’-region could pose a challenge due to the sterically congested environment imposed by the presence of the base and a 2’-β-methyl group. Thus, we devised a synthetic plan that begins with a 2’-α-azido-2’-β-methyl ribose lactone (1)

15 (Scheme 1). We

envisioned that key to our synthesis was the preparation of the precursor compound for the β-lactam formation

step, since the intramolecular β-lactam formation should be a favored process kinetically.

O

HO

HO

N3

N

X

N

N

NH2

CO2MeO

HO

HO

NH

N

X

N

N

NH2

O

O

HO

HO

N31

O

X = CH, NX = CH, N

Scheme 1 Retrosynthetic Analysis to access 1’,2’-β-lactam nucleosides

The starting lactone 1 was readily prepared in

six steps using a patent procedure15

and the forward synthesis of the key nucleoside 8 is summarized in Scheme 2.

Scheme 2 Synthesis of compound 8. Reagents and conditions: (a) TBSCl

(5.0 eq.), imidazole (6.0 eq.), DMF, 50 ºC, 16h, 77%; (b) LDA (1.1 eq.),

4-(MeS)-imidazo[2,1-f][1,2,4]triazine (1.0 eq.), THF, -78 ºC to -20 ºC, 40

min, 82% (c) MeOH, sulfuric acid (0.5 eq.), 5-10 ºC, 1h, 85%; (d)

TMSCN (2.0 eq.), BF3-OEt2 (1.2 eq.), THF, -78 ºC, 3 h, 58%; (e) NH3

(sat.), iPrOH, 60 ºC, 40 h, 75%; (f) NaOMe, MeOH, 25 ºC, 4d, 60%; (g)

PMe3 (3.1 eq.), THF-water, 25 ºC, 2h, 67%.

Protection of the 3’- and 5’-hydroxy groups as

TBS ethers, which is required for subsequent base-coupling reactions, provided compound 2 in 77% yield. Lithiation of 4-(methylsulfanyl)pyrrolo[2,1-f][1,2,4]triazine using LDA followed by reaction with lactone 2 gave compound 3 in 82% yield. To introduce a cyano group at the 1’-position, we first tried to prepare the acetate and benzoate derivatives of the 1’-OH, but both esters were found to have poor stability and would hydrolyze back to compound 3 during work up. Thus, a more stable methyl ether approach was selected. Treatment of compound 3 with methanol in the presence of a catalytic amount of sulfuric acid generated

compound 4 in 85% yield. One of the two TBS groups fell off during the reaction with methanol under acidic conditions, and we hyporthesized that it was the 5’-TBS ether since it is well precedented that 5’-TBS could be removed selectively in the presence of a 3’-TBS ether under acidic conditions.

16 Introduction of a 1’-CN group

was achieved via the treatment of compound 4 with TMSCN in the presence of borontrifluoride etherate generating compound 5 in 58% yield. Aminolysis of compound 5 and separation of the 1’-anomeric isomers gave compound 6 in 75% yield. A NMR experiment of compound 6 showed that two of the base protons (δ = 7.91 and 7.08) have strong NOE interactions with the 2’-β-Me group (δ = 1.19), indicating that the base is in the β-face (which could be inferred to compound 5 as a β-nucleoside as well). The fact that compound 6 was isolated in good yield indicates that the addition to the lactone by the lithiated base for step two was preferentially from the β-face. Treatment of compound 6 with sodium methoxide in methanol gave compound 7 in 60% yield, which is the key intermediate to study the β-lactam formation reactions. Initially, we reasoned that given that the intramolecular β-lactam formation reaction is a kinetically favored process, conversion of the 2’-azido group to its corresponding amino group might result in the spontaneous cyclization to give us the desired β-lactam. Surprisingly, treatment of compound 7 with trimethylphosphine only produced compound 8 in 67% yield; no β-lactam formation was detected. This observation suggested that the highly sterically congested environment around the 1’- and 2’-positions might pose a challenge for the desired β-lactam formation. Indeed, all attempts to effect the β-lactam formation directly from compound 8 were not successful. No reaction was observed after refluxing its toluene solution overnight; only starting material remained. Hydrolysis of compound 8 to its corresponding acid, followed by various amide bond formation conditions such as DCC, EDCI did not produce the desired lactam formation. No lactam formation was observed when compound 8 was treated with sodium hydride in THF (0

oC to reflux). Therefore,

a new strategy for the intramolecular β-lactam formation was developed and is summarized in Scheme 3.

a

b

c

O

TBSO

TBSO

NH2

NN

N

NH2

CO2Me

O

TBSO

HO

NH2

CO2Me

NN

N

NH2

9

8

O

TBSO

TBSO

NH

NN

N

NH2

10

O

O

HO

HO

NH

NN

N

NH2

11

O

X

Scheme 3 Synthesis of compound 11. Reagents and conditions: (a)

TBSCl (5.0 eq.), imidazole (6.0 eq.), DMF, 50 ºC, 16h, 79%; (b) LDA

(3.5 eq.), THF, -70 ºC to 25 ºC, 12 h, 85%; (c) TBAF (8.1 eq.), CHCl3, -

15 ºC, 30 min, 58%.

