Design, synthesis of orally bioavailable novel anaplastic lymphoma kinase (ALK) inhibitor diphenylaminopyrimidine analogs and efficacy study on NCI-H2228 xenografts mice model

Article abstract of DOI:10.1016/j.bmcl.2019.04.012

Anaplastic lymphoma kinase (ALK) is an attractive therapeutic target for the treatment of non-small cell lung cancer (NSCLC). Within our ALK drug discovery program, we identified novel deuterated 2,4-diarylamino pyrimidine compounds as potent ALK inhibitors. The compound 11 showed better in vitro activity and in vivo efficacy with good pharmacokinetic profile. In vivo efficacy of compound 11 was better than standard drug ceritinib in NCI-H2228 xenograft mice model.

Full text of DOI:10.1016/j.bmcl.2019.04.012

Accepted Manuscript
Design, synthesis of orally bioavailable novel anaplastic lymphoma kinase
(ALK) inhibitor diphenylaminopyrimidine analogs and efficacy study on NCI-
H2228 xenografts mice model
Debasis Das, Jingbing Wang, Yong Li, Jingli Shi, Jian Hong
PII:
S0960-894X(19)30221-5
https://doi.org/10.1016/j.bmcl.2019.04.012
BMCL 26379
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 November 2018
29 March 2019
6 April 2019
Please cite this article as: Das, D., Wang, J., Li, Y., Shi, J., Hong, J., Design, synthesis of orally bioavailable novel
anaplastic lymphoma kinase (ALK) inhibitor diphenylaminopyrimidine analogs and efficacy study on NCI-H2228
xenografts mice model, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.04.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis of orally bioavailable novel anaplastic lymphoma kinase
(ALK) inhibitor diphenylaminopyrimidine analogs and efficacy study on
NCI-H2228 xenografts mice model
Debasis Das*, Jingbing Wang, Yong Li, Jingli Shi, Jian Hong*
Discovery Chemistry Research, Arromax Pharmatech Co. Ltd.
Sangtian Island Innovation Park, No. 1 Huayun Road, SIP, Suzhou 215123, P. R. China.
*Corresponding authors:
Email: debasis.das@arromax.com (D. Das) & eric.hong@arromax.com (J. Hong).
Abstract
Anaplastic lymphoma kinase (ALK) is an attractive therapeutic target for the treatment of
non-small cell lung cancer (NSCLC). Within our ALK drug discovery program, we
identified novel deuterated 2,4-diarylamino pyrimidine compounds as potent ALK inhibitors.
The compound 11 showed better in vitro activity and in vivo efficacy with good
pharmacokinetic profile. In vivo efficacy of compound 11 was better than standard drug
ceritinib in NCI-H2228 xenograft mice model.
Key words: Anaplastic lymphoma kinase, ALK, non-small cell lung cancer, NSCLC, kinase,
inhibitor, ceritinib, deuterated drug

Graphical Abstract
Cl
N
Cl
N
HN
N
NH
O
HN
N
NH
O
S
O
O
O
S
O
D3C
N
H
N
H
Ceritinib
11
IC50 = 4.0 nM (NCI-H3122)
IC50 = 41.3 nM (NCI-H2228)
IC50 = 27.2 nM (NCI-H3122)
IC50 = 97.0 nM (NCI-H2228)

Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor tyrosine kinase
super family. Currently more than twenty ALK fusion proteins arisen from different
chromosomal rearrangements have been found, which are involved in the development of
anaplastic large cell lymphoma, diffuse large B-cell lymphoma, inflammatory
myofibroblastic tumor, neuroblastoma, and so on. AlK is a well validated target based on
preclinical and clinical studies.1 Activated, oncogenic form of ALK has been first found in a
subset of anaplastic large cell lymphoma (ALCL), namely the chimeric protein NPM-ALK.
Subsequently, additional ectopically expressed, activated fusion forms of ALK have been
identified and recognized as potent oncogenic drivers in other tumor types such as
inflammatory myofibroblastic tumors (IMT). ALK fusion proteins play a fundamental role
in the development of approximately 5-10% of non-small cell lung cancers (NSCLC).2 The
majority of ALK-positive NSCLC harbors the same intrachromosomal rearrangement that
results in the expression of EML4−ALK chimeric protein, generated from the fusion of the
N-terminal portion of echinoderm microtubule- associated protein-like-4 (EML4) and the
ALK kinase domain.3
Cl
N
NH2
O
N
HN
N
NH
O
O
N
N
O
S
Cl
Cl
Crizotinib
Ceritinib (LDK378)
HN
F
Cl
NH
N
H
N
O
N
HN
N
N
O
P
H
O
N
N
NC
O
Alectinib (CH5424802)
Brigatinib (AP26113)
N
N
Figure 1. ALK inhibitor drugs

