Replacing the terminal piperidine in ceritinib with aliphatic amines confers activities against crizotinib-resistant mutants including G1202R

Article abstract of DOI:10.1016/j.ejmech.2016.11.046

The piperidine fragment in ceritinib was replaced with diverse aliphatic amines to improve inherent resistance issues of ceritinib. While most of the prepared compounds exhibit as similar in?vitro activities as ceritinib, compound 10 shows encouraging act

Full text of DOI:10.1016/j.ejmech.2016.11.046

European Journal of Medicinal Chemistry 126 (2017) 536e549
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Replacing the terminal piperidine in ceritinib with aliphatic amines
confers activities against crizotinib-resistant mutants including
G1202R
,
,
Gangadhar Rao Mathi a b, Chung Hyo Kang a c, Heung Kyoung Lee a,
Raghavendra Achary a b, Ha-Yeon Lee a d, Joo-Youn Lee e, Jae Du Ha a, Sunjoo Ahn a
,
,
,
, b
Chi Hoon Park a b, Chong Ock Lee a, Jong Yeon Hwang a b, Chang-Soo Yun a
,
,
,
, b
Hee Jung Jung a b, Sung Yun Cho a b, Hyoung Rae Kim a *, Pilho Kim a
,
,
,
, b, *
a Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114, South Korea
b Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, South Korea
c College of Pharmacy, Chungnam National University, Daejeon 34134, South Korea
d College of Pharmacy, Chungbuk National University, Cheongju 28644, South Korea
e Korea Chemical Bank, Korea Research Institute of Chemical Technology, Daejeon 34114, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The piperidine fragment in ceritinib was replaced with diverse aliphatic amines to improve inherent
resistance issues of ceritinib. While most of the prepared compounds exhibit as similar in vitro activities
as ceritinib, compound 10 shows encouraging activities against wild-type ALK as well as crizotinib-
resistant mutants including extremely resistant G1202R mutant with an IC50 of 1.8 nM. Furthermore,
pharmacokinetic profiles of 10 is apparently better than that of ceritinib. In murine xenograft studies,
compound 10 turns out to be as active as ceritinib, suggesting that further optimization of 10 may lead to
clinical candidates overcoming ALK mutant issues.
Received 24 August 2016
Received in revised form
1 November 2016
Accepted 21 November 2016
Available online 24 November 2016
Keywords:
© 2016 Elsevier Masson SAS. All rights reserved.
Anaplastic lymphoma kinase (ALK)
Non-small cell lung cancer (NSCLC)
Ceritinib
Crizotinib-resistant
G1202R
1. Introduction
of NSCLC and 70% of ALCL [4]. Moreover, it has been reported that
the amplification or point mutations of ALK is involved in the
Anaplastic lymphoma kinase (ALK), one of the receptor tyrosine
kinases, was first identified as a fused form with nucleophosmin
(NPM) in anaplastic large cell lymphoma (ALCL) and a subclass of T-
cell non-Hodgkin lymphomas [1]. In 2007, echinoderm
microtubule-associated protein-like-4 (EML4)-ALK, found in over
6% of non-small cell lung cancers (NSCLC), was recognized as a
promising oncogenic drug target [2]. Although the specific physi-
ological function of ALK in cancer remains unclear, ALK fusion
protein has been found in various human cancers [3], such as
breast, colorectal, inflammatory myofibroblastic tumors (IMT),
diffuse large B-cell lymphoma (DLBCL), and most notably in 2e7%
development of neuroblastoma (NB), anaplastic thyroid cancer, and
ovarian cancer [5].
In 2011, crizotinib was approved as the first drug to treat ALK-
positive advanced NSCLC patients (Fig. 1) [6]. It is remarkable in a
sense that the clinical development of crizotinib took only six years
from the ALK rearrangement identification in NSCLC patients.
However, relapse inevitably occurs usually in a year of crizotinib
treatment. It has been known that crizotinib resistance occurs
through various mechanisms, such as secondary mutations,
amplification of the EML4-ALK fusion gene, and EGFR activation
[7,8]. To overcome the advent of crizotinib-resistant mutants,
second-generation ALK inhibitors were developed, such as ceritinib
and alectinib. Ceritinib is known to be active against most of the
crizotinib-resistant mutants except G1202R and F1174C [9].
Considering ALK mutations occurred in 5e7% of NB cases, one of
* Corresponding authors. Bio & Drug Discovery Division, Korea Research Institute
of Chemical Technology, Daejeon 34114, South Korea.
E-mail addresses: hyungrk@krict.re.kr (H.R. Kim), pkim@krict.re.kr (P. Kim).
http://dx.doi.org/10.1016/j.ejmech.2016.11.046
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
537
Fig. 1. Known ALK inhibitors and design strategies.
the most significant drawbacks of ceritinib is unfavorable blood-
brain barrier (BBB) penetration abilities. On the other hand, alec-
tinib has superb BBB penetration properties as well as activities
against the ALK mutants except G1202R [10,11]. It is noteworthy to
mention that relapsed tumors from ceritinib and alectinib often
express the G1202R mutant [9,11]. Lately, tremendous efforts were
devoted to discover ALK inhibitors fortified with G1202R activities,
giving rise to the discovery of a macrocyclic molecule mimicking
crizotinib (PF-06463922; Clinical Phase II) [12] and an alectinib
analog (JH-VIII-157-02) [13].
starting with 4-methyl-2-nitroanisole 1. Introduction of acetoni-
trile group para to the nitro group of anisole 1 was carried out using
phenylthioacetonitrile and powdered sodium hydroxide in DMSO
to afford nitrile 2 [25]. Nitro group of 2 was reduced under hy-
drogenation conditions using Pd/C in MeOH to facilitate amine 3.
Commercially available pyrimidine 4 was coupled with amine 3 to
facilitate compound 5 in the presence of diluted HCl [16]. Similarly,
compound 8 was prepared from dimethylated nitrile 7, which was
generated from tandem NaH-mediated dimethylation of the active
methylene group of 2 and subsequent reduction of the nitro group
in 6.
The 2-position of pyrimidine in ceritinib has been explored
extensively to find out novel ALK inhibitors by introducing either
bicyclic scaffolds, such as tetrahydrothiazoloazepine [14], benza-
zepines [15], tetrahydroisoquinolines [16], and tetrahydronaph-
thalenes [17], or flexible side chains, such as amino acid analogs
[18], phosphamides or carbamates [19], and hydrazones [20]. Cer-
itinib has a secondary amine at the piperidine part as a key func-
tional group known to interact with Glu1210 of ALK [21]. A recent
literature suggested that modification of the terminal secondary
amine in ceritinib could be tolerable to maintain efficacies [19], as
one may envision flexible accommodation nature of this terminal
amine pocket from the structures of AZD3463 [22] and brigatinib
[5,23], recently developed ALK inhibitors with terminal primary
and tertiary amines, respectively (Fig. 1). A previous report revealed
that presence of a methyl group para to the alkoxy group on the
aromatic ring helps slow down the metabolism of the phenyl group
by blocking the formation of reactive metabolites [24]. With the
above information in mind and aiming to achieve challenging
G1202R mutant activities, KRCA compounds were designed and
synthesized in which the methyl group was retained while the
piperidine was replaced with diverse aliphatic amine groups.
To examine substitution effects of the terminal amine group,
primary, secondary, and tertiary amines were prepared (Scheme 2).
Reduction of the nitrile group in 5 and 8 with BH3$THF proceeded
effectively to give primary amines 9 and 13, respectively. Tertiary
amines were prepared by using two different methods, viz.
reductive alkylation of 9 with formaldehyde and NaBH3CN for 10,
and N-alkylation of 13 using iodomethane and DIPEA for 14. Pri-
mary amines 9 and 13 were also transformed to afford formamides
11 and 15 under refluxing ethyl formate conditions [26], and sub-
sequent reduction with LiAlH4 gave secondary amines 12 and 16 in
low yields, respectively.
With a view to understanding the effect of carbon chain length,
compounds 22e23 and 29e30, one carbon shorter version of 9e10
and 13e14, respectively, were prepared (Scheme 3). Nitrile 2 was
treated with conc. HCl to facilitate amide 17. While acid-catalyzed
hydrolysis of 2 in H2SO4 (aq) under refluxing conditions resulted
in the complete hydrolysis of nitrile to acid, the same reaction
condition with nitrile 6 delivered amide 24. Treatment of 17 and 24
with (bis(trifluoroacetoxy)iodo)benzene gave Hofmann rear-
rangement products 18 and 25, respectively [27]. Tandem protec-
tion of amines 18 and 25 with trifluoroacetyl and reduction of the
nitro groups afforded amides 19 and 26, and anilines 20 and 27,
respectively. Anilines 20 and 27 were coupled with 4 in the pres-
ence of HCl to give compounds 21 and 28, respectively. Compounds
2. Chemistry
Compounds 5 and 8 were synthesized as outlined in Scheme 1,

