Design, synthesis and biological evaluation of WZ4002 analogues as EGFR inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.09.048

A series of thirty two anilinopyrimidines derived from WZ4002 has been synthesized and evaluated for percentage inhibition of six different EGFR kinases using LanthaScreen binding assay method (EGFR d746 – 750) or Z'LYTE assay method (EGFR-WT, EGFR d746 – 750, EGFR T790M, EGFR T790M L858R, EGFR C797S and EGFR T790M L858R C797S). Ortho-hydroxyacetamide 10 exhibited complete inhibition of all the six kinases at 10 μM. Against the triple mutant, EGFR T790M C797S L858R, compounds 9–12 exhibited complete inhibition at 10 μM and nearly complete inhibition at 1 μM. The target compounds were also evaluated using the MTT assay to determine their cytotoxic activity against human non-small cell lung cancer cells (PC9, PC9GR and H460) and mouse leukemic cells (Ba/F3 WT and Ba/F3T 3151). Overall, 7, 9–12, 30 and 31 were found to be the most potent compounds across all five cell lines.

Full text of DOI:10.1016/j.bmcl.2017.09.048

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of WZ4002 analogues as
EGFR inhibitors
⇑
Aireen A. Romu, Zining Lei, Bin Zhou, Zhe-Sheng Chen, Vijaya Korlipara
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY 11439 United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thirty two anilinopyrimidines derived from WZ4002 has been synthesized and evaluated for
percentage inhibition of six different EGFR kinases using LanthaScreen binding assay method (EGFR d746
– 750) or Z’LYTE assay method (EGFR-WT, EGFR d746 – 750, EGFR T790M, EGFR T790M L858R, EGFR
C797S and EGFR T790M L858R C797S). Ortho-hydroxyacetamide 10 exhibited complete inhibition of
all the six kinases at 10 mM. Against the triple mutant, EGFR T790M C797S L858R, compounds 9–12
exhibited complete inhibition at 10 mM and nearly complete inhibition at 1 mM. The target compounds
were also evaluated using the MTT assay to determine their cytotoxic activity against human non-small
cell lung cancer cells (PC9, PC9GR and H460) and mouse leukemic cells (Ba/F3 WT and Ba/F3T 3151).
Overall, 7, 9–12, 30 and 31 were found to be the most potent compounds across all five cell lines.
Ó 2017 Elsevier Ltd. All rights reserved.
Received 25 July 2017
Revised 20 September 2017
Accepted 25 September 2017
Available online xxxx
Keywords:
EGFR
WZ4002
Anilinopyrimidine
NSCLC
TKIs
EGFR triple mutant
Epidermal growth factor receptor (EGFR) is a transmembrane
glycoprotein of 170 kDa that belongs to the ErbB/HER family of
RTKs which plays a critical role in vital cellular processes, including
proliferation, differentiation, migration, survival, and angiogene-
sis.1,2 Dysregulation of EGFR (overexpression or mutations) can
lead to various types of cancers, including head and neck, ovarian,
breast and lung, where it promotes and induces angiogenic growth
and survival of malignant cells.3,4 Presence of EGFR mutations
which include deletion in exon 19 or L858R substitution in exon
21 has been identified in NSCLC patients.5,6 NSCLC constitutes
about 85% of all lung cancer. Approximately 10–15% of patients
with NSCLC in the United States and 30–35% in East Asia have
tumor associated EGFR mutations.7,8
Three generations of the EGFR-TKIs have been developed for the
treatment of NSCLC thus far. Currently, the first-generation EGFR-
TKIs gefitinib and erlotinib, second-generation EGFR-TKI afatinib
and third-generation EGFR-TKI osimeritinib have been approved
by FDA for the treatment of metastatic NSCLC (Fig. 1).8,9 The first
