Discovery of (S)-2-amino-N-(5-(6-chloro-5-(3-methylphenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-yl)-3-methylbutanamide (CHMFL-PI3KD-317) as a potent and selective phosphoinositide 3-kinase delta (PI3Kδ) inhibitor

Article abstract of DOI:10.1016/j.ejmech.2018.07.036

PI3Kδ which is mainly expressed in leukocytes, plays a critical role in B-cell receptor mediated signaling pathway and has been extensively studied as a drug discovery target for B cell malignances such as AML, CLL etc. In this manuscript, we report the discovery, SAR optimization and pharmacological evaluation of a novel series of aminothiazole-pyridine containing PI3Kδ inhibitors. Among them compound 15i (CHMFL-PI3KD-317) displays an IC₅₀ of 6 nM against PI3Kδ in the ADP-Glo biochemical assays. It also exhibits over 10–1500 fold selectivity over other class I, II and III PIKK family isoforms. In addition, in the cellular context, 15i can selectively and potently inhibit PI3Kδ mediated phosphorylation of Akt T308 but not PI3Kα β γ mediated Akt phosphorylation. 15i also exhibits an excellent selectivity profile in the protein kinases including 468 kinases/mutants at the concentration of 1 μM. 15i has acceptable pharmacokinetic properties and can dose-dependently inhibit the tumor growth of AML cell line MOLM14 inoculated xenograft mouse model. The high selectivity and potency makes 15i a potential valuable addition to the current PI3Kδ armory.

Full text of DOI:10.1016/j.ejmech.2018.07.036

Accepted Manuscript
Discovery of (S)-2-amino-N-(5-(6-chloro-5-(3-methylphenylsulfonamido)pyridin-3-
yl)-4-methylthiazol-2-yl)-3-methylbutanamide (CHMFL-PI3KD-317) as a potent and
selective phosphoinositide 3-kinase delta (PI3Kδ) inhibitor
Xiaofei Liang, Feng Li, Cheng Chen, Zongru Jiang, Aoli Wang, Xiaochuan Liu, Juan
Ge, Zhenquan Hu, Kailin Yu, Wenliang Wang, Fengming Zou, Qingwang Liu, Beilei
Wang, Li Wang, Shanchun Zhang, Yuxin Wang, Qingsong Liu, Jing Liu
PII:
S0223-5234(18)30592-0
10.1016/j.ejmech.2018.07.036
EJMECH 10571
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2018
Revised Date: 5 July 2018
Accepted Date: 15 July 2018
Please cite this article as: X. Liang, F. Li, C. Chen, Z. Jiang, A. Wang, X. Liu, J. Ge, Z. Hu, K. Yu, W.
Wang, F. Zou, Q. Liu, B. Wang, L. Wang, S. Zhang, Y. Wang, Q. Liu, J. Liu, Discovery of (S)-2-amino-N-
(
(
5-(6-chloro-5-(3-methylphenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-yl)-3-methylbutanamide
CHMFL-PI3KD-317) as a potent and selective phosphoinositide 3-kinase delta (PI3Kδ) inhibitor,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.036.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery
of
(S)-2-amino-N-(5-(6-chloro-5-(3-
methylphenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-yl)-3-
methylbutanamide (CHMFL-PI3KD-317) as a Potent and Selective
Phosphoinositide 3-kinase Delta (PI3Kδ) Inhibitor
1
,3,8
1,2,8
1,2,8
1,2,8
1,3,8
Xiaofei Liang , Feng Li , Cheng Chen , Zongru Jiang , Aoli Wang , Xiaochuan
1
,3
4
1,3
1,3
1,2
1,3
Liu , Juan Ge , Zhenquan Hu , Kailin Yu , Wenliang Wang , Fengming Zou , Qingwang
4
1,2
1,2
3,5
7
1,2,3,4,6,7*
Liu , Beilei Wang , Li Wang , Shanchun Zhang , Yuxin Wang , Qingsong Liu
, Jing
1
,3,4,7*
Liu
1
. High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam
Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei,
Anhui, 230031, P. R. China
2
3
. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
. CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei, Anhui
2
30031, P. R. China
4
. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei
Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R.
China
5
6
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui 230031,
P. R. China
. Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui
2
30601, P. R. China
1

ACCEPTED MANUSCRIPT
7
8
. Precision Medicine Research Laboratory of Anhui Province, Hefei, Anhui, 230088, P. R.
China
. These authors contribute equally
AUTHOR INFORMATION
Corresponding Authors
*
E-mail address: qsliu97@hmfl.ac.cn (Q. Liu), E-mail address: jingliu@hmfl.ac.cn (J. Liu).
ABSTRACT
PI3Kδ, which is mainly expressed in leukocytes, plays a critical role in B-cell receptor mediated
signaling pathway and has been extensively studied as a drug discovery target for B cell
malignances such as AML, CLL etc. In this manuscript, we report the discovery, SAR
optimization and pharmacological evaluation of a novel series of aminothiazole-pyridine
containing PI3Kδ inhibitors. Among them compound 15i (CHMFL-PI3KD-317) displays an IC50
of 6 nM against PI3Kδ in the ADP-Glo biochemical assays. It also exhibits over 10-1500 fold
selectivity over other class I, II and III PIKK family isoforms. In addition, in the cellular context,
1
5i can selectively and potently inhibit PI3Kδ mediated phosphorylation of Akt T308 but not
PI3Kα, β, γ mediated Akt phosphorylation. 15i also exhibits an excellent selectivity profile in the
protein kinases including 468 kinases/mutants at the concentration of 1 µM. 15i has acceptable
pharmacokinetic properties and can dose-dependently inhibit the tumor growth of AML cell line
MOLM14 inoculated xenograft mouse model. The high selectivity and potency makes 15i a
potential valuable addition to the current PI3Kδ armory.
Keywords
PI3Kδ, Kinase inhibitor, Acute myeloid leukemia, Structure-activity relationship
2