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G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
Scheme 1. Reagents and conditions: (a) PhSCH2CN, NaOH, DMSO, 0 ꢀC to rt, 1 h (78%); (b) Pd/C, H2 (g), MeOH, rt, 12 h (91% for 3; 89% for 7); (c) 4, 0.08 M HCl in EtOEtOH, 80 ꢀC, 12 h
(81%); (d) NaH, MeI, DMF, 0 ꢀC to rt, 8 h (94%).
Scheme 2. Reagents and conditions: (a) 1.0 M BH3 in THF, THF, rt, 12 h (45%); (b) formaldehyde (37 wt. % in H2O), AcOH (cat.), NaBH3CN, MeOH, rt, 2 h (77%); (c) MeI, DIPEA, DMF,
m
W, 80 ꢀC, 10 min (47%); (d) HCO2Et, 60 ꢀC, 4 h (76% for 11; 94% for 15); (e) LiAlH4, THF, 60 ꢀC, 12 h (17% for 12; 20% for 16).
22 and 29 were obtained from hydrolysis of 21 and 28 with LiOH
(aq), respectively. The N,N-dimethylation was achieved using
formaldehyde for 22 and iodomethane for 29 to furnish tertiary
amines 23 and 30, respectively.
With 18 and 25 in hand, tandem formylation, nitro reduction,
coupling with 4, and amide reduction gave rise to 31e34 and
35e38, respectively (Scheme 4).
ALK inhibitory activity in both enzymatic and cell-based assays.
Enzymatic assays include the ALK wild-type and crizotinib-
resistant L1196M mutant enzymes. The cell lines harboring EML4-
ALK fusion genes were utilized for the assay, such as H3122, Ba/
F3 L1196M, and Ba/F3 EML4-ALK wild-type.
Nitriles 5 and 8 are not active most likely due to the absence of
terminal amines. All of the other compounds tested exhibited ac-
tivities against both ALK wild-type and crizotinib-resistant L1196M
mutant enzymes with IC50 values of single-digit nano molars. In cell
assays, non-substituted primary amines, 9 and 22, are less active
than ceritinib against at least two of the three cell lines tested.
Among phenethylamines without substitution at the benzylic car-
bon (9e12), tertiary amine 10 is more active than primary (9) and
secondary (12) amines as well as formamide 11. Likewise, when the
benzylic position was substituted with dimethyl (13e16), tertiary
3. Results and discussion
3.1. In vitro biological evaluations
Overall 18 KRCA compounds were synthesized with varying
alkyl chain lengths and substitutions. To investigate the anti-
proliferative activities, these compounds were evaluated for their

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
539
Scheme 3. Reagents and conditions: (a) conc. HCl, 30 ꢀC, 48 h (70%); (b) H2SO4, H2O, 100 ꢀC, 4 h (78%); (c) PhI(OCOCF3)2, CH3CN/H2O (1/1), rt, 12 h (76% for 18; 89% for 25); (d) TFAA,
Et3N, DCM, 0 ꢀC to rt, 30 min (76% for 19; 83% for 26); (e) Pd/C, H2 (g), MeOH, rt, 2 h (82% for 20; 63% for 27); (f) 4, 0.08 M HCl in EtOEtOH, 80 ꢀC, 12 h (82% for 21; 74% for 28); (g)
LiOH$H2O, THF/MeOH/H2O (4/2/1), rt, 12 h (78% for 22; 40% for 29); (h) formaldehyde (37 wt. % in H2O), AcOH (cat.), NaBH3CN, MeOH, rt, 2 h (57%); (i) MeI, DIPEA, DMF, m
W, 80 ꢀC,
10 min (47%).
Scheme 4. Reagents and conditions: (a) HCO2Et, 60 ꢀC, 12 h (52% for 31; 78% for 35); (b) Pd/C, H2 (g), MeOH, rt, 12 h (85% for 32; 87% for 36); (c) 4, 0.08 M HCl in EtOEtOH, 80 ꢀC, 12 h
(42% for 33; 55% for 37); (d) LiAlH4, THF, 60 ꢀC, 12 h (17% for 34; 27% for 38).
amine 14 was the most active. Substitution at the benzylic position
does not seem go give significant influences on the activity. Com-
pound 10 is 2- to 7-fold more active than ceritinib in the cell assays.
In case of the benzyl amines prepared (22e38), all of them showed
moderate to good activities in the cell assays except primary amine
22. Compound 29, primary amine with dimethyl substituted at the
benzylic position, turns out to be the most active among the benzyl
amines. Considering both enzyme and cell assay results, compound
10 (KRCA-764) was eventually selected for further in-depth
investigation.
G1202R is one of the most crucial mutants to overcome in pa-
tients with crizotinib- or ceritinib-resistant NSCLC [9,11]. Due to the
incapability of first- and second-generation ALK inhibitors against
G1202R, discovery of novel ALK inhibitors conferring G1202R ac-
tivities is urgently needed. Thus, the activity of selected compound
10 was further evaluated using several crizotinib-resistant ALK
mutant enzymes (Table 2). Overall, compound 10 exhibited similar
or better activities than those of ceritinib against the mutants
tested. In case of G1202R, compound 10 showed seven-fold higher
activity than that of ceritinib. Additionally, the activity of com-
pound 10 against G1269A, one of the most frequently observed
secondary mutations along with L1196M [9], is three-fold more
active than ceritinib.
mutant ALKs, docking studies were performed using the x-ray co-
crystal structure of wild-type ALK and ceritinib (PDB code 4MKC)
[9] as well as L1196M mutant ALK complexed with crizotinib (PDB
code 2YFX) [28]. In case of wild-type ALK, the proposed binding
mode of 10 is similar to that of ceritinib (Fig. 2a). Compound 10 has
hinge region interactions with Met1199 backbone amide as cer-
itinib does. Moreover, oxygen of the sulfonyl group in 10 has a
hydrogen bond with Lys1150 through water-mediated interactions.
5-Chloropyrimidine core structure, located between Ala1148 and
Leu1256 at the active site, is bound to a hydrophobic pocket
composed of gatekeeper residue Leu1196. Methoxyphenyl group of
10 forms hydrophobic interactions with Leu1122. In particular, the
tertiary amine group of 10, heading for the solvent exposed area,
makes a hydrogen bond with Ser1206 as well as electrostatic in-
teractions with Asp1203 and Glu1210.
In Fig. 2b, predicted binding modes of both ceritinib and 10 in
L1196M mutant ALK appear to be virtually identical, as observed in
wild-type ALK. It is worthwhile to point out that there is a weak
steric repulsion adjacent to Met1196 in case of 10. Using the x-ray
structure of ALK with ceritinib (PDB code 4MKC), a G1202R mutant
ALK model was generated (Fig. 2c). The G1202R mutant model
shows that the bulky isopropoxy group of ceritinib has a significant
amount of steric collision interactions with Arg1202 side chain [29],
while the methoxy group of 10 has much less steric effect,
providing a rationale for the improved G1202R mutant activity of
10 over ceritinib [13,16].
3.2. Docking studies
To decipher the binding mode of 10 for wild-type and L1196M

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G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
Table 1
ALK inhibitory activities against enzymes and cells.
Compounds
Enzymes (IC50 nM)
ALK (wt)
Cells (CC50 nM)
H3122
ALK L1196M
Ba/F3 L1196M
Ba/F3 EML4-ALK wt
ceritinib
5
8
14
21
29
36
25
74
250
60
13
68
20
53
26
63
41
110
37
12
35
28
25
52
29
75
75
61
250
68
10
60
13
36
14
38
19
120
21
12
20
15
15
24
15
190
440
300
35
260
58
96
66
99
68
39
66
55
91
62
54
81
66
200
2.7
1.4
4.0
1.5
4.9
2.2
7.8
1.5
1.9
3.4
1.5
2.8
4.7
1.7
2.1
1.2
570
1.7
1.3
4.4
1.9
3.4
3.6
6.1
1.7
2.1
2.1
1.6
1.5
2.8
2.2
2.5
1.4
9
10
11
12
13
14
15
16
22
23
29
30
33
34
37
38
Table 2
Inhibitory activities against ALK mutant enzymes.
Compounds
G1202R (nM)
G1269A (nM)
T1151-L1152 insT (nM)
F1174L (nM)
C1156Y (nM)
ceritinib
10
13
1.8
1.4
0.4
0.63
0.63
1.5
0.56
2.0
1.1
3.3. Pharmacokinetic profiles
4. Conclusion
Pharmacokinetic profiles of compound 10 and ceritinib was
investigated using male rats with a dose of 10 mg/kg. Compared
with ceritinib, compound 10 exhibited over two times of improved
Considerably potent ALK inhibitors against wild-type as well as
crizotinib-resistant mutants were discovered through replacing
piperidine in ceritinib with various alkyl amines. In particular,
compound 10 displayed significantly better ALK inhibitory activ-
ities in both enzymes and cell assays, compared to those of cer-
itinib. Intriguingly, highly resistant G1202R mutant is also sensitive
to compound 10. Docking studies support that more compact
methoxy group in 10 could be favorable for more effective binding
to the active site of G1202R mutant. With these encouraging bio-
logical data in hand, further optimization of 10 is ongoing and the
progress will be reported shortly.
C
max and AUC values (Fig. 3).
3.4. In vivo xenograft evaluations
Female nude mice were utilized for the xenograft assay using
H3122 cells which were inoculated subcutaneously into the right
flanks of the mice. When the tumor volume became as big as
200 mm3, drug treatment began. Ceritinib and 10 were orally
administered with a dose of 50 mg/kg each for 14 days (Fig. 4).
Although compound 10 exhibited better in vitro activities and PK
profiles, compared with ceritinib, xenograft assay results showed
that 10 and ceritinib have similar potency, indicating that drug-like
properties of 10 should be improved to escalate its efficacy.
Furthermore, no substantial body weight changes were observed
during this xenograft studies (Fig. 5).
5. Experimental
5.1. Materials and methods for chemistry
Solvents and reagents were obtained from commercial vendors
and used as received. Compound 4 was acquired from Chemlin