Given that the intramolecular β-lactam

formation is a kinetically favored process, we chose to generate the anion of the 2’-amino group under kinetically controlled conditions in order to facilitate the formation of the desired β-lactam. Protection of the 5’-hydroxy group as a TBS ether gave compound 9 in 79% yield. To increase the nucleophilicity of the 2’-amino group, compound 9 was treated with LDA, which indeed led to the desired β-lactam formation, producing compound 10 in excellent yield (85%). A final TBS ethers removal using TBAF gave the desired 1’,2’- β-lactam-containing nucleoside 11 in 58% yield. With the novel nucleoside 11 in hand, we set out to demonstrate that this novel nucleoside scaffold is suitable for prodrug synthesis as well. In this regard, the classical McGuigan phosphoramidate prodrugs were prepared stereo-selectively using the method developed by Ross et al

17, as illustrated in Scheme 4. Treatment of

nucleoside 11 with the two chiral phosphoramidites of phenol, pentafluorophenol and L-alanine isopropyl ester in the presence of tetramethylpiperidinide magnesium chloride-lithium chloride (TMPMgCl-LiCl)

18 gave the

corresponding McGuigan prodrugs 12 and 13, 18% and 20% respectively.

Scheme 4 Synthesis of Prodrugs 12 and 13

To further demonstrate the utility of this

synthetic strategy, we aimed to prepare a 7-aza analog of C-nucleoside 11 , which should help to expand the SAR for this series of nucleosides. The synthesis of C-nucleoside 22 is illustrated in Scheme 5.

aO

TBSO

TBSO

N3

O bO

TBSO

TBSO

N3

N

N

N

N

SMe

OH

cO

TBSO

TBSO

N3

N

N

N

N

SMe

OAc

dO

TBSO

TBSO

N3

N

N

N

N

SMe

CN

eO

HO

HO

N3

N

N

N

N

SMe

CN

fO

HO

HO

N3

N

N

N

N

NH2

CN

gO

HO

HO

N3

N

N

N

N

NH2

CO2MeO

TBSO

TBSO

NH2

CO2Me

N

N

N

N

NH2

h

2

15

14

16

1718

19 20

iO

TBSO

TBSO

NH

N

N

N

N

NH2

O

HO

HO

NH

N

N

N

N

NH2

21 22

OO

Scheme 5 Synthesis of compound 20. Reagents and conditions: (a) LDA

(1.1 eq.), 4-(MeS)-imidazo[2,1-f][1,2,4]triazine (1.0 eq.), THF, -78 ºC to -

20 ºC, 40 min, 59% (b) Ac2O (5.0 eq.), DMAP (0.2 eq.), pyridine, 50 ºC,

30 min, 95%; (c) TMS-CN (2.0 eq.), BF3-OEt2 (1.2 eq.), CH2Cl2, -78 ºC,

3 h, 79%; (d) TBAF, THF, 35%; (e) NH3(sat.), iPrOH, 40 ºC, 40 h, 90%;

(f) NaOMe (10.0 eq.), MeOH, 25 ºC, 14h, 78%; (g) i. H2, Pd-C, MeOH; ii.

TBSCl, imidazole, DMF, 25 ºC, 2h, 60%; (h) LDA (1.1 eq.), THF, -78 ºC

to -20 ºC, 40 min, 91%; (i) TBAF (8.1 eq.), CHCl3, -15 ºC, 30 min, 58%.

Lithiation of 4-(methylsulfanyl)imidazo-[2,1-

f][1,2,4]triazine using LDA followed by reaction with

lactone 2 gave compound 14 in 59% yield. Given the

perceived higher reactivity of the 1’-acetate versus the

1’-methyl ether of compound 14, we first tried to

prepare the acetate. In contrast to the case with

compound 3, acetylation of compound 14 gave

compound 15 in 95% yield, and it is stable for isolation.

Treatment of compound 15 with TMSCN in the

presence of borontrifluoride etherate introduced the 1’-

CN group leading to compound 16 in 79% yield.

Removal of TBS ethers using TBAF and separation of

the anomeric isomers gave compound 17 in 35% yield,

which has the desired 1’-CN group in the α-face

required for β-lactam formation. Aminolysis of

compound 17 gave compound 18 in 90% yield, and

treatment of compound 18 with sodium methoxide in

methanol gave compound 19 in 78% yield, which was

then ready for the key β-lactam formation reaction.

Similar to the observation with compound 7, reduction

of the azido group in compound 19 under hydrogenation

conditions did not lead to β-lactam formation, but

instead only produced the corresponding 2’-amino

nucleoside. Protection of the 3’,5’-hydroxy groups gave

compound 20 in 60% over the two steps. Therefore, the

strategy developed for the synthesis of compound 11

(Scheme 3) was applied to compound 20 in order to

form the desired 1’,2’-β-lactam. Treatment of

compound 20 with LDA led to the desired β-lactam

formation, generating compound 21 in excellent yield

(91%). Removal of the 3’ and 5’-TBS ethers using

TBAF gave the desired 1’,2’-β-lactam-containing

nucleoside 22 in 58% yield. In summary, nucleosides with a 1’,2’-β-lactam

moiety were designed as a hybrid scaffold of MK-608 and GS-6620, which potentially could be HCV NS5B polymerase inhibitors. Synthetic strategies were successfully developed to prepare two series of C-nucleosides with a 1’,2’-β-lactam moiety, leading to novel nucleosides 11, 17, 18 and 22. Moreover, McGuigan prodrugs were also prepared for nucleoside 11. Biological testing of these novel nucleosides should enhance our understanding of the HCV nucleoside field, and results will be disclosed in due course.

