Design, synthesis and biological evaluation of novel, orally bioavailable pyrimidine-fused heterocycles as influenza PB2 inhibitors

Accepted Manuscript Design, synthesis and biological evaluation of novel, orally bioavailable pyrimidinefused heterocycles as influenza PB2 inhibitors Jian Xiong, Jingjing Wang, Guoping Hu, Weili Zhao, Jianqi Li PII:

S0223-5234(18)30969-3

DOI:

https://doi.org/10.1016/j.ejmech.2018.11.015

Reference:

EJMECH 10872

To appear in:

European Journal of Medicinal Chemistry

Received Date: 2 September 2018 Revised Date:

29 October 2018

Accepted Date: 6 November 2018

Please cite this article as: J. Xiong, J. Wang, G. Hu, W. Zhao, J. Li, Design, synthesis and biological evaluation of novel, orally bioavailable pyrimidine-fused heterocycles as influenza PB2 inhibitors, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Design, synthesis and biological evaluation of novel, orally bioavailable pyrimidine-fused heterocycles as influenza PB2 inhibitors Jian Xionga,c, Jingjing Wangc, Guoping Huc, Weili Zhaoa,**, Jianqi Lib,* a

School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China

b

Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, China State

Institute of Pharmaceutical Industry, 285 Gebaini Road, Shanghai 201203, P.R. China WuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, P. R. China

RI PT

c

Abstract:

With the aim to identify novel influenza PB2 inhibitors with high potency and excellent

SC

pharmacokinetic parameters, we have designed and synthesized two new series of pyrimidine-fused heterocycle derivatives based on two generations of co-crystal structures. Docking studies with the newly disclosed PDB structure guided the second round of rational design and led to the discovery

M AN U

of 25m, 25o and 25p as representative compounds with improved potency (EC50 < 1 nM). After pinpointing the metabolic labile site, the C-N replacement of compound 25p successfully produced compound 29c, which demonstrated highly improved PK properties (Cl = 1.3 mL/min/kg, PO AUC = 152 µM.h at 10 mpk in mouse, F = 57%) and improved potency, emerging as a promising lead compound for the treatment of influenza A infection.

*Corresponding author. **Corresponding author.

TE D

Keywords: Influenza, polymerase inhibitor, PB2, drug design, metabolic stability

1. Introduction:

EP

E-mail address: [email protected] (J.Q. Li), [email protected] (W.L. Zhao),

Influenza (IFV) infection remains a significant challenge to the worldwide public healthcare

AC C

system. Epidemics of influenza can seriously affect all populations, and the highest risk of complications exists among pregnant women, children aged 6–59 months, the elderly, individuals with specific chronic medical conditions such as HIV/AIDS, asthma, and chronic heart or lung disease, and health-care workers [1]. The currently marketed anti-influenza medications consist of 4 drugs from 2 classes: amantadine and rimantadine, which are M2-ion channel blockers, and the oral drug oseltamivir and the inhaled medication zanamivir, which target influenza neuraminidase (Fig. 1) [2]. The main concern with these antivirals is drug resistance and their only having a moderate impact on the severity of symptoms, as well as the duration of the sickness in clinical practice. As outlined above, a critical need exists for new antivirals with targets other than the neuraminidase and M2-ion 1 / 41

ACCEPTED MANUSCRIPT

channel. As the key structural protein, the viral polymerase emerges as a promising target for influenza infection treatment. It contains three subunits, PB1, PB2, and PA, that are responsible for the replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected host cells [2]. Other researchers have proposed focusing on the host factors involved in the viral life cycle. This novel type of inhibitor, discovered by Tonelli’s group, is expected to possess a markedly higher barrier for selecting drug-resistant viruses and furthermore

RI PT

may display broad-spectrum antiviral activity [5,6].

In February 2018, baloxavir marboxil (S-033188) from Shionogi was approved in Japan as the first drug that targets the PA subunit of the influenza virus polymerase complex [5]. VX-787, developed by Vertex, was put into phase 3 clinical trials for the treatment of IFV A infection in late

SC

2017 and showed strong superiority to the standard of care (oseltamivir) in a preclinical study [8]. It was reported to block the PB2 cap-snatching activity of the influenza viral polymerase complex [2].

M AN U

However, VX-787 is administered 600 mg b.i.d. in clinical use, which indicates that its potency and half-life could be optimized to improve patient compliance. Some organizations have attempted to design and synthesize analogues of VX-787. Most of them focused on isosteres of aza-indole [10] and the carboxylic acid motif [12] or the replacement of the pyrimidine ring or the 6-substituent of pyrimidine [13] to boost the antiviral potency. However, no comprehensive study has been reported with the aim of improving both the potency and metabolic stability.

TE D

Herein, we report medicinal chemistry research on VX-787 by means of rational structure based drug design [2] that afforded a few lead compounds, 25m, 25o and 25p, with decent properties. Further optimization to block a potential metabolism site improved the PK profile dramatically and

AC C

EP

led to 29c as a promising lead compound.

Fig. 1. Structures of representative anti-influenza compounds 2. Chemistry The preparation of compound 8a was accomplished by applying a linear synthetic route, as outlined in Scheme 1. The displacement of the dichloride 1 with a chiral amine afforded the 4substituted amine 2, which was followed by Suzuki cross-coupling to make the enol ether 3. The subsequent cyclization reaction to prepare 4 exhibited a moderate yield. In the presence of NIS in 2 / 41

ACCEPTED MANUSCRIPT

DMF, compound 4 was converted into compound 5, followed by a one-step cyanation with iPrMg.LiCl and TosCN to prepare compound 6. With the chlorides 4 and 6 in hand, a successive Suzuki coupling with an aza-indole boronate moiety successfully assembled compounds 7a and 7b,

TE D

M AN U

SC

RI PT

which led to the final carboxylic acids 8a and 8b under basic conditions.

Scheme 1. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, DMF, rt, overnight; (b) 2-[(E)-2-Ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2dioxaborolane,Pd(dppf)Cl2, K2CO3, dioxane, 80°C; (c) AcOH, 120°C; (d) NIS, DMF; (e) i-

EP

PrMgCl.LiCl, TosCN, THF, -78°C; (f) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1tosyl-1H-pyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (g) LiOH.H2O, MeOH,

AC C

H2O

An alternative strategy to make the analogue 14 is outlined in Scheme 2. The displacement reaction of the dichloride 9 afforded intermediate 10 under basic conditions, followed by a reduction to give the amine 11. Intermediate 12 was prepared from the dehydration reaction of compound 11, followed by a Suzuki cross-coupling with an aza-indole boronate moiety and subsequent hydrolysis to yield 14.

3 / 41

RI PT

ACCEPTED MANUSCRIPT

SC

Scheme 2. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2-

carboxylate, DIEA, THF, rt; (b) Fe, NH4Cl, MeOH, THF, H2O, 60°C; (c) Trimethyl orthoformate, formic acid; (d) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3-

M AN U

b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (e) LiOH.H2O, MeOH, H2O For the synthesis of another analogue 21, intermediate 16 was prepared from the dichloride 15 via amination, followed by demethylation to prepare intermediate 17. The esterification of 17 in the presence of SOCl2 in MeOH afforded compound 18, and a one-step cyclization successfully

TE D

synthesized intermediate 19. The subsequent Suzuki coupling of 19 prepared intermediate 20,

AC C

EP

followed by hydrolysis to make compound 21.

Scheme 3. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, THF, rt; (b) BBr3, DCM, 0°C; (c) SOCl2, MeOH, 70°C; (d) 1,2-Dibromoethane, K2CO3, DMF, 80°C; (e) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1Hpyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (f) LiOH.H2O, MeOH, H2O

4 / 41

ACCEPTED MANUSCRIPT

RI PT

Scheme 4. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, THF; (b) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1Hpyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (c) LiOH.H2O, MeOH, H2O Starting from the commercially available dichlorides 22a-22v, a substitution reaction afforded compounds 23a-23v under mild conditions. The subsequent Suzuki cross coupling with an aza-

M AN U

SC

indole boronate moiety gave 24a-24v, followed by hydrolysis to smoothly lead to 25a-25v.

TE D

Scheme 5. Reagents and conditions: (a) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1-trityl-1H-pyrazolo[3,4-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (b) TFA, Et3SiH, DCM; (c) LiOH.H2O, MeOH, H2O

Following a similar synthetic strategy outlined in Scheme 5, the final carboxylic acids 29a, 29b,

EP

and 29c were prepared smoothly starting from 26a-c. 3. Design of PB2 inhibitors and biological evaluation and discussion

AC C

The goal of this research was to develop novel inhibitors for the treatment of influenza with excellent potency and metabolic stability both in vitro and in vivo. The initial structure-based design was performed based on the co-crystal structures of influenza A PB2 and its inhibitor VX-787 (PDB: 4P1U) [8]. In addition to the hydrogen bonds formed between aza-indole and the side chains of Glu361 and Lys376, π-stacking was observed between the pyrimidine ring and Phe323 in the cap-binding site, as well as the stabilization of the aza-indole between His357 and Phe404 and the packing of the cyclohexyl group with Phe325, as observed in the co-crystal structure. The carboxylic group of the tail part makes two water-mediated interactions with the nitrogen of His357 and with Gln406 as well as with the main chain carbonyl of Arg355 [8]. However, the pyrimidine moiety around the C-5 position has been underexplored, and it has the potential to boost the potency 5 / 41

ACCEPTED MANUSCRIPT

by appropriate design.

a t-h

de si

SC

RI PT

h rig

nd

Fig. 2. Structures of VX-787 and three series of compounds

M AN U

It is speculated that the incorporation of C-5 with the NH on C-4 from the right-hand side or with C-6 into a fused ring system will increase the molecule rigidity, and this could potentially reinforce the π-π stacking and improve the potency of the newly designed ligands. On the basis of this hypothesis, we designed two series of compounds (Fig. 2) from the right-hand side (8a, 8b, 14 and 21, Series 1) and left-hand side (25a-25l, Series 2) of the pyrimidine moiety after docking with

TE D

4P1U.

The Series 1 compounds showed no improvement, or even a decrease in potency (Table 1). The newly released PDB structures (6EUY) shed light on this [14], in which another important domain the midlink domain - was proven to be crucial in positioning the capped RNA outside of the known

EP

cap-binding domain. Accordingly, the linker NH on the C-4 position of VX-787 formed an important hydrogen bond with the main chain Val511. However, this important interaction was lost

AC C

in the Series 1 compounds that incorporated the NH into a new ring, which led to dramatically decreased potency. For instance, compound 8a exhibited a good binding mode in 4P1U, but its potency was far less than that of VX-787, which could be explained using the newly reported PDB 6EUY (Fig. 3a and 3b) by the loss of the hydrogen bond with the main chain Val511. Table 1

Structures and biological activities of the inhibitors in the right-hand side series 1

6 / 41

ACCEPTED MANUSCRIPT Ring A/B

EC50 (nM)a

CC50 (µM)b

VX-787

-

0.6

12.0

8a

12.0

4.0

8b

70.0

5.4

14

190.0

21

24.0

SC

21.7

> 50.0

M AN U

a

RI PT

Compd No

The compound concentration required to reduce the virus-induced cell death by 50% was defined

as EC50. bThe compound concentration required to reduce the cell viability to 50% of the tested

AC C

EP

TE D

control culture was defined as CC50.

Fig. 3 a) The predicted binding mode of compound 8a on the PB2 cap domain alone (4P1U used as docking template), where the top is open to the solvent; b) Co-crystal structure of VX-787 with a cap-midlink double domain (PDB: 6EUY). Red dash lines stand for hydrogen bonds. After taking this new finding into account, we then focused on the left-hand side Series 2 and re-performed docking for the representative compounds designed. The six-membered (25a) and five-membered ring fused compounds (25i) were well tolerated in the new PB2 cap-midlink double domain 6EUY (Fig. 4a and 4b).

7 / 41

RI PT

ACCEPTED MANUSCRIPT

SC

Fig. 4 a) The predicted binding mode of compound 25i on the PB2 cap-midlink double domain (6EUY was used as a docking template). Red dash lines stand for hydrogen bonds. b) The interactions between compound 25i and the PB2 cap-midlink double domain depicted in a 2D

M AN U

diagram. Pink arrows stand for hydrogen bonds, green lines stand for π-π stacking, hydrophobic residues are coloured green, negative residues are coloured red, positive residues are coloured purple, and polar residues are coloured blue.

The six-membered ring fused compounds showed in vitro antiviral potency in the range of 10 nM to 20 nM, no matter whether the ring was aromatic or non-aromatic (Table 2). Interestingly, the five-membered ring fused compounds exhibited high potency. Among them, the furan analogue 25h

TE D

(EC50 = 5.3 nM), pyrrole analogue 25i (EC50 = 4.5 nM), thiazole analogue 25l (EC50 = 3.0 nM) and cyclopentane analogue 25k (EC50 = 5.5 nM) showed very decent potencies. Inspired by the improved potency achieved by the introduction of the five-membered ring, more analogues bearing

EP

the same pyrimidine moiety were designed and synthesized (25m-25v). As expected, most of the five-membered ring fused analogues displayed higher potency, in which the EC50 could be maintained at less than 1 nM, 10-fold more potent than the right-hand side

AC C

series compounds (8a, 21). 25u/25v, the oxidized states of the sulfide compounds 25p/25q, lost their potency, which indicates that the narrow pocket of PB2 might not tolerate the sulfone functionality.

Table 2 Structures and biological activities of the inhibitors in the left-hand side series 2

8 / 41

ACCEPTED MANUSCRIPT

Ring C/B

EC50 (nM)a

VX-787

-

0.6

25b

14.0

25c

25g

25h

EP AC C

25f

TE D

25d

25e

1.8

SC

20.0

12.0

M AN U

25a

CC50 (µM)b

RI PT

Compd No

25i

9 / 41

7.0

11.0

4.9

11.0

4.7

10.0

1.4

13.0

46.0

12.0

> 50.0

5.3

6.2

4.5

15.0

ACCEPTED MANUSCRIPT 0.6

5.5

10.0

25l

3.0

1.5

25m

0.5

25n

1.4

N

N

25o

25s

25t

EP AC C

25r

TE D

25p

25q

1.5

M AN U

25k

RI PT

9.6

SC

25j

25u

10 / 41

>50.0

0.2

0.6

0.4

4.5

2.6

4.4

2.8

5.6

4.0

0.7

10.0

3.3

110.0

>50.0

ACCEPTED MANUSCRIPT 25v

2000

>50.0

a

The compound concentration required to reduce the virus-induced cell death by 50% was defined as EC50. bThe compound concentration required to reduce the cell viability to 50% of the tested control culture was defined as CC50. The four most potent compounds (25m, 25o, 25p and 25n) were selected for further profiling.