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Abbreviations used
PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PI4K, phosphatidylinositol 4-kinase;
CML, chronic myeloid leukemia; AML, acute myeloid leukemia; MCL, mantel cell lymphoma;
TGI, tumor growth inhibition; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]-pyridinium 3-oxid hexafluorophosphate; DIPEA, N,N-diisopro-pylethylamine; DMF,
dimethylformamide; DCM, dichloromethane.
1
. Introduction
Phosphoinositide 3-kinase delta (PI3Kδ) is a lipid kinase, which belongs to a large family of
phosphoinositide 3-kinases. This family of kinases is classified into four classes (I, II, III and IV)
based on their sequence homology. Class I is further divided into class IA and class IB based on
1
their different regulatory subunits and upstream activators . Class IA contains three isoforms
(PI3Kα, PI3Kβ and PI3Kδ) with the respective p110 catalytic subunit (p110α, p110β and p110δ)
2
bound to the p85 regulatory subunit , which are activated by receptor tyrosine kinases (RTKs).
Class IB is activated by G protein-coupled receptors (GPCRs), which consists of PI3Kγ with
3
p110γ bound to p101 or p84 . Class I PI3K catalyzes the phosphorylation of phosphatidylinositol
4
,5-bisphosphate to produce the cellular secondary messenger phosphatidyinositol 3,4,5-
triphosphate, subsequently initiating a cascade of downstream events to modulate a variety of
4
-7
biological processes including cell growth, proliferation, survival and differention . Among
these, PI3Kα and PI3Kβ are ubiquitously expressed in all of the mammalian tissues while PI3Kδ
8
-10
and PI3Kγ are predominately expressed in leukocytes . PI3Kδ has been shown to be involved
in B-cell related malignances such as chronic lymphocytic leukemia (CLL), acute myelogenous
leukemia (AML), follicular lymphoma (FL) and small lymphocytic lymphoma (SLL). Therefore
it has been extensively studied as a therapeutic target in hematologic malignances.
3

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CAL-101 (1, Idelalisib, Figure 1) is the first selective PI3Kδ inhibitor that has been approved
by FDA for relapsed FL after two lines of therapy, relapsed CLL in combination with rituximab
1
1
and relapsed SLL after two lines of therapy . More recently, BAY 80-6946 (2, Copanlisib),
which is a pan-PI3K inhibitor with preferential activity against PI3Kα and PI3Kδ, was approved
1
2
by FDA for relapsed follicular lymphoma . Several other small molecules that inhibit the PI3Kδ
1
3
14-15
16
activity, including AMG-319 (3) , TGR-1202 (4)
, GSK-2269557 (5) and UCB5857 (6,
1
7
Seletalisib) , are currently in clinical trials for the treatment of different cancers or respiratory
conditions and psoriasis/Sjögrens syndrome. In this manuscript, we describe the discovery and
optimization of a novel aminothiazole scaffold of PI3Kδ selective inhibitors and their potential
application in the treatment of B-cell malignances.
Figure 1. Chemical structures of representative PI3Kδ inhibitors.
2
. Results and discussion
.1. Chemistry
2
4

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General procedures to synthesize compounds 11a and 12a-s were shown in scheme 1 which
1
8-19
were similar to methods described previously
. Heck reaction of aryl bromides and N-(4-
2
0-23
methylthiazol-2-yl)acetamide provided the intermediates 8a-d
, which were sulfonated to give
2
0-24
9
a-d
. The intermediates 9a-d were then hydrolyzed under acidic condition to yield 10a-e.
Intermediates 11a-t were obtained from 10a-e and R -substituted carboxylic acid under amide
2
coupling condition. The following N-Boc deprotection reaction of intermediates 11b-t provided
the final products 12a-s.
The syntheses of 15a-p were carried out in 4 steps as depicted in scheme 2. Compounds 8a and
8
e were hydrolyzed under acidic condition to provide the intermediates 13a-b, which were
coupled with R -substituted carboxylic acid to give the intermediates 14a-b. Then 14a-b were
2
sulfonated under basic condition followed by the N-Boc deprotection to afford the final products
1
5a-p.
5

ACCEPTED MANUSCRIPT
2 3 2 3
Scheme 1. Reagents and conditions: (a) aryl bromide, Pd(OAc) , t-Bu P, Cs CO , 130 °C, 14 h;
(b) sulfonyl chloride, pyridine, 50 °C, 2 h; (c) for 10a-d: 6 M HCl, EtOH, 100 °C, 6 h; for 10e: 6
M HCl, EtOH, 100 °C, 12 h; (d) carboxylic acid, HATU, DIPEA, DMF, rt, 12 h; (e) 4 M HCl in
MeOH, MeOH, rt, 2 h.
6

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O
O
N
N
N
R2
a
H N
2
NH2
b
NH
H N
NH
S
2
H N
2
S
S
R1
N
R1
N
R1
N
8
13
14
8
a: R =Cl
13a: R1=Cl
13b: R1=CH3
14a: R1=Cl, R2=CH3CHCH3CHNHBoc(S)
14b: R1=CH3, R2=CH2NHBoc
1
8e: R =CH
1
3
1
5a: R =CH , R =CH NH , R =Ph
1 3 3 2 2 4
O
S
O
R4
O
15b: R1=Cl, R3=(CH3)2CHCHNH2(S), R4=4-CF3Ph
15c: R =Cl, R =(CH ) CHCHNH (S), R =4-(CH ) CHPh
N
R3
c
NH
1
3
3 2
2
4
3 2
HN
S
15d: R1=Cl, R3=(CH3)2CHCHNH2(S), R4=4-(CH3)3CPh
5e: R =Cl, R =(CH ) CHCHNH (S), R =4-PhPh
1
1
3
3 2
2
4
R1
N
15f: R =Cl, R =(CH ) CHCHNH (S), R =2,6-difluoroPh
1
3
3 2
2
4
1
1
1
1
1
1
1
1
1
1
5g: R =Cl, R =(CH ) CHCHNH (S), R =2-CH Ph
1 3 3 2 2 4 3
15
5h: R =Cl, R =(CH ) CHCHNH (S), R =4-CH Ph
1
3
3 2
2
4
3
5i: R =Cl, R =(CH ) CHCHNH (S), R =3-CH Ph
1
3
3 2
2
4
3
5j: R =Cl, R =(CH ) CHCHNH (S), R =2-ClPh
1
3
3 2
2
4
5k: R =Cl, R =(CH ) CHCHNH (S), R =3-ClPh
1
3
3 2
2
4
5l: R =Cl, R =(CH ) CHCHNH (S), R =4-ClPh
1
3
3 2
2
4
5m: R =Cl, R =(CH ) CHCHNH (S), R =2,3-dichloroPh
1
3
3 2
2
4
5n: R =Cl, R =(CH ) CHCHNH (S), R =2,4-dichloroPh
1
3
3 2
2
4
5o: R =Cl, R =(CH ) CHCHNH (S), R =2,5-dichloroPh
1
3
3 2
2
4
5p: R =Cl, R =(CH ) CHCHNH (S), R =3,4-dichloroPh
1
3
3 2
2
4
Scheme 2. Reagents and conditions: (a) 6 M HCl, EtOH, 100 °C, 6 h; (b) carboxylic acid,
HATU, DIPEA, DMF, 0 °C to rt, 14 h; (c) i: sulfonyl chloride, pyridine, 50 °C, 6 h; ii: 4 M HCl
in MeOH, MeOH, rt, 2 h.
2
.2. Structure-activity relationship (SAR)
The aminothiazol-pyridine compound 7 (Figure 2A) has been reported as a multi-target
2
2
inhibitor against PI3Kα, PI4Kα, PI4Kβ and PIP5Kγ ，another closely structure compound was
2
5
also developed as selective phosphatidylinositol 4 kinase IIIβ for antiviral . However, we found
that it could also potently inhibit PI3Kδ, PI3Kβ and PI3Kγ (IC50 = 1 nM, 1.28 nM and 6 nM
respectively). After careful analysis of the molecular modeling results, we envisioned that 7
adopts a flat conformation in the ATP binding pocket and occupies a deeper binding pocket that
2
6-28
is potentially accessible by ATP
(Figure 2B). The aminothiazole forms two hydrogen bonds
2
9-30
with Val828 in the hinge binding region
(Figure 2B). The nitrogen of pyridine forms a
7