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
541
(Nanjing, China). TLC was carried out on precoated silica gel F254
plate (Merck, art. 5715) and column chromatography was per-
formed using silica gel (Merck, mesh 230e400 mm). Melting point
was detected on OptiMelt-Automated Melting Point System. 1H and
13C NMR spectra were recorded with Bruker Avance 300 and 500,
using CDCl3 or other deuterated solvents as an internal standard.
LC/MS analysis was performed on the Waters Acquity UPLC system
with electrospray ionization in positive ion mode. HRMS was
detected on JMS-700 (JEOL, Tokyo, Japan). Most of the final com-
pounds in Table 1 have purities over 95% based on LC/MS.
5.1.1. 2-(4-Nitro-5-methoxy-2-methylphenyl) acetonitrile (2)
To a suspension of powdered sodium hydroxide (5.74 g,
144 mmol) in anhydrous DMSO (15 mL) was added a mixture of 4-
methyl-2-nitroanisole (1, 2.40 g, 14.4 mmol) and phenyl-
thioacetonitrile (2.14 g, 14.4 mmol) in anhydrous DMSO (10 mL).
The reaction mixture was stirred at rt for 1 h, and then poured into
ice water and 6 N HCl (aq) solution. The mixture was extracted with
DCM (2 ꢁ 100 mL). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated to give the crude mixture
which was purified by silica gel column chromatography using
EtOAc/hexanes (2/3) to afford the title compound (2.31 g,
11.2 mmol, 78%) as a yellow solid. Mp 126.9e129.4 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
7.73 (s, 1H), 7.15 (s, 1H), 3.99 (s, 3H), 3.73 (s, 2H),
151.57, 138.75, 135.14,
2.32 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
128.20, 127.41, 116.44, 113.88, 56.80, 22.23, 18.21; LC/MS m/z 207.1
[MþHþ].
5.1.2. 2-(4-Amino-5-methoxy-2-methylphenyl) acetonitrile (3)
To a solution of 2 (450 mg, 2.18 mmol) in MeOH (25 mL) was
added Pd/C (45.0 mg, 0.422 mmol) at rt. The reaction mixture was
stirred under a hydrogen balloon at rt for 12 h, filtered through a
bed of celite, and evaporated under reduced pressure. The crude
mixture was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (350 mg,
1.98 mmol, 91%) as an off white solid. Mp 86.5e88.1 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
6.72 (s,1H), 6.53 (s,1H), 3.83 (s,1H), 3.77 (s, 1H),
145.68, 136.07,
3.55 (s, 1H), 2.18 (s, 1H); 13C NMR (125 MHz, CDCl3)
d
128.38, 118.37, 117.31, 117.07, 111.11, 55.76, 21.36, 18.50; LC/MS m/z
177.1 [MþHþ].
5.1.3. 2-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)acetonitrile (5)
To a solution of 3 (200 mg, 1.13 mmol) in 0.08 M HCl in ethox-
yethanol (1.0 mL) was added 2,5-dichloro-N-(2-(isopropylsulfonyl)
phenyl)pyrimidin-4-amine (4, 472 mg, 1.36 mmol) at rt and the
reaction mixture was stirred at 80 ꢀC for 12 h. The solid precipitated
in the reaction mixture was filtered and washed with ethanol
(2.0 mL). The filtered solid was then dissolved in EtOAc (50 mL) and
washed with sat. NaHCO3 (aq). The combined organic layers were
concentrated under reduced pressure to give the crude mixture
which was purified by silica gel column chromatography using
MeOH/DCM (1/9) to afford the title compound (450 mg,
0.926 mmol, 81%) as a white solid. Mp 152.8e154.6 ꢀC; 1H NMR
Fig. 2. Molecular docking studies of ceritinib and 10 with wild-type, L1196M, and
G1202R mutant ALKs. (a) Proposed binding mode of 10 (yellow, ball and stick style)
and overlapped x-ray co-crystal structure of ceritinib (pink, stick style) in wild type
ALK (grey ribbon); (b) Predicted binding modes of both ceritinib and 10 in L1196M
mutant ALK (blue ribbon); (c) Overlapping 10 and ceritinib docking model in G1202R
mutant ALK generated (green ribbon). *Notation: water (red sphere), hydrogen bonds
(green dashed lines), water-mediated interactions (sky blue dashed lines), electrostatic
interactions (orange dashed lines). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

542
G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
Fig. 3. Pharmacokinetic profiles of ceritinib and compound 10 in male rats.
Fig. 4. Effects of compounds on growth of H3122 human lung cancer in nude mice. *P < 0.001 vs. control group on the final day using Student's t-test.
(300 MHz, CDCl3)
d
9.53 (s, 1H), 8.53 (d, J ¼ 8.3 Hz, 1H), 8.18 (s, 1H),
(sept, J ¼ 6.9 Hz, 1H), 2.18 (s, 4H), 1.32 (d, J ¼ 6.9 Hz, 7H); 13C NMR
8.13 (s, 1H), 7.94 (d, J ¼ 7.9 Hz, 1H), 7.65 (t, J ¼ 7.9 Hz, 1H), 7.55 (s,
(125 MHz, CDCl3) d 157.29, 155.37, 155.25, 146.54, 138.35, 134.64,
1H), 7.32e7.25 (m, 1H), 6.86 (s, 1H), 3.91 (s, 3H), 3.64 (s, 2H), 3.26
131.35, 128.68, 127.90, 125.01, 123.59, 123.33, 121.17, 120.62, 117.84,

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
543
Fig. 5. Effects of ceritinib and 10 on body weight of nude mice bearing H3122.
110.48, 106.49, 56.02, 55.53, 21.69, 18.94, 15.37; LC/MS m/z 485.9
5.1.6. 2-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)-2-
methylpropanenitrile (8)
[MþHþ]; HRMS (EI) m/z calcd for C23H24ClN5O3S [Mþ] 485.1288,
found 485.1289.
To a solution of 7 (200 mg, 0.979 mmol) in 0.08 M HCl in
ethoxyethanol (1.0 mL) and 2,5-dichloro-N-(2-(isopropylsulfonyl)
phenyl)pyrimidin-4-amine (4, 407 mg, 1.17 mmol) was added at rt
and the reaction mixture was stirred at 80 ꢀC for 12 h. The solid
precipitated was filtered and washed with ethanol (2 mL). The solid
filtered was then dissolved in EtOAc (50 mL) and washed with sat.
NaHCO3 (aq). The combined organic layers were concentrated un-
der reduced pressure to obtain the crude product which was pu-
rified by silica gel column chromatography using MeOH/DCM (1:9)
to afford the title compound (433 mg, 0.875 mmol, 94%) as a white
5.1.4. 2-(5-Methoxy-2-methyl-4-nitrophenyl)-2-
methylpropanenitrile (6)
To a suspension of 60% NaH in mineral oil (0.804 g, 20.2 mmol)
in DMF (15 mL) was added a solution of 2 (1.66 g, 8.05 mmol) in
DMF (10 mL) at 0 ꢀC. After stirring for 20 min, to the mixture was
added CH3I (1.10 mL, 17.7 mmol) at 0 ꢀC. The reaction mixture was
stirred at rt for 8 h, quenched with ice, extracted with EtOAc
(2 ꢁ 150 mL). The combined organic layers were dried over anhy-
drous Na2SO4 to get the crude mixture which was recrystallized
using water. The solid precipitated was filtered to afford the title
compound (1.77 g, 7.57 mmol 94%) as an off white solid. Mp
solid. Mp 207.4e209.6 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.53 (s, 1H),
8.53 (d, J ¼ 8.3 Hz, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 7.94 (dd, J ¼ 7.9,
1.3 Hz,1H), 7.66 (t, J ¼ 7.3 Hz,1H), 7.53 (s, 1H), 7.30 (t, J ¼ 7.9 Hz, 1H),
6.83 (s, 1H), 3.90 (s, 3H), 3.26 (sept, J ¼ 6.9 Hz, 1H), 2.45 (s, 3H), 1.79
(s, 6H), 1.32 (d, J ¼ 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3)
d 157.32,
116.5e118.4 ꢀC; 1H NMR (300 MHz, CDCl3)
d 7.74 (s, 1H), 7.06 (s, 1H),
155.40, 155.33, 146.15, 138.34, 134.76, 131.36, 131.11, 128.33, 125.00,
124.58, 123.55, 123.38, 122.45, 107.24, 106.48, 55.93, 55.52, 35.00,
28.27, 20.78, 15.37; LC/MS m/z 513.8 [MþHþ]; HRMS (EI) m/z calcd
for C25H28ClN5O3S [Mþ] 513.1601, found 513.1607.
3.97 (s, 3H), 2.62 (s, 3H), 1.83 (s, 6H); 13C NMR (125 MHz, CDCl3)
d
151.16, 144.51, 129.46, 128.67, 123.20, 111.09, 56.67, 35.84, 27.85,
20.23; LC/MS m/z 235.1 [MþHþ].
5.1.7. N2-(4-(2-Aminoethyl)-2-methoxy-5-methylphenyl)-5-chloro-
N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (9)
5.1.5. 2-(4-Amino-5-methoxy-2-methylphenyl)-2-
methylpropanenitrile (7)
To a solution of 5 (200 mg, 0.411 mmol) in THF (2 mL) was added
1.0 M BH3$THF in THF (2.06 mL, 2.06 mmol) at 0 ꢀC. The reaction
mixture was stirred at rt for 12 h, quenched with methanol, and
concentrated to dryness. To the mixture was added 1.5 N HCl (aq)
(25 mL) and extracted with EtOAc. The aqueous layer was basified
with NaOH (aq) and extracted with EtOAc (2 ꢁ 50 mL). The com-
bined organic layers were dried over anhydrous Na2SO4, concen-
trated to obtain the crude mixture which was purified by silica gel
column chromatography using MeOH/DCM (1/9) to afford the title
compound (90.0 mg, 0.185 mmol, 45%) as a pale yellow solid. Mp
To a solution of 6 (450 mg, 1.92 mmol) in MeOH (25 mL) was
added Pd/C (45.0 mg, 0.422 mmol) at rt. The reaction mixture was
stirred under a hydrogen balloon at rt for 12 h, filtered through a
bed of celite, and evaporated under reduced pressure. The crude
mixture was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (350 mg,
1.71 mmol, 89%) as an off white solid. Mp 87.2e88.9 ꢀC; 1H NMR
(500 MHz, CDCl3)
d
6.75 (s, 1H), 6.59 (s, 1H), 3.86 (s, 3H), 3.78 (s, br,
145.22,
2H), 2.51 (s, 3H), 1.78 (s, 6H); 13C NMR (125 MHz, CDCl3)
d
135.66, 128.79, 127.46, 125.01, 119.06, 108.00, 55.77, 34.78, 28.45,
94.1e96.4 ꢀC; 1H NMR (300 MHz, CDCl3)
J ¼ 8.4 Hz, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.92 (dd, J ¼ 8.0, 1.3 Hz, 1H),
d 9.51 (s, 1H), 8.57 (d,
20.30; LC/MS m/z 205.1 [MþHþ].