RI PT

Compound 25n suffered from high clearance (Cl = 73.7 mL/min/kg, PO AUC = 48.8 nM.h at 1 mpk in mouse, F = 10%) while 25m, 25o and 25p displayed much better metabolic stability and showed

M AN U

SC

comparable AUCs to VX-787.

Fig. 5. Potent anti-influenza compound and its possible metabolite by AO All these analogues contained the same bulky carboxylic acid moiety, which would be metabolized by conjugation with water-soluble groups (phase II). Except for this metabolic pathway, CYPs are the major enzymes involved in phase I metabolism, accounting for

TE D

approximately 75% of the total metabolism [15]. Of note, drug molecules bearing this classical azaindole skeleton tend to be metabolized by aldehyde oxidase (AO) at the C-2’ position to form 2’hydroxy-7-aza-indole (Fig. 5) [16]. Recent research has indicated that AO oxidation could lead to metabolic products that can cause liver injury and renal toxicity through the formation of reactive

EP

oxygen species or toxic metabolites [17]. The next step is to block the labile site of the in-hand hit compounds to further improve the metabolic stability.

AC C

One strategy is to block the potential metabolic hot spot with a 2’-substituent in the aza-indole motif, but this might lead to a potency loss [16]. Another strategy is the replacement of C with N at C-2’ position to generate an aza-indazole ring that would eliminate the labile site as well. Therefore, the counterparts of 25m, 25o and 25p were designed accordingly. The predicted binding modes of compound 25p (EC50 = 0.4 nM) and 29c were evaluated with PDB 6EUY (Fig. 6a, 6b). For the two compounds, both the aza-indole and aza-indazole moieties would form π-stacking between His357 and Phe404, and the carboxyl group formed a salt bridge with Arg355 and a hydrogen bond with the main chain Val511. Of note, compound 29c could induce Arg332 to form additional hydrogen bonds with the two N atoms of the pyrazole-pyrimidine ring concurrently after the C-N replacement, which would potentially benefit the binding affinity. 11 / 41

ACCEPTED MANUSCRIPT

Meanwhile, dihydrothieno[3,2-d]pyrimidine might be able to enhance the hydrophobic interactions with Phe323 and Leu512 and the Van der Waals force interaction with Asn510 compared with VX787. Compounds 29a and 29c displayed high potency, and surprisingly compound 29b showed extremely high potency (EC50 = 0.057 nM) (Table 3), with the potency correlating well with the predicted binding mode.

RI PT

Regarding the metabolic stability, VX-787 showed the best liver microsome stability in all three species (human/rat/mouse) (Table 3), although its clearance rate from mouse plasma is not optimal (30.9 mL/min/kg), which indicates that another metabolic pathway exists besides the CYPmediated metabolism. Of note, the data shown in Table 3 indicates that 29c indeed benefited from

SC

this C-N replacement, as the T1/2 in the mouse liver microsome was prolonged from 51.3 min to 102.8 min. However, the liver microsome stabilities of 29a and 29b were not improved compared

M AN U

with those of 25m and 25o. This might be explained by the existing thieno[3,2-d]pyrimidine ring systems of 29a and 29b, which tend to be oxidized in the aromatic ring system via CYP or AO as well. For 29c, the 6,7-dihydrothieno[3,2-d]pyrimidine system is less fragile from the metabolic point of view.

Consistent with the LMS data, this trend was also reflected in the much lower clearance of compound 29c (Cl = 1.3 mL/min/kg) versus 25p (Cl = 13.8 mL/min/kg) after oral dosage.

TE D

Fortunately, the absorption of 29c was also improved, with the plasma exposure reaching 152 uM.h with moderate oral bioavailability versus 25p (AUC = 19.5 µM.h) and VX-787 (AUC = 13.4 µM.h) at 10 mpk po, as shown in Table 4. The PK profile of compound 29c (low clearance and high exposure) shows its potential to support a dosage with a longer interval in the clinic compared with

EP

VX-787. Table 3

AC C

Structures and biological activities of the inhibitors in the C-N replacement series 3

Compd #

VX-787

R1/R2

-

A

-

EC50 (nM)a

CC50 (µM)b

0.6

12.0 12 / 41

Liver Microsome Stability T1/2 (min)c Human

Rat

Mouse

>145.0

>145.0

>145.0

ACCEPTED MANUSCRIPT 25m

H

0.5

1.5

49.7

52.2

48.5

29a

N

0.5

0.2

77.0

73.0

41.0

25o

H

0.2

0.6

104.3

90.9

61.1

29b

N

0.06

2.7

71.0

65.0

33.0

H

0.4

4.5

99.6

115.2

51.3

N

0.2

4.9

>145.0

>145.0

102.8

RI PT

S

25p S

29c a

The compound concentration required to reduce the virus-induced cell death by 50% was

SC

defined as EC50. bThe compound concentration required to reduce the cell viability to 50% of the

TE D

M AN U

tested control culture was defined as CC50. cT1/2 is the half-life

Fig. 6 a) The predicted binding mode of compound 25p on the PB2 cap-midlink double domain

EP

(6EUY was used as the docking template). b) The predicted binding mode of compound 29c on the

Table 4

AC C

PB2 cap-midlink double domain (6EUY was used as the docking template).

In Vivo Mouse PK Properties VX-787

25na

25ma

25o a

25pb

29cb

1 mpk

0.5 mpk

0.5 mpk

-

1 mpk

1 mpk

Cl (mL/min/kg)

30.9

73.7

18.9

-

13.8

1.3

T1/2 (h)

1.9

1.3

2.0

-

2.3

3.0

Vd (L/kg)

7.1

5.7

2.4

-

2.3

0.2

1357

252

1008

-

2785

28686

10 mpk

1 mpk

1 mpk

1 mpk

10 mpk

10 mpk

Compd No IV dose

AUC (nM.h) PO dose

13 / 41

Cmax (nM) Tmax (h) AUC (nM.h) %F

ACCEPTED MANUSCRIPT

2053

29.2

172

509

14967

32600

2.5

0.33

0.40

0.83

0.25

0.67

13381

48.8

690

1350

19519

151686

100

10

37

-

71

57

Data reported are the means from the dosing cohorts (Male CD-1 mouse, n=3/dose). aDosed at 0.5 mg/kg iv (20% Solutol in water) and 1 mpk PO (20% Solutol in water), cassette PK; bDosed at 1

RI PT

mg/kg iv (20% Solutol in water) and 10 mpk PO (20% Solutol in water). Cl is the clearance. Vd is the volume of distribution. Cmax represents the highest observed concentration. Tmax is the time taken to reach the maximum concentration. T1/2 is the half-life of the compound exposure in plasma. AUC is the area under the curve. %F is the percent bioavailability.

SC

4. Conclusion

In summary, we have developed two series of structures of fused pyrimidine skeletons based on

M AN U

the newly released crystal structure of the influenza PB2 domain. More than 30 compounds were designed and synthesized after three rounds of SAR optimization. Compound 25p was determined to maintain the same potency (IC50 = 0.4 nM, CC50 = 4.5 µM) and PK profile (Cl = 13.8 mL/min/kg, AUC = 19.5 µM.h) as VX-787 (IC50 = 0.6 nM, CC50 = 12 µM, Cl = 30.9 mL/min/kg, AUC = 13.4 µM.h). The subsequent PK-driven optimization to block a potential metabolic site led to the identification of compound 29c, which showed excellent antiviral potency (IC50 = 0.2 nM,

TE D

CC50 = 4.9 µM) and far superior PK properties (Cl = 1.3 mL/min/kg, AUC = 152 µM.h), with lower clearance and higher plasma exposure. Together with the data set, 29c was proven to be a potential influenza PB2 inhibitor for further drug development. 5. Experimental section

EP

5.1.Computational methods

The molecular docking procedure was performed using GlideSP in Maestro (Schrödinger

AC C

version 10.7) with the default option. OPLS3 Force Field minimization algorithm was used to produce low-energy conformers. The co-crystal structure of influenza A (Flu) virus PB2 bound to VX-787 (PDB ID code: 6EUV and 6EUY) were selected as the docking template. PDB protein and VX-787 were prepared by adding hydrogen and adding missing residues using protein preparation Wizard module of Maestro. Ligand molecules were prepared in 3D format using LigPrep, and the most abundant protonation state of each molecule was used. A total of 15 conformers for each ligand were generated by Confgen to mimic the compound flexibility. A 30 Å docking grid was generated using centroid of ligand in crystal structure. Then the ligand was removed and compound was placed during the molecular docking procedure. The docking procedure was carried out for the unchanged conformation of the receptor and flexible ligand molecules. Types of interaction of the 14 / 41

ACCEPTED MANUSCRIPT

docked PB2 with ligand were analyzed and then the docking conformations were selected and saved based on calculated Glide docking energy score. 5.2.Chemistry 5.2.1. Material and measurements Commercially available starting materials, reagents, and solvents were used as supplied, without further purification. All reactions were monitored by thin-layer chromatography with 13

C NMR spectra) were

RI PT

precoated silica gel F254. Proton magnetic resonance (1H NMR and

recorded on a Bruker Avance 400 (400 MHz). Chemical shifts were recorded in ppm and the spectral data are consistent with the assigned structures. 5.2.2

The

synthesis

of

(2S,3S)-3-[2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrrolo[2,3-

SC

d]pyrimidin-7-yl]bicyclo[2.2.2]octane-2-carboxylic acid (8a) and (2S,3S)-3-(5-cyano-2-(5-fluoro1H-pyrrolo[2,3-b]pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane-2-carboxylic

5.2.2.1 Synthesis

M AN U

acid (8b) of

methyl

(2S,3S)-3-[(5-bromo-2-chloro-pyrimidin-4-

yl)amino]bicyclo[2.2.2]octane-2-carboxylate (2)

To a solution of methyl (2S,3S)-3-aminobicyclo[2.2.2]octane-2-carboxylate (400.00 mg, 2.18 mmol) and compound 1 (497.40 mg, 2.18 mmol) in DMF (5.00 mL) was added DIEA (846.32 mg, 6.55 mmol). The mixture was stirred at room temperature overnight. The mixture was treated with

TE D

saturated NH4Cl solution (10 mL) and extracted with EtOAc (20 mL×2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography with PE:EtOAc (15:1 to 10:1) to 1

HNMR (400 MHz,

EP

afford compound 2 (450.00 mg, 1.20 mmol, 55.10% yield) as white solid.

CHLOROFORM-d) δ: 8.10 (s, 1H), 5.58-5.59 (m, 1H), 4.44-4.46 (m, 1H), 3.75 (m, 3H), 2.36-2.38 (m, 1H), 1.97-1.98 (m, 1H), 1.58-1.66 (m, 8H). MS (ESI) m/z calc. for C14H17BrClN3O2: [M+H]+:

AC C

374.0; found: 374.2 5.2.2.2 Synthesis

of

methyl

(2S,3S)-3-[[2-chloro-5-[(E)-2-ethoxyvinyl]pyrimidin-4-

yl]amino]bicyclo[2.2.2]octane -2-carboxylate (3) The a solution of compound 2 (60.00 mg, 160.15 umol, 1.00 eq) and 2-[(E)-2-ethoxyvinyl]-4,4,5,5tetramethyl-1,3,2-dioxaborolane (34.89 mg, 176.17 umol, 1.10 eq) in dioxane (2.00 mL) was added Pd(dppf)Cl2 (11.72 mg, 16.02 umol, 0.10 eq) and K2CO3 (44.27 mg, 320.30 umol, 2.00 eq) under N2, the mixture was heated to 80°C for 2 hours. The mixture was concentrated under reduced pressure and the residue was purified by Prep-TLC with PE:EtOAc(2:1) to afford compound 3 (30.00 mg, 82.00 umol, 51.20% yield) as colorless oil. 1HNMR (400 MHz, CHLOROFORM-d) δ: 15 / 41

ACCEPTED MANUSCRIPT

7.80 (s, 1H), 6.68 (d, J=12.4 Hz, 1H), 5.34 (d, J=12.8 Hz, 1H), 4.99-5.01 (m, 1H), 4.40 (br s, 1H),3.93(q, J=7.0 Hz ,2H), 3.77 (s, 3H), 2.34-2.36 (m, 1H), 1.83-1.95 (m, 1H), 1.54-1.68 (m, 8H), 1.36(t, J=7.0 Hz ,3H). MS (ESI) m/z calc. for C18H24ClN3O3: [M+H]+: 366.2; found: 366.0 5.2.2.3 Synthesis of methyl (2S,3S)-3-(2-chloropyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane2-carboxylate (4)

RI PT

A solution of compound 3 (250.00 mg, 683.34 umol, 1.00 eq) in HOAc (3.00 mL) was stirred at 120°C for 2 hr. The reaction mixture was concentrated in vacuum and the residue was treated with saturated NaHCO3 solution (10 mL). The aqueous phase was extracted with EtOAc (10 mL ×2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and

SC

concentrated in vacuum. The residue was purified by Prep-TLC with PE:EtOAc (2:1) to afford compound 4 (95.00 mg, 297.07 umol, 43.47% yield) as yellow oil.