ACCEPTED MANUSCRIPT
hydrogen bond with the side chain of Tyr813 (Figure 2C). The sulfonamide moiety forms two
hydrogen bonds with the side chain of residues Asp911 and Ser754 in the P-loop (Figure 2C).
Alignment of PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ revealed that although the structures of these
lipid kinases are highly similar in the ATP binding pocket, there are indeed several amino acid
residues that are different among them. These residues include Val 827 in PI3Kδ (α, β, δ=Val,
γ=Ile), Ser831 (α, β, δ, γ=Ala) and Leu829 (α=Arg, β=Ser, γ=Lys) adjacent to the hinge binding
area, which provide potential opportunities to explore these regions to achieve the selectivity
(
Figure 2B). Based on these analyses, we decided to optimize three regions of the molecule
including the central pyridine ring (in blue), the sulfonamide moiety (in cyan) and the acetamide
moiety (in magenta) as shown in Figure 2A to obtain a full spectrum of the SAR with protein
enzymatic ADP-Glo assay as the primary readout.
8

ACCEPTED MANUSCRIPT
Figure 2. Illustration of the SAR exploration rationale. (A) Structure of compound 7. (B) - (D)
Docking of compound 7 into PI3Kδ kinase (PDB: 5DXU).
The acetyl group of 7 was replaced with either a propionyl group (11a) or aminoacetyl group
(12a), which all led to activity loss against PI3Kδ (Table 1). We then paid attention to the R
2
moiety. The docking results showed that the chloro substituent directed into a affinity pocket,
which was formed with the side chains of Tyr813, Ile825, Asp911 and Asp787 residues (Figure
2
D). Replacement of the Cl atom with a methoxy group (12b) retained the potency against
9

ACCEPTED MANUSCRIPT
PI3Kδ and gained an increase in selectivity over PI3Kα, PI3Kβ and PI3Kγ compared to 12a.
Further changes at R position with the hydroxyl group (12c), methyl group (15a) or hydrogen
2
atom (12d) all led to activity loss against PI3Kδ. Switching the nitrogen atom of the pyridine
ring in 12a to carbon (12e) at Y position caused loss of potency against PI3Kδ, which
reconfirmed the docking results that the pyridine ring forms a critical hydrogen bond with
2
2
Tyr813 .
a
Table 1. SAR results of R /R /Y modifications (data represents IC values in nM) .
1
2
50
Compd.
R
1
R
2
Y
N
N
N
N
N
N
N
PI3K
2.1
α
PI3K
β
PI3K
6
γ
PI3K
1
δ
7
Me
Et
Cl
Cl
1.28
6.6
1
1
1a
2a
2.3
17
6.8
CH
NH
Cl
2.3
17
28
25.8
21
2
2
2
2
2
2
2
2
2
2
1
2b
CH
CH
CH
CH
NH
NH
NH
NH
OMe
OH
Me
H
222
290
303
236
1
2c
1017
>10000
604.5
1539
8769
422.1
109.4
420.4
375.9
1
5a
>10000
1590
1
2d
10

ACCEPTED MANUSCRIPT
1
2e
CH
NH
2
2
Cl
C
1448
>10000
1207
2134
a
Average of triplet testings.
Because of the sulfonamide, Cl and pyridine moieties are critical for the binding, we then
further explored the aminoacetyl moiety (R , R ) and the phenyl group (R ) (Table 2 and Table
3
4
5
3
). Introduction of a methyl group (12f, 12g) to the R /R position of 12a enhanced both
3 4
inhibitory activity against PI3Kδ and selectivity over other PI3Ks. Unfortunately, the dimethyl
substitute (12h) at this position resulted in both activity and selectivity loss. Introduction of a
cyclopropane ring (12i) retained the potency against PI3Kδ and increased the selectivity over
PI3Kγ (>500 fold). Installment of a methyl hydroxyl group (12j) and 2-hydroxy ethyl substituent
3
(12k) group at R both resulted in activity loss against PI3Kδ. Interestingly, introduction of an
ethyl group (12l-12m) led to both good activity against PI3Kδ and selectivity over other PI3Ks.
In addition, the S-isomer configuration (12l) displayed better activity against PI3Kδ than the R-
2
3
isomer configuration (12m). Similarly, the S-isomer of isopropyl substituent (12n) also
displayed stronger potency against PI3Kδ and selectivity over other PI3Ks than the R-isomer
(12o). The racemic cyclopropyl substituent (12p) resulted in slight decrease of the activity
against PI3Kδ but better selectivity over PI3Kγ (>100 fold). The isobutane moiety (12q) retained
the potency against PI3Kδ and selectivity over PI3Ks. However, the 2-methylbutane moiety
(
12r) and larger group at this position such as benzyl group (12s) both led to activity loss against
PI3Kδ.
4 5
When R and R moieties were fixed as in 12n, para-substituted benzene derivatives at R
3
position (15b-15e) all caused activity loss to PI3Kδ (Table 3). This suggested that the
hydrophobic pocket created by Met900, Ile910 and Asp911 in PI3Kδ could only tolerate
11