544
G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
7.63 (t, J ¼ 7.8 Hz, 1H), 7.49 (s, 1H), 7.29e7.22 (m, 1H), 6.70 (s, 1H),
3.87 (s, 3H), 3.26 (sept, J ¼ 6.8 Hz,1H), 2.94 (t, J ¼ 6.8 Hz, 2H), 2.75 (t,
J ¼ 7.0 Hz, 2H), 2.18 (s, 5H), 1.31 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 489.8
[MþHþ]; HRMS (EI) m/z calcd for C23H28ClN5O3S [Mþ] 489.1601,
found 489.1601.
5.1.11. N2-(4-(1-Amino-2-methylpropan-2-yl)-2-methoxy-5-
methylphenyl)-5-chloro-N4-(2-(isopropylsulfonyl)phenyl)
pyrimidine-2,4-diamine (13)
To a solution of 8 (100 mg, 0.194 mmol), 1.0 M BH3$THF in THF
(0.972 mL, 0.972 mmol) was added at 0 ꢀC. The reaction mixture
was stirred at rt for 12 h, quenched with methanol, and concen-
trated to dryness. To the mixture was added 1.5 N HCl (aq) (25 mL)
and extracted with EtOAc. The aqueous layer was basified with
NaOH (aq) and extracted with EtOAc (2 ꢁ 50 mL). The combined
organic layers were dried over anhydrous Na2SO4, concentrated to
obtain the crude mixture which was purified by silica gel column
chromatography using MeOH/DCM (1/9) to afford the title com-
5.1.8. 5-Chloro-N2-(4-(2-(dimethylamino)ethyl)-2-methoxy-5-
methylphenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-
diamine (10)
To a solution of 9 (25.0 mg, 0.051 mmol) in MeOH (2.0 mL) was
added acetic acid (catalytic), formalin (35%) (4.37 mg, 0.051 mmol)
at 0 ꢀC. After 30 min, NaBH3CN (4.80 mg, 0.076 mmol) was added to
the mixture and stirred at rt for 30 min. The reaction mixture was
quenched with NaHCO3 (aq) and extracted with EtOAc (2 ꢁ 15 mL).
The combined organic layers were dried over anhydrous Na2SO4,
concentrated to obtain the crude mixture which was purified by
silica gel column chromatography using MeOH/DCM (1/9) to afford
the title compound (20.0 mg, 0.039 mmol, 77%) as a white solid. Mp
pound (45.0 mg, 0.087 mmol, 45%) as
a
white solid. Mp
152.3e154.1 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.51 (s, 1H), 8.57 (d,
J ¼ 8.4 Hz, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.93 (dd, J ¼ 8.0, 1.4 Hz, 1H),
7.63 (t, J ¼ 7.8 Hz, 1H), 7.47 (s, 1H), 7.31e7.21 (m, 1H), 6.87 (s, 1H),
3.89 (s, 3H), 3.26 (sept, J ¼ 6.9 Hz, 1H), 2.97 (s, 2H), 2.34 (s, 3H), 1.40
(s, 6H), 1.32 (d, J ¼ 6.9 Hz, 6H), 1.26 (s, br, 2H); 13C NMR (125 MHz,
140.4e142.1 ꢀC; 1H NMR (500 MHz, CDCl3)
d
9.54 (s, 1H), 8.59 (d,
CDCl3) d 157.40, 155.36, 155.19, 146.08, 138.41, 136.19, 134.76, 131.29,
J ¼ 8.3 Hz, 1H), 8.18 (s, 1H), 8.05 (s, 1H), 7.95 (dd, J ¼ 8.0, 1.5 Hz, 1H),
7.66 (t, J ¼ 8.5 Hz, 1H), 7.52 (s, 1H), 7.33e7.28 (m, 1H), 6.73 (s, 1H),
3.91 (s, 3H), 3.28 (sept, J ¼ 6.9 Hz, 1H), 2.86 (dd, J ¼ 10.2, 6.5 Hz, 2H),
2.68 (dd, J ¼ 10.2, 6.4 Hz, 2H), 2.52 (s, 6H), 2.20 (s, 3H), 1.34 (d,
128.14, 127.05, 124.80, 123.46, 123.33, 123.20, 110.49, 106.16, 55.82,
55.49, 50.43, 40.44, 27.35, 22.80, 15.37; LC/MS m/z 517.7 [MþHþ];
HRMS (EI) m/z calcd for C25H32ClN5O3S [Mþ] 517.1914, found
517.1916.
J ¼ 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3)
d 157.57, 155.33, 155.32,
146.49, 138.47, 134.68, 131.96, 131.25, 127.66, 126.68, 124.80, 123.62,
123.11, 120.78, 111.33, 105.87, 60.43, 55.87, 55.45, 45.43, 31.73, 18.91,
15.37; LC/MS m/z 518.0 [MþHþ]; HRMS (EI) m/z calcd for
5.1.12. 5-Chloro-N2-(4-(1-(dimethylamino)-2-methylpropan-2-yl)-
2-methoxy-5-methylphenyl)-N4-(2-(isopropylsulfonyl)phenyl)
pyrimidine-2,4-diamine (14)
C
25H32ClN5O3S [Mþ] 517.1914, found 517.1909.
To a solution of 13 (40.0 mg, 0.077 mmol) in DMF (1.0 mL) was
added DIPEA (24.9 mg, 0.192 mmol) and CH3I (21.9 mg, 0.154 mmol)
at rt and the reaction mixture was heated under microwave at 80 ꢀC
for 10 min. The reaction was quenched with water (10 mL) and
extracted with EtOAc (2 ꢁ 15 mL). The combined organic layers
were washed with brine solution, concentrated to obtain the crude
mixture which was purified by silica gel column chromatography
using MeOH/DCM (1/9) to afford the title compound (20.0 mg,
0.036 mmol, 47%) as a pale yellow solid. Mp 134.2e136.4 ꢀC; 1H
5.1.9. N-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenethyl)formamide
(11)
A solution of 9 (250 mg, 0.510 mmol) in ethyl formate (10 mL)
was heated at 60 ꢀC for 4 h. The reaction mixture was concentrated
under reduced pressure to obtain the crude mixture which was
purified by silica gel column chromatography using MeOH/DCM (1/
9) to afford the title compound (200 mg, 0.386 mmol, 76%) as a
white solid. Mp 145.5e147.4 ꢀC; 1H NMR (300 MHz, CDCl3, rota-
NMR (300 MHz, CDCl3)
d
9.49 (s, 1H), 8.57 (d, J ¼ 8.3 Hz, 1H), 8.15 (s,
1H), 7.98e7.88 (m, 2H), 7.62 (t, J ¼ 7.8 Hz,1H), 7.47 (s, 1H), 7.32e7.21
(m, 1H), 6.94 (s, 1H), 3.89 (s, 3H), 3.26 (sept, J ¼ 6.9 Hz, 1H), 2.60 (s,
2H), 2.36 (s, 3H), 2.11 (s, 6H), 1.44 (s, 6H), 1.31 (d, J ¼ 6.9 Hz, 6H); LC/
MS m/z 545.6 [MþHþ]; HRMS (EI) m/z calcd for C27H36ClN5O3S [Mþ]
545.2227, found 545.2225.
meric mixture)
d
9.52 (s, 1H), 8.57 (d, J ¼ 8.4 Hz, 1H), 8.18 (s, 1H),
8.16 (s, 1H), 8.07 (s, 0.2H), 8.05 (s, 0.8H), 7.94 (dd, J ¼ 7.9, 1.3 Hz, 1H),
7.64 (t, J ¼ 7.8 Hz, 1H), 7.49 (s, 1H), 7.31e7.22 (m, 1H), 6.68 (s, 0.8H),
6.61 (s, 0.2H), 5.54 (s, br, 1H), 3.87 (s, 3H), 3.53 (q, J ¼ 6.8 Hz, 1.7H),
3.44e3.42 (m, 0.4H), 3.26 (sept, J ¼ 6.8 Hz, 1H), 2.82 (t, J ¼ 7.2 Hz,
2H), 2.18 (s, 3H), 1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 517.7 [MþHþ];
HRMS (EI) m/z calcd for C24H28ClN5O4S [Mþ] 517.1551, found
517.1551.
5.1.13. N-(2-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)-2-
methylpropyl)formamide (15)
A solution of 13 (80.0 mg, 0.154 mmol) in ethyl formate (3.0 mL)
was heated at 60 ꢀC for 4 h. The reaction mixture was concentrated
under reduced pressure to obtain the crude mixture which was
purified by silica gel column chromatography using MeOH/DCM (1/
9) to afford the title compound (80.0 mg, 0.146 mmol, 94%) as a
white solid. Mp 89.4e91.3 ꢀC; 1H NMR (300 MHz, CDCl3, rotameric
5.1.10. 5-Chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-
5-methyl-4-(2-(methylamino)ethyl)phenyl)pyrimidine-2,4-diamine
(12)
To a solution of 11 (180 mg, 0.347 mmol) in THF (15 mL) was
added LiAlH4 (132 mg, 3.47 mmol) at 0 ꢀC. The reaction mixture was
stirred at 60 ꢀC for 12 h, quenched with water and NaOH (aq), and
filtered. The filtrate was extracted with EtOAc (2ꢁ) and the com-
bined organic layers were concentrated under reduced pressure to
obtain the crude mixture which was purified by silica gel column
chromatography using MeOH/DCM (1/9) to afford the title com-
pound (30.0 mg, 0.059 mmol, 17%) as a pale yellow solid. Mp
mixture)
d
9.54 (s, 0.7H), 9.50 (s, 0.2H), 8.56 (d, J ¼ 8.4 Hz, 1H), 8.17
(s, 1H), 8.12 (s, 1H), 8.05 (s, 1H), 7.93 (d, J ¼ 7.8 Hz, 1H), 7.64 (t,
J ¼ 7.9 Hz, 1H), 7.48 (s, 1H), 7.36e7.21 (m, 1H), 6.86 (s, 0.8H), 6.79 (s,
0.2H), 5.20 (s, 1H), 3.89 (s, 2H), 3.88 (s, 1H), 3.67 (d, J ¼ 5.9 Hz, 1.6H),
3.48 (d, J ¼ 6.6 Hz, 0.4H), 3.26 (sept, J ¼ 6.9 Hz, 1H), 2.37 (s, 2.3H),
2.34 (s, 0.7H), 1.44 (s, 6H), 1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 546.2
[MþHþ]; HRMS (EI) m/z calcd for C26H32ClN5O4S [Mþ] 545.1864,
found 545.1866.
123.2e125.2 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.51 (s, 1H), 8.57 (d,
J ¼ 8.3 Hz, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.63
(t, J ¼ 7.9 Hz, 1H), 7.48 (s, 1H), 7.34e7.21 (m, 1H), 6.70 (s, 1H), 3.87 (s,
3H), 3.26 (sept, J ¼ 6.8 Hz, 1H), 2.80 (s, 4H), 2.48 (s, 3H), 2.18 (s, 3H),
1.78 (s, br, 1H), 1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 504.0 [MþHþ];
HRMS (EI) m/z calcd for C24H30ClN5O3S [Mþ] 503.1758, found
503.1729.
5.1.14. 5-Chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-
5-methyl-4-(2-methyl-1-(methylamino)propan-2-yl)phenyl)
pyrimidine-2,4-diamine (16)
To a solution of 15 (54.0 mg, 0.098 mmol) in THF (5.0 mL) was