1

HNMR (400 MHz,

CHLOROFORM-d) δ: 8.77 (s, 1H), 7.34 (d, J=3.2 Hz, 1H), 6.56 (d, J=3.6 Hz, 1H), 5.37-5.39 (m,

M AN U

1H), 3.69 (s, 3H), 3.26-3.28 (m, 1H), 2.24-2.25 (m, 1H), 1.54-1.91 (m, 8H). MS (ESI) m/z calc. for C16H18ClN3O2 [M+H]+: 320.1; found: 320.2 5.2.2.4 Synthesis

of

methyl

yl)bicyclo[2.2.2]octane-2-carboxylate (5)

(2S,3S)-3-(2-chloro-5-iodo-pyrrolo[2,3-d]pyrimidin-7-

To a solution of compound 4 (30.00 mg, 93.81 umol, 1.00 eq) in DMF (1.00 mL) was added NIS

TE D

(31.66 mg, 140.72 umol, 1.50 eq) at 0°C, then was stirred at room temperature overnight. The reaction solution was diluted with EtOAc (50 mL), washed with saturated aqueous solution of NaHSO3 (20 mL), water (20 mL×3), brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column with PE:EtOAc (2:1) to give

EP

compound 5 (30.00 mg, 67.31 umol, 71.75% yield) as yellow solid. MS (ESI) m/z calc. for C16H17BrClN3O2 [M+H]+: 398.0; found: 398.2 of

AC C

5.2.2.5 Synthesis

methyl

(2S,3S)-3-(2-chloro-5-cyano-pyrrolo[2,3-d]pyrimidin-7-

yl)bicyclo[2.2.2]octane-2-carboxylate (6) To a solution of i-PrMgCl.LiCl (1.3 M, 258.90 uL, 3.00 eq) in THF (2.50 mL) was added compound 5 (50.00 mg, 112.19 umol, 1.00 eq) in THF (0.5 mL) dropwise at -78°C under N2, then was stirred at -78°C for 30 minutes. Then tosyl cyanide(44.73 mg, 246.82 umol, 2.20 eq) in THF (0.5 mL) was added and the reaction was stirred at -78°C for another 30 minutes. HOAc (0.1 mL) was added to the reaction solution and stirred at room temperature for 30 minutes, Then water (10 mL) was added and extracted with EtOAc (20 mL×3), the combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The 16 / 41

ACCEPTED MANUSCRIPT

residue was purified by silica gel column with PE:EtOAc (3:1) to give compound 6 (35.00 mg, 101.51 umol, 90.48% yield) as white solid. MS (ESI) m/z calc. for C17H17ClN4O2 [M+H]+:345.1; found: 345.1 5.2.2.6 Synthesis of methyl (2S,3S)-3-[2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3yl]pyrrolo[2,3-d]pyrimidin-7-yl]bicyclo [2.2.2]octane-2-carboxylate (7a) a

solution

of

5-fluoro-1-(p-tolylsulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

RI PT

To

yl)pyrrolo[2,3-b]pyridine (117.15 mg, 281.43 umol, 1.00 eq) and compound 6 (90.00 mg, 281.43 umol, 1.00 eq) in H2O (1.00 mL) and 2-MeTHF (5.00 mL) was added K3PO4 (11.95 mg, 56.29 umol, 0.20 eq), xantphos (16.10 mg, 33.77 umol, 0.12 eq) and Pd2(dba)3 (5.15 mg, 5.63 umol, 0.02 eq). The reaction mixture was stirred at 115°C overnight. The reaction mixture was filtered and the

SC

filtrate was treated with H2O (10 mL) and extracted with EtOAc (20 mL ×3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced

M AN U

pressure. The residue was purified by Prep-TLC with PE:EtOAc (4:1) to afford compound 7a (60.00 mg, 104.60 umol, 37.17% yield) as yellow oil. MS (ESI) m/z calc. for C30H28FN5O4S [M+H]+:574.2; found:574.2

5.2.2.7 Synthesis of (2S,3S)-methyl 3-(5-cyano-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)7H-pyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane-2-carboxylate (7b)

TE D

7b was synthesized with the experimental protocols described for 7a. White solid (29.21% yield). MS (ESI) m/z calc. for C31H27FN6O4S [M+H]+:599.2; found:599.2 5.2.2.8 Synthesis

of

(2S,3S)-3-[2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrrolo[2,3-

EP

d]pyrimidin-7-yl]bicyclo[2.2.2]octane-2-carboxylic acid (8a) To a solution of compound 7a (57.00 mg, 99.37 umol, 1.00 eq) in THF (2.00 mL) and H2O (1.00

AC C

mL) was added LiOH.H2O (12.51 mg, 298.11 umol, 3.00 eq). The mixture was stirred at 50°C overnight. The reaction mixture was treated with H2O (3 mL) and washed with EtOAc (5 mL×2). Then the aqueous phase was purified by Prep-HPLC (TFA) to afford compound 8a (10.00 mg, 24.67 umol, 24.82% yield) as a white solid.

1

H NMR (400 MHz, DMSO-d6) δ 12.83 (br s, 1H),

9.25 (s, 1H), 8.96 (br s, 1H), 8.65 (dd, J=2.89, 9.66 Hz, 1H), 8.43 (d, J=1.26 Hz, 1H), 8.24 (br d, J=2.76 Hz, 1H), 6.92 (d, J=3.76 Hz, 1H), 5.65 (br d, J=7.28 Hz, 1H), 3.46 (br d, J=7.78 Hz, 1H), 2.26 (br s, 1H), 1.99-2.10 (m, 1H), 1.82-1.96 (m, 2H), 1.42-1.78 (m, 6H).

13

C NMR (400 MHz,

DMSO-d6) δ: 175.0, 167.0, 160.9, 157.6, 155.2, 151.6, 146.3, 133.1, 131.7, 118.5, 115.8, 55.3, 46.2, 31.1, 28.7, 25.1, 21.5, 19.9 ppm; MS (ESI) m/z calc. for C22H20FN5O2 [M+H]+:406.2; found:406.2 5.2.2.9 Synthesis

of

(2S,3S)-3-(5-cyano-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-7H17 / 41

ACCEPTED MANUSCRIPT

pyrrolo[2,3-d]pyrimidin-7-yl)bicyclo[2.2.2]octane-2-carboxylic acid (8b) 8b was synthesized with the experimental protocols described for 8a. White solid (18.55% yield). 1

H NMR (400MHz, METHANOL-d4) δ 9.13 (s, 1H), 8.80-8.82 (m, 1H), 8.56 (s, 1H), 8.45 (s, 1H),

8.24 (s, 1H), 5.37 (s, 1H), 2.38 (s, 1H), 2.21 (br. s,2H), 1.60 - 1.80 (m, 8H); MS (ESI) m/z calc. for C23H19FN6O2 [M+H]+:431.2; found:431.2 5.2.3

Synthesis of compound (2S,3S)-3-[2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)purin-9-

5.2.3.1 Synthesis

of

RI PT

yl]bicyclo[2.2.2]octane-2-carboxylic acid (14)

(2S,3S)-3-[(2-chloro-5-nitro-pyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-

carboxylate (10)

To a mixture of methyl (2S,3S)-3-aminobicyclo[2.2.2]octane-2-carboxylate (300.00 mg, 1.64

SC

mmol, 1.00 eq) and compound 9 (317.57 mg, 1.64 mmol, 1.00 eq) in THF (5.00 mL) was added DIEA (1.06 g, 8.19 mmol, 1.43 mL, 5.00 eq) and the reaction solution was stirred at room

M AN U

temperature overnight. Then the reaction solution was diluted with EtOAc (80 mL), washed with H2O (30 mL) and brine (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column with PE:EtOAc (100:1 to 8:1) to give compound 10 (380.00 mg, 1.12 mmol, 68.00% yield) as a light yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 9.04 (s, 1H), 8.59(s, 1H), 4.67 (s, 1H), 3.77(s, 3H), 2.43-2.45 (m, 1H), 2.03-2.04 (m, 1H), 1.91-1.92 (m, 1H), 1.57-1.72 (m,8H). MS (ESI) m/z calc. for C14H17ClN4O4

TE D

[M+H]+: 341.1; found: 341.1

5.2.3.2 Synthesis of (2S,3S)-3-[(5-amino-2-chloro-pyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2carboxylate (11)

EP

To a mixture of compound 10 (190.00 mg, 557.58 umol, 1.00 eq) in MeOH (3.00 mL), THF (2.00 mL) and H2O (1.00 mL) was added Fe (155.70 mg, 2.79 mmol, 5.00 eq) and NH4Cl (149.12 mg, 2.79 mmol, 97.47 uL, 5.00 eq) at 25°C. The suspension was stirred at 60°C for 5 hours. The

AC C

reaction mixture was diluted with EtOAc (20 mL) and filtered, the filtrate was concentrated in vacuum to afford compound 11 (170.00 mg, 547.01 umol, 98.10% yield) as a light yellow solid. MS (ESI) m/z calc. for C14H19ClN4O2 [M+H]+:311.1; found:311.1 5.2.3.3 Synthesis of compounds methyl (2S,3S)-3-(2-chloropurin-9-yl)bicyclo[2.2.2]octane-2carboxylate (12) Compound 11 (20.00 mg, 64.35 umol, 1.00 eq) and trimethoxymethane (68.29 mg, 643.50 umol, 70.40 uL, 10.00 eq) was taken up into a microwave tube and then formic acid (1.00 mL) was added. The sealed tube was heated at 140°C for 30 min under microwave. The reaction mixture was diluted with EtOAc (20 mL), washed with H2O (10 mL) and brine (10 mL). The organic layer was 18 / 41

ACCEPTED MANUSCRIPT

dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by Prep-TLC with PE:EtOAc (1:1) to give compound 12 (10.00 mg, 31.17 umol, 48.44% yield) as yellow oil. MS (ESI) m/z calc. for C15H17ClN4O2 [M+H]+:321.1; found:321.1 5.2.3.4 Synthesis of methyl (2S,3S)-3-[2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3yl]purin-9-yl]bicyclo[2.2.2]octane-2-carboxylate (13)

RI PT

To a solution of compound 12 (28.00 mg, 87.29 umol, 1.00 eq) , 5-fluoro-1-(p-tolylsulfonyl)-3(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine (36.34 mg, 87.29 umol, 1.00 eq) , Pd2(dba)3 (7.99 mg, 8.73 umol, 0.10 eq), XPhos (8.32 mg, 17.46 umol, 0.20 eq), and K3PO4 (37.06 mg, 174.58 umol, 2.00 eq) in 2-MeTHF (2.00 mL) and H2O (800.00 uL) was de-gassed and

SC

then heated to 80°C overnight. The reaction mixture was diluted with EtOAc (40 mL), washed with H2O (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by Prep-TLC with PE:EtOAc (1:1) to give

M AN U

compound 13 (20.00 mg, 34.81 umol, 39.88% yield) as a yellow gum. MS (ESI) m/z calc. for C29H27FN6O4S [M+H]+:575.2; found:575.2 5.2.3.5 Synthesis

of

(2S,3S)-3-[2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)purin-9-

yl]bicyclo[2.2.2]octane-2-carboxylic acid (14)

To a solution of compound 13 (15.00 mg, 26.10 umol, 1.00 eq) in THF (1.00 mL), MeOH (1.00

TE D

mL) and H2O (500.00 uL) was added LiOH.H2O (5.48 mg, 130.50 umol, 5.00 eq) at 0°C. The mixture was stirred at 50°C overnight. Then the reaction mixture was concentrated and the residue was adjusted to pH 5 by the addition of HCl (1N). The residue was purified by Prep-HPLC (TFA) to give compound 14 (2.20 mg, 4.02 umol, 15.39% yield, 95% purity, TFA salt) as light yellow

EP

solid. 1H NMR (400MHz, METHANOL-d4) δ 8.81 - 8.73 (m, 1H), 8.51 - 8.38 (m, 1H), 8.11 - 8.05 (m, 1H), 7.69 - 7.60 (m, 1H), 7.33 (s, 1H), 4.22 - 4.17 (m, 1H), 3.96 - 3.89 (m, 1H), 2.75 - 2.68 (m,

AC C

1H), 2.43 - 2.34 (m, 1H), 2.18 - 2.02 (m, 2H), 1.90 - 1.62 (m, 4H), 1.39 - 1.24 (m, 2H). MS (ESI) m/z calc. for C21H19FN6O2 [M+H]+:407.2; found: 407.2 5.2.4

Synthesis

of

(2S,3S)-3-(2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6H-pyrimido[5,4-

b][1,4]oxazin-8(7H)-yl)bicyclo[2.2.2] octane-2-carboxylic acid (21) 5.2.4.1 Synthesis

of

methyl

(2S,3S)-3-[(2-chloro-5-methoxy-pyrimidin-4-

yl)amino]bicyclo[2.2.2]octane-2-carboxylate (16) To a solution of 2,4-dichloro-5-methoxy-pyrimidine (146.52 mg, 818.55 umol, 1.00 eq) and methyl (2S,3S)-3-aminobicyclo[2.2.2]octane-2-carboxylate (150.00 mg, 818.55 umol, 1.00 eq) in dioxane (3.00 mL) was added TEA (91.11 mg, 900.40 umol, 124.81 uL, 1.10 eq), then the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into H2O (15 mL). The 19 / 41

ACCEPTED MANUSCRIPT

aqueous phase was extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL), dried with Na2SO4, filtered and concentrated in vacuum. The residue was purified by Prep-TLC with PE:EtOAc(1:1) to afford compound 16 (150.00 mg, 451.21 umol, 55.12% yield, 98% purity) as yellow solid. MS (ESI) m/z calc. for C15H20ClN3O3 [M+H]+:326.1; found:326.1 5.2.4.2 Synthesis of (2S,3S)-3-[(2-chloro-5-hydroxy-pyrimidin-4-yl)amino]bicycle [2.2.2]octane-2-

RI PT

carboxylic acid (17)

To a solution of compound 16 (75.00 mg, 230.21 umol, 1.00 eq) in DCM (2.00 mL) at 0°C was added BBr3 (259.52 mg, 1.04 mmol, 99.82 uL, 4.50 eq) dropwise. The mixture was allowed to warm to room temperature and stirred overnight. To the mixture was added H2O (3 mL). The

SC

mixture was stirred for 0.5 hr, and the solid was filtered, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC with EtOAc to afford compound 17 (120.00 mg,

5.2.4.3 Synthesis

M AN U

crude) as light yellow solid . MS (ESI) m/z calc. for C13H16ClN3O3 [M+H]+:298.1; found:298.1 of

methyl

(2S,3S)-3-[(2-chloro-5-hydroxy-pyrimidin-4-

yl)amino]bicyclo[2.2.2]octane-2-carboxylate (18)

To a solution of compound 17 (120.00 mg, 403.04 umol, 1.00 eq) in MeOH (2.00 mL) was added SOCl2 (95.90 mg, 806.07 umol, 58.47 uL, 2.00 eq), then the reaction mixture was heated to 70°C

TE D

and stirred for 2 hr. The reaction mixture was concentrated in vacuo to afford compound 18 (128.00 mg, crude) as colourless gum which was used in the next step without further purification. MS (ESI) m/z calc. for C14H18ClN3O3 [M+H]+:312.1; found: 312.0 of

methyl

(2S,3S)-3-(2-chloro-6,7-dihydropyrimido[5,4-b][1,4]oxazin-8-

EP

5.2.4.4 Synthesis

yl)bicyclo[2.2.2]octane-2-carboxylate (19)