ACCEPTED MANUSCRIPT
medium-size hydrophobic moiety. Introduction of a methyl or chloro substitute to the benzene
ring at the 2-position (15g, 15j), 3-position (15i, 15k) and 4-position (15h, 15l) all resulted in
similar or slightly better activities against PI3Kδ and much better selectivity over PI3Kγ
compared with 12n. Interestingly, the dichlorobenzene substitutes (15m-15o) and
difluorobenzene substitute (15f) at R retained the potency against PI3Kδ and the selectivity over
5
PI3Kγ except 15p.
a.
Table 2. SAR results of R and R modifications (data represents IC values in nM)
3
4
50
Compd.
R
3
R
4
PI3K
7.5
α
PI3K
β
PI3K
47
γ
PI3K
3
δ
1
2f
Me
H
H
Me
Me
-
19.5
27.5
23.4
50
1
2g
2h
15.6
29.5
<10
8.2
62
2.1
1
Me
31
53.9
1.3
1
2i
708
41
1
2j
H
85.6
57.2
17.4
751
1
2k
H
28
751
12

ACCEPTED MANUSCRIPT
1
2l
Et
H
H
Et
H
41.2
66
82.1
480.5
98.8
255.7
408
1.4
11.5
5.3
12m
1
2n
2o
2p
2q
62.1
328.5
52.8
33.9
75.9
78.2
120.5
1036
2628
456.7
893.1
358.3
1
H
228.8
124.2
119.3
84.2
135.2
20.5
8.6
1
1
H
H
H
H
(
rac)
1
2r
100
1
2s
148.5
156.5
a
Average of triplet testings
a.
5
Table 3. SAR results of R modifications (data represents IC50 values in nM)
Compd.
R
5
PI3K
α
PI3K
386
β
PI3K
256
γ
PI3Kδ
1
5b
17.7
87.8
13

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1
5c
59.8
129.1
1224
105.8
43
317.1
766.4
1761
62.5
602.1
1898
1591
118.1
245.5
193.7
202.7
110
25.6
100.4
291.5
6.2
1
5d
1
5e
1
5f
1
5g
243.7
155.9
284
11.5
3.2
1
5h
22.8
62.6
14.8
27.7
48.3
53.5
19.9
1
5i
6.0
1
5j
34.7
3.3
1
5k
307.5
201.5
28.4
596.3
146
10
1
5l
12.3
2
1
5m
64
1
5n
92.3
140
2
1
5o
24.7
7.3
76
1
14

ACCEPTED MANUSCRIPT
1
5p
73.2
109.3
170
15
a
Average of triplet testings
.3. Biochemical and cellular property evaluation
Since a number of compounds displayed good inhibitory activity against PI3Kδ as well as
2
selectivity over other class I PI3K isoforms, we then chose a panel of them (15g-15i) for further
selectivity testing among class I/II/III and PI4KIII family proteins (Table 4). The selective PI3Kδ
3
1
inhibitor 1 and pan-PI3K inhibitor GDC-0941 (16) were used as control compounds. The
results revealed that compounds 15g and 15h exhibited selectivity among class I PI3K isoforms
and PI4KIII except PI3Kα. Compound 15i inhibited PI3Kδ with an IC of 6 nM, meanwhile it
5
0
exhibited 10-200 fold selectivity over PI3Kα, PI3Kβ, PI3Kγ, VPS34, PI4KIIIA and PI4KIIIB.
These data suggested that overall compound 15i achieved the best selectivity.
Table 4. Activities of a panel of selected PI3Kδ inhibitors against class I/II/III PI3K and PI4KIII
a
(
data represents IC values in nM)
50
Compd. P3IK
α
P3IK
β
P3IK
5.9
δ
P I3K
67
γ
PIK3C2A PIK3C2B VPS34 PI4KIIIA PI4KIIIB
1
391.3
845.5
243.7
155.9
284
>10000
2675
>10000
1096
4417
834
>10000
130.6
>10000
114.5
68
1
5g
5h
43.0
22.8
62.6
6.5
11.5
3.23
6.0
245.5
193.7
202.7
110.5
1
1518
247.9
882.3
900.9
405
163.7
1
5i
>10000
6039
1801.7
2837
574.1
300.2
>10000
1
6
181
15.7
>10000
a
Average of triplet testings.
15

ACCEPTED MANUSCRIPT
In order to further confirm the selectivity of 15i among the class I PI3Ks, we then examined its
inhibitory efficacy in the cellular background (Figure 3). Upon anti-IgM stimulation, the PI3Kδ-
3
2
mediated Akt T308 phosphorylation in Raji cells was inhibited by 15i with an EC50 value of
4
.3 nM. However, 15i did not exhibit apparent inhibitory effect on Akt phosphorylation
controlled by PI3Kα (NIH3T3 cell, PDGFBB stimulation), PI3Kβ (NIH3T3 cell, LPA
stimulation) and PI3Kγ (RAW264.7 cell, C5a stimulation) (EC >3000 nM). These cellular data
5
0
combining with the biochemical assay results demonstrated that 15i was a highly potent and
selective PI3Kδ inhibitor.
Figure 3. Inhibitory efficacy of compound 15i against class I PI3K in cellular background.
We then moved forward to examine the selectivity of 15i profile against other protein kinases.
In the DiscoverX KINOMEscan selectivity testing platform, 15i (at 1 µM) exhibited high
selectivity among 468 kinases/mutants tested with a S score(1)=0.0 (Figure 4 and Supplemental
table 1). Only PI3Kδ was hit with a percent control number less than 1 and none of other kinases
was affected.
16

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Figure 4. Kinome-wide selectivity profiling of compound 15i with DiscoverX KINOMEscan
technology.
In order to better understand the binding mode of compound 15i, we then docked it into PI3Kδ
(PDB ID: 5DXU) (Figure 5). The results showed that the aminothiazole moiety formed two
hydrogen bonds with and Ser831 (Figure 5A). The NH group of 15i formed two hydrogen
2
bonds with the side chain of Asp832 and backbone of Ser831 (Figure 5B). The two oxygens of
the sulfonamide moiety also formed hydrogen bonds with the side chain of conserved residue
Lys779 located in the roof β-sheet and Ser754 of P-loop (Figure 5B). Furthermore, the isopropyl
moiety formed close hydrophobic interaction with the side chain of Val827 and Leu829 (Figure
5
C), which could explain the activity difference between 12n and 12r/12s. In addition, the side
chains of Met900, Ile910 and Asp911 formed a small hydrophobic pocket, which could
accommodate the tolyl group of 15i, and this could explain the reason why 15d and 15e started
to lose the activity to PI3Kδ.
17