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
545
added LiAlH4 (37.5 mg, 0.988 mmol) at 0 ꢀC. The reaction mixture
3H), 3.83 (s, br, 2H), 2.21 (s, 3H); LC/MS m/z 263.1 [MþHþ].
was stirred at 60 ꢀC for 12 h, quenched with water and NaOH (aq),
and filtered. The filtrate was extracted with EtOAc (2ꢁ) and the
combined organic layers were concentrated under reduced pres-
sure to obtain the crude mixture which was purified by silica gel
column chromatography using MeOH/DCM (1/9) to afford the title
compound (10.0 mg, 0.018 mmol, 20%) as a white solid. Mp
5.1.19. N-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylbenzyl)-2,2,2-
trifluoroacetamide (21)
To a solution of 20 (100 mg, 0.381 mmol) in 0.08 M HCl in
ethoxyethanol (1.0 mL) was added 4 (145 mg, 0.419 mmol) at rt and
the reaction mixture was stirred at 80 ꢀC for 12 h. The reaction
mixture was then basified with sat. NaHCO3 (aq) and extracted with
EtOAc (2 ꢁ 25 mL). The combined organic layers were concentrated
under reduced pressure to obtain the crude mixture which was
purified by silica gel column chromatography using EtOAc/Hexane
(3/7) to afford the title compound (180 mg, 0.314 mmol, 82%) as a
124.9e127.2 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.51 (s, 1H), 8.57 (d,
J ¼ 8.3 Hz, 1H), 8.16 (s, 1H), 7.98 (s, 1H), 7.92 (dd, J ¼ 7.9, 1.3 Hz, 1H),
7.63 (t, J ¼ 7.8 Hz, 1H), 7.46 (s, 1H), 7.32e7.19 (m, 1H), 6.90 (s, 1H),
3.88 (s, 3H), 3.26 (sept, J ¼ 6.8 Hz, 1H), 2.86 (s, 2H), 2.39 (s, 3H), 2.35
(s, 3H),1.45 (s, 6H),1.32 (d, J ¼ 6.9 Hz, 7H),1.25 (s, br 1H); LC/MS m/z
531.7 [MþHþ]; HRMS (EI) m/z calcd for C26H34ClN5O3S [Mþ]
531.2071, found 531.1993.
white solid. 1H NMR (300 MHz, CDCl3)
d 9.54 (s, 1H), 8.54 (d,
J ¼ 8.3 Hz, 1H), 8.18 (s, 1H), 8.14 (s, 1H), 7.94 (dd, J ¼ 7.9, 1.5 Hz, 1H),
7.64 (t, J ¼ 7.8 Hz, 1H), 7.56 (s, 1H), 7.35e7.22 (m, 1H), 6.76 (s, 1H),
6.30 (s, 1H), 4.49 (d, J ¼ 5.3 Hz, 2H), 3.89 (s, 3H), 3.25 (sept,
J ¼ 6.9 Hz, 1H), 2.18 (s, 3H), 1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 572.1
[MþHþ].
5.1.15. 2-(5-Methoxy-2-methyl-4-nitrophenyl)acetamide (17)
To a solution of 2 (900 mg, 4.36 mmol) was added HCl (10 mL) at
rt and the reaction mixture was stirred at 30 ꢀC for 48 h. The re-
action mixture was diluted with water and extracted with EtOAc
(2 ꢁ 150 mL). The combined organic layers were concentrated
under reduced pressure to obtain the crude product which was
recrystallized using EtOAc to afford the title compound (690 mg,
3.08 mmol, 70%) as a white solid. Mp 188.1e189.4 ꢀC; 1H NMR
5.1.20. N2-(4-(Aminomethyl)-2-methoxy-5-methylphenyl)-5-
chloro-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine
(22)
(300 MHz, CDCl3)
d
7.75 (s, 1H), 6.98 (s, 1H), 5.53 (s, br, 1H), 5.37 (s,
To a solution of 21 (170 mg, 0.297 mmol) in THF (4.0 mL), MeOH
(2.0 mL) and H2O (2.0 mL) was added LiOH$H2O (62.4 mg,
1.49 mmol) at rt. The reaction mixture was stirred at rt for 12 h and
concentrated. The reaction was quenched with water (15 mL) and
extracted with EtOAc (2 ꢁ 35 mL). The combined organic layers
were concentrated under reduced pressure to obtain the crude
mixture which was purified by silica gel column chromatography
using MeOH/DCM (1/9) to afford the title compound (110 mg,
0.231 mmol, 78%) as an off white solid. Mp 91.6e93.8 ꢀC; 1H NMR
br, 1H), 3.97 (s, 3H), 3.65 (s, 2H), 2.33 (s, 3H); 13C NMR (125 MHz,
DMSO-d6)
d 171.35, 150.62, 143.22, 137.55, 129.83, 126.16, 116.62,
56.99, 40.64, 18.37; LC/MS m/z 224.9 [MþHþ].
5.1.16. (5-Methoxy-2-methyl-4-nitrophenyl)methanamine (18)
To a solution of 17 (600 mg, 2.68 mmol) in CH3CN/H2O (1/1) was
added [bis(trifluoroacetoxy)iodo]benzene (1.15 g, 2.67 mmol) at rt.
The reaction mixture was stirred at rt for 12 h, diluted with water
(5.0 mL) and NaHCO3 (aq), and extracted with EtOAc (2 ꢁ 75 mL).
The combined organic layers were concentrated under reduced
pressure to obtain the crude product which was purified by silica
gel column chromatography using MeOH/DCM (1/9) to afford the
title compound (400 mg, 2.04 mmol, 76%) as a pale yellow solid. 1H
(500 MHz, CDCl3)
d
9.53 (s, 1H), 8.59 (d, J ¼ 8.3 Hz, 1H), 8.18 (s, 1H),
8.05 (s, 1H), 7.95 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.66 (t, J ¼ 7.8 Hz, 1H), 7.54
(s, 1H), 7.32e7.25 (m, 1H), 6.92 (s, 1H), 3.92 (s, 3H), 3.85 (s, 2H), 3.28
(sept, J ¼ 6.8 Hz, 1H), 2.21 (s, 3H), 1.68 (s, br 2H), 1.34 (d, J ¼ 6.9 Hz,
6H); LC/MS m/z 475.9 [MþHþ]; HRMS (EI) m/z calcd for
NMR (300 MHz, CDCl3)
d
7.69 (s, 1H), 7.23 (s, 1H), 3.98 (s, 3H), 3.91
C
22H26ClN5O3S [Mþ] 475.1445, found 475.1438.
(s, 2H), 2.27 (s, 3H), 1.46 (s, br, 2H); LC/MS m/z 197.1 [MþHþ].
5.1.21. 5-Chloro-N2-(4-((dimethylamino)methyl)-2-methoxy-5-
methylphenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-
diamine (23)
5.1.17. 2,2,2-Trifluoro-N-(5-methoxy-2-methyl-4-nitrobenzyl)
acetamide (19)
To a solution of 18 (150 mg, 0.764 mmol) in DCM (15 mL) was
added trifluoroacetic anhydride (193 mg, 0.917 mmol) and Et3N
(193 mg, 1.91 mmol) dropwise 0 ꢀC. The reaction mixture was
stirred at rt for 30 min, quenched with water (15 mL), and extracted
with DCM (2 ꢁ 30 mL). The combined organic layers were washed
with water and brine solution, dried over anhydrous Na2SO4, and
concentrated under reduced pressure to obtain the crude product
which was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (150 mg,
To a solution of 22 (50.0 mg, 0.105 mmol) in MeOH (2.0 mL) was
added acetic acid (catalytic), formalin (35%) (19.8 mg, 0.051 mmol)
at 0 ꢀC. After 30 min, NaBH3CN (9.90 mg, 0.157 mmol) was added to
the mixture and stirred at rt for 30 min. The reaction mixture was
quenched with NaHCO3 (aq) and extracted with EtOAc (2 ꢁ 20 mL).
The combined organic layers were dried over anhydrous Na2SO4,
concentrated to obtain the crude mixture which was purified by
silica gel column chromatography using MeOH/DCM (1/9) to afford
the title compound (30.0 mg, 0.059 mmol, 57%) as a white solid. Mp
0.581 mmol, 76%) as a white solid. 1H NMR (300 MHz, CDCl3)
d
7.71
148.1e150.4 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.50 (s, 1H), 8.57 (d,
(s, 1H), 6.96 (s, 1H), 6.53 (s, br, 1H), 4.56 (d, J ¼ 5.9 Hz, 2H), 3.94 (s,
J ¼ 8.4 Hz, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.64
(t, J ¼ 7.8 Hz, 1H), 7.53 (s, 1H), 7.38e7.19 (m, 1H), 6.87 (s, 1H), 3.89 (s,
3H), 3.36 (s, 2H), 3.25 (sept, J ¼ 6.9 Hz, 1H), 2.27 (s, 6H), 2.19 (s, 3H),
1.31 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 503.9 [MþHþ]; HRMS (EI) m/z
calcd for C24H30ClN5O3S [Mþ] 503.1758, found 503.1732.
3H), 2.32 (s, 3H); LC/MS m/z 293.0 [MþHþ].
5.1.18. N-(4-Amino-5-methoxy-2-methylbenzyl)-2,2,2-
trifluoroacetamide (20)
To a solution of 19 (150 mg, 0.513 mmol) in MeOH (20 mL) was
added Pd/C (15.0 mg, 0.140 mmol) at rt. The reaction mixture was
stirred under a hydrogen balloon at rt for 2 h, filtered through a bed
of celite, and evaporated under reduced pressure. The crude
mixture was purified by silica gel column chromatography using
EtOAc/Hexane (3/7) to afford the title compound (110 mg,
5.1.22. 2-(5-Methoxy-2-methyl-4-nitrophenyl)-2-
methylpropanamide (24)
To a solution of 6 (500 mg, 2.13 mmol) in H2O (8.0 mL) was
added H2SO4 (7.0 mL) at rt. The reaction mixture was stirred at
100 ꢀC for 4 h and extracted with EtOAc (2 ꢁ 100 mL). The com-
bined organic layers were concentrated under reduced pressure to
obtain the crude product which was purified by silica gel column
0.419 mmol, 82%) as a white solid. 1H NMR (300 MHz, CDCl3)
(s, 1H), 6.58 (s, 1H), 6.25 (s, br, 1H), 4.45 (d, J ¼ 5.2 Hz, 2H), 3.85 (s,
d 6.68