AC C

A mixture of compound 18 (108.00 mg, 346.42 umol, 1.00 eq), 1,2-dibromoethane (65.08 mg, 346.42 umol, 26.14 uL, 1.00 eq) and K2CO3 (239.39 mg, 1.73 mmol, 5.00 eq) was suspened in DMF (5.00 mL), the reaction mixture was heated to 80°C and stirred overnight. The mixture was poured into water (20 mL).The aqueous phase was extracted with EtOAc (25 mL×3).The combined organic layers were washed with water (10 mL×2) and brine (10 mL), dried over Na2SO4, filtered and concentrated in vacuum. The residue was purified by Prep-TLC with PE:EtOAc (1:1) to afford compound 19 (25.00 mg, 43.66 umol, 12.60% yield, 59% purity) as white solid. MS (ESI) m/z calc. for C16H20ClN3O3 [M+H]+:338.1; found:338.1 5.2.4.5 Synthesis of methyl (2S,3S)-3-[2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]6,7-dihydropyrimido[5,4-b][1,4]oxazin-8-yl]bicyclo[2.2.2]octane-2-carboxylate (20) 20 / 41

ACCEPTED MANUSCRIPT

To a solution of compound 19 (25.00 mg, 74.01 umol, 1.00 eq) and 5-fluoro-1-(p-tolylsulfonyl)-3(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine (30.81 mg, 74.01 umol, 1.00 eq) in THF (2.00 mL) and H2O (500.00 uL) was added K3PO4 (47.13 mg, 222.03 umol, 3.00 eq), XPhos (7.06 mg, 14.80 umol, 0.20 eq) and Pd2(dba)3 (6.78 mg, 7.40 umol, 0.10 eq), then the reaction mixture was heated to 80°C overnight. The reaction mixture was purified by Prep-TLC with PE:EtOAc (1:1) to afford compound 20 (10.00 mg, 16.39 umol, 22.15% yield, 97% purity) as

5.2.4.6 Synthesis

of

RI PT

white solid. MS (ESI) m/z calc. for C30H30FN5O5S [M+H]+:592.2; found:592.2

(2S,3S)-3-(2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6H-pyrimido[5,4-

b][1,4]oxazin-8(7H)-yl)bicyclo[2.2.2] octane-2-carboxylic acid (21)

To a solution of compound 20 (10.00 mg, 16.90 umol, 1.00 eq) in THF (500.00 uL), MeOH (500.00

SC

uL) and H2O (1.00 mL) was added LiOH.H2O (7.09 mg, 169.00 umol, 10.00 eq), then the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo,

M AN U

the residue was adjusted to pH 5 with 1 N citric acid solution. The mixture was purified by PrepHPLC (column: Boston Green ODS 150*30 5u; mobile phase: [water(0.1%TFA)-ACN];B%: 20%50%,8min) to afford compound 21 (5.16 mg, 9.41 umol, 55.67% yield, 98% purity, TFA salt) as white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (br s, 1H), 8.63 (dd, J=2.76, 9.79 Hz, 1H), 8.49 (d, J=2.76 Hz, 1H), 8.34-8.41 (m, 1H), 8.05 (s, 1H), 5.70 (br d, J=7.53 Hz, 1H), 4.20-4.40 (m, 2H), 3.91-4.03 (m, 1H), 3.74-3.83 (m, 1H), 3.74-3.83 (m, 1H), 3.03 (br d, J=7.53 Hz, 2H), 2.16 (br

TE D

s, 1H), 1.90 (br s, 1H), 1.43-1.86 (m, 8H). 13C NMR (400 MHz, DMSO-d6) δ: 175.1, 160.7, 154.8, 152.8, 150.9, 145.6, 133.8, 118.0, 114.8, 109.8, 106.1, 51.9, 46.6, 28.8, 28.1, 26.1, 24.9, 23.9, 21.6, 21.0, 20.5, 20.1,19.0 ppm; MS (ESI) m/z calc. for C22H22FN5O3 [M+H]+:424.2; found:424.2

5.2.5.1

Synthesis of compound 23a-23v Synthesis

EP

5.2.5

of

(2S,3S)-methyl

3-((2-chloro-5-fluoroquinazolin-4-

AC C

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23a) To a solution of 2,4-dichloro-5-fluoroquinazoline (22a) (100.00 mg, 460.77 umol, 1.00 eq) in THF (3.00 mL) was added methyl (2S,3S)-3-aminobicyclo[2.2.2]octane-2-carboxylate (84.44 mg, 460.77 umol, 1.00 eq) and DIPEA (238.20 mg, 1.84 mmol, 4.00 eq) at room temperature overnight. H2O (20 mL) was added to the mixture and extracted with EtOAc (10 mL×3). The combined organic layers were dried over Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel with PE:EtOAc (10:1 to 5:1) to give compound 23a (130.00 mg, 357.33 umol, 77.55% yield) as a yellow solid. 1HNMR (400 MHz, CHLOROFORM-d) δ 7.59-7.68 (m, 1H), 7.51-7.58 (m, 1H), 7.10 (dd, J=8.03, 12.05 Hz, 1H), 6.98 (dd, J=4.27, 16.31 Hz, 1H), 4.60 (br. s., 1H), 3.80 (s, 3H), 2.42-2.50 (m, 1H), 1.97 (dd, J=2.51, 16.06 Hz, 2H), 1.81-1.91 (m, 1H), 21 / 41

ACCEPTED MANUSCRIPT

1.57-1.79 (m, 7H), 1.39-1.50 (m, 1H). MS (ESI) m/z calc. for C18H19ClFN3O2 [M+H]+:364.1; found:364.0 5.2.5.2

Synthesis

of

(2S,3S)-methyl

3-((2-chloropyrido[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23b) Compound 23b was synthesized with the experimental protocols described for 23a. (Yellow solid,

RI PT

80.74 % yield). 1H NMR (400 MHz, CHLOROFORM-d) δ 8.65 (d, J=, 2.80 Hz, 1H), 8.00-8.02 (m, 1H), 7.63-7.66 (m, 1H), 7.39 (s, 1H), 4.63-4.66 (br. s., 1H), 3.78 (s, 3H), 2.52-2.54 (m, 1H), 2.002.01 (m, 1H), 1.30-1.72 (m, 8H).MS (ESI) m/z calc. for C17H19ClN4O2 [M+H]+: 347.1; found:347.0 5.2.5.3

Synthesis

of

(2S,3S)-methyl

3-((2-chloro-7-methylpyrido[3,2-d]pyrimidin-4-

SC

yl)amino)bicycle [2.2.2] octane-2-carboxylate (23c), via two-step synthesis from 22c

Step 1: The intermediate - (2S,3S)-methyl 3-((7-bromo-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3yl)pyrido[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate was synthesized with the

M AN U

experimental protocols described for 23a with 7-bromo-2,4-dichloro-pyrido[3,2-d]pyrimidine. (White solid, 65.52% yield).

Step 2: To a mixture of (2S,3S)-methyl 3-((7-bromo-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3yl)pyrido[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (50.00 mg, 117.45 umol, 1.00 eq) and methylboronic acid (21.09 mg, 352.35 umol, 3.00 eq) in dioxane (3.00 mL) and H2O

TE D

(500.00 uL) was added Pd(dppf)Cl2 (8.59 mg, 11.75 umol, 0.10 eq) and Na2CO3 (37.35 mg, 352.35 umol, 3.00 eq). The suspension was degassed and purged with N2 for three times. The mixture was stirred at 60°C overnight. The mixture was concentrated in vacuum, and the residue was purified by column chromatography on silica gel with PE:EtOAc (10:1 to 5:1) to give compound 23c (30.00

EP

mg, 81.48 umol, 69.37% yield,) as a yellow solid. MS (ESI) m/z calc. for C18H21ClN4O2 [M+H]+:361.1; found:361.1 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-5-methylquinazolin-4-

AC C

5.2.5.4

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23d) 23d was synthesized with the experimental protocols described for 23a. Yellow solid (87.92% yield). MS (ESI) m/z calc. for C19H22ClN3O2 [M+H]+:360.1; found: 360.1 5.2.5.5

Synthesis

of

(2S,3S)-methyl

3-((2-chloro-7-fluoropyrido[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2] octane-2-carboxylate (23e) 23e was synthesized with the experimental protocols described for 23a. Light yellow solid (71.72% yield). 1H NMR (400MHz, CHLOROFORM-d) δ 8.53 (d, J = 2.8 Hz, 1H), 7.63 (dd, J = 8.8, 2.5 Hz, 1H), 7.28 (br. s., 1H), 4.59-4.67 (m, 1H), 3.77 (s, 3H), 2.52 (dt, J = 5.8, 1.9 Hz, 1H), 1.96-2.06 (m, 2H), 1.56-1.92 (m, 8H), 1.40-1.51 (m, 1H). MS (ESI) m/z calc. for C17H18ClFN4O2 [M+H]+:365.1; 22 / 41

ACCEPTED MANUSCRIPT

found:365.1 5.2.5.6

Synthesis

of

(2S,3S)-methyl

3-((2-chloropteridin-4-yl)amino)bicyclo[2.2.2]octane-2-

carboxylate (23f) 23f was synthesized with the experimental protocols described for 23a. Light yellow oil (11.32% yield). MS (ESI) m/z calc. for C16H18ClN5O2 [M+H]+:348.1; found:347.9 5.2.5.7 Synthesis

of

(2S,3S)-ethyl

3-((2-chloro-5,6,7,8-tetrahydroquinazolin-4-

RI PT

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23g)

23g was synthesized with the experimental protocols described for 23a. Light solid (27.85% yield). 1

H NMR (400MHz, CHLOROFORM-d) δ 4.73 (br d, J=5.52 Hz, 1H), 4.45 (br t, J=5.77 Hz, 1H),

4.18-4.28 (m, 2H), 2.67 (t, J=5.77 Hz, 2H), 2.32 (br d, J=5.52 Hz, 1H), 2.27 (t, J=6.02 Hz, 2H),

SC

1.97 (br d, J=2.51 Hz, 1H), 1.50-1.89 (m, 12H), 1.36-1.47 (m, 1H), 1.27 (t, J=7.28 Hz, 3H). MS (ESI) m/z calc. for C19H26ClN3O2 [M+H]+:364.2; found:364.2 Synthesis

of

(2S,3S)-methyl

3-((2-chlorofuro[3,2-d]pyrimidin-4-

M AN U

5.2.5.8

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23h)

23h was synthesized with the experimental protocols described for 23a. Colorless oil (64.62% yield). 1H NMR (400MHz, CHLOROFORM-d) δ 7.74 (s, 1H), 6.78 (d, J=2.00 Hz, 1H),5.38 (s, 1H),4.66 (s,1H), 3.75 (s, 1H), 2.43 (s, 1H), 1.91 (s, 1H), 1.64-1.84 (m, 8H).

5.2.5.9

Synthesis

of

TE D

MS (ESI) m/z calc. for C16H18ClN3O3 [M+H]+:336.1; found: 335.9 (2S,3S)-methyl

3-((2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23i), via two-step synthesis from 22i Step 1: The intermediate - methyl (2S, 3S)-3-[[2-chloro-7-(p-tolylsulfonyl)pyrrolo[2, 3-

EP

d]pyrimidin-4-yl]amino]bicyclo[2.2.2]octane-2-carboxylate was synthesized with the experimental protocols described for 23a. Colorless oil (80.48% yield).

AC C

Step 2: To a solution of intermediate methyl (2S, 3S)-3-[[2-chloro-7-(p-tolylsulfonyl)pyrrolo[2, 3d]pyrimidin-4-yl]amino]bicyclo[2.2.2]octane-2-carboxylate (100.00 mg, 204.50 umol, 1.00 eq) in MeOH(2.00 mL) was added K2CO3 (56.53 mg, 409.00 umol, 2.00 eq). The mixture was stirred at room temperature overnight. The mixture was diluted by water (5 mL) and extracted with EtOAc (5 mL×3). The organic layer was dried over Na2SO4, filtered and concentrated in vacuum to afford compound 23i (68.00 mg, crude) as a light yellow oil which was used for the next step directly without purification. MS (ESI) m/z calc. for C16H19ClN4O2 [M+H]+:335.1; found: 335.0 5.2.5.10 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23j) 23j was synthesized with the experimental protocols described for 23a. Yellow solid (61.78% 23 / 41

ACCEPTED MANUSCRIPT

yield). 1H NMR (400MHz, CHLOROFORM-d) δ 6.55 (s, 1H), 4.69-4.74 (m, 1H), 3.67 (s, 3H), 3.64 (s, 3H), 3.08-3.10 (m, 1H), 1.45-1.98 (m, 10H). MS (ESI) m/z calc. for C17H20ClFN4O2 [M+H]+:367.1; found:367.1 5.2.5.11 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23k) 23k was synthesized with the experimental protocols described for 23a. Light yellow solid (17.59%

RI PT

yield). 1H NMR (400MHz, CHLOROFORM-d) δ 4.60 (s, 1H), 4.43-4.46 (m, 1H), 3.77 (s, 3H), 2.85-2.89 (m, 2H), 2.63-2.67 (m, 2H), 2.35-2.36 (m, 1H), 2.11-2.15 (m, 2H), 1.41-1.85 (m, 10H). MS (ESI) m/z calc. for C17H22ClN3O2 [M+H]+:336.1; found:336.1 of

compound

(2S,3S)-methyl

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23l)

3-((5-chlorothiazolo[5,4-d]pyrimidin-7-

SC

5.2.5.12 Synthesis

23l was synthesized with the experimental protocols described for 23a. Yellow solid (58.40%

M AN U

yield). 1H NMR (400MHz, CHLOROFORM-d) δ 8.75 (s, 1H), 6.42 (d, J=5.02 Hz, 1H), 4.63 (br. s., 1H), 3.77 (s, 3H), 2.49 (d, J=5.52 Hz, 1H), 2.02 (d, J=2.01 Hz, 1H), 1.96 (d, J=2.01 Hz, 1H), 1.521.90 (m, 7H), 1.40-1.50 (m, 1H).MS (ESI) m/z calc. for C15H17ClN4O2S[M+H]+:353.1; found:353.1 5.2.5.13 Synthesis

of

(2S,3S)-methyl

3-((2-chlorothieno[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23m)

TE D

23m was synthesized with the experimental protocols described for 23a. Colorless oil (41.23% yield). MS (ESI) m/z calc. for C16H18ClN3O2S [M+H]+:352.1; found: 352.0 5.2.5.14 Synthesis

of

(2S,3S)-methyl

3-((2-chlorothieno[3,4-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23n)

EP

23n was synthesized with the experimental protocols described for 23a. Yellow solid (75.77% yield). 1H NMR (400MHz, CHLOROFORM-d) δ 7.79 (s, 1H), 7.55 (d, J=3.2 Hz, 1H), 5.77 (s,

AC C

1H), 4.65 (br. s., 1H), 3.79 (s, 3H), 2.51 (s, 1H), 1.59-2.02 (m, 10H). MS (ESI) m/z calc. for C16H18ClN3O2S [M+H]+:352.1; found:352.2 5.2.5.15 Synthesis

of

(2S,3S)-methyl

3-((2-chlorothieno[2,3-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23o) 23o was synthesized with the experimental protocols described for 23a. White solid (50.33% yield). MS (ESI) m/z calc. for C16H18ClN3O2S [M+H]+:352.1; found: 352.2 5.2.5.16 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-6,7-dihydrothieno[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23p) 23p was synthesized with the experimental protocols described for 23a. Colorless oil (29.26% yield). MS (ESI) m/z calc. for C16H20ClN3O2S [M+H]+:354.1; found:354.2 24 / 41

5.2.5.17 Synthesis

ACCEPTED MANUSCRIPT

of

(2S,3S)-methyl

3-((2-chloro-5,7-dihydrothieno[3,4-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23q) 23q was synthesized with the experimental protocols described for 23a. Yellow solid (68.82% yield). MS (ESI) m/z calc. for C16H20ClN3O2S [M+H]+:354.1; found:354.0 5.2.5.18 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-5,6-dihydrothieno[2,3-d]pyrimidin-4-

RI PT

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23r) 23r was synthesized with the experimental protocols described for 23a. Yellow solid (47.55% yield). MS (ESI) m/z calc. for C16H20ClN3O2S [M+H]+:354.1; found:353.9 5.2.5.19 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-7-cyanothieno[3,2-d]pyrimidin-4-

SC

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23s), via two-step synthesis from 22s

Step 1: The intermediate - (2S,3S)-methyl 3-((7-bromo-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate was synthesized with the

M AN U

experimental protocols described for 23a. Yellow solid (86.14% yield). MS (ESI) 432.3 [M+H]+ Step 2: To a stirred solution of intermediate - (2S,3S)-methyl 3-((7-bromo-2-(5-fluoro-1Hpyrrolo[2,3-b]pyridin-3-yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (100.00 mg, 232.15 umol, 1.00 eq) in THF (2.00 mL) was added n-BuLi (2.6 M, 223.22 uL, 2.50 eq) dropwise at -78°C.