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Figure 5. Binding mode illustration of compound 15i with PI3Kδ (PDB ID: 5DXU) in (A) and
(B) catoon mode and (B) solid phase.
Since PI3Kδ is specifically over-expressed/aberrantly activated in a variety of B-cell
malignances such as CLL and AML, we then screened compound 15i against a panel of B-cell
malignancies derived cancer cell lines using compounds 1 and 16 as positive control (Table 4).
1
5i exhibited moderate antiproliferative effects against most of the cell lines including AML and
ALL cell lines with GI50 values between 3.0-4.8 µM. The well-established highly selective
PI3Kδ inhibitor 1 didn’t exhibit apparent inhibitory against most of the cell lines. In contrast, the
pan-PI3K inhibitor 16 displayed more potent antiproliferative efficacies than 15i and 1. This
again might reflect the fact that PI3Kδ inhibitor might contribute to the antitumor efficacy
3
3
through microenvironment rather than killing the cells directly .
Table 5. The antiproliferative effects of compounds 1, 16 and 15i against a panel of B-cell
a
malignances related cancer cell lines (data represents GI values in
µ
M) .
5
0
Compd. 1
Compd. 16
Compd. 15i
Cell line
PF382
Cell type
ALL
(
GI : µM)
(GI : µM)
(GI : µM)
5
0
50
50
>10
0.14±0.01
3.5±0.8
18

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NALM-6
MV4-11
ALL
>10
>10
0.15±0.03
4.0±0.9
4.8±0.2
3.3±0.2
3.0±0.4
AML
AML
AML
0.88±0.06
0.21±0.15
MOLM-14
6.4±1.0
1.7±0.4
MOLM-13
0.048±0.012
a
Average of triplet testings.
2
.4. In vivo PK/PD evaluation
We next evaluated the pharmacokinetics profile of compound 15i in Sprague-Dawley rats
Table 5). At an oral dose of 10 mg/kg, 15i exhibited good systemic exposure (AUC0-t=1216
h·ng/mL, Cmax=2063 ng/mL), favorable oral bioavailability (F%=22.8%), acceptable half-life
T =3.28 h) and fast absorption (Tmax=0.25 h). These data suggested that 15i might be suitable
(
(
1
/2
for oral administration for the in vivo efficacy study.
Table 6. Pharmacokinetic profile of compound 15i in Sprague-Dawley Rats
IV (1 mg/kg)
549±68
PO (10 mg/kg)
AUC0-t (h·ng/mL)
AUC0-∞ (h·ng/mL)
1216±222
1257±209
2063±736
0.25±0
551±69
C
max (ng/mL)
Tmax (h)
2467±142
0.033±0
Vz (mL/kg)
Cl (mL/h/kg)
MRT (h)
6210±421
1833±225
0.33±0.03
0.38±0.04
37248±12023
8103±1317
1.18±0.41
1.72±1.00
0-t
MRT0-∞ (h)
19

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T (h)
2.36±0.20
-
3.28±1.20
1
/2
F %
22.8%±3.8%
Mean±SD, n=3.
Based on the favorable potency, selectivity and pharmacokinetics profile of compound 15i, we
finally tested its in vivo antitumor efficacy in the MOLM-14 cell-inoculated xenograft animal
model. All dosages (25, 50 and 100 mg/kg/day) of 15i were well tolerated with no mortality and
no significant body weight loss observed (Figure 6 A). After 14 days of continuous treatment,
1
5i dose-dependently inhibited the growth of the MOLM14 tumor at 25, 50 and 100 mg/kg/day
dosages (Figure 6 B-C). 15i displayed obvious antitumor efficacy with a TGI of 77% at 100
mg/kg/day dosage which was better than 1 at the same dosage. In addition, the
immunohistochemistry (IHC) stain revealed that the tumor proliferation was effectively inhibited
(
Ki-67 lane, blue arrow, Figure 6 D) and significant apoptosis was induced (TUNEL lane, blue
arrow, Figure 6 D) in the tumor.
20

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Figure 6. Efficacy of compound 15i on MOLM-14 cell-inoculated xenograft mouse model.
Female nu/nu mice bearing an established control group and MOLM-14 tumor xenografts were
treated with 15i at 25 mg/kg/day, 50 mg/kg/day, 100 mg/kg/day or vehicle. Daily oral
3
administration was initiated when MOLM-14 tumors had reached a size of 300 mm . Each group
contained 5 animals. Data = mean ± SD. (A) Body weight measurement from MOLM-14
xenograft mice after 15i administration. Initial body weight was set as 100%. (B) Tumor size
measurement from MOLM-14 xenograft mice after 15i administration. Initial tumor size was set
as 100%. (C) Comparison of the final tumor weight in each group after a 14-day treatment
period. (D) Representative micrographs of hematoxylin and eosin (HE), Ki-67 and TUNEL
staining of tumor tissues with 15i treatment compared to the vehicle group and 1.
21

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3
. Conclusions
In summary, starting from a pan-PI3K inhibitor 7, through a focused medicinal chemistry
approach guided by structure-aided drug design, we have discovered a aminothiaozle-pyridine
scaffold based compound 15i via 5 steps of chemical syntheses (24% overall yield). 15i had an
IC value of 6 nM against PI3Kδ, and it exhibited 10 to 300 fold selectivity against isoforms of
5
0
PI3Ks and PI4Ks as well as excellent selectivity over other protein kinases. 15i potently
inhibited the phosphorylation of Akt with an EC50 of 4.3 nM in anti-IgM-stimulated PI3Kδ
active Raji cell line, but displayed far less activity against PI3Kα, PI3Kβ and PI3Kγ. In addition,
1
5i exhibited better antiproliferative effects against the majority of B-cell related malignancies
that were tested (ALL and AML) compared to 1. The suitable pharmacokinetic profile and good
in vivo antitumor efficacy suggested that 15i might be a potential drug candidate for B-cell
related malignances.
4
. Experimental section
4
.1. Chemistry. All reagents and solvents were purchased from commercial sources and were
used as received unless specified otherwise, or prepared as described in the literature. All
moisture sensitive reactions were carried out using dry solvents under ultrapure argon protection.
Glassware was dried in an oven at 140 °C for at least 12 h prior to use and then assembled
quickly while hot, sealed with rubber septa, and allowed to cool under a stream of argon.
Reactions were stirred magnetically using Teflon-coated magnetic stirring bars. Commercially
available disposable syringes were used for transferring the reagents and solvents. LC/MS were
performed on an Agilent 6224 TOF using an ESI source coupled to an Agilent 1260 Infinity
HPLC system operating in reverse mode with an Agilent XDB-C18 column (4.6 × 50 mm, 1.8
µm) using a water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a flow rate at 0.4
22