546
G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
chromatography using MeOH/DCM (1/9) to afford the title com-
pound (420 mg, 1.66 mmol, 78%) as a yellow solid. 1H NMR
5.1.27. N2-(4-(2-Aminopropan-2-yl)-2-methoxy-5-methylphenyl)-
5-chloro-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine
(29)
(300 MHz, CDCl3)
d 7.70 (s, 1H), 7.14 (s, 1H), 5.33 (s, br, 1H), 5.18 (s,
br, 1H), 3.99 (s, 3H), 2.34 (s, 3H), 1.62 (s, 6H); LC/MS m/z 253.1
To a solution of 28 (73.0 mg, 0.121 mmol) in THF (4.0 mL), MeOH
(2.0 mL) and H2O (2.0 mL) was added LiOH$H2O (127 mg,
3.03 mmol) at rt. The reaction mixture was stirred at rt for 12 h and
concentrated. The reaction was quenched with water (5 mL) and
extracted with EtOAc (2 ꢁ 15 mL). The combined organic layers
were concentrated under reduced pressure to obtain the crude
mixture which was purified by silica gel column chromatography
using MeOH/DCM (1/9) to afford the title compound (25.0 mg,
0.049 mmol, 40%) as an off white solid. Mp 178.8e181.2 ꢀC; 1H NMR
[MþHþ].
5.1.23. 2-(5-Methoxy-2-methyl-4-nitrophenyl)propan-2-amine
(25)
To a solution of 24 (100 mg, 0.396 mmol) in CH3CN/H2O (1/1)
was
added
[bis(trifluoroacetoxy)iodo]benzene
(171
mg,
0.396 mmol) at rt. The reaction mixture was stirred at rt for 12 h,
diluted with water (5.0 mL) and NaHCO3 (aq), and extracted with
EtOAc (2 ꢁ 25 mL). The combined organic layers were concentrated
under reduced pressure to obtain the crude product which was
purified by silica gel column chromatography using MeOH/DCM (1/
9) to afford the title compound (80.0 mg, 0.356 mmol, 89%) as an off
(300 MHz, CDCl3)
d
9.50 (s, 1H), 8.57 (d, J ¼ 8.3 Hz, 1H), 8.16 (s, 1H),
8.00 (s, 1H), 7.92 (d, J ¼ 7.9 Hz, 1H), 7.64 (t, J ¼ 7.2 Hz, 1H), 7.49 (s,
1H), 7.31e7.22 (m, 1H), 7.15 (s, 1H), 3.89 (s, 3H), 3.26 (sept,
J ¼ 6.9 Hz, 1H), 2.43 (s, 3H), 1.76 (s, br, 2H), 1.58 (s, 6H), 1.32 (d,
J ¼ 6.9 Hz, 6H); LC/MS m/z 504.0 [MþHþ]; HRMS (EI) m/z calcd for
white solid. 1H NMR (300 MHz, CDCl3)
d 7.69 (s, 1H), 7.46 (s, 1H),
C
24H30ClN5O3S [Mþ] 503.1758, found 503.1758.
3.96 (s, 3H), 2.57 (s, 3H), 1.58 (s, 6H), 1.54 (s, br, 2H); LC/MS m/z
225.1 [MþHþ].
5.1.28. 5-Chloro-N2-(4-(2-(dimethylamino)propan-2-yl)-2-
methoxy-5-methylphenyl)-N4-(2-(isopropylsulfonyl)phenyl)
pyrimidine-2,4-diamine (30)
5.1.24. 2,2,2-Trifluoro-N-(2-(5-methoxy-2-methyl-4-nitrophenyl)
propan-2-yl)acetamide (26)
To a solution of 29 (10.0 mg, 0.019 mmol) in DMF (1.0 mL) was
added DIPEA (6.41 mg, 0.049 mmol) and CH3I (5.63 mg,
0.039 mmol) at rt and the reaction mixture was heated under mi-
crowave at 80 ꢀC for 10 min. The reaction was quenched with water
(10 mL) and extracted with EtOAc (2 ꢁ 15 mL). The combined
organic layers were washed with brine solution, concentrated to
obtain the crude mixture which was purified by silica gel column
chromatography using MeOH/DCM (1/9) to afford the title com-
pound (5.0 mg, 0.0090 mmol, 47%) as a pale yellow solid. Mp
To a solution of 25 (80.0 mg, 0.356 mmol) in DCM (5 mL) was
added trifluoroacetic anhydride (89.9 mg, 0.428 mmol) and Et3N
(90.1 mg, 0.892 mmol) dropwise 0 ꢀC. The reaction mixture was
stirred at rt for 30 min, quenched with water (5 mL), and extracted
with DCM (2 ꢁ 10 mL). The combined organic layers were washed
with water and brine solution, dried over anhydrous Na2SO4, and
concentrated under reduced pressure to obtain the crude product
which was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (95.0 mg,
141.9e144.4 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.50 (s, 1H), 8.59 (d,
0.296 mmol, 83%) as a white solid. 1H NMR (300 MHz, CDCl3)
d
7.69
J ¼ 8.3 Hz, 1H), 8.15 (s, 1H), 7.92 (d, J ¼ 8.3 Hz, 2H), 7.63 (t, J ¼ 7.9 Hz,
1H), 7.46 (s, 1H), 7.32e7.18 (m, 1H), 6.91 (s, 1H), 3.87 (s, 3H), 3.26
(sept, J ¼ 6.9 Hz, 1H), 2.47 (s, 3H), 2.16 (s, 6H), 1.38 (s, 6H), 1.31 (d,
J ¼ 6.9 Hz, 6H); LC/MS m/z 532.3 [MþHþ]; HRMS (EI) m/z calcd for
(s, 1H), 7.10 (s, 1H), 6.48 (s, br, 1H), 3.96 (s, 3H), 2.41 (s, 3H), 1.83 (s,
6H); LC/MS m/z 321.1 [MþHþ].
C
26H34ClN5O3S [Mþ] 531.2071, found 531.2070.
5.1.25. N-(2-(4-Amino-5-methoxy-2-methylphenyl)propan-2-yl)-
2,2,2-trifluoroacetamide (27)
5.1.29. N-(5-methoxy-2-methyl-4-nitrobenzyl)formamide (31)
A solution of 18 (250 mg, 1.27 mmol) in ethyl formate (10 mL)
was heated at 65 ꢀC for 4 h. The reaction mixture was concentrated
under reduced pressure to obtain the crude mixture which was
purified by silica gel column chromatography using MeOH/DCM (1/
9) to afford the title compound (150 mg, 0.669 mmol, 52%) as a
To a solution of 26 (95.0 mg, 0.296 mmol) in MeOH (10 mL) was
added Pd/C (10.0 mg, 0.094 mmol) at rt. The reaction mixture was
stirred under a hydrogen balloon at rt for 2 h, filtered through a bed
of celite, and evaporated under reduced pressure. The crude
mixture was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (54.0 mg,
0.186 mmol, 63%) as an off white solid. 1H NMR (300 MHz, CDCl3)
white solid. 1H NMR (300 MHz, CDCl3, rotameric mixture)
d 8.34 (s,
0.86H), 8.20 (d, J ¼ 11.8 Hz, 0.1H), 7.70 (s, 0.12H), 7.68 (s, 0.82H), 7.00
(s, 0.85H), 6.97 (s, 0.12H), 5.88 (s, 1H), 4.50 (d, J ¼ 6.0 Hz, 1.78H),
4.44 (d, J ¼ 6.6 Hz, 0.25H), 3.94 (s, 0.35H), 3.93 (s, 2.59H), 2.31 (s,
2.46H), 2.30 (s, 0.43H); LC/MS m/z 225.1 [MþHþ].
d
6.82 (s, 1H), 6.53 (s, 1H), 6.37 (s, br, 1H), 3.84 (s, 3H), 3.74 (s, 2H),
2.33 (s, 3H), 1.83 (s, 6H); LC/MS m/z 291.1 [MþHþ].
5.1.26. N-(2-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)propan-2-yl)-
2,2,2-trifluoroacetamide (28)
5.1.30. N-(4-Amino-5-methoxy-2-methylbenzyl) formamide (32)
To a solution of 31 (150 mg, 0.668 mmol) in MeOH (15 mL) was
added Pd/C (15.0 mg, 0.140 mmol) at rt. The reaction mixture was
stirred under a hydrogen balloon at rt for 12 h, filtered through a
bed of celite, and evaporated under reduced pressure. The crude
mixture was purified by silica gel column chromatography using
EtOAc/Hexane (2/3) to afford the title compound (110 mg,
0.566 mmol, 85%) as an off white solid. 1H NMR (300 MHz, CDCl3)
To a solution of 27 (54.0 mg, 0.186 mmol) in 0.08 M HCl in
ethoxyethanol (0.5 mL) was added 4 (77.3 mg, 0.223 mmol) at rt
and the reaction mixture was stirred at 80 ꢀC for 12 h. The reaction
mixture was then basified with sat. NaHCO3 (aq) and extracted with
EtOAc (2 ꢁ 25 mL). The combined organic layers were concentrated
under reduced pressure to obtain the crude mixture which was
purified by silica gel column chromatography using EtOAc/Hexane
(2/3) to afford the title compound (83.0 mg, 0.138 mmol, 74%) as a
d
8.20 (s, 1H), 6.67 (s, 1H), 6.54 (s, 1H), 5.54 (s, br, 1H), 4.37 (d,
J ¼ 5.3 Hz, 2H), 3.82 (s, 3H), 3.78 (s, br, 2H), 2.20 (s, 3H); LC/MS m/z
white solid. 1H NMR (500 MHz, CDCl3)
d 9.54 (s, 1H), 8.55 (d,
194.1 [MþHþ].
J ¼ 8.4 Hz, 1H), 8.20 (s, 1H), 8.09 (s, 1H), 7.96 (dd, J ¼ 7.9, 1.5 Hz, 1H),
7.64 (t, J ¼ 7.2 Hz, 1H), 7.54 (s, 1H), 7.37e7.23 (m, 1H), 6.94 (s, 1H),
6.43 (s, 1H), 3.93 (s, 3H), 3.28 (sept, J ¼ 6.9 Hz, 1H), 2.30 (s, 3H), 1.87
(s, 6H), 1.34 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 599.7 [MþHþ].
5.1.31. N-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
pyrimidin-2-yl)amino)-5-methoxy-2-methylbenzyl)formamide (33)
To a solution of 32 (100 mg, 0.514 mmol) in 0.08 M HCl in