The mixture was stirred at -78°C for 30 min then a solution of p-

TE D

tolylsulfonylformonitrile (50.48 mg, 278.58 umol, 1.20 eq) in THF (1.00 mL) was added. The mixture was stirred at -78 °C for 1.5 hr then warmed to 10°C and stirred for another 10 hr. The mixture was diluted with water (5 mL), extracted with EtOAc (10 mL x 3). The combined organic layer were washed with brine (10 mL), dried over Na2SO4, filtered, concentrated in vacuum. The

EP

residue was purified by Prep-TLC with PE:EtOAc (3:1) to afford the title compound 23s (40.00 mg, 84.91 umol, 36.58% yield, 80% purity) as colorless oil. MS (ESI) m/z calc. for C17H17ClN4O2S

AC C

[M+H]+:377.1; found: 376.9 5.2.5.20 Synthesis

of

(2S,3S)-methyl

3-((2-chloro-6-(difluoromethyl)thieno[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (23t) 23t was synthesized with the experimental protocols described for 23a. Yellow oil (63.47% yield). MS (ESI) m/z calc. for C17H18ClF2N3O2S [M+H]+:402.1; found:401.9 5.2.6

Synthesis of compound 24a-24v

5.2.6.1 Synthesis of (2S,3S)-methyl 3-((5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3yl)quinazolin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (24a) To a solution of compound 23a (130.00 mg, 357.33 umol, 1.00 eq) in 2-methyltetrahydrofuran (4.00 mL) and H2O (1.00 mL) was added 5-fluoro-1-(p-tolylsulfonyl)-3-(4,4,5,5-tetramethyl-1,3,225 / 41

ACCEPTED MANUSCRIPT

dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine (148.75 mg, 357.33 umol, 1.00 eq), K3PO4 (151.70 mg, 714.66 umol, 2.00 eq) , Pd2(dba)3 (16.36 mg, 17.87 umol, 0.05 eq) and XPhos (34.07 mg, 71.47 umol, 0.20 eq) at 25°C. The mixture was stirred at 80°C overnight. The reaction mixture was cooled to room temperature and filtered. H2O (20 mL) was added to the mixture and extracted with EtOAc (10 mL×3). The combined organic layers were dried over Na2SO4 and concentrated in vacuum. The residue was purified by column chromatography on silica gel with PE:EtOAc (20:1 to 6:1) to give

RI PT

compound 24a (40.00 mg, 64.76 umol, 18.12% yield) as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 8.76-8.85 (m, 2H), 8.34 (d, J=1.76 Hz, 1H), 8.18 (d, J=8.28 Hz, 2H), 7.607.71 (m, 2H), 7.31 (d, J=8.53 Hz, 2H), 7.03-7.12 (m, 1H), 6.94 (dd, J=6.78, 16.81 Hz, 1H), 4.98 (br. s., 1H), 3.77 (s, 3H), 2.52 (br. s., 1H), 2.10 (br. s., 2H), 1.75 (br. s., 3H), 1.45-1.55 (m, 6H). MS

5.2.6.2

Synthesis

of

(2S,3S)-methyl

SC

(ESI) m/z calc. for C32H29F2N5O4S [M+H]+:618.2; found:618.2

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

M AN U

yl)pyrido[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24b） 24b was synthesized with the experimental protocols described for 24a. Yellow solid (55 mg, crude). MS (ESI) m/z calc. for C31H29FN6O4S [M+H]+:601.2; found: 601.1 5.2.6.3

Synthesis of

(2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-7-

methylpyrido[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2] octane-2-carboxylate（24c）

TE D

24c was synthesized with the experimental protocols described for 24a. Yellow solid (72.01% yield). MS (ESI) m/z calc. for C32H31FN6O4S [M+H]+:615.2; found: 615.2 5.2.6.4

Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-

EP

methylquinazolin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24d） 24d was synthesized with the experimental protocols described for 24a. Yellow solid (41.05% yield). MS (ESI) m/z calc. for C33H32FN5O4S [M+H]+:614.2; found: 614.2 Synthesis of (2S,3S)-methyl 3-((7-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

AC C

5.2.6.5

yl)pyrido[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24e） 24e was synthesized with the experimental protocols described for 24a. Yellow solid (19.90% yield). MS (ESI) m/z calc. for C31H28F2N6O4S [M+H]+:619.2; found: 619.1 5.2.6.6

Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

yl)pteridin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24f） 24f was synthesized with the experimental protocols described for 24a. Yellow solid (23.30% yield). MS (ESI) m/z calc. for C30H28FN7O4S [M+H]+:602.2; found: 602.1 5.2.6.7

Synthesis of (2S,3S)-ethyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,6,7,826 / 41

ACCEPTED MANUSCRIPT

tetrahydroquinazolin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24g） 24g was synthesized with the experimental protocols described for 24a. Yellow solid (78.54% yield). MS (ESI) m/z calc. for C33H36FN5O4S [M+H]+:618.3; found:618.2 5.2.6.8

Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

yl)furo[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24h）

RI PT

24h was synthesized with the experimental protocols described for 24a. Colorless oil (31.89% yield). MS (ESI) m/z calc. for C30H28FN5O5S [M+H]+:590.2; found: 590.1 5.2.6.9

Synthesis of (2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-7H-

pyrrolo[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24i）

SC

24i was synthesized with the experimental protocols described for 24a. Yellow oil (25.09% yield). MS (ESI) m/z calc. for C30H29FN6O4S [M+H]+:589.2; found:589.2

M AN U

5.2.6.10 Synthesis of (2S,3S)-methyl 3-((5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3yl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24j） 24j was synthesized with the experimental protocols described for 24a. Light yellow solid (16.60% yield). MS (ESI) m/z calc. for C31H30F2N6O4S [M+H]+:621.2; found:621.0 5.2.6.11 Synthesis of (2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-6,7-

TE D

dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)bicycle [2.2.2]octane-2-carboxylate（24k） 24k was synthesized with the experimental protocols described for 24a. Light yellow solid (65.61% yield). MS (ESI) m/z calc. for C31H32FN5O4S [M+H]+:590.2; found: 590.6 5.2.6.12 Synthesis

of

(2S,3S)-methyl

3-((5-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

EP

yl)thiazolo[5,4-d]pyrimidin-7-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24l） 24l was synthesized with the experimental protocols described for 24a. Yellow solid (58.16%

AC C

yield). 1H NMR (400 MHz, CHLOROFORM-d) 8.73 (d, J=7.03 Hz, 2H), 8.69 (dd, J=3.01, 9.03 Hz, 1H), 8.32 (d, J=2.01 Hz, 1H), 8.14 (d, J=8.03 Hz, 2H), 7.29 (d, J=8.03 Hz, 2H), 6.27 (d, J=6.53 Hz, 1H), 4.95 (br. s., 1H), 3.73 (s, 3H), 2.53 (d, J=6.02 Hz, 1H), 2.38 (s, 3H), 2.10 (d, J=2.51 Hz, 2H), 1.60-2.03 (m, 9H). MS (ESI) m/z calc. for C29H27FN6O4S2 [M+H]+:607.2; found:607.0 5.2.6.13 Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24m） 24m was synthesized with the experimental protocols described for 24a. Colorless oil (27.39% yield). MS (ESI) m/z calc. for C30H28FN5O4S2 [M+H]+:606.2; found: 606.2 5.2.6.14 Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-327 / 41

ACCEPTED MANUSCRIPT

yl)thieno[3,4-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24n） 24n was synthesized with the experimental protocols described for 24a. Yellow oil (66.35% yield). MS (ESI) m/z calc. for C30H28FN5O4S2 [M+H]+:606.2; found: 606.1 5.2.6.15 Synthesis

of

(2S,3S)-methyl

3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

yl)thieno[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24o）

RI PT

24o was synthesized with the experimental protocols described for 24a. Yellow solid (14.38% yield). MS (ESI) m/z calc. for C30H28FN5O4S2 [M+H]+:606.2; found: 606.1

5.2.6.16 Synthesis of (2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-6,7dihydrothieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24p）

SC

24p was synthesized with the experimental protocols described for 24a. Colorless oil (34.94% yield). MS (ESI) m/z calc. for C30H30FN5O4S2 [M+H]+:608.2; found: 608.1

M AN U

5.2.6.17 Synthesis of (2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,7dihydrothieno[3,4-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24q） 24q was synthesized with the experimental protocols described for 24a. Yellow solid (37.91% yield). MS (ESI) m/z calc. for C30H30FN5O4S2 [M+H]+:608.2; found: 608.1 5.2.6.18 Synthesis of (2S,3S)-methyl 3-((2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,6-

TE D

dihydrothieno[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24r） 24r was synthesized with the experimental protocols described for 24a. Colorless oil (24.60% yield). MS (ESI) m/z calc. for C30H30FN5O4S2 [M+H]+:608.2; found: 608.1 5.2.6.19 Synthesis of (2S,3S)-methyl 3-((7-cyano-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-

EP

yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate（24s） 24s was synthesized with the experimental protocols described for 24a. Yellow solid (51.98%

AC C

yield). MS (ESI) m/z calc. for C31H27FN6O4S2 [M+H]+:631.2; found: 631.1 5.2.6.20 Synthesis of (2S,3S)-methyl 3-((6-(difluoromethyl)-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3b]pyridin-3-yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2] octane-2-carboxylate（24t） 24t was synthesized with the experimental protocols described for 24a. Yellow solid (45.96% yield).MS (ESI) m/z calc. for C31H28F3N5O4S2 [M+H]+:656.2; found:656.1 5.2.6.21 Synthesis of ((2S,3S)-3-[[2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]-5,5dioxo-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl]amino]bicycle [2.2.2]octane-2-carboxylate（24u） To a solution of compound 24p (10.00 mg, 16.45 umol, 1.00 eq) in DCM (2.00 mL) was added mCPBA (10.02 mg, 49.36 umol, 85% purity, 3.00 eq). The mixture was stirred at room temperature overnight and the mixture was concentrated in vacuum. The residue was purified by Prep-HPLC 28 / 41

ACCEPTED MANUSCRIPT

(column: Boston Green ODS 150*30 5u; mobile phase:[water(0.1%TFA)-ACN];B%: 35%65%,8min) to afford compound 24u (10.00 mg, 15.63 umol, 95.03% yield, 100% purity) as white solid. MS (ESI) m/z calc. for C30H30FN5O6S2 [M+H]+:640.2; found: 640.1 5.2.6.22 Synthesis of methyl (2S,3S)-3-[[2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]6,6-dioxo-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]amino]bicyclo[2.2.2]octane-2-carboxylate（24v） To a solution of compound 24q (70.00 mg, 115.18 umol, 1.00 eq) in Acetone (2.00 mL), H2O (2.00

RI PT

mL) was added Oxone (26.29 mg, 172.78 umol, 1.50 eq) slowly at 0°C. The reaction mixture was warmed to room temperature and stirred overnight. The mixture was quenched with saturated Na2SO3 solution (10 mL) and the aqueous phase was extracted with EtOAc (15 mL×2). The combined organic phase was washed with brine (10 mL), dried with Na2SO4, filtered and

SC

concentrated in vacuum. The residue was purified by Prep-TLC (PE:EtOAc = 2:1) to afford compound 24v (40.00 mg, 59.40 umol, 51.57% yield, 95% purity) as yellow solid. MS (ESI) m/z

5.2.7

Synthesis of compound 25a-25v

M AN U

calc. for C30H30FN5O6S2 [M+H]+:640.2; found:640.1

5.2.7.1 Synthesis of (2S,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)quinazolin-4yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid (25a)

To a solution of compound 24a (40.00 mg, 64.76 umol, 1.00 eq) in MeOH (1.00 mL) and H2O (1.00

TE D

mL) was added LiOH.H2O (13.59 mg, 323.80 umol, 5.00 eq) at 25 °C. The mixture was stirred at 50 C overnight. The solvent was removed under reduced pressure. 1 M HCl (0.5 mL) was added to the mixture. The mixture was filtered and the filter cake was collected. The crude product was purified by Prep-HPLC (HCl) to give compound 25a (5.00 mg, 10.29 umol, 15.89% yield, 100%

EP

purity, HCl salt) as a yellow solid. 1HNMR (400MHz, METHANOL-d4) δ 8.80 (s, 1H), 8.74 (dd, J=2.76, 9.29 Hz, 1H), 8.39 (s, 1H), 7.98-8.06 (m, 1H), 7.68 (d, J=8.28 Hz, 1H), 7.48 (dd, J=8.16,