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1
13
mL/min. H and C spectra were recorded with a Bruker 400 MHz NMR spectrometer and
referenced to deuterated methanol (CD OD), deuterated dimethyl sulfoxide (DMSO-d ) or
3
6
deuterated chloroform (CDCl ). Chemical shifts are expressed in ppm. In the NMR tabulation, s
3
indicates singlet; d, doublet; t, triplet; q, quartet and m, multiplet. Flash column chromatography
was conducted using silica gel (Silicycle40−64 µm). The purities of all final compounds were
determined to be above 95% by HPLC.
4
.1.1. Compounds 8a-d were prepared following the synthetic procedure of 8a-c as described
2
0-23
previously
.
1
4
.1.1.1 N-(5-(3-Amino-4-chlorophenyl)-4-methylthiazol-2-yl)acetamide (8d). Yield 75%. H
NMR (400 MHz, DMSO-d ) δ 7.21 (s, 1H), 6.91 (s, 1H), 6.61 (s, 1H), 5.48 (s, 2H), 2.33 (s, 3H),
6
1
3
2
.14 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 168.84, 155.39, 145.35, 142.22, 132.04, 129.79,
6
+
1
24.01, 117.33, 116.54, 115.44, 22.90, 16.62. LC-MS (ESI, m/z): 282.0453 [M+H] .
4
.1.2. Compounds 9a-d were prepared following the synthetic procedure of 9a-b as described
2
0-24
previously
.
4
6
7
.1.2.1. N-(4-Methyl-5-(5-(phenylsulfonamido)pyridin-3-yl)thiazol-2-yl)acetamide (9c). Yield
1
6%. H NMR (400 MHz, DMSO-d
6
) δ 12.22 (s, 1H), 10.76 (s, 1H), 8.35 (s, 1H), 8.25 (s, 1H),
1
3
.82 (s, 2H), 7.63 (s, 1H), 7.59 (s, 2H), 7.51 (s, 1H), 2.25 (s, 3H), 2.15 (s, 3H). C NMR
(
100MHz, CDCl ) δ 169.05, 156.53, 144.56, 144.40, 140.18, 139.42, 134.82, 133.80, 130.00,
3
+
1
29.12, 127.17, 126.51, 119.83, 22.89, 16.41. LC-MS (ESI, m/z): 389.0764 [M+H] .
4
.1.2.2. N-(5-(4-Chloro-3-(phenylsulfonamido)phenyl)-4-methylthiazol-2-yl)acetamide (9d).
1
Yield 64%. H NMR (400 MHz, DMSO-d ) δ 12.16 (s, 1H), 10.18 (s, 1H), 7.79 (s, 2H), 7.64 (s,
6
1
3
1
H), 7.58 (s, 2H), 7.43 (s, 1H), 7.25 (s, 2H), 2.23 (s, 3H), 2.15 (s, 3H). C NMR (100 MHz,
23

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DMSO-d
6
) δ 168.94, 155.99, 143.35, 140.70, 134.36, 133.51, 132.20, 130.85, 129.79, 127.86,
+
1
4
4
27.54, 127.13, 126.76, 122.45, 22.89, 16.43. LC-MS (ESI, m/z): 422.0392 [M+H] .
.1.3. Compounds 10b-e were prepared following the synthetic procedure of 10a.
.1.3.1. N-(5-(2-Amino-4-methylthiazol-5-yl)-2-chloropyridin-3-yl)benzenesulfonamide (10a). To
a solution of 9a (211 mg, 0.5 mmol, 1.00 eq.) in EtOH (4 mL), 6 M HCl (0.7 mL) was added at
room temperature under argon. Then it was heated to reflux for 6 h. The reaction mixture was
cooled to room temperature and filtered to afford the product 10a as yellow solid (169 mg, 89%).
1
H NMR (400 MHz, DMSO-d
6
) δ 9.75 (s, 1H), 8.32 (s, 1H), 7.78 (d, J = 7.1 Hz, 2H), 7.69 (d, J
1
3
=
6.7 Hz, 1H), 7.65 (s, 1H), 7.61 (d, J = 7.3 Hz, 2H), 2.21 (s, 3H). C NMR (100 MHz, DMSO-
d6) δ 168.27, 146.02, 145.36, 140.17, 134.71, 133.89, 131.30, 130.00, 127.28, 126.52, 111.42,
+
9
9.99, 13.11. LC-MS (ESI, m/z): 381.0225 [M+H] .
4
.1.3.2. N-(5-(2-Amino-4-methylthiazol-5-yl)-2-methoxypyridin-3-yl)benzenesulfonamide (10b).
1
Yield 85%. H NMR (400 MHz, DMSO-d ) δ 7.95 (s, 1H), 7.76 (s, 2H), 7.56 (s, 3H), 7.25 (s,
6
+
1
H), 3.67 (s, 3H), 2.12 (s, 3H). LC-MS (ESI, m/z): 377.0741 [M+H] .
4
.1.3.3. N-(5-(2-Amino-4-methylthiazol-5-yl)pyridin-3-yl)benzenesulfonamide (10c). Yield 89%.
1
6
H NMR (400 MHz, DMSO-d ) δ 8.47 (s, 2H), 7.90 (s, 1H), 7.87 (s, 3H), 7.65 (d, J = 6.7 Hz,
1
3
1
1
1
4
9
7
H), 7.61 (s, 1H), 7.59 (s, 1H), 7.58 (s, 1H), 2.21 (s, 3H). C NMR (100 MHz, DMSO-d ) δ
6
68.39, 141.13, 140.32 , 139.12, 136.50, 135.05, 133.97, 130.06, 129.34, 128.14, 127.26, 111.51,
+
3.14. LC-MS (ESI, m/z): 347.0624 [M+H] .
.1.3.4. N-(5-(2-Amino-4-methylthiazol-5-yl)-2-chlorophenyl)benzenesulfonamide (10d). Yield
1
0%. H NMR (400 MHz, DMSO-d ) δ 10.34 (s, 1H), 9.71 (s, 2H), 7.74 (s, 2H), 7.65 (s, 1H),
6
1
3
.59 (s, 2H), 7.49 (s, 1H), 7.24 (s, 1H), 7.17 (s, 1H), 2.15 (s, 3H). C NMR (100 MHz, DMSO-
24