Supplementary Material

The supplementary material contains the experimental procedures and characterization data for all new compounds.

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(3R,4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]methyl]-

3-methyloxolan-2-one (2) Into a 250-mL 3-necked round-bottom flask purged and maintained with an

inert atmosphere of argon, was placed a solution of compound 1 (10 g, 53.43 mmol, 1.00 eq.) in DMF

(20 mL), imidazole (21.85 g, 6.00 eq.), TBDMSCl (48.17 g, 321.00 mmol, 6.00 eq.). After the

resulting solution was stirred for 4 h at 50 oC, the cooled reaction mixture was quenched with 200 mL

of water/ice, extracted with 3 x 100 mL of ethyl acetate. The organic layers were combined, washed

with 2 x 150 mL of sodium chloride, dried over anhydrous sodium sulfate and concentrated under

vacuum. The residue was purified by chromatography (silica gel column, ethyl acetate-petroleum ether

(1-30)) to give compound 2 (17 g, 77%). as colorless oil. 1H NMR: (400MHz, CDCl3, ppm): � 4.23-

4.15 (m, 2H), 3.94-3.90 (m, 1H), 3.73-3.70 (m, 1H), 1.48 (s, 3H), 0.86-0.80 (m, 18H), 0.09-0.08 (m,

12H)

(3R,4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]methyl]-

3-methyl-2-[4-(methylsulfanyl)pyrrolo[2,1-f][1,2,4]triazin-7-yl]oxolan-2-ol (3) Into a 500-mL 3-

necked round-bottom flask purged and maintained with an inert atmosphere of argon, was placed a

solution of 4-(methylsulfanyl)pyrrolo[2,1-f][1,2,4]triazine (4.33 g, 26.21 mmol, 1.00 eq.) in THF (150

mL). This was followed by the addition of LDA (20 mL, 1.50 eq.) dropwise with stirring at -78 0C in

10 min. The resulting solution was stirred for an additional 15 min while the temperature was

maintained at -78 0C. To this was added a solution of compound 2 (13 g, 31.27 mmol, 1.20 eq.) in THF

(50 mL) dropwise with stirring at -78 0C in 10 min. The resulting solution was stirred for 30 min at -78

0C, and then quenched by the addition of 200 mL of aq. NH4Cl. The resulting solution was diluted with

200 mL of H2O, extracted with 3 x 200 mL of ethyl acetate. The organic layers were combined, dried

over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by

chromatography (silica gel column, ethyl acetate-petroleum ether, gradient 1:50 to 1:5) to give

compound 3 (12.5 g, 82%) of as a light yellow solid. LC-MS: (ES, m/z): 581.20 [M+H]+,

1H NMR:

(400MHz, CDCl3, ppm): � 8.24 (s, 1H), 7.07 (d, J = 4.8Hz, 1H), 6.82 (d, J = 4.8Hz, 1H), 4.66 (d, J =

8.4Hz, 1H), 4.29-4.26 (m, 1H), 4.01-3.97 (m, 1H), 3.72-3.69 (m, 1H), 2.70 (s, 3H), 1.03 (s, 3H), 0.98-

0.96 (m, 18H), 0.19 (s, 6H), 0.16 (s, 6H).

[(2R,3S)-4-azido-3-[(tert-butyldimethylsilyl)oxy]-5-methoxy-4-methyl-5-[4-(methylsulfanyl)-

pyrrolo[2,1-f][1,2,4]triazin-7-yl]oxolan-2-yl]methanol (4) Into a 500-mL 3-necked round-bottom

flask purged and maintained with an inert atmosphere of argon, was placed a solution of compound 3

(12 g, 20.66 mmol, 1.00 eq.) in methanol (300 mL). This was followed by the addition of con. sulfuric

acid (2.04g, 20.66 mmol, 1.00 eq.) at 0-10 0C in 5 min. The resulting solution was stirred for 2 h at 0-

10 0C. The pH value of the solution was adjusted to 7 with triethylamine and then concentrated under

vacuum. The residue was diluted with of DCM, washed with H2O, dried over anhydrous sodium sulfate

and concentrated under vacuum. The residue was purified by chromatography (silica gel column, ethyl

acetate-petroleum ether, gradient 1:50 to1:5) to give compound 4 (8.4 g , 85%) as a white solid. LC-

MS: (ES, m/z): 481.10, [M+H]+,

1H NMR: (400MHz, CDCl3, ppm): � 8.32 (s, 1H), 7.03 (d, J = 4.8Hz,

1H), 6.87 (d, J = 4.8Hz, 1H), 4.51 (d, J = 8.4Hz, 1H), 4.30-4.28 (m, 1H), 3.97-3.95 (m, 1H), 3.81-3.79

(m, 1H), 3.10 (s, 3H), 2.70 (s, 3H), 1.03 (s, 3H), 0.98-0.94 (m, 9H), 0.20 (s, 3H), 0.18 (s, 3H).