AC C

12.17 Hz, 1H), 5.50 (br. s., 1H), 3.04 (d, J=6.02 Hz, 1H), 2.26 (br. s., 1H), 2.20 (br. s., 1H), 1.622.04 (m, 3H).

13

C NMR (400 MHz, DMSO-d6) δ: 176.1, 157.3, 154.9, 152.6, 146.4, 140.6, 140.0,

138.8, 138.0, 132.0, 131.7, 131.3, 130.2, 129.7, 129.3, 128.5, 126.0, 118.8, 118.7, 115.6, 115.4, 113.9, 51.2, 48.3, 31.7, 28.8, 28.7, 25.8, 24.3, 22.6, 21.6, 19.5, 14.4 ppm. MS (ESI) m/z calc. for C24H21F2N5O2 [M+H]+:450.2; found:450.0 5.2.7.2

Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrido[3,2-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25b） 25b was synthesized with the experimental protocols described for 25a. White solid (17.21% yield). 1

HNMR (400MHz, METHANOL-d4) δ 8.88 (d, J=3.26 Hz, 1 H) 8.77 (dd, J=9.29, 2.76 Hz, 1 H)

8.70 (s, 1 H) 8.34 (s, 1 H) 8.17 (dd, J=8.53, 1.25 Hz, 1 H) 7.94 (dd, J=8.53, 4.27 Hz, 1 H) 5.31 (d, 29 / 41

ACCEPTED MANUSCRIPT

J=6.27 Hz, 1 H) 2.23 (s., 1 H) 2.16 (s., 1 H) 1.56 - 2.08 (m, 9 H). 13C NMR (400 MHz, DMSO-d6) δ: 175.9, 159.0, 158.6, 157.4, 155.0, 153.7, 153.6, 146.4, 138.1, 136.9, 134.0, 132.8, 132.2, 131.9, 131.4, 125.2, 120.0, 118.6, 118.5, 115.4, 115.2, 113.2, 113.1, 51.6, 47.9, 28.8, 28.7, 25.7, 21.5, 19.5 ppm. MS (ESI) m/z calc. for C23H21FN6O2 [M+H]+:433.2; found: 433.1 5.2.7.3

Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-7-methylpyrido[3,2-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25c）

RI PT

25c was synthesized with the experimental protocols described for 25a. Off-white solid (82.26% yield). 1HNMR (400 MHz, DMSO-d6) δ 13.13 (br s, 1H), 10.03 (br s, 1H), 8.89 (br s, 1H), 8.77 (d, J=1.76 Hz, 1H), 8.61 (dd, J=2.76, 9.54 Hz, 1H), 8.45 (d, J=1.51 Hz, 1H), 8.03 (s, 1H), 5.11 (br t, J=7.15 Hz, 1H), 3.33 (br d, J=6.02 Hz, 1H), 2.58 (s, 3H), 2.10 (br s, 1H), 2.04 (br s, 1H), 1.37-1.92 13

C NMR (400 MHz, DMSO-d6) δ: 175.3, 159.3, 158.2, 157.9, 157.4, 155.2, 154.9, 149.9,

SC

(m, 8H).

145.8, 141.0, 133.2, 126.3, 118.5, 115.3, 52.1, 46.4, 36.3, 28.8, 28.0, 24.8, 24.0, 21.0, 19.2, 18.6

5.2.7.4

M AN U

ppm. MS (ESI) m/z calc. for C24H23FN6O2 [M+H]+:447.2; found:447.1

Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-methylquinazolin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25d）

25d was synthesized with the experimental protocols described for 25a. White solid (67.12% yield). 1

HNMR (400 MHz, DMSO-d6) δ 13.07 (br s, 1H), 8.88 (br s, 1H), 8.57 (dd, J=2.76, 9.54 Hz, 1H),

TE D

8.52-8.63 (m, 1H), 8.41 (dd, J=1.25, 2.51 Hz, 1H), 7.99 (br s, 1H), 7.67-7.85 (m, 2H), 7.43 (br d, J=7.03 Hz, 1H), 5.11 (br s, 1H), 2.98 (br d, J=5.27 Hz, 1H), 2.90 (s, 3H), 2.06-2.23 (m, 2H), 1.401.90 (m, 8H).

13

C NMR (400 MHz, DMSO-d6) δ: 175.4, 161.0, 158.8, 158.5, 156.5, 154.6, 150.6,

139.9, 116.4, 113.8, 51.3, 47.6, 36.3, 29.9, 29.2, 28.8, 25.6, 24.3, 21.6, 19.4 ppm. MS (ESI) m/z

5.2.7.5

EP

calc. for C25H24FN5O2 [M+H]+:446.2; found:446.2 Synthesis of (2S,3S)-3-((7-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrido[3,2-

AC C

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25e） 25e was synthesized with the experimental protocols described for 25a. Light yellow solid (21.75% yield). 1HNMR (400MHz, METHANOL-d4) δ 8.88 - 8.79 (m, 2H), 8.70 (dd, J = 2.8, 9.0 Hz, 1H), 8.35 (br. s., 1H), 7.98 (dd, J = 2.5, 8.5 Hz, 1H), 5.34 (d, J = 6.8 Hz, 1H), 3.16 (d, J = 6.5 Hz, 1H), 2.25 (br. s., 1H), 2.14 (br. s., 1H), 2.09 - 1.54 (m, 9H).

13

C NMR (400 MHz, DMSO-d6) δ: 176.0,

157.0, 154.9, 154.6, 152.4, 152.3, 141.6, 141.3, 139.0, 134.5, 116.5, 116.3, 115.7, 113.9, 51.0, 47.9, 29.0, 28.8, 24.3, 21.6, 19.4 ppm. MS (ESI) m/z calc. for C23H20F2N6O2 [M+H]+:451.2; found: 451.1 5.2.7.6

Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pteridin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25f） 30 / 41

ACCEPTED MANUSCRIPT

25f was synthesized with the experimental protocols described for 25a. Yellow solid (9.38% yield). 1

HNMR (400MHz, METHANOL-d4) δ 8.97 (s, 1H), 8.80 – 8.81 (m, 1H), 8.71 (s, 1H), 8.29 (s, 1H),

5.23 – 5.24 (m, 2H), 3.04 – 3.06 (m, 1H), 2.15 – 2.21 (m, 1H), 1.62 – 1.93 (m, 9H). MS (ESI) m/z calc. for C22H20FN7O2 [M+H]+:434.2; found: 434.3 5.2.7.7

Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,6,7,8-

tetrahydroquinazolin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25g）

RI PT

25g was synthesized with the experimental protocols described for 25a. White solid (81.78% yield). 1

HNMR (400 MHz, DMSO-d6) δ 13.17-13.63 (m, 1H), 13.04 (br d, J=2.76 Hz, 1H), 8.81 (d, J=3.26

Hz, 1H), 8.38-8.51 (m, 2H), 8.08 (br d, J=7.03 Hz, 1H), 4.99 (br t, J=6.90 Hz, 1H), 3.11 (br d, J=6.78 Hz, 1H), 2.78 (br s, 2H), 2.46 (br s, 2H), 2.08 (br s, 1H), 1.89 (br s, 1H), 1.26-1.85 (m, 12H). 13

SC

C NMR (400 MHz, DMSO-d6) δ: 175.1, 160.7, 158.3, 158.0, 157.2, 154.8, 152.8, 150.9, 145.6,

133.8, 133.2, 132.9, 118.0, 115.0, 114.8, 109.8, 106.1, 51.9, 46.6, 28.8, 28.1, 24.9, 23.9, 21.6, 21.0,

5.2.7.8

M AN U

20.5, 20.1, 19.0 ppm. MS (ESI) m/z calc. for C24H26FN5O2 [M+H]+:436.2; found: 436.2 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)furo[3,2-d]pyrimidin-

4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25h）

25h was synthesized with the experimental protocols described for 25a. White solid (38.25% yield). 1

HNMR (400MHz, METHANOL-d4) δ 8.67 (s., 1 H), 8.49 (s, 1 H), 8.32 (s., 1 H), 8.25 (s., 1 H),

TE D

7.02 (d, J=2.01 Hz, 1 H), 5.25 (s., 1 H), 2.90 (d, J=5.52 Hz, 1 H), 2.20 (s., 1 H), 1.53 - 2.12 (m, 9 H). MS (ESI) m/z calc. for C22H20FN5O3 [M+H]+:422.2; found: 422.1 5.2.7.9

Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-7H-pyrrolo[2,3-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25i）

EP

25i was synthesized with the experimental protocols described for 25a. Yellow solid (20.58% yield). 1HNMR (400MHz, METHANOL-d4) δ 8.42 - 8.78 (m, 2H), 8.33 (br. s., 1H), 7.24 (br. s.,

AC C

1H), 6.90-7.04 (m, 1H), 2.87 (br. s., 1H), 2.19 - 2.35 (m, 1H), 1.84-2.10 (m, 3H), 1.57-1.83 (m, 4H). MS (ESI) m/z calc. for C22H21FN6O2 [M+H]+:421.2; found:421.0 5.2.7.10 Synthesis of (2S,3S)-3-((5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-7-methyl-7Hpyrrolo[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25j） 25j was synthesized with the experimental protocols described for 25a. Light yellow solid (12.26% yield). 1HNMR (400 MHz, DMSO-d6) δ 12.86 (br. s., 1H), 12.24 (br. s., 1H), 8.58 (br. s., 1H), 8.24 - 8.40 (m, 1H), 7.50- 7.82 (m, 1H), 7.10 (br. s., 1H), 6.64 - 6.83 (m, 1H), 4.78 (br. s., 1H), 3.85 (br. s., 2H), 3.08 - 3.19 (m, 1H), 1.34-2.04 (m, 8H). 13C NMR (400 MHz, DMSO-d6) δ: 176.0, 157.0, 154.8, 154.6, 152.4, 152.3, 141.4, 139.6, 139.1, 139.0, 138.9, 132.3, 132.2, 116.5, 116.3, 116.1, 116.0, 113.9, 113.8, 61.9, 51.0, 47.9, 29.0, 28.8, 25.7, 24.3, 21.6, 19.4 ppm. MS (ESI) m/z calc. for 31 / 41

ACCEPTED MANUSCRIPT

C23H22F2N6O2 [M+H]+:453.2; found: 453.0 5.2.7.11 Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25k） 25k was synthesized with the experimental protocols described for 25a. Light yellow solid (33.04% yield). 1H NMR (400 MHz, METHANOL-d4) δ 8.63 (dd, J=2.76, 9.03 Hz, 1H), 8.52 (s, 1H), 8.33 (br s, 1H), 5.17 (d, J=6.78 Hz, 1H), 3.10 (t, J=7.53 Hz, 2H), 2.86-2.94 (m, 3H), 2.30 (q, J=7.34 Hz, 13

C NMR (400 MHz, DMSO-d6) δ: 176.0, 159.0, 158.7,

RI PT

2H), 2.19 (br s, 1H), 1.50-2.05 (m, 10H).

158.4, 157.6, 157.3, 154.9, 154.5, 152.2, 152.0, 151.1, 146.4, 131.9, 131.4, 118.7, 115.6, 114.3, 61.9, 51.0, 48.1, 28.8, 25.7, 24.3, 21.6, 19.5 ppm. MS (ESI) m/z calc. for C23H24FN5O2 [M+H]+:422.2; found: 422.2 of

(2S,3S)-3-((5-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)thiazolo[5,4-

SC

5.2.7.12 Synthesis

d]pyrimidin-7-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25l）

M AN U

25l was synthesized with the experimental protocols described for 25a. Yellow solid (22.23% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.71 (br s, 1H), 9.22 (s, 1H), 8.99 (br s, 1H), 8.76 (br s, 1H), 8.60 (dd, J=2.76, 9.79 Hz, 1H), 8.36 (s, 1H), 4.99 (br t, J=6.40 Hz, 1H), 3.12 (br d, J=6.53 Hz, 1H), 2.04 (br d, J=12.55 Hz, 2H), 1.21-1.91 (m, 8H).

13

C NMR (400 MHz, DMSO-d6) δ: 175.9,

157.6, 156.8, 155.7, 155.2, 151.8, 133.3, 132.8, 132.5, 129.2, 118.9, 115.6, 115.6, 111.6, 51.6, 47.5,

TE D

29.0, 28.7, 25.5, 24.4, 21.6, 19.6 ppm. MS (ESI) m/z calc. for C21H19FN6O2S [M+H]+:439.1; found: 439.1

5.2.7.13 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)thieno[3,2-d]pyrimidin4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25m）

EP

25m was synthesized with the experimental protocols described for 25a. White solid (20.42% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.89 (br s, 1H), 8.96-9.22 (m, 1H), 8.71 (d, J=2.76 Hz,

AC C

1H), 8.58 (br d, J=8.78 Hz, 1H), 8.27-8.44 (m, 2H), 7.51 (d, J=5.52 Hz, 1H), 5.05 (br s, 1H), 2.933.01 (m, 1H), 2.10 (br s, 1H), 2.02 (br s, 1H), 1.35-1.88 (m, 8H).

13

C NMR (400 MHz, DMSO-d6)

δ: 175.1, 158.4, 158.1, 157.1, 156.7, 154.7, 154.4, 145.7, 137.1, 133.0, 132.7, 119.7, 118.3, 115.3, 112.1, 108.5, 103.3, 51.7, 47.2, 45.7, 28.7, 28.1, 25.1, 23.7, 20.9, 19.0 ppm. MS (ESI) m/z calc. for C22H20FN5O2S [M+H]+:438.1; found: 438.1 5.2.7.14 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)thieno[3,4-d]pyrimidin4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25n） 25n was synthesized with the experimental protocols described for 25a. Light yellow solid (44.24 % yield).

1

H NMR (400 MHz, METHANOL-d4) δ 8.88 (d, J=3.01 Hz, 1H), 8.72 (dd,

J=2.64, 8.91 Hz, 1H), 8.66 (s, 1H), 8.36 (s, 1H), 7.66 (d, J=3.01 Hz, 1H), 5.40 (br d, J=6.78 Hz, 32 / 41

ACCEPTED MANUSCRIPT 13

1H), 3.00 (br d, J=7.03 Hz, 1H), 1.59-2.30 (m, 12H).