ACCEPTED MANUSCRIPT
d
1
4
6
) δ 167.87, 140.51, 134.66, 133.62, 132.70, 131.23, 129.83, 129.41, 129.37, 127.51, 127.14,
+
26.80, 114.83, 13.07. LC-MS (ESI, m/z): 380.0298 [M+H] .
.1.4. N-(5-(2-Amino-4-methylthiazol-5-yl)-2-hydroxypyridin-3-yl)benzenesulfonamide (10e). To
a solution of 9b (211 mg, 0.5 mmol, 1.00 eq.) in EtOH (4 mL), 6 M HCl (0.7 mL) was added at
room temperature under argon. Then it was heated to reflux for 12 h. The reaction mixture was
cooled to room temperature and filtered to afford the product 10e as yellow solid (65 mg, 40%).
1
H NMR (400 MHz, DMSO-d
6
) δ 7.86 (s, 1H), 7.63 (s, 2H), 7.50 (s, 3H), 7.25 (s, 1H), 2.07 (s,
+
3
4
4
H). LC-MS (ESI, m/z): 363.0582 [M+H] .
.1.5. Compounds 11a and 11c-t were prepared following the synthetic procedure of 11b.
.1.5.1.
tert-Butyl(2-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
yl)amino)-2-oxoethyl)carbamate (11b). To a solution of 10a (40 mg, 0.1 mmol, 1.00 eq.) in
anhydrous DMF (2 mL), Boc-glycine (33 mg, 0.15 mmol, 1.50 eq.), HATU (57 mg, 1.50 eq.)
and DIPEA (0. 89 mL, 0.50 mmol, 5.0 eq.) were added at 0 °C under argon. The reaction
mixture was stirred at 0 °C for 1 h, and then it was allowed to warm to room temperature for 20
h. The resulting mixture was concentrated to dryness. The residue was diluted with water (30
mL) and then extracted with EtOAc (3×30 mL). The combined organic layers were washed with
water (50 mL), brine (50 mL) and dried over anhydrous Na SO . Evaporation of the solvent
2
4
provided the crude product, which was purified by flash chromatography (eluting with MeOH in
1
DCM 0-1.5 %) to give the title compound 11b as yellow solid (44 mg, 76%). H NMR (400
MHz, CDCl3) δ 8.19 (s, 1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.85 (s, 1H), 7.63 (t, J = 7.4 Hz, 1H),
7
.53 (t, J = 7.5 Hz, 2H), 4.14 (s, 2H), 2.38 (s, 3H), 1.48 (s, 9H). LC-MS (ESI, m/z): 538.0952
+
[
M+H] .
25

ACCEPTED MANUSCRIPT
4
.1.5.2. N-(5-(6-Chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-yl)propionamide
1
(
11a). Yield 70%. H NMR (400 MHz, CDCl ) δ 8.21 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 2.2 Hz,
3
1
H), 7.88 (s, 1H), 7.86 (d, J = 1.3 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 2.55
1
3
(
q, J = 7.5 Hz, 2H), 2.39 (s, 3H), 1.31 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, CDCl ) δ 171.69,
3
1
2
4
57.02, 144.57, 144.37, 140.57, 138.51, 133.96, 130.53, 129.55, 129.50, 129.01, 127.21, 120.05,
+
9.49, 16.11, 9.07. LC-MS (ESI, m/z): 437.0498 [M+H] .
.1.5.3.
tert-Butyl(2-((5-(6-methoxy-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-2-oxoethyl)carbamate (11c). Yield 76%. H NMR (400 MHz, DMSO-d
6
) δ 12.21 (s,
1
2
1
4
4
H), 10.10 (s, 1H), 8.01 (s, 1H), 7.81 (s, 2H), 7.73 – 7.51 (m, 4H), 3.86 (s, 2H), 3.70 (s, 3H),
1
3
.25 (s, 3H), 1.40 (s, 9H). C NMR (100 MHz, DMSO-d ) δ 169.16, 156.39, 156.01, 155.61,
6
43.31, 142.42, 140.60, 133.45, 131.98, 129.66, 127.15, 122.12, 121.20, 120.23, 78.74, 54.00,
+
3.46, 28.68, 16.12. LC-MS (ESI, m/z): 534.1476 [M+H] .
.1.5.4.
tert-Butyl(2-((5-(6-hydroxy-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-2-oxoethyl)carbamate (11d). Yield 77%. H NMR (400 MHz, DMSO-d ) δ 12.17 (s,
6
2
H), 9.80 (s, 1H), 7.92 (s, 2H), 7.78 – 7.54 (m, 3H), 7.33 (s, 1H), 7.21 (s, 2H), 3.85 (s, 2H), 2.20
1
3
(
s, 3H), 1.40 (s, 9H). C NMR (100 MHz, DMSO-d
6
) δ 169.02, 156.90, 156.36, 154.98, 142.69,
1
1
4
40.22, 133.63, 129.72, 129.12, 128.35, 127.28, 126.33, 120.24, 109.78, 78.75, 43.48, 28.65,
+
5.98. LC-MS (ESI, m/z): 520.1316 [M+H] .
.1.5.5. tert-Butyl(2-((4-methyl-5-(5-(phenylsulfonamido)pyridin-3-yl)thiazol-2-yl)amino)-2-
1
oxoethyl)carbamate (11e). Yield 76%. H NMR (400 MHz, CDCl ) δ 8.39 (s, 2H), 7.85 (d, J =
3
1
3
6
.7 Hz, 2H), 7.54 (d, J = 6.1 Hz, 1H), 7.46 (s, 3H), 4.19 (s, 2H), 2.24 (s, 3H), 1.47 (s, 9H). C
NMR (100 MHz, CDCl ) δ 168.72, 156.44, 156.20, 145.17, 143.96, 140.58, 138.90, 134.03,
3
26