(4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)-3-methyl-2-[4-

(methylsulfanyl)pyrrolo[2,1-f][1,2,4]triazin-7-yl]oxolane-2-carbonitrile (5) Into a 250-mL 3-necked

round-bottom flask with an inert atmosphere of argon, was placed a solution of compound 4 (7 g, 14.56

mmol, 1.00 eq.) in DCM (150 mL). This was followed by the additional of TMSCN (7.23 g, 5.00 eq.)

dropwise with stirring at -5-10 0

C in 2 min and then BF3Et2O (3.11 g, 1.50 eq.) was added dropwise.

After the resulting solution was stirred for 0.5 h at -5-10 0

C, the pH of the solution was adjusted to 7

with triethylamine. The resulting mixture was washed with 3 x 50 ml H2O, dried over anhydrous

sodium sulfate and concentrated under vacuum. The residue was purified by chromatography (silica gel

column, ethyl acetate-petroleum ether gradient 1:20 to 1:5) to give compound 5 (4.0 g, 58%) as a light

yellow solid. LC-MS: (ES, m/z): 476.00 [M+H]+,

1H NMR: (400MHz, CDCl3, ppm): � 8.32, (s, 1H),

7.18 (d, J = 4.8Hz, 1H), 6.85 (d, J = 4.8Hz, 1H), 4.59 (d, J = 8.4Hz, 1H), 4.29-4.28 (m, 1H), 4.16-4.14

(m, 1H), 3.88-3.85 (m, 1H), 2.74 (s, 3H), 1.16 (s, 3H), 0.99-0.96 (m, 9H), 0.22 (s, 3H), 0.18 (s, 3H).

(2R,3R,4S,5R)-2-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-3-azido-4-[(tert-

butyldimethylsilyl)oxy]-5-(hydroxymethyl)-3-methyloxolane-2-carbonitrile (6) Into a 250-mL

sealed tube, was placedcompound 5 (4.0 g, 8.41 mmol, 1.00 eq.) in NH3 saturated i-PrOH (200 mL) at

-78 0 C. The resulting solution was stirred overnight at 60

0 C and then concentrated under vacuum. The

residue was purified by chromatography (silica gel column, methanol-DCM gradient 1:50 to 1:20) to

give compound 6 (2.8 g, 75%) as a white solid. LC-MS: (ES, m/z): 445.40 [M+H]+,

1H NMR:

(300MHz, CD3OD, ppm): � 7.91, (s, 1H), 7.08 (d, J = 4.8Hz, 1H), 6.93 (d, J = 4.8Hz, 1H), 4.33-4.30

(m, 1H), 4.18-4.13 (m, 1H), 4.05-4.00 (m, 1H), 3.84-3.78 (m, 1H), 1.19 (s, 3H), 0.98 (s, 9H), 0.19 (s,

3H), 0.15 (s, 3H). NOE interactions observed between the 2-H (�= 7.91) and 8-H (�= 7.08) from

nucleobase and the 2'-Me (�= 1.19); between the 8-H (�= 7.08) and the two proton of 5'-H (�= 4.05-4.00

(m, 1H), 3.84-3.78 (m, 1H).

Methyl (2R,4S,5R)-2-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-3-azido-4-[(tert-

butyldimethylsilyl)oxy]-5-(hydroxymethyl)-3-methyloxolane-2-carboxylate (7) Into a 100-mL

round-bottom flask purged and maintained with an inert atmosphere of argon, was placed a solution of

compound 6 (2.7 g, 6.08 mmol, 1.00 eq.) in methanol (50 mL). To the resulting solution, NaOMe (1.65

g, 2.5 eq.) was added at room temperature. The resulting solution was stirred for 72 h at 40 0 C and then

concentrated under vacuum. The residue was purified by chromatography (silica gel column, methanol-

DCM gradient 1:50 to 1:5) to give crude of compound 7 (1.75 g, 60%) as a white solid, which was

used for the next reaction directly without further purification.

Methyl (2R,4S,5R)-3-amino-2-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-hydroxy-5-

(hydroxymethyl)-3-methyloxolane-2-carboxylate (8) Into a 50-mL round-bottom flask, was placed a

solution of compound 7 (1.7 g, 3.56 mmol, 1.00 eq.) in THF/H2O(4:1) (20 mL), Me3P (0.3253 g, 1.20

eq.). The resulting solution was stirred for 4 h at 22 0C in an oil bath. The resulting mixture was

concentrated under vacuum. The residue was purified by chromatography (silica gel column,

dichloromethane-methanol gradient 20:1 to 5:1) to give crude of compound 8 (1.3 g, 67%) as a yellow

solid, which was used for the next reaction directly without further purification.