C NMR (400 MHz, DMSO-d6) δ: 177.5,

157.4, 157.2, 154.8, 152.9, 152.8, 146.4, 141.7, 140.8, 140.7, 139.1, 136.3, 136.2, 131.7, 131.4, 128.4, 128.3, 127.9, 127.0, 118.9, 118.8, 115.6, 115.4, 114.4, 52.5, 50.0, 29.0, 28.8, 26.2, 24.5, 22.0, 19.9 ppm. MS (ESI) m/z calc. for C22H20FN5O2S [M+H]+:438.1; found: 438.1 5.2.7.15 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)thieno[2,3-d]pyrimidin4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25o）

RI PT

25o was synthesized with the experimental protocols described for 25a. White solid (26.85% yield). 1

H NMR (400 MHz, DMSO-d6) δ 12.50 (br d, J=2.01 Hz, 1H), 8.61 (dd, J=2.76, 9.54 Hz, 1H), 8.43

(d, J=2.76 Hz, 1H), 8.33 (dd, J=1.38, 2.64 Hz, 1H), 8.29-8.37 (m, 1H), 8.08 (br d, J=6.27 Hz, 1H), 7.76 (d, J=6.02 Hz, 1H), 7.53 (d, J=6.02 Hz, 1H), 4.92 (br t, J=6.90 Hz, 1H), 2.85 (br d, J=6.78 Hz, 13

CNMR (400 MHz, DMSO-d6) δ: 175.3, 158.5, 158.1,

SC

1H), 2.01-2.11 (m, 2H), 1.33-1.94 (m, 8H).

156.9, 156.4, 154.5, 145.9, 131.9, 131.6, 131.5, 121.5, 119.8, 118.4, 118.3, 117.0, 115.3, 115.1,

M AN U

113.9, 112.3, 50.8, 47.8, 28.3, 28.2, 25.4, 23.7, 21.0, 19.0 ppm. MS (ESI) m/z calc. for C22H20FN5O2S [M+H]+:438.1; found: 438.1

5.2.7.16 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6,7-dihydrothieno[3,2d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25p） 25p was synthesized with the experimental protocols described for 25a. White solid (9.22% yield). H NMR (400 MHz, DMSO-d6) δ 12.71 (br s, 1H), 8.44-8.57 (m, 2H), 8.36 (dd, J=1.25, 2.51 Hz,

TE D

1

1H), 7.83 (br s, 1H), 4.87 (br t, J=6.90 Hz, 1H), 3.44-3.55 (m, 2H), 3.30-3.42 (m, 2H), 2.96 (br d, J=7.03 Hz, 1H), 2.04 (br s, 1H), 1.91 (br s, 1H), 1.28-1.82 (m, 8H).

13

C NMR (400 MHz, DMSO-

d6) δ: 175.3, 158.3, 158.0, 157.0, 156.1, 154.6, 145.7, 132.5, 132.2, 131.9, 118.1, 115.1, 115.0,

EP

112.2, 109.4, 51.4, 47.0, 35.0, 29.8, 28.7, 28.2, 25.1, 23.8, 21.0, 19.0 ppm. MS (ESI) m/z calc. for C22H22FN5O2S [M+H]+:440.1; found: 440.1

AC C

5.2.7.17 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,7-dihydrothieno[3,4d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25q） 25q was synthesized with the experimental protocols described for 25a. White solid (26.23% yield). 1

H NMR (400 MHz, DMSO-d6) δ 12.79 (br s, 1H), 8.59 (br d, J=2.26 Hz, 1H), 8.50 (dd, J=2.89,

9.41 Hz, 1H), 8.37 (s, 1H), 7.93-8.08 (m, 1H), 4.87-4.95 (m, 1H), 4.27 (br s, 2H), 4.07 (br s, 2H), 2.88-2.94 (m, 1H), 2.06 (br s, 1H), 1.94 (br s, 1H), 1.77 (br d, J=2.76 Hz, 8H).

13

C NMR (400

MHz, DMSO-d6) δ: 175.7, 159.2, 158.9, 158.6, 157.6, 157.3, 155.2, 146.2, 133.3, 133.1, 132.8, 118.7, 118.6, 115.7, 115.5, 111.6, 109.6, 51.8, 47.7, 46.2, 36.7, 32.7, 29.0, 28.7, 25.6, 24.3, 21.5, 19.4 ppm. MS (ESI) m/z calc. for C22H22FN5O2S [M+H]+:440.1; found: 440.4 5.2.7.18 Synthesis of (2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,6-dihydrothieno[2,333 / 41

ACCEPTED MANUSCRIPT

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25r） 25r was synthesized with the experimental protocols described for 25a. White solid (8.19% yield). 1

H NMR (400 MHz, METHANOL-d4) δ8.59 (br. s., 1 H), 8.26 (br. s., 1 H), 8.10 (s, 1 H), 4.62 (br.

s., 1 H), 3.56 (d, J=7.03 Hz, 2 H), 3.50 (d, J=7.03 Hz, 2 H), 2.60 (d, J=6.02 Hz, 1 H), 1.42 - 2.11 (m, 10 H).

13

C NMR (400 MHz, DMSO-d6) δ: 176.1, 157.1, 155.3, 155.2, 154.7, 151.9, 151.8, 145.8,

143.5, 141.7, 132.7, 132.4, 132.0, 128.2, 127.2, 118.9, 116.3, 107.9, 51.0, 48.0, 28.8, 28.7, 25.8,

5.2.7.19 Synthesis

of

RI PT

24.2, 21.6, 19.5, 15.9 ppm. MS (ESI) m/z calc. for C22H22FN5O2S [M+H]+:440.1; found: 440.0 (2S,3S)-3-((7-cyano-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)thieno[3,2-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25s）

SC

25s was synthesized with the experimental protocols described for 25a. Light yellow solid (5.56% yield). 1H NMR (400 MHz, METHANOL-d4) δ 8.73 (dd, J=9.54, 2.76 Hz, 1 H), 8.37 (s, 1 H), 8.23 (d, J=5.52 Hz, 1 H), 7.55 (d, J=5.52 Hz, 1 H), 5.64 (d, J=6.78 Hz, 1 H), 3.19 (d, J=6.53 Hz, 1 H),

M AN U

2.33 (br. s., 1 H), 2.20 (d, J=2.51 Hz, 1 H), 1.59 - 2.13 (m, 8 H). MS (ESI) m/z calc. for C23H19FN6O2S [M+H]+:463.1; found: 463.0 5.2.7.20 Synthesis

of

(2S,3S)-3-((6-(difluoromethyl)-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-

yl)thieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25t） 25t was synthesized with the experimental protocols described for 25a. White solid (1.67% yield).

5.2.7.21 Synthesis

of

TE D

MS (ESI) m/z calc. for C23H20F3N5O2S [M+H]+:488.1; found: 488.0

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-5,5-dioxido-6,7-

dihydrothieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25u）

1

EP

25u was synthesized with the experimental protocols described for 25a. White solid (20.54% yield). H NMR (400 MHz, METHANOL-d4) δ 8.69 (br d, J=9.54 Hz, 1H), 8.46 (s, 1H), 8.23 (s, 1H), 5.08

(br s, 2H), 3.60-3.65 (m, 2H), 3.37-3.43 (m, 2H), 2.78 (br d, J=4.77 Hz, 1H), 1.55-2.15 (m, 10H).

AC C

MS (ESI) m/z calc. for C22H22FN5O4S [M+H]+:472.1; found: 472.1 5.2.7.22 Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-6,6-dioxido-5,7-

dihydrothieno[3,4-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid（25v） 25v was synthesized with the experimental protocols described for 25a. White solid (7.57% yield). 1

H NMR (400 MHz, METHANOL-d4) δ 8.63 (dd, J=9.29, 2.51 Hz, 1 H), 8.41 (s, 1 H), 8.26 (s, 1

H), 5.07 (d, J=6.53 Hz, 1 H), 4.45 - 4.52 (m, 2 H), 4.36 (s, 2 H), 2.81 (d, J=7.03 Hz, 1 H), 1.54 2.07 (m, 10 H). MS (ESI) m/z calc. for C22H22FN5O4S [M+H]+:472.1; found: 472.0 5.2.8

Synthesis of compound 29a-c

5.2.8.1 Synthesis

of

(2S,3S)-ethyl 34 / 41

3-((2-chlorothieno[3,2-d]pyrimidin-4-

ACCEPTED MANUSCRIPT

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (26a)

26a was synthesized with the experimental protocols described for 23a. White solid (85.09% yield). MS (ESI) m/z calc. for C17H20ClN3O2S [M+H]+:366.1; found: 366.0 5.2.8.2 Synthesis

of

(2S,3S)-ethyl

3-((2-chlorothieno[2,3-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (26b) MS (ESI) m/z calc. for C17H20ClN3O2S [M+H]+:366.1; found: 366.0 5.2.8.3 Synthesis

of

(2S,3S)-ethyl

RI PT

26b was synthesized with the experimental protocols described for 23a. White solid (89.86% yield).

3-((2-chloro-6,7-dihydrothieno[3,2-d]pyrimidin-4-

yl)amino)bicyclo[2.2.2]octane-2-carboxylate (26c)

26c was synthesized with the experimental protocols described for 23a. White solid (56.28%

5.2.9

SC

yield). MS (ESI) m/z calc. for C17H22ClN3O2S [M+H]+:368.1; found: 368.1 Synthesis of compound 27a-c

M AN U

5.2.9.1 Synthesis of compound ethyl (2S,3S)-3-[[2-(5-fluoro-1-trityl-pyrazolo[3,4-b]pyridin-3yl)thieno[3,2-d]pyrimidin-4-yl]amino]bicyclo[2.2.2]octane-2-carboxylate(27a) To a mixture of compound 26a (450.00 mg, 1.23 mmol, 1.00 eq) and 5-fluoro-3-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1-trityl-pyrazolo[3,4-b]pyridine (1.04 g, 1.23 mmol, 1.00 eq) in H2O (2.00 mL) and 2-MeTHF (8.00 mL) was added K3PO4 (783.22 mg, 3.69 mmol, 3.00 eq) ,XPhos (117.26 mg, 245.98 umol, 0.20 eq) and Pd2(dba)3 (112.63 mg, 122.99 umol, 0.10 eq),

TE D

the mixture was degassed in vacuo and purged with N2 for three times and the mixture was stirred at 80°C for 3hr. The mixture was concentrated in vacuo. The residue was purified by Combi-Flash column (PE:EtOAc = 20:1 to 3:1) to give compound 27a (400.00 mg, 564.30 umol, 45.88% yield) as a yellow solid was obtained. MS (ESI) m/z calc. For C42H37FN6O2S [M+Na]+:731.3;

EP

found: 731.2 5.2.9.2 Synthesis

of

(2S,3S)-ethyl

3-((2-(5-fluoro-1-trityl-1H-pyrazolo[3,4-b]pyridin-3-

AC C

yl)thieno[2,3-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (27b) 27b was synthesized with the experimental protocols described for 27a. Yellow solid (58.15% yield). MS (ESI) m/z calc. for C42H37FN6O2S [M+Na]+:731.3; found: 731.0 5.2.9.3 Synthesis of

(2S,3S)-ethyl 3-((2-(5-fluoro-1-trityl-1H-pyrazolo[3,4-b]pyridin-3-yl)-6,7-

dihydrothieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (27c) 27c was synthesized with the experimental protocols described for 27a. Yellow solid (25.88% yield). MS (ESI) m/z calc. for C42H39FN6O2S [M+Na]+:733.3; found: 733.2 5.2.10 Synthesis of compound 28a-c 5.2.10.1 Synthesis

of

ethyl

(2S,3S)-3-[[2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)thieno[2,3-

d]pyrimidin-4-yl]amino]bicyclo[2.2.2]octane-2-carboxylate (28a) 35 / 41

ACCEPTED MANUSCRIPT

To a mixture of compound 27a (600.00 mg, 846.44 umol, 1.00 eq) in DCM (6.00 mL) was added TFA (965.11 mg, 8.46 mmol, 626.69 uL, 10.00 eq) and triethylsilane(984.24 mg, 8.46 mmol, 1.35 mL, 10.00 eq) , and the mixture was stirred at 25°C for 12hr. The mixture was concentrated in vacuo. The residue was purified by Combi Flash column with DCM:MeOH ( 50:1 to 20:1) to give compound 28a (240.00 mg, 514.44 umol, 60.78% yield, 100% purity) as yellow solid. MS (ESI) m/z calc. for C23H23FN6O2S [M+H]+:467.2; found: 467.2 of

(2S,3S)-ethyl

3-((2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)thieno[2,3-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate (28b)

RI PT

5.2.10.2 Synthesis

28b was synthesized with the experimental protocols described for 28a. Yellow solid (100% yield). MS (ESI) m/z calc. for C23H23FN6O2S [M+H]+:467.2; found: 467.2 of

(2S,3S)-ethyl

3-((2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)-6,7-

SC

5.2.10.3 Synthesis

dihydrothieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylate(28c) 28c was synthesized with the experimental protocols described for 28a. Yellow solid (61.01%

5.2.11 Synthesis of compound 29a-c 5.2.11.1 Synthesis

of

M AN U

yield). MS (ESI) m/z calc. for C23H25FN6O2S [M+H]+:469.2; found: 469.1

(2S,3S)-3-((2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)thieno[3,2-

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid(29a) 29a was synthesized with the experimental protocols described for 25a. 1

H NMR (400 MHz, DMSO-d6) δ 14.93 (br s, 1H), 9.34 (br s, 1H), 8.76 (s, 1H),

TE D

(93.66 % yield).

Light yellow solid

8.55 (br d, J=7.28 Hz, 1H), 8.32-8.47 (m, 1H), 7.57 (d, J=5.27 Hz, 1H), 5.09 (br s, 2H), 3.04 (br d, J=5.52 Hz, 2H), 2.10 (br s, 1H), 1.93-2.06 (m, 1H), 1.83 (br d, J=8.28 Hz, 4H), 1.44-1.73 (m, 5H). 13

C NMR (400 MHz, DMSO-d6) δ: 175.7, 157.4, 155.0, 152.7, 150.5, 140.3, 138.0, 121.1, 115.8,

EP

114.1, 52.2, 47.5, 29.2, 28.7, 25.6, 24.4, 21.5, 19.6 ppm. MS (ESI) m/z calc. for C21H19FN6O2S [M+H]+:439.1; found: 439.2 of

(2S,3S)-3-((2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)thieno[2,3-

AC C

5.2.11.2 Synthesis

d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid (29b) 29b was synthesized with the experimental protocols described for 25a. (77.58 % yield).

1

Light yellow solid

H NMR (400 MHz, DMSO-d6) δ 8.99-9.46 (m, 1H), 8.73 (br s, 1H), 8.54 (br d,

J=7.03 Hz, 1H), 8.00-8.31 (m, 1H), 7.72 (br d, J=5.52 Hz, 1H), 5.04 (br s, 1H), 3.06-3.27 (m, 1H), 2.10 (br s, 1H), 2.02 (br s, 1H), 1.29-1.94 (m, 8H).