generation of EGFR-TKIs are ATP-competitive and reversible inhibi-
tors designed to treat patients harboring deletion of exon 19 (del
E746_A750) and the L858R point mutation in exon 21 in the EGFR
domain.7,10 Due to the development of the secondary mutation
(T790M) in the catalytic domain of EGFR exon 20, resistance to the
first generation inhibitors is acquired after repeated treatments.11,12
The second generation inhibitor, afatinib, is an irreversible EGFR
inhibitor containing a quinazoline scaffold similar to the first gener-
ation along with an acrylamide warhead that can undergo a Michael
addition reaction with Cys797 at the active site to overcome the
T790M mutation related resistance.13–15 However, the major draw-
back for afatinib is dose-limiting toxicity.16 The third generation
inhibitors are anilinopyrimidines related to WZ4002 which exhibits
30–100 times greater potency than anilinoquinazoline derivatives
against EGFR T790M, but is 100-fold less effective in inhibiting
phosphorylation of WT EGFR.17
Osimeritinib (AZD9291, TAGRISSOTM) is the first third-genera-
tion EGFR inhibitor approved by FDA for the treatment of NSCLC
in November 2015. It is an irreversible EGFR inhibitor of both EGFR
mutation sensitizing and T790M resistant mutants with selectivity
over the wild type EGFR.18,19 However, as with the first generation
EGFR inhibitors, clinical use of the third generation EGFR inhibitors
also induced resistance and led to disease progression.20,21
The goal of this project was to introduce electrophilic and non-
electrophilic groups with varied hydrophilic and lipophilic charac-
teristics to the anilinopyrimidine scaffold to further explore the
SAR of WZ4002. The substituents were selected to explore the
binding site of EGFR providing a class of new EGFR-TKIs which
can overcome the resistance of both T790M and C797S mutations.
A series of WZ4002 analogues has been synthesized and their EGFR
kinase inhibitory activity against six EGFR kinases, and antiprolif-
eration against several cell lines has been evaluated.
⇑
Corresponding author.
E-mail address: korlipav@stjohns.edu (V. Korlipara).
https://doi.org/10.1016/j.bmcl.2017.09.048
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Romu A.A., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.09.048

2
A.A. Romu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. FDA approved reversible and irreversible EGFR TKIs for the treatment of
NSCLC.
Scheme 2. Synthesis of acetamide, isothiocyanate, azido and acrylamide analogues.
(a) Acetic anhydride, Et3N, DCM, rt, under N2, 2 h, 28–30%; (b) Di-2-pyridyl
thionocarbonate, DCM, rt, 2 h, 23–26%; (c) NaNO2, NaN3, HCl/H2O, 0 °C to rt, 2 h,
20–24%; (d) Acryloyl chloride, DIEA, DCM, 0 °C, 2 h, 31–36%.
Scheme 1. Synthesis of nitro and amino analogues. Reagents and conditions: (a)
K2CO3, DMF, 60 °C, 2 h, 78–83%; (b) TFA, 2-BuOH, reflux, 4 h, 72–79%; (c) Iron
powder, NH4Cl, THF/H2O (1/1, v/v), reflux, 4 h, 73–77%.
Acetylation of the amines using acetic anhydride led to the forma-
tion of the acetamide analogues 5, 15 and 26 (Scheme 2).22 The
amino analogues were reacted with di-2-pyridyl thionocarbonate
in dichloromethane to obtain the corresponding isothiocyanates
6, 16 and 27 (Scheme 2).23 Azide derivatives 7, 17 and 28 were
obtained by diazotization of the corresponding amines followed
by the reaction with sodium azide (Scheme 2).23 Acylation of
amino analogues by acryloyl chloride in presence of N,N-diiso-
propylethylamine led to the formation of the acrylamide ana-
logues, 1, 2 and 23 (Scheme 2).18
Preparation of 8, 18 and 29 required three steps (Scheme 3).