Methyl (2R,4S,5R)-3-amino-2-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-[(tert-

butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]methyl]-3-methyloxolane-2-carboxylate

(9) Into a 25-mL round-bottom flask purged and maintained with an inert atmosphere of argon, was

placed a solution of compound 8 (300 mg, 0.67 mmol, 1.00 eq., 75% purity) in N,N-

dimethylformamide (2 mL), imidazole (303.05 mg, 5.00 eq.), TBDMSCl (667.9 mg, 5.00 eq.). The

resulting solution was stirred for 4 h at 50 0C, then quenched by the addition of water/ice. The resulting

solution was extracted with 3 x 15 ml of ethyl acetate, washed with brine, dried over anhydrous sodium

sulfate and concentrated under vacuum. The residue was purified by chromatography (silica gel

column, dichloromethane-methanol gradient 20:1 to 10:1) to give compound 9 (300 mg, 79%) as a

white solid. LC-MS: (ES, m/z): 566.30 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 7.95 (s, 1H),

6.94 (d, J = 4.8, 1H), 6.82 (d, J = 4.8, 1H), 4.24-4.29 (m, 2H), 3.96 (d, J = 3.9, 2H), 3.71 (s, 3H), 1.23

(s, 3H), 0.98-0.94 (m, 18H), 0.22 (s, 6H), 0.14 (s, 6H).

(1R,3R,4S,5R)-1-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-[(tert-butyldimethylsilyl)oxy]-

3-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-2-oxa-6-azabicyclo[3.2.0]heptan-7-one (10) Into

a 25-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of argon, was

placed a solution of compound 9 (150 mg, 0.27 mmol, 1.00 eq.) in THF (10 mL). This was followed by

the addition of Lithium diisopropylamide (85.22 mg, 0.80 mmol, 3.00 eq.) dropwise with stirring at –

78 oC in 2 min. The resulting solution was stirred for 2 h at -78

oC and then quenched by water. The

resulting solution was diluted with EA, extracted with 3 x 20 ml of ethyl acetate, dried over anhydrous

sodium sulfate and concentrated under vacuum to give crude of compound 10 (120 mg, 85%) as yellow

oil, which was used to the next reaction directly.

(1R,3R,4S,5R)-1-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-hydroxy-3-(hydroxymethyl)-5-

methyl-2-oxa-6-azabicyclo[3.2.0]heptan-7-one (11) Into a 50-mL round-bottom flask purged and

maintained with an inert atmosphere of argon, was placed a solution of compound 10 (120 mg, 0.22

mmol, 1.00 eq.) in THF (20 mL), tetrabutylammonium fluoride (117.73 mg, 0.45 mmol, 2.00 eq.). The

resulting solution was stirred for 15 min at 15 oC and then concentrated under vacuum. The residue was

applied onto a silica gel column with dichloromethane/methanol (20:1-5:1) to give crude product. The

crude product was purified by Prep-HPLC with the following conditions (1#-Pre-HPLC-011(Waters)):

Column, SunFire Prep C18 OBD Column,5um,19*150mm; mobile phase, water

(10mmol/L ammonium acid carbonate) and acetonitrile (5% acetonitrile up to 21.5% in 5.5 min, up to

100% in 1.5 min, down to 5% in 1 min; Detector, UV 254 & 220 nm to give compound 11 (40 mg

(58%) as a white solid. LC-MS-EBR: (ES, m/z): 306.20 [M+H]+,

1H NMR: (400MHz, CD3OD, ppm): �

7.99 (s, 1H), 7.32 (d, J = 4.8, 1H), 7.08 (d, J = 4.4, 1H), 4.07-4.05 (m, 1H), 3.98-3.94 (m, 2H), 3.85-

3.81 (m, 1H), 1.30 (s, 3H).

Propan-2-yl (2S)-2-[[(R)-[[(1R,3R,4S,5R)-1-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-

hydroxy-5-methyl-7-oxo-2-oxa-6-azabicyclo[3.2.0]heptan-3-

yl]methoxy](phenoxy)phosphoryl]amino]propanoate (12) Into a 25-mL round-bottom flask purged

and maintained with an inert atmosphere of argon, was placed a solution of compound 11 (80 mg, 0.26

mmol, 1.00 eq.) in N,N-dimethylformamide (9 mL). This was followed by the addition of

lithio(3,3,7,7-tetramethyl-1-aza-2-magnesacycloheptan-2-yl)-1,3-dichlorane (0.262 mL, 1.00 eq., 1.0

M in THF/toluene) dropwise with stirring at 0 oC. The mixture was stirred for 1h at 0

oC. To this was

added a solution of propan-2-yl (2S)-2-[[(R)-pentafluorophenoxy(phenoxy)phosphoryl]-

amino]propanoate (119 mg, 0.26 mmol, 1.00 eq.) in N,N-dimethylformamide (1 mL) at 0 oC. The

resulting solution was stirred overnight at 0 oC. The reaction was then quenched by the addition of

methanol, and then concentrated under vacuum. The crude product (50 mg) was purified by Prep-

HPLC with the following conditions (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, SunFire

Prep C18 OBD Column,5um,19*150mm,; mobile phase, water with 0.05% trifluoroacetic acid and

acetonitrile (20% acetonitrile up to 35% in 10 min); Detector, UV 254 & 220 nm to give compound 12

(16.0 mg, 11%) of as a white solid. LC-MS (ES, m/z): 575.15[M+H]+,

1H NMR: (300MHz, CD3OD,

ppm): � 7.91 (s, 1H), 7.30-7.18 (m, 5H), 7.14-7.08 (m, 1H), 7.01 (d, J = 4.5Hz, 1H), 4.90-4.79 (m, 1H),

4.50-4.44 (m, 1H), 4.35-4.29 (m, 1H), 4.03 (s, 2H), 3.85-3.74 (m, 1H), 1.28-1.21 (m, 6H), 1.18-1.15 (m,

6H), P-NMR (121MHz, CD3OD, ppm): � 3.595.