13

C NMR (400 MHz, DMSO-d6) δ: 175.8,

157.3, 154.9, 152.7, 150.6, 140.4, 138.9, 124.8, 121.1, 116.3, 115.8, 114.1, 51.9, 47.5, 29.1, 28.7, 25.5, 24.4, 21.5, 19.6 ppm. MS (ESI) m/z calc. for C21H19FN6O2S [M+H]+:439.1; found: 439.2 5.2.11.3 Synthesis

of

(2S,3S)-3-((2-(5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-yl)-6,7-

dihydrothieno[3,2-d]pyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid (29c) 36 / 41

ACCEPTED MANUSCRIPT

29c was synthesized with the experimental protocols described for 25a. Yellow solid (36.28% yield). 1H NMR (400 MHz, DMSO-d6) δ 14.15-15.01 (m, 1H), 11.82-12.79 (m, 1H), 8.67 (br s, 1H), 8.52 (br d, J=7.78 Hz, 1H), 7.14-7.65 (m, 1H), 7.14-7.65 (m, 1H), 4.84 (br s, 1H), 3.43-3.47 (m, 2H), 3.31-3.39 (m, 3H), 2.94 (br d, J=6.27 Hz, 1H), 2.02 (br s, 1H), 1.89 (br s, 1H), 1.39-1.80 (m, 8H).

13

C NMR (400 MHz, DMSO-d6) δ: 176.0, 158.6, 158.3, 151.1, 156.5, 154.6, 150.6, 139.9,

C21H21FN6O2S [M+H]+:441.1; found: 441.1 5.3.MDCK cell protection and cytotoxicity assays

RI PT

116.4, 113.8, 51.3, 47.6, 36.3, 29.9, 29.2, 28.8, 25.6, 24.3, 21.6, 19.4 ppm. MS (ESI) m/z calc. for

MDCK cells were seeded into 384-well plates at a density of 2×103 cells per well and then cultured at 37°C and 5% CO2 overnight. The medium in each well was replaced with aliquots of

SC

VGM containing diluted compounds (final DMSO concentration of 0.5%) and influenza virus strain A/Weiss/43 (H1N1) at a concentration of 100 50% tissue culture infective doses (TCID50)/well. The

M AN U

cells were cultured at 37°C and 5% CO2 for additional 5 days. Cytotoxicity of the compounds was assessed under the same conditions, but without virus infection, in parallel. Cell viability was measured with Cell Counting Kit 8 (CCK8, Dojindo or Biolite) according to the manufacturer’s instructions using a microtiter plate reader (Molecular device). The 50% effective concentration (EC50) and 50% cytotoxicity concentration (CC50) were calculated by fitting the compound dose versus the response data by GraphPad Prism.

TE D

5.4.Microsomal stability assay

The metabolic stability profiles of test compound were assessed by monitoring the disappearance of test compounds in the presence of liver microsomes. A typical incubation mixture (100 uL total volume) for metabolic stability studies contained 1 uM test compounds, 0.5 mg/mL

EP

microsomal protein (CD-1 mouse, SD rat and human liver microsomes), 100 mM potassium phosphate buffer (pH 7.4), and NADPH generating system (1 mM). After preincubation at 37 °C for

AC C

10 min, the reactions were started by addition of NADPH and further incubated for another 0, 5, 10, 20, 30 and 60 min. For negative control, NADPH was omitted from these incubations and incubated for 60 min. The reactions were terminated by adding 300 uL of ice-cold acetonitrile containing 100 ng/mL Tolbutamide and 100 ng/mL Labetalol as internal standard, after shaked 5 min, then centrifuged at 4000 rpm/min for 20 min to obtain the supernatant. Aliquots (10 uL) were then analyzed for substrate disappearance using LC-MS/MS.

Abbreviations Influenza virus (flu), Mardin-Darby Canine Kidney (MDCK); Polymerase Acidic Protein (PA); Polymerase Basic Protein 1 (PB1); Polymerase Basic Protein 2 (PB2); Structure-Activity 37 / 41

ACCEPTED MANUSCRIPT

Relationship (SAR), Liver Microsome Stability (LMS), Clearance (CL), Pharmacokinetic (PK), Volume of Distribution (Vd), Per Os (PO), Cytochrome P450 (CYP450), Percent of Bioavailability (%F), Area Under the Curve (AUC), Protein Data Bank (PDB), Dichloromethane (DCM), NBromosuccinimide (NBS), Diisopropylethylamine (DIEA), Dimethyl formamide (DMF), Acetic Acid (AcOH), Tetrahydrofuran (THF), Trifluoroacetic Acid (TFA).

RI PT

Conflict of interest The authors declare no conflict of interest Acknowledgements

The authors thank Peng Li, Dr. Cheng Xie, Dr. Jianfeng Mou, Jianwei Wu for helpful discussions.

SC

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

M AN U

Reference and notes

[1] The pathogen and epidemiology of Influenza. http://www.who.int/mediacentre/factsheets/ fs211/en/, 2018

[2] Tonelli M., Cichero E. Fight against H1N1 Influenza A Virus: Recent Insights towards the Development of Druggable Compounds. Curr. Med. Chem. 2016, 23, 1802-1817 [3] Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T.; Sehr, P., Lewis, J.,

TE D

Ruigrok, R. W., Ortin, J., Hart, D. J., Cusack, S. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat. Struct. Mol. Biol. 15 (2008) 500−506 (DOI: 10.1038/nsmb.1421)

[4] Fechter, P., Mingay, L., Sharps, J., Chambers, A., Fodor, E., Brownlee, G.G. Two aromatic

EP

residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J. Biol. Chem. 278 (2003) 20381−20388. (DOI: 10.1074/jbc.M300130200)

AC C

[5] Tonelli M., et al. Host dihydrofolate reductase (DHFR)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus. Eur. J. Med. Chem. 2017, 135, 467-478 [6] Francesconi V. et al. Synthesis, biological evaluation and molecular modeling of novel azaspiro dihydrotriazines as influenza virus inhibitors targeting the host factor dihydrofolate reductase (DHFR). Eur. J. Med. Chem. 2018, 155, 229-243 [7] XOFLUZA (Baloxavir Marboxil) Tablets 10mg/20mg Approved for the Treatment of Influenza Types A and B in Japan. http://www.shionogi.co.jp/en/company/news/2018/pmrltj0000003nx1-att/e180223.pdf (201802-23) 38 / 41

ACCEPTED MANUSCRIPT

[8] Michael P., Clark, Mark W., Ledeboer, et al. Discovery of a Novel, First-in-Class, Orally Bioavailable Azaindole Inhibitor (VX-787) of Influenza PB2. J. Med. Chem. 57 (2014) 6668−6678 (DOI: dx.doi.org/10.1021/jm5007275) [9] Byrn R.A., Jones S.M., Bennett HB, Bral C, Clark M.P., Jacobs M.D., Kwong A.D., Ledeboer M.W., Leeman J.R., McNeil C.F., Murcko M.A., Nezami A., Perola E., Rijnbrand R., Saxena K., Tsai A.W., Zhou Y., Charifson P.S. Preclinical activity of VX-787, a first-in-class, orally

RI PT

bioavailable inhibitor of the influenza virus polymerase PB2 subunit. Antimicrob Agents Chemother. 59 (2015) 1569–1582. (DOI:10.1128/AAC.04623-14)

[10] Tang Changhua, Ren Qingyun, Yin Junjun, Yi Kai, Zhang Yingjun. WO2018/041091

[11] Liu Yongfu, Wu Jun, Zbinden Katrin Groebke, Kuhn Bernd, Tang Guozhi, Wu Guodong,

SC

Wang Lisha, Shen Hong, Shi Tianlai, Qiu Zongxin, WO2017/133670

[12] Michael J. Boyd, Upul K. Bandarage, et al. Isosteric replacements of the carboxylic acid of

M AN U

drug candidate VX-787: Effect of charge on antiviral potency and kinase activity of azaindolebased influenza PB2 inhibitors. Bioorg. Med. Chem. Lett. 25 (2015) 1990–1994 (DOI: http://dx.doi.org/10.1016/j.bmcl.2015.03.013)

[13] Luc J. Farmer, Michael P. Clark, Michael J. Boyd, Emanuele Perola, Steven M. Jones, Alice Tsai, Marc D. Jacobs, Upul K. Bandarage, Mark W. Ledeboer, Tiansheng Wang, Hongbo Deng, Brian Ledford, Wenxin Gu, John P. Duffy, Randy S. Bethiel, Dean Shannon, Randal A.

TE D

Byrn, Joshua R. Leeman, Rene Rijnbrand, Hamilton B. Bennett, Colleen O’Brien, Christine Memmott, Kwame Nti-Addae, Youssef L. Bennani, Paul S. Charifson. Discovery of Novel, Orally Bioavailable β-Aminoacid Azaindole Inhibitors of Influenza PB2. ACS Med. Chem. Lett. 8 (2017) 256-260 (DOI: 10.1021/acsmedchemlett.6b00486)

EP

[14] This research disclosed that the both the cap domain and the midlink domain in PDB 6EUY played important role in positioning the capped RNA, therefore the PB2 structure shown in

AC C

PDB 6EUY is more reliable to guide compound design. One fact is that, this research disclosed that VX-787 yielded a KD of 2.4 nM to the A/H3N2 cap-midlink double domain, compared to 24 nM reported previously for the cap-binding domain alone. Alexander Pflug, Stephanie Gaudon, Patricia Resa-Infante, Mathilde Lethier, Stefan Reich, Wiebke M. Schulze and Stephen Cusack. Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors. Nucleic Acids Research, 46 (2018) 956-971 (doi: 10.1093/nar/gkx1210) [15] Guengerich F.P. Cytochrome p450 and chemical toxicology. Chem. Res. Toxicol. 21 (2008) 70– 83 (DOI:10.1021/tx700079z) [16] Upul K. Bandarage, Michael P. Clark , Emanuele Perola, Huai Gao, Marc D. Jacobs, Alice Tsai, 39 / 41

ACCEPTED MANUSCRIPT

Jeffery Gillespie, Joseph M. Kennedy, François Maltais, Mark W. Ledeboer, Ioana Davies, Wenxin Gu, Randal A. Byrn, Kwame Nti Addae, Hamilton Bennett, Joshua R. Leeman, Steven M. Jones, Colleen O’Brien, Christine Memmott, Youssef Bennani, and Paul S. Charifson. Novel 2-Substituted 7-Azaindole and 7-Azaindazole Analogues as Potential Antiviral Agents for the Treatment of Influenza. ACS Med. Chem. Lett. 8 (2017) 261–265 (DOI: 10.1021/acsmedchemlett.6b00487)

RI PT

[17] Pryde, D.C., Dalvie, D., Hu, Q., Jones, P., Obach, R.S., Tran, T.D. Aldehyde oxidase: an enzyme of emerging importance in drug discovery. J. Med. Chem. 53 (2010) 8441-8460 (DOI: 10.1021/jm100888d)

SC

Highlights

Novel pyrimidine fused heterocycles were synthesized as influenza A PB2 inhibitor

M AN U

Target compounds were evaluated in vitro antiviral potency (H1N1) via CPE assay 25p showed excellent in vitro antiviral potency (EC50 = 0.4 nM) 29c showed excellent in vitro antiviral potency (EC50 = 0.2 nM)

29c displayed favorable PK parameters (Cl = 1.3 mL/min/kg, F = 57%)

Figure captions

TE D

Fig. 1. Structures of representative anti-influenza compounds Fig. 2. Structures of VX-787 and three series of compound

Fig. 3 a) The predicted binding mode of compound 8a on the PB2 cap domain alone (4P1U used as docking template), the upside area is open to the solvent; b) Co-crystal structure of VX-787 with

EP

cap-midlink double domain (PDB: 6EUY). Red dash lines stand for the hydrogen bonds. Fig. 4 a) The predicted binding mode of compound 25i on the PB2 cap-midlink double domain

AC C

(6EUY was used as docking template). Red dash lines stand for the hydrogen bonds. b) The interactions between compound 25i and the PB2 cap-midlink double domain were depicted in 2D diagram. Pink arrows stand for the hydrogen bonds, green lines stand for the π-π stacking, hydrophobic residues are colored green, negative residues are colored red, positive residues are colored purple, polar residues are colored blue. Fig. 5. Potent anti-influenza compound and its possible metabolite by AO Fig. 6 a) The predicted binding mode of compound 25p on the PB2 cap-midlink double domain (6EUY was used as docking template). b) The predicted binding mode of compound 29c on the PB2 cap-midlink double domain (6EUY was used as docking template).

40 / 41

ACCEPTED MANUSCRIPT Scheme captions Scheme 1. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, DMF, rt, overnight; (b) 2-[(E)-2-Ethoxyvinyl]-4,4,5,5-tetramethyl-1,3,2dioxaborolane,Pd(dppf)Cl2, K2CO3, dioxane, 80°C; (c) AcOH, 120°C; (d) NIS, DMF; (e) iPrMgCl.LiCl, TosCN, THF, -78°C; (f) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-

RI PT

tosyl-1H-pyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (g) LiOH.H2O, MeOH, H2O

Scheme 2. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2-

carboxylate, DIEA, THF, rt; (b) Fe, NH4Cl, MeOH, THF, H2O, 60°C; (c) Trimethyl orthoformate,

SC

formic acid; (d) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-pyrrolo[2,3b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (e) LiOH.H2O, MeOH, H2O

M AN U

Scheme 3. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, THF, rt; (b) BBr3, DCM, 0°C; (c) SOCl2, MeOH, 70°C; (d) 1,2-Dibromoethane, K2CO3, DMF, 80°C; (e) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1Hpyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (f) LiOH.H2O, MeOH, H2O Scheme 4. Reagents and conditions: (a) (2S,3S)-Methyl 3-aminobicyclo[2.2.2]octane-2carboxylate, DIEA, THF; (b) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-1H-

TE D

pyrrolo[2,3-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (c) LiOH.H2O, MeOH, H2O Scheme 5. Reagents and conditions: (a) 5-Fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1-trityl-1H-pyrazolo[3,4-b]pyridine, Pd2(dba)3, Xphos, K3PO4, 2-MeTHF, H2O; (b) TFA, Et3SiH,

Table captions

EP

DCM; (c) LiOH.H2O, MeOH, H2O

Table 1 Structures and biological activities of the inhibitors in the right-hand side series 1

AC C

Table 2 Structures and biological activities of the inhibitors in the left-hand side series 2 Table 3 Structures and biological activities of the inhibitors in the C-N replacement series 3 Table 4 In Vivo Mouse PK Properties

41 / 41