Nucleophilic substitution of 4-bromomethylbut-2-enoate with
dimethylamine hydrochloride followed by base catalyzed hydroly-
sis of the ester 38 resulted in the formation of the corresponding
carboxylic acid hydrochloride 39. The carboxylic acid hydrochlo-
ride 39 was then treated with oxalyl chloride to produce the corre-
sponding acid chloride which then reacted with the amines to
Compounds 1–33 were synthesized as illustrated in Schemes 1–
4. The nitro (3, 13, 24) and amino (4, 14, 25) analogues were syn-
thesized as shown in Scheme 1. Reacting ortho, meta or para nitro-
phenol with 2,4,5-trichloropyrimidine led to the formation of
pyrimidine derivatives 34–36. N-alkylation at the C2 position on
the pyrimidine ring of 34–36 with 4-(4-methylpiperazin-1-yl) ani-
line 37 was accomplished in the presence of trifluoroacetic acid
under reflux conditions to yield the nitro analogues 3, 13 and 24.
Compound 37 was obtained by reacting commercially available
5-fluoro-2-nitroanisole with N-methylpiperazine in DMF followed
by reduction using 10% Pd/C in presence of hydrogen. The nitro
derivatives 3, 13 and 24 were reduced using Fe/NH4Cl to obtain
the corresponding amino analogues 4, 14 and 25.17
The amino derivatives 4, 14 and 25 served as the key interme-
diates for the synthesis of the remaining target compounds.
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A.A. Romu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
EGFR C797S and EGFR T790M L858R C797S protein tyrosine
kinases by Z’-LYTE assay method. The compounds were tested
against these six kinases at 1 mM and 10 mM concentrations to
determine the percent inhibition. WZ4002 1 served as the refer-
ence compound. Summary of kinase inhibition results is presented
in Table 1.
At both 1 mM and 10 mM concentrations, WZ4002 1 produced
complete inhibition of EGFR-WT, EGFR d746 – 750, EGFR T790M
and EGFR T790M L858R, 77% inhibition of EGFR T790M C797S
L858R kinase, and no inhibition of EGFR C797S at 10 mM. Changing
the position of the acrylamide group from meta (1) to the ortho (2)
lowered the percentage inhibition at both concentrations (66% at
1 mM and 82% at 10 mM) against EGFR-WT kinase. The percent inhi-
bition of compound 2 also decreased against EGFR d746 – 750,
EGFR T790M and EGFR T790M L858R kinases at 1 mM (75–90%),
however, at 10 mM 2 produced complete inhibition of these three
kinases.
Replacement of the acrylamide chain at the ortho position with
amino (4), acetamide (5), isothiocyanate (6) and azido (7) group
also produced complete inhibition at 10 mM concentration against
EGFR d746 – 750, EGFR T790M and EGFR T790M L858R kinases.
However, inhibition against EGFR-WT kinase for these compounds
reduced to 55–87% at 10 mM.
Scheme 3. Synthesis of dimethylaminopropanamide analogues. (a) Dimethylamine
hydrochloride, HCl, TEA, THF, rt, overnight, 43%; (b) NaOH, MeOH, rt, 4 h, 56%; (c)
Oxalyl chloride, DMF, THF, 0 °C, under N2; (d) 0 °C to rt, overnight, under N2, 20–
21%.
Similar to WZ4002 1, the ortho series of benzyloxy and hydroxy
analogues 9–12 produced complete inhibition of EGFR d746 – 750,
EGFR T790M and EGFR T790M L858R kinases at both 1 mM and
10 mM concentrations and complete inhibition of EGFR-WT at
10 mM. The corresponding meta substituted analogues 19–22
exhibited good inhibition of these four kinases ranging from 70
to 82% at 1 mM and 75 to 95% at 10 mM. Corresponding para substi-
tuted benzyloxy and hydroxy analogues 30–33 exhibited good
inhibition of EGFR d746 – 750, EGFR T790M and EGFR T790M
L858R kinases with percent inhibitions ranging from 79 to 91% at
10 mM.