ACCEPTED MANUSCRIPT
1
5
4
33.40, 129.35, 128.94, 128.07, 127.15, 121.43, 81.22, 50.75, 28.32, 15.97. LC-MS (ESI, m/z):
+
04.1366 [M+H] .
.1.5.6. tert-Butyl(2-((5-(4-chloro-3-(phenylsulfonamido)phenyl)-4-methylthiazol-2-yl)amino)-2-
1
oxoethyl)carbamate (11f). Yield 81%. H NMR (400 MHz, DMSO-d ) δ 12.24 (s, 1H), 10.21 (s,
6
1
1
9
1
H), 7.79 (d, J = 7.5 Hz, 2H), 7.69 – 7.63 (m, 1H), 7.59 (t, J = 7.1 Hz, 2H), 7.47 (d, J = 8.1 Hz,
H), 7.31 – 7.24 (m, 2H), 7.21 (t, J = 5.4 Hz, 1H), 3.87 (d, J = 5.3 Hz, 2H), 2.24 (s, 3H), 1.40 (s,
1
3
3
H). C NMR (100 MHz, CDCl ) δ 169.17, 162.76, 156.36, 143.49, 140.72, 134.43, 133.51,
32.07, 130.82, 129.72, 127.94, 127.47, 127.13, 126.77, 122.64, 78.72, 38.69, 28.64, 16.42. LC-
+
MS (ESI, m/z): 537.1043 [M+H] .
4
.1.5.7.
tert-Butyl(R)-(1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-1-oxopropan-2-yl)carbamate (11g). Yield 74%. H NMR (400 MHz, CD OD) δ 8.17
3
(s, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.83 (s, 1H), 7.82 (s, 1H), 7.65 – 7.59 (m, 1H), 7.52 (t, J = 7.7
1
3
Hz, 2H), 4.37 (d, J = 6.4 Hz, 1H), 2.38 (s, 3H), 1.45 (d, J = 5.4 Hz, 9H), 1.43 (s, 3H). C NMR
(
100 MHz, CD OD) δ 172.32, 169.59, 156.83, 144.80, 144.52, 142.44, 139.59, 133.32, 132.84,
3
1
5
4
31.32, 129.17, 128.91, 126.91, 119.80, 80.00, 50.14, 27.58, 17.25, 15.43. LC-MS (ESI, m/z):
+
52.1161 [M+H] .
.1.5.8.
tert-butyl(S)-(1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-1-oxopropan-2-yl)carbamate (11h). Yield 75%. H NMR (400 MHz, DMSO-d ) δ
6
1
1
1
7
2.34 (s, 1H), 10.54 (s, 1H), 8.32 (s, 1H), 7.80 (s, 2H), 7.72 – 7.52 (m, 4H), 7.28 (s, 1H), 4.25 (s,
1
3
H), 2.28 (s, 3H), 1.36 (s, 9H), 1.28 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 172.90, 156.76,
6
55.67, 145.60, 145.21, 144.03, 140.36, 134.22, 133.76, 131.24, 129.93, 128.87, 127.12, 118.89,
+
8.70, 50.21, 28.63, 17.96, 16.32. LC-MS (ESI, m/z): 552.1154 [M+H] .
27

ACCEPTED MANUSCRIPT
4
.1.5.9.
tert-Butyl(1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-2-methyl-1-oxopropan-2-yl)carbamate (11i). Yield 78%. H NMR (400 MHz, CDCl )
3
δ 8.18 (s, 1H), 8.02 (s, 1H), 7.86 (d, J = 7.9 Hz, 2H), 7.62 (t, J = 7.0 Hz, 1H), 7.53 (d, J = 7.2 Hz,
1
3
2
1
2
4
H), 2.37 (s, 3H), 1.60 (s, 6H), 1.44 (s, 9H). C NMR (100 MHz, CDCl ) δ 156.90, 154.92,
3
44.82, 144.34, 138.72, 133.83, 130.82, 129.50, 129.32, 129.15, 127.22, 120.05, 57.11, 29.71,
+
8.27, 16.05. LC-MS (ESI, m/z): 566.1286 [M+H] .
.1.5.10.
tert-Butyl(1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)carbamoyl)cyclopropyl)carbamate (11j).Yield 81%. H NMR (400 MHz, CDCl
3
) δ 8.38 (s,
1
2
1
1
4
H), 8.16 (s, 1H), 8.03 (d, J = 7.4 Hz, 2H), 7.83 (d, J = 7.1 Hz, 1H), 7.74 (t, J = 7.2 Hz, 2H),
1
3
.58 (s, 3H), 1.69 (s, 9H), 1.47 (s, 4H). C NMR (100 MHz, CDCl ) δ 172.96, 158.11, 145.45,
3
43.54, 140.51, 134.30, 133.82, 132.25, 130.12, 129.79, 127.80, 120.50, 61.13, 30.17, 28.56,
+
6.12, 14.30. LC-MS (ESI, m/z): 564.1153 [M+H] .
.1.5.11. tert-Butyl(S)-(1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-
1
yl)amino)-3-hydroxy-1-oxopropan-2-yl)carbamate (11k). Yield 76%. H NMR (400 MHz,
CD
3
OD) δ 8.74 (d, J = 3.9 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 7.96 (d, J = 1.9 Hz,
1
2
1
7
4
2
H), 7.83 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.2 Hz, 1H), 7.56 (s, 1H), 4.44 (s, 1H), 3.98 – 3.82 (m,
1
3
H), 2.37 (s, 3H), 1.46 (d, J = 13.2 Hz, 9H). C NMR (100 MHz, CD OD) δ 169.97, 156.82,
3
50.81, 144.56, 142.51, 139.86, 135.15, 133.27, 131.21, 129.13, 128.51, 126.84, 120.72, 119.62,
+
9.76, 61.69, 56.59, 27.29, 14.79. LC-MS (ESI, m/z): 568.1105 [M+H] .
.1.5.12. tert-Butyl((2S,3R)-1-((5-(6-chloro-5-(phenylsulfonamido)pyridin-3-yl)-4-methylthiazol-
1
-yl)amino)-3-hydroxy-1-oxobutan-2-yl)carbamate (11l). Yield 77%. H NMR (400 MHz,
CDCl
3
) δ 8.05 (d, J = 1.6 Hz, 1H), 7.93 (s, 1H), 7.85 (t, J = 6.5 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H),
7
.52 (t, J = 7.7 Hz, 2H), 4.69 – 4.59 (m, 1H), 4.46 (d, J = 5.9 Hz, 1H), 2.35 (s, 3H), 1.52 (s, 9H),
28