Propan-2-yl (2S)-2-[[(S)-[[(1R,3R,4S,5R)-1-[4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl]-4-

hydroxy-5-methyl-7-oxo-2-oxa-6-azabicyclo[3.2.0]heptan-3-

yl]methoxy](phenoxy)phosphoryl]amino]propanoate (13) Into a 25-mL round-bottom flask purged

and maintained with an inert atmosphere of argon, was placed a solution of compound 11 (80 mg, 0.26

mmol, 1.00 eq.) in N,N-dimethylformamide (9 ml). This was followed by the addition of lithio(3,3,7,7-

tetramethyl-1-aza-2-magnesacycloheptan-2-yl)-1,3-dichlorane (0.262 mL, 1.00 eq., 1.0 M in

THF/toluene) at 0 oC. The mixture was stirred for 1h at 0

oC. To this was added a solution of propan-2-

yl (2S)-2-[[(S)-pentafluorophenoxy(phenoxy)phosphoryl]amino]propanoate (119 mg, 0.26 mmol, 1.00

eq.) in N,N-dimethylformamide (1mL ) at 0 oC. The resulting solution was stirred overnight at 0

oC.

The reaction was then quenched by the addition of methanol. The resulting mixture was concentrated

under vacuum. The crude product (50 mg) was purified by Prep-HPLC with the following conditions

(Prep-HPLC-011): Column, SunFire Prep C18 OBD Column, 5um,19*150mm,; mobile phase, water

with 0.05% trifluoroacetic acid and acetonitrile (20% acetonitrile up to 35% in 10 min); Detector, UV

254 & 220 nm to give compound 13 (7.8 mg, 5%) as a white solid. LC-MS (ES, m/z): 575.15 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 7.87 (s, 1H), 7.28-7.22 (m, 3H), 7.17-7.08 (m, 3H), 7.03 (d, J =

4.8Hz, 1H), 4.92-4.83 (m, 1H), 4.56-4.49 (m, 1H), 4.37-4.29 (m, 1H), 4.12-4.09 (m, 1H), 4.05-4.05 (m,

1H), 3.86-3.80 (m, 1H), 1.25-1.15 (m, 12H).

(3R,4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-[[(tert-

butyldimethylsilyl)oxy]methyl]-3-methyl-2-[4-(methylsulfanyl)imidazo[2,1-f][1,2,4]triazin-7-

yl]oxolan-2-ol (14) Into a 500-mL 3-necked round-bottom flask purged and maintained with an inert

atmosphere of argon, was placed a solution of 4-(methylsulfanyl)imidazo[2,1-f][1,2,4]triazine (4.33 g,

26.05 mmol, 1.00 eq.) in THF (100 mL). This was followed by the addition of LDA (4.17 g, 38.93

mmol, 1.50 eq.) dropwise with stirring at -78 0 C in 5 min. The resulting solution was allowed to react,

with stirring, for an additional 15 min while the temperature was maintained at -78 0 C. To this was

added a solution of compound 2 (13 g, 31.27 mmol, 1.20 eq.) in THF (30 mL) dropwise with stirring at

-78 0 C in 2 min. The resulting solution was stirred for 30 min at -78

0 C. The reaction was then

quenched by the addition of 200 mL of NH4Cl. The resulting solution was diluted with 100 mL of H2O.

The resulting solution was extracted with 3 x 250 mL of ethyl acetate, dried over anhydrous sodium

sulfate and concentrated under vacuum. The residue was purified by chromatography (silica gel

column, ethyl acetate-petroleum ether gradient 1:50 to 1:5) to give compound 14 (9 g, 59%) as a white

solid. LC-MS: (ES, m/z): 582.2 [M+H]+,

1H NMR: (300MHz, CDCl3, ppm) : � 8.38, 8.33 (s, 1H), 7.76,

7.62 (s, 1H), 4.56-4.54 (m, 1H), 4.14-4.11 (m, 1H), 3.82-3.61 (m, 2H), 2.57 (s, 3H), 1.50 (s, 3H), 0.80

(s, 18H), 0.05-0.02 (m, 9H).

(3R,4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]methyl]-

3-methyl-2-[4-(methylsulfanyl)imidazo[2,1-f][1,2,4]triazin-7-yl]oxolan-2-yl acetate (15) Into a 250-

mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of argon, was placed

a solution of compound 14 (9 g, 15.47 mmol, 1.00 eq.) in pyridine (100 mL), 4-dimethylaminopyridine

(940 mg, 7.69 mmol, 0.50 eq.). This was followed by the addition of acetyl acetate (9 mL) dropwise

with stirring at 25 0

C in 5 min. The resulting solution was stirred for 2 h at 50 0

C and then

concentrated under vacuum. The residue was purified by chromatography (silica gel column, ethyl

acetate-petroleum ether gradient 1:50 to 1:5) to give compound 15 (9.2 g, 95%) as a white solid. 1H

NMR: (300MHz, CDCl3, ppm) : � 8.40 (s, 1H), 7.85 (s, 1H), 4.66-4.64 (m, 1H), 4.31-4.27 (m, 1H),

4.05 (d, J = 15Hz, 1H), 3.80 (d, J = 15Hz, 1H), 2.70 (s, 3H), 2.08 (s, 3H), 1.68 (s, 3H), 0.93 (m, 18H),

0.23-0.15 (m, 12H).