Against EGFR C797S kinase, the target compounds were, in gen-
eral, more active than the lead compound 1 which did not exhibit
any inhibition of this kinase at 1 mM and 10 mM concentrations. It is
noteworthy that compounds 4, 9–12 and 20–22 exhibited inhibi-
tions ranging from 64 to 91% at 10 mM concentration. The target
compounds were also more active against EGFR T790M C797S
L858R kinase than the lead (<40% at 1 mM and 77% at 10 mM con-
centrations). In particular compounds 4 and 9–12 produced com-
plete inhibition of this triple mutant at 10 mM concentration and
83–94% inhibition at 1 mM concentration. Compounds 2, 3, 5–7,
15 and 19–22 produced good inhibition with percentage inhibition
ranging from 71 to 90% at 10 mM concentration. For the para sub-
stituted benzyloxy and hydroxy analogues 30–33 the percent inhi-
bition ranged from 57 to 76% at 10 mM concentration. Ortho-
hydroxyacetamide derivative was found to produce complete inhi-
bition of all six kinases at 10 mM concentration.
Scheme 4. Synthesis of benzyloxy and hydroxy analogues. (a) TEA, acetone, 0 °C to
rt, overnight, 37–40%; (b) H2, Pd/C, anhydrous EtOH, rt, overnight, 38–45%.
The target compounds were evaluated for their antiproliferative
activity against PC9, PC9-GR, H460, Ba/F3 WT and Ba/F3-T3151 cell
lines using the MTT assay. PC9 cells (EGFR E746_A750) were used
to model an activated allele of EGFR while PC9 growth-resistant GR
cells (EGFR E746_A750/T790M) were used to model the T790M
resistance allele. H460 is a human non-small cell lung cancer cell
line, whereas Ba/F3 WT and Ba/F3 T315I are mouse leukemic cell
lines. The IC50 values obtained are presented in Table 2.
All the target compounds in the ortho series (2–12) exhibited
higher IC50 values than the lead compound WZ4002 1 (0.11 mM)
and gefitinib (0.012 mM) against the PC9 cell line. The azido
(0.23 mM) and the hydroxyacetamide (0.20 mM) analogues 7 and
10 were closer in activity to that of WZ4002 in PC9 cell line. The
ortho analogues exhibited similar or better activity against PC9-
GR in comparison to 1 (8.60 mM) and the positive control gefitinib
(7.68 mM). The azido 7, the benzyloxyacetamide 9 and hydroxyac-
obtain the corresponding dimethylaminobutenamide 8, 18 and
29.24,25
2-Hydroxyacetamides 10, 20 and 31 and 3-hydrox-
ypropanamides 12, 22 and 33 were synthesized from the
corresponding benzyloxyacetamide analogues (9, 19 and 30) and
benzyloxypropanamide analogues (11, 21 and 32) as shown in
Scheme 4. The amino analogues 4, 14 and 25 were treated with com-
mercially available acid chlorides and triethylamine in anhydrous
acetone to obtain the desired benzyloxyamide analogues. Debenzy-
lation using 10% Pd/C in methanol with hydrogen pressure led to the
formation of the corresponding hydroxy analogues 10, 20, 31, 12, 22
and 33.26,27
Compounds 1–12, 15, 19–22 and 30–33 were tested against
EGFR (ErbB1) d746 – 750 kinase by LanthaScreen binding assay
method, and against EGFR-WT, EGFR T790M, EGFR T790M L858R,
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4
A.A. Romu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1
Percent inhibition of compounds against various kinases at 1 mM and 10 mM concentrations.