G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
547
ethoxyethanol (1.0 mL) was added 4 (214 mg, 0.617 mmol) at rt and
5.1.35. N-(2-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)
the reaction mixture was stirred at 80 ꢀC for 12 h. The solid
precipitated in the reaction mixture was filtered and washed with
ethanol (2.0 mL). The filtered solid was then dissolved in EtOAc
(50 mL) and washed with sat. NaHCO3 (aq). The combined organic
layers were concentrated under reduced pressure to give the crude
mixture which was purified by silica gel column chromatography
using MeOH/DCM (1/9) to afford the title compound (110 mg,
0.218 mmol, 42%) as an off white solid. Mp 94.8e97.2 ꢀC; 1H NMR
pyrimidin-2-yl)amino)-5-methoxy-2-methylphenyl)propan-2-yl)
formamide (37)
To a solution of 36 (150 mg, 0.674 mmol) in 0.08 M HCl in
ethoxyethanol (1.0 mL) was added 4 (280 mg, 0.809 mmol) at rt and
the reaction mixture was stirred at 80 ꢀC for 12 h. The solid
precipitated in the reaction mixture was filtered and washed with
ethanol (2.0 mL). The filtered solid was then dissolved in EtOAc
(50 mL) and washed with sat. NaHCO3 (aq). The combined organic
layers were concentrated under reduced pressure to give the crude
mixture which was purified by silica gel column chromatography
using MeOH/DCM (1/9) to afford the title compound (197 mg,
0.371 mmol, 55%) as an off white solid. Mp 113.4e115.6 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
9.53 (s, 1H), 8.54 (d, J ¼ 8.3 Hz, 1H), 8.25 (s, 1H),
8.17 (s, 1H), 8.09 (s, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.64 (t, J ¼ 7.8 Hz,
1H), 7.53 (s, 1H), 7.29e7.24 (m, 1H), 6.78 (s, 1H), 5.60 (s, br, 1H), 4.44
(d, J ¼ 5.5 Hz, 2H), 3.88 (s, 3H), 3.25 (sept, J ¼ 6.9 Hz, 1H), 2.19 (s,
3H),1.31 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 503.6 [MþHþ]; HRMS (EI) m/
z calcd for C23H26ClN5O4S [Mþ] 503.1394, found 503.1398.
(300 MHz, CDCl3, rotameric mixture) d 9.52 (s, 0.6H), 9.50 (s, 0.3H),
8.53 (t, J ¼ 7.7 Hz,1H), 8.17 (s, 0.6H), 8.16 (s, 0.4H), 8.13 (s, 0.4H), 8.09
(s, 0.6H), 8.02 (d, J ¼ 6.6 Hz, 0.7H), 7.97 (s, 0.3H), 7.93 (d, J ¼ 8.0 Hz,
1H), 7.63 (t, J ¼ 7.9 Hz, 1H), 7.53 (s, 0.6H), 7.51 (s, 0.4H), 7.29e7.28
(m, 0.5H), 7.24e7.23 (m, 0.5H), 6.94 (s, 0.4H), 6.93 (s, 0.6H), 6.03 (d,
J ¼ 12.4 Hz, 0.6H), 5.63 (s, br, 0.4H), 3.90 (s, 1.9H), 3.89 (s, 1.1H), 3.26
(sept, J ¼ 6.8 Hz, 1H), 2.32 (s, 1H), 2.30 (s, 2H), 1.81 (s, 2H), 1.73 (s,
4H),1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 532.0 [MþHþ]; HRMS (EI) m/
z calcd for C25H30ClN5O4S [Mþ] 531.1707, found 531.1708.
5.1.32. 5-Chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-
5-methyl-4-((methylamino)methyl)phenyl)pyrimidine-2,4-diamine
(34)
To a solution of 33 (50 mg, 0.099 mmol) in THF (15 mL) was
added LiAlH4 (37.6 mg, 0.992 mmol) at 0 ꢀC. The reaction mixture
was stirred at 60 ꢀC for 12 h, quenched with water and NaOH (aq),
and filtered. The filtrate was extracted with EtOAc (2ꢁ) and the
combined organic layers were concentrated under reduced pres-
sure to obtain the crude mixture which was purified by silica gel
column chromatography using MeOH/DCM (1/9) to afford the title
compound (8.0 mg, 0.016 mmol,17%) as a pale yellow solid. 1H NMR
5.1.36. 5-Chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-
5-methyl-4-(2-(methylamino)propan-2-yl)phenyl)pyrimidine-2,4-
diamine (38)
To a solution of 37 (170 mg, 0.319 mmol) in THF (15 mL) was
added LiAlH4 (121 mg, 3.19 mmol) at 0 ꢀC. The reaction mixture was
stirred at 60 ꢀC for 12 h, quenched with water and NaOH (aq), and
filtered. The filtrate was extracted with EtOAc (2ꢁ) and the com-
bined organic layers were concentrated under reduced pressure to
obtain the crude mixture which was purified by silica gel column
chromatography using MeOH/DCM (1/9) to afford the title com-
pound (45.0 mg, 0.086 mmol, 27%) as a pale yellow solid. Mp
(300 MHz, CDCl3)
d
9.51 (s, 1H), 8.56 (d, J ¼ 8.4 Hz, 1H), 8.16 (s, 1H),
8.04 (s, 1H), 7.93 (d, J ¼ 6.7 Hz, 1H), 7.64 (t, J ¼ 7.9 Hz, 1H), 7.52 (s,
1H), 7.38e7.17 (m, 1H), 6.91 (s, 1H), 3.88 (s, 3H), 3.73 (s, 2H), 3.25
(sept, J ¼ 6.8 Hz,1H), 2.52 (s, 3H), 2.19 (s, 3H), 2.02 (s, br,1H),1.31 (d,
J ¼ 6.9 Hz, 6H); LC/MS m/z 490.1 [MþHþ]; HRMS (EI) m/z calcd for
C
23H28ClN5O3S [Mþ] 489.1601, found 489.1595.
170.2e172.4 ꢀC; 1H NMR (300 MHz, CDCl3)
d 9.51 (s, 1H), 8.58 (d,
J ¼ 8.3 Hz, 1H), 8.16 (s, 1H), 7.98 (s, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.63
(t, J ¼ 7.8 Hz,1H), 7.48 (s, 1H), 7.30e7.20 (m, 1H), 6.93 (s, 1H), 3.88 (s,
3H), 3.26 (sept, J ¼ 6.8 Hz, 1H), 2.41 (s, 3H), 2.17 (s, 3H), 1.75 (s, br,
1H), 1.52 (s, 6H), 1.32 (d, J ¼ 6.9 Hz, 6H); LC/MS m/z 520.0 [MþHþ];
HRMS (EI) m/z calcd for C25H32ClN5O3S [Mþ] 517.1914, found
517.1915.
5.1.33. N-(2-(5-methoxy-2-methyl-4-nitrophenyl)propan-2-yl)
formamide (35)
A solution of 25 (250 mg, 1.11 mmol) in ethyl formate (10 mL)
was heated at 65 ꢀC for 12 h. The reaction mixture was concen-
trated under reduced pressure to obtain the crude mixture which
was purified by silica gel column chromatography using MeOH/
DCM (1/9) to afford the title compound (220 mg, 0.872 mmol, 78%)
5.2. In vitro enzyme assays
as a white solid. 1H NMR (300 MHz, CDCl3, rotameric mixture)
d 8.15
Experimental procedure was followed by the manufactured
instruction (Cisbio, France). The reaction was initiated by ATP
addition to a mixture containing the kinases, peptide substrates,
and inhibitors. After 30 min, EDTA containing solution was added to
stop the reaction mixture. EDTA containing solution has europium
conjugated anti-phospho residue antibody and streptavidin-XL665
(SA-XL665) for the detection of the phosphorylated peptide prod-
uct. After 1 h incubation, fluorescence was measured with excita-
tion at 337 nm and dual emission at 665 and 620 nm of the Envision
reader. IC50 was calculated using GraphPad Prism version 5 for
Windows. The curves were fit using a nonlinear regression model
with a log (inhibitor) versus response formula.
(s, 0.6H), 7.99 (d, J ¼ 12.4 Hz, 0.3H), 7.74 (s, 0.4H), 7.70 (s, 0.6H), 7.15
(s, 1H), 6.09 (d, J ¼ 12.4 Hz, 0.3H), 5.73 (s, br, 0.6H), 3.99 (s, 1H), 3.98
(s, 2H), 2.50 (s, 1H), 2.47 (s, 2H), 1.80 (s, 4H), 1.79 (s, 2H); LC/MS m/z
253.1 [MþHþ].
5.1.34. N-(2-(4-Amino-5-methoxy-2-methylphenyl)propan-2-yl)
formamide (36)
To a solution of GM-15-029 (35) (220 mg, 0.872 mmol) in MeOH
(25 mL) was added Pd/C (25.0 mg, 0.234 mmol) at rt. The reaction
mixture was stirred under a hydrogen balloon at rt for 12 h, filtered
through a bed of celite, and evaporated under reduced pressure.
The crude mixture was purified by silica gel column chromatog-
raphy using EtOAc/Hexane (2/3) to afford the title compound
(170 mg, 0.764 mmol, 87%) as an off white solid. 1H NMR (300 MHz,
5.3. Cell cytotoxicity assays
For viability experiments, Ba/F3 cells were tested by WST-1
assay. Cells were seeded in 96-well plates at 30% confluency and
exposed to chemicals the next day. After 72 h, WST-1 reagent was
added and absorbance at 450 nm was measured on a Spectramax
spectrophotometer (Molecular Devices, US) according to the
manufacturer's instructions. H3122, Ba/F3 L1196M, and Ba/F3
CDCl3, rotameric mixture)
d
8.11 (s, 0.3H), 7.99 (d, J ¼ 12.4 Hz, 0.7H),
6.84 (s, 0.3H), 6.82 (s, 0.7H), 6.54 (s, 0.7H), 6.52 (s, 0.3H), 5.90 (d,
J ¼ 12.6 Hz, 0.7H), 5.56 (s, 0.3H), 3.84 (s, 3H), 3.73 (s, br, 2H), 2.37 (s,
1H), 2.33 (s, 2H), 1.81 (s, 1.9H), 1.69 (s, 4.1H); LC/MS m/z 223.2
[MþHþ].