(3R,4S,5R)-3-azido-4-[(tert-butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]-

methyl]-3-methyl-2-[4-(methylsulfanyl)imidazo[2,1-f][1,2,4]triazin-7-yl]oxolane-2-carbonitrile

(16) Into a 500-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of

argon, was placed a solution of compound 15 (9 g, 14.43 mmol, 1.00 eq.) in dichloromethane (250 mL).

This was followed by the addition of trimethylsilylcyanide (17.2 g, 12.00 eq.) dropwise with stirring at

0 oC in 5 min. To this was added boron trifluoride etherate (6.16 g, 3.00 eq.) dropwise with stirring at 0

oC in 10 min. After the resulting solution was stirred for 1 h at 0

oC, the reaction was quenched by the

addition of 20 mL of aq. ammonium chloride. The resulting solution was diluted with of

dichloromethane, washed with 2 x 100 mL of sodium chloride, dried over anhydrous sodium sulfate

and concentrated under vacuum. The residue was purified by chromatography (silica gel column, ethyl

acetate-petroleum ether gradient 1:30 to 1:5) to give compound 16 (6.7 g, 79%) as a white solid, which

was used for next reaction directly. LC-MS: (ES, m/z): 591.3 [M+H]+,

1H NMR: (300MHz, CDCl3,

ppm) : � 8.63, 8.56 (s, 1H), 7.99, 7.86 (s, 1H), 4.27-4.05 (m, 3H), 3.91-3.85 (m, 1H), 2.75 (s, 3H), 1.28

(s, 3H), 0.98-0.96 (s, 18H), 0.24-0.20 (m, 12H).

(2R,3R,4S,5R)-3-azido-4-hydroxy-5-(hydroxymethyl)-3-methyl-2-[4-(methylsulfanyl)-

imidazo[2,1-f][1,2,4]triazin-7-yl]oxolane-2-carbonitrile (17) Into a 100-mL round-bottom flask

purged and maintained with an inert atmosphere of argon, was placed a solution of compound 16 (5.5 g,

9.31 mmol, 1.00 eq.) in THF (60 mL), TBAF (4.87 g, 18.63 mmol, 2.00 eq.). The resulting solution

was stirred for 0.5 h at 25 oC and then concentrated under vacuum. The residue was purified by

chromatography (silica gel column, ethyl acetate-petroleum ether gradient 1:10 to 2:1) to give

compound 17 (1.17 g, 35%) as a white solid. LC-MS: (ES, m/z): 363.0 [M+H]+,

1H NMR: (400MHz,

CDCl3, ppm): � 8.62 (s, 1H), 8.08 (s, 1H), 4.33-4.25 (m, 2H), 4.21-4.09 (m, 1H), 4.00 (d, J = 32Hz, 1H),

2.76 (s, 3H), 1.56 (s, 3H).

(2R,3R,4S,5R)-2-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-3-azido-4-hydroxy-5-

(hydroxymethyl)-3-methyloxolane-2-carbonitrile (18) Into a 80-mL sealed tube, was placed

compound 17 (850 mg, 2.35 mmol, 1.00 eq.). This was followed by the addition of NH3 saturated i-

PrOH (80 mL) at -78 0 C in a liquid nitrogen bath. The resulting solution was stirred for 5 h at 40

0 C.

The resulting mixture was concentrated under vacuum and the residue was purified by chromatography

(silica gel column, ethyl acetate-petroleum ether gradient 1:10 to 2:1) to give compound 18 (700 mg,

90%) as a white solid. LC-MS: (ES, m/z): 332 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 8.17 (s,

1H), 7.94 (s, 1H), 4.15 (s, 2H), 4.02 (d, J = 12Hz, 1H), 3.84 (d, J = 12Hz, 1H), 1.33 (s, 3H).

Methyl (2R,4S,5R)-2-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-3-azido-4-hydroxy-5-

(hydroxymethyl)-3-methyloxolane-2-carboxylate (19) Into a 50-mL round-bottom flask purged and

maintained with an inert atmosphere of argon, was placed a solution of compound 18 (700 mg, 2.11

mmol, 1.00 eq.) in methanol (20 mL), NaOMe (0.46 g, 4.00 eq.). The resulting solution was stirred

overnight at 20 0 C and then concentrated under vacuum. The residue was purified by chromatography

(silica gel column, dichloromethane-methanol gradient 50:1 to 10:1) to give compound 19 (600 mg,

78%) as a white solid. LC-MS (ES, m/z): 365.0 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 8.06 (s,

1H), 7.70 (s, 1H), 4.89 (s, 1H), 4.11-4.05 (m, 1H), 3.94 (s, 3H), 3.88-3.84 (m, 2H), 1.48 (s, 3H).