Comp#
R
% Inhibition
Conc. (mM) EGFR-
EGFR d746-750 EGFR T790M EGFR T790M L858R EGFR C797S EGFR T790M C797S L858R
WT
1 WZ4002 30-NHCOCH@CH2
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
10
1
94
112
66
104
100
75
96
31
63
84
96
67
94
47
94
73
98
67
79
97
99
103
82
99
63
80
96
99
88
92
84
98
72
97
70
72
96
101
91
99
95
99
96
99
80
83
71
85
70
88
71
77
78
79
<40
81
<40
81
<40
79
<40
81
99
107
90
98
66
82
94
98
88
95
87
96
84
98
69
71
96
100
93
98
95
97
98
99
81
84
74
91
73
95
71
81
82
83
54
91
53
93
53
82
49
85
<0
<0
<0
<40
77
65
88
<40
71
85
97
58
88
64
90
45
88
<40
55
94
96
90
99
83
96
89
100
65
81
60
74
<40
83
78
78
68
80
2
20-NHCOCH@CH2
20-NO2
82
<40
<40
<40
<40
70
<40
<40
<40
<40
<40
<40
<40
<40
<40
70
3
<40
<40
59
87
70
73
<40
83
<40
55
61
64
63
98
79
94
82
98
83
100
70
70
71
75
77
80
70
76
4
20-NH2
5
20-NHCOCH3
20-NCS
6
7
20-N3
8
20-
NHCOCH@CHCH2NMe2
9
20-NHCOCH2OBn
106
98
99
10
11
12
15
19
20
21
22
30
31
32
33
20-NHCOCH2OH
68
91
<40
74
50
20-NHCO(CH2)2Bn
20- NHCO(CH2)2OH
30- NHCOCH3
93
104
100
106
71
83
72
90
78
92
70
80
78
81
<40
91
<40
91
<40
80
<40
80
83
<40
<40
<40
57
<40
68
<40
64
<40
71
<40
<40
<40
<40
<40
<40
<40
57
30- NHCOCH2OBn
30- NHCOCH2OH
30- NHCO(CH2)2 OBn
30- NHCO(CH2)2 OH
40- NHCOCH2OBn
40- NHCOCH2OH
40- NHCO(CH2)2 OBn
40- NHCO(CH2)2 OH
10
1
10
1
10
1
10
1
74
80
<40
<40
<40
<40
<40
<40
<40
<40
<40
62
<40
76
<40
57
<40
63
10
1
10
etamide 10 were found to be the most potent with IC50 values
<0.6 mM against PC9-GR. All ortho analogues except for the nitro
3, acetamide 5 and isothiocyanate 6 exhibited better activity than
both WZ4002 (18.2 mM) and gefitinib (22.8 mM) against H460 cell
line. Compounds 9–12 exhibited 6–10-fold greater activity than
WZ4002 and gefitinib in H460 cell line. In the mouse leukemia cell
lines, imatinib with IC50 value of 0.73 mM against Baf3-WT and
20.03 mM against Baf3-T3151 was used as a positive control. The
ortho series of compounds exhibited higher IC50 values than ima-
tinib against Baf3-WT, however, these compounds exhibited better
activity compared to WZ4002 (9.69 mM). In comparison to both
WZ4002 and imatinib, all ortho analogues have lower IC50 values
ranging from 1.33 to 11.57 mM against Baf3-T3151. The azido ana-
logue 7 was found to be the most potent in this cell line with an
IC50 value of 1.33 mM.
Similar trend was observed with the meta and the para series
against all the cell lines. In both series, the benzyloxy and hydroxy
analogues (19–22 and 30–32) were found to be the most potent
against PC9 with IC50 values in the range of 1.06–2.7 mM. Against
PC9-GR also, the benzyloxyacetamide and hydroxyacetamide ana-
logues (19–22 and 30–32) were most active with IC50 values in the
range of 0.94–2.08 mM. Compounds 14 (3-amino), meta hydroxy
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A.A. Romu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Table 2
Cytotoxicity of WZ4002 analogues using the MTT assay.