548
G.R. Mathi et al. / European Journal of Medicinal Chemistry 126 (2017) 536e549
EML4-ALK wild-type were tested by SRB (SulfoRhodamine B) assay.
Cells were seeded in 96-well plates at 30% confluency and exposed
to chemicals the next day. After 72 h, cells were fixed with 10%
CCl3CO2H and stained with 0.05% SRB solutions. Then, 10 mM Tris
base was added to solubilize the protein-bound SRB dye. Absor-
bance at 564 nm was measured on each well. IC50 and curve fitting
were performed at the methods described above.
using Mann-Whitney U test (a ¼ 0.01).
Acknowledgements
This work was supported by Korea Research Institute of
Chemical Technology (SI-1607-01) and by the National Research
Foundation of Korea (NRF-2012M3A9A9054902).
5.4. Molecular docking
Appendix A. Supplementary data
€
A flexible docking study was performed using the Schrodinger
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.046.
€
Glide v.7.0 program with standard precision settings (Schrodinger,
LLC, http://www.schrodinger.com). The x-ray crystal structure of
the human wild type ALK (PDB code 4MKC) and L1196M mutant
ALK (PDB code 2YFX) were obtained from the Protein Data Bank
(http://www.rcsb.org). The G1202R mutant ALK model was built
using 4MKC PDB as a template. The ligands were minimized using a
MMFF force field with a dielectric constant of 80.0 using the Mac-
roModel v.11.1 program. The binding models were visualized using
Discovery Studio 2016 (Biovia, http://www.accelrys.com).
References
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5.5. PK studies
In vivo PK of compounds was examined in male rats (6e7 weeks,
250e280 g). The rats were cannulated with polyethylene tubing
(PE-50, Intramedic, BD Bioscience, MD) in the femoral vein under
anesthesia by a mixture of zoletil and rompun solution (3:1, 1 mL/
kg). Animals were fasted overnight after the cannulation surgery.
Then, the test compound was administered orally using oral
gavage. Dosing vehicles were composed of 5% DMSO and 40%
PEG400 in water and dosing volume was 2 mL/kg in p.o. adminis-
tration. Blood samples were collected at different time points (n ¼ 3
rats per time point) from the femoral vein. After centrifugation
(13000 g, 3 min, 37 ꢀC), the plasma samples were obtained and
stored at ꢂ20 ꢀC until the analysis. The area under the plasma
concentration-time curve (AUC∞) was calculated by the trapezoidal
rule with extrapolation to time infinity. The maximum plasma
concentration (Cmax) was obtained by visual inspection of the
plasma concentration-time curve. The concentrations of test com-
pounds were determined by LC/MS/MS on an Agilent 1200 HPLC
system coupled to an Agilent 6460 triple quadrupole mass spec-
trometer equipped with ESI source (Agilent, Santa Clara, CA, USA)
using imipramine as an internal standard. The peak areas for all
components were automatically integrated using Agilent 6460
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