Methyl (2R,3R,4S,5R)-3-amino-2-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-4-[(tert-

butyldimethylsilyl)oxy]-5-[[(tert-butyldimethylsilyl)oxy]methyl]-3-methyloxolane-2-carboxylate

(20) Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of argon, was

placed a solution of compound 19 (350 mg, 0.96 mmol, 1.00 eq.) in methanol (20 mL), 10% Palladium

carbon (350 mg) under hydrogen. The resulting solution was stirred overnight at 20 0

C. The solids

were filtered out and the filtrate was concentrated under vacuum to give 200 mg (62%) of methyl

(2R,3R,4S,5R)-3-amino-2-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-4-hydroxy-5-(hydroxymethyl)-3-

methyloxolane-2-carboxylate as a white solid, which was used for the next reaction directly without

further purification. LC-MS: (ES, m/z): 339.0 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 8.18 (s,

1H), 7.74 (s, 1H), 4.33-4.28 (m,1H), 4.21-4.18 (m, 1H), 3.91-3.80 (m, 2H), 3.76 (s, 3H), 1.28 (s, 3H).

Into a 25-mL round-bottom flask purged and maintained with an inert atmosphere of argon, was placed

a solution of methyl (2R,3R,4S,5R)-3-amino-2-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-4-hydroxy-

5-(hydroxymethyl)-3-methyloxolane-2-carboxylate (200 mg, 0.59 mmol, 1.00 eq.) in DMF (2 mL),

imidazole (201.58 mg, 5.00 eq.), TBDMSCl (443.96 mg, 5.00 eq.). After the resulting solution was

stirred for 4 h at 50 oC in an oil bath, the reaction was quenched by the addition of 20 mL of water/ice.

The resulting solution was extracted with of ethyl acetate, washed with brine, dried over anhydrous

sodium sulfate and concentrated under vacuum. The residue was purified by chromatography (silica gel

column, ethyl acetate-petroleum ether gradient 1:30 to 1:1) to give compound 20 (201 mg, 60 %) as a

white solid. LC-MS: (ES, m/z): 567.7 [M+H]+,

1H NMR: (300MHz, CD3OD, ppm): � 8.11 (s, 1H), 7.58

(s, 1H), 4.31-4.28 (m, 1H), 4.18-4.17 (m, 1H), 3.89-3.82 (m, 2H), 3.71 (s, 3H), 1.26 (s, 3H), 0.80-0.90

(m, 18H), 0.18-0.02 (m, 12H).

(1R,3R,4S,5R)-1-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-4-[(tert-butyldimethylsilyl)oxy]-

3-[[(tert-butyldimethylsilyl)oxy]methyl]-5-methyl-2-oxa-6-azabicyclo[3.2.0]heptan-7-one (21) Into

a 50-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of argon, was

placed a solution of compound 20 (70 mg, 0.12 mmol, 1.00 eq.) in THF (20 mL). This was followed by

the addition of Lithium diisopropylamide (39 mg, 0.36 mmol, 3.00 eq.) dropwise with stirring at -60 oC

in 5 min. After the resulting solution was stirred for 2 h at -60 oC in a liquid nitrogen bath, it was

concentrated under vacuum, and then diluted with 50 ml of ethyl acetate. The resulting mixture was

washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue

was purified by chromatography (silica gel column, dichloromethane-methanol gradient 50:1 to 10:1)

to give compound 21 (60 mg, 91%) as a white solid. LC-MS: (ES, m/z): 535.5 [M+H]+,

1H NMR:

(300MHz, MeOD, ppm): � 7.95 (s, 1H), 7.63 (s, 1H),7.30-7.21 (m, 2H), 3.81-3.75 (m, 2H), 3.51-3.35

(m, 2H), 1.24 (s, 3H), 0.89, 0.87 (s, 18H), 0.12-0.01 (m, 12H).

(1R,3R,4S,5R)-1-[4-aminoimidazo[2,1-f][1,2,4]triazin-7-yl]-4-hydroxy-3-(hydroxymethyl)-5-

methyl-2-oxa-6-azabicyclo[3.2.0]heptan-7-one (22) Into a 50-mL round-bottom flask purged and

maintained with an inert atmosphere of argon, was placed a solution of compound 21 (60 mg, 0.11

mmol, 1.00 eq.) in THF (20 mL), TBAF (58.76 mg, 0.22 mmol, 2.00 eq.). The resulting solution was

stirred for 15 min at 20 oC and then concentrated under vacuum. The residue was purified by

chromatography (silica gel column, dichloromethane-methanol gradient 20:1 to 5:1). The crude

product (23 mg) was purified by Prep-HPLC with the following conditions (1#-Pre-HPLC-

011(Waters)): Column, SunFire Prep C18 OBD Column,5um,19*150mm; mobile phase,

water(10mmol/L ammonium acid carbonate) and acetonitrile (5% acetonitrile up to 21.5% in 5.5 min,

up to 95% in 1.5 min, down to 5% in 1 min; Detector, UV 254 & 220 nm to give compound 22 (20 mg,

58%) as a white solid. LC-MS: (ES, m/z): 329.3 [M+Na]+,

1H NMR: (300MHz, DMSO-d6, ppm): �

8.76 (bs, 1H), 8.32 (bs, 1H), 8.24 (bs, 1H), 8.07 (s, 1H), 7.63 (s, 1H), 5.61 (s, 1H), 4.71 (s, 1H), 3.75 (s,

3H), 3.62-3.58 (d, J = 12Hz, 1H), 1.15 (s, 3H).