Comp#
R
aIC50
PC9
(lM) SD
PC9-GR
H460
Baf3-WT
Baf3-T315I
1
2
3
4
5
6
7
8
30-NHCOCH@CH2
20-NHCOCH@CH2
20-NO2
0.11 0.01
8.42 0.83
7.59 0.86
1.80 0.13
4.21 0.49
1.82 0.18
0.23 0.03
6.07 0.51
0.39 0.05
0.20 0.03
1.98 0.25
0.65 0.05
8.87 0.83
6.49 0.77
3.27 0.47
5.61 0.62
7.43 0.78
5.01 0.49
2.55 0.37
1.50 0.17
1.41 0.13
1.59 0.13
2.82 0.18
9.36 1.02
7.37 0.88
3.08 0.48
9.04 1.02
9.76 0.97
8.07 0.95
2.70 0.31
1.06 0.16
1.09 0.19
6.12 0.65
0.012 0.003
–
8.60 0.98
9.04 0.82
9.35 1.12
1.16 0.13
5.64 0.65
3.07 0.44
0.52 0.07
7.80 0.85
0.26 0.04
0.10 0.02
2.14 0.36
1.01 0.23
5.70 0.66
3.15 0.35
6.10 0.75
4.78 0.51
6.22 0.72
3.80 0.27
1.07 0.15
1.23 0.11
2.08 0.23
1.18 0.18
6.50 0.74
9.97 1.08
7.39 0.70
3.61 0.45
21.00 2.52
15.13 1.70
5.80 0.49
0.94 0.14
0.86 0.07
1.67 0.30
3.25 0.40
7.68 0.78
–
18.21 1.59
6.98 0.61
19.63 1.45
9.78 1.05
33.21 3.06
20.12 1.98
6.34 0.58
12.02 1.31
3.53 0.40
2.01 0.28
3.06 0.39
2.15 0.27
23.57 2.53
6.68 0.58
17.41 1.69
22.33 2.31
6.02 0.71
10.02 1.11
9.50 1.09
15.83 1.73
17.93 1.62
18.30 1.69
2.48 0.25
12.03 1.44
14.01 1.35
8.55 1.10
15.27 1.41
26.87 2.24
5.02 0.66
4.79 0.39
3.87 0.41
16.97 1.68
23.31 2.57
22.80 1.96
–
9.69 1.19
2.92 0.35
6.81 0.59
6.73 0.73
8.49 0.88
5.56 0.47
1.45 0.16
9.10 1.01
1.37 0.22
2.07 0.19
3.12 0.45
2.12 0.36
6.80 0.71
1.90 0.37
2.91 0.34
8.41 0.92
3.87 0.50
7.02 0.81
2.44 0.18
1.48 0.14
2.47 0.31
2.07 0.21
6.39 0.59
3.48 0.39
2.81 0.38
2.08 0.30
7.50 0.77
13.06 1.75
4.72 0.62
2.11 0.31
6.68 0.73
1.43 0.21
3.25 0.47
–
16.28 1.85
2.37 0.19
11.57 1.30
5.74 0.50
4.73 0.44
8.99 0.99
1.33 0.25
8.12 0.91
3.50 0.41
4.18 0.56
3.06 0.29
3.86 0.33
9.15 1.20
1.96 0.23
5.56 0.66
4.99 0.44
4.67 0.61
6.02 0.76
2.87 0.20
1.12 0.15
2.49 0.37
1.39 0.17
7.70 0.73
6.89 0.72
5.47 0.63
2.89 0.28
9.53 1.16
15.13 1.60
3.41 0.45
3.62 0.33
2.74 0.21
1.50 0.14
3.35 0.45
–
20-NH2
20-NHCOCH3
20-NCS
20-N3
20-NHCOCH@CHCH2NMe2
20-NHCOCH2OBn
20-NHCOCH2OH
20-NHCO(CH2)2OBn
20-NHCO(CH2)2OH
30-NO2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Gefitinib
Imatinib
30-NH2
30-NHCOCH3
30-NCS
30-N3
30-NHCOCH@CHCH2NMe2
30-NHCOCH2OBn
30-NHCOCH2OH
30-NHCO(CH2)2OBn
30-NHCO(CH2)2OH
40-NHCOCH@CH2
40-NO2
40-NH2
40-NHCOCH3
40-NCS
40-N3
40-NHCOCH@CHCH2NMe2
40-NHCOCH2OBn
40-NHCOCH2OH
40-NHCO(CH2)2OBn
40-NHCO(CH2)2OH
0.73 0.12
20.03 2.06
a
IC50 values are represented as mean
SD of three independent experiments.
and benzyloxy analogues 19–21, 26 (4-acetamide), 30 (4-benzy-
loxyacetamide) and 32 (4-benzyloxypropionamide) were most
potent against Baf3-WT and Baf3-T3151 exhibiting IC50 values in
the range of 1.12–3.62 mM. An extension on the acrylamide chain
with dimethylamine 8, 18 and 29 resulted in a decrease in potency
across all cell lines. The hydroxyacetamide analogues 10, 20 and 31
were in general similar or more potent than the corresponding
homologues 12, 22 and 33 with an extra carbon.
In summary, a series of thirty-two WZ4002 analogues (2–33)
was successfully designed, synthesized, characterized and evalu-
ated in enzyme and cell based assays.
The target compounds were tested against EGFR-WT, EGFR
d746 – 750, EGFR T790M, EGFR T790M L858R, EGFR C797S and
EGFR T790M L858R C797S protein tyrosine kinases by Z’-LYTE
assay method at 1 mM and 10 mM concentrations.
bition against these three kinases at both 1 mM and 10 mM are the
benzyloxy and hydroxy analogues in the ortho series (9–12). Com-
pounds 9–12 also showed the best inhibition (70–91%) against
EGFR C797S at 10 mM concentration in comparison to WZ4002
(no inhibition) suggesting a potential H-bonding interaction with
the mutated serine 797 residue found at the covalent binding site.
The H-bonding interaction between the oxygen atom of the benzy-
loxy or hydroxy group of these analogues and the serine side chain
may play an important role in their activity against the third gener-
ation of mutation (C797S). Majority of the other analogues except
for compounds with H-bonding potential such as 4, 19–22 and 33
did not show significant inhibition of the EGFR C797S kinase at
10 mM concentration. Against the triple mutant, EGFR T790M
C797S L858R, compounds 9–12 exhibited complete inhibition at
10 mM and nearly complete inhibition at 1 mM. Compound 10 exhib-
ited complete inhibition against all the six kinases at 10 mM.
In the cytotoxicity assay, majority of the target compounds
were found to exhibit similar or better potency against PC9-GR
All the tested compounds inhibited EGFR d746 – 750, EGFR
T790M and EGFR T790M L858R in percent inhibitions >60% at
10 mM concentrations. The analogues that exhibited complete inhi-
Please cite this article in press as: Romu A.A., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.09.048

6
A.A. Romu et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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and H460 cell lines in comparison to gefitinib. They also exhibited
better potency against Ba/F3 T3151 in comparison to imatinib. In
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get compounds have similar or lower IC50 values against PC9-GR,
H460, Ba/F3 WT and Ba/F3-T3151 cell lines. Overall, 7, 9–12, 30
and 31 were found to be the most potent compounds across all five
cell lines. Among all the target compounds, the benzyloxy and
hydroxy analogues (9–12, 19–22 and 30–33) exhibited greater
potency against PC9-GR cell line.
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Acknowledgement
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27. Synthesis and NMR characterization of Compound 10: Benzyloxyacetamide 9
(200 mg, 0.34 mmol) was dissolved in methanol (5 mL) followed by the
addition of catalytic amount of 10% Pd/C. The reaction mixture was stirred
overnight at room temperature under hydrogen pressure in Parr apparatus. The
completion of the reaction was monitored by TLC. The reaction mixture was
then filtered through celite and the filtrate was evaporated under reduced
pressure. The crude product was recrystallised from a mixture of chloroform:
methanol to obtain pure hydroxyacetamide 10 as white solid in 38% yield. 1H
NMR (DMSO-d6, d ppm): 9.39 (s, 1H), 8.33 (s, 1H), 8.04 (s, 1H), 7.90 (d, J = 1.7
Hz, 1H), 7.25 (m, 3H), 6.55 (s, 1H), 6.21 (br s, 1H), 5.75 (s, 1H), 4.01 (s, 2H) 3.75
(s, 3H), 3.04 (m, 4H), 2.42 (m, 4H), 2.20 (s, 3H).
The authors thank Dr. Brian J. Druker at the Oregon Health &
Science University, Drs. Eric B. Haura and Fumi Kinose at the Mof-
fitt Cancer Center for providing the cancer cell lines. Support for
this research was provided by Saint John’s University.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.09.048.
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Please cite this article in press as: Romu A.A., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.09.048