Crizotinib (PF2341066, Pfizer) was designed and developed as a c-Met/ALK dual
inhibitor and it showed good antitumor efficacy in ALK-dependent xenograft models.4 In
2011, Crizotinib (Xalkori) was approved by FDA as the first ALK inhibitor for treating non-
small cell lung cancer (Figure 1).5 Crizotinib demonstrated response rate of about 60% and a
median progression free survival of approximately 8 to 11 months. After the initial dramatic
responses, however, the patients eventually developed drug resistance.6 These resistance
mechanisms include secondary ALK kinase domain mutations,7 bypass downstream
signalling,8 ALK gene amplification.9 The mutations were observed within a year at ALK
kinase domain, including L1196M, F1174L, C1156Y, S1206Y, G1269A, G1202R after
crizotinib treatment.10
To overcome mutation limitations, several 2,4-diarylamino pyrimidine analogs (DAAPs)
are explored extensively. Novel ALK inhibitors such as alectinib (CH5424802),11 brigatinib
(AP26113),12 ceritinib (LDK378)13 have been studied. These second-generation ALK kinase
inhibitor drugs are selective ALK kinase inhibitors for treating cancers, cell proliferative
disorders and other related diseases. These pyrimidine ALK inhibitors reportedly retain
potency over crizotinib-resistant ALK mutants. Either a sulfone or phosphine oxide at the
aniline ortho position of the C4 pyrimidine inhibitors gives a selective hydrogen interaction
to ALK Lys1150. Ceritinib and alectinib14 have been approved by FDA for the treatment of
patients with ALK-specific non-small cell lung cancer who are resistant to crizotinib.
Although the above listed commercially available ALK kinase inhibitors can alleviate the
problems of treatment of ALK-specific lung cancer and drug resistance therein to some
extent, there are still problems of insufficient activity and efficacy, which result in a large
dose of these drugs during use.
Deuterated compounds have been studied to improve metabolic stability or
pharmacokinetic (PK) profiles.15,16 The incorporation of deuterium into pharmacologically
active agents offers potential benefits, such as improved exposure profiles and the decreased
production of toxic metabolites that could yield improvements in efficacy, tolerability, or
safety.16,17 Deutetrabenazine, the first deuterated drug got approval by FDA recently.18 As a
part of our ongoing NSCLC research, our group reported the efficacy studies of tyrosine
kinase inhibitors on mice model.19
We identified novel deuterated diphenylamino-
pyrimidine derivatives as ALK kinase inhibitors.20 In this article, we have discussed the
synthesis of deuteratd alkoxy derivatives and the in vivo efficacy study on mice NSCLC
xenografts.

The synthesis of deuterated diphenylaminopyrimidine 11 is shown in Scheme 1. In
presence of variety of bases (eg, K2CO3, Cs2CO3, NaH) deutertaed methanol was reacted
with fluoronitro compound 2 which was easily prepared form commercially available 1 by
nitration reaction, to give compound 3 in good yield (63-77%). The Suzuki reaction of 3 with
4-pyridine boronic acid led to 4, followed by hydrogenation of pyridine ring gave the amine
compound 5 in god yield (70%). Sulfonyl amine 7 was reacted with trichloropyrimidine 8 to
give the intermediate 9 which was allowed to react with compound 6 to give intermediate 10.
It was then deprotected to give compound 11 in good yield (97%). The syntheses of
compounds 12 - 16 were achieved following the similar synthetic scheme and known
literature methods. The formations of the compounds were characterized by 13CNMR and
mass spectra analysis. The incorporation of D3CO- unit in 11 was clearly detected by careful
analysis of 13C NMR. Detection septet peaks of D3CO-group in DMSO-d6 solvent were quite
13
difficult. The expected splitting of D3CO- peak was observed on C NMR at  54.97 ppm
and J = 21.7 Hz in CDCl3 solvent after overnight scanning of the sample.21

N
Cl
Cl
Cl
c
a
b
O2N
O2N
O2N
O
O
F
1
F
2
CD3
CD3
3
4
NH
NBoc
d
e
H2N
H2N
O
O
CD3
CD3
5
6
Cl
N
Cl
+
g
N
f
N
N
Cl
NH2
H
Cl
N
Cl
S O
O
S O
O
7
8
9
Cl
Cl
NH
N
N
h
HN
N
NH
HN
N
O
S
O
S
O
O
O
O
D3C
D3C
11
N
Boc
N
H
10
Scheme 1. Reagent and conditions: (a) HNO3/H2SO4, 0-5oC, 80%;
(b) K2CO3/CD3OD, DMSO, 50-60oC, 77%; (c) 4-pyridineboronic
acid, Pd2(dba)3, K3PO4, dioxane, water, reflux, 69%; (d) PtO2/H2,
AcOH, 70%; (e) Boc2O,TEA, DCM; (f) NaH, 60%, DMSO, (g) 6,
Pd(OAc)2, Xantphos, Cs2CO3, THF, 69%; (h) TFA, DCM, rt, 2h, 97%.
Firstly we studied some selected compounds for in cell based assay on lung cancer cell lines
and observed promising results. The results are summarized in Table 1. We found that the
deturated alkoxy compounds showed better activity in cell models. Compound 11 and 12
were better than standard drug ceritinib and compound 11 showed best among the deuterated
alkoxy compounds. Compound 13 with deuterated isopropyl group was inferior to compound
11. Compound 11 was very potent to cell line H3122 (IC50 = 4.0 nM). The deuturated
substitution on the aromatic ring (compound 14 and 15) did not help to improve the activity.
Both the compounds 14 and 15 showed similar result to ceritinib.

Table 1. In vitro cell assay
Cl
Cl
Cl
N
N
N
HN
N
NH
O
S
HN
N
D
NH
O
HN
N
NH
O
S
O
O
R
R
S
R
O
D
15
14
11 -13, 16
N
H
N
H
N
H
Compounds
R
NCI-H2228
IC50 (nM)
41.3
NCI-H3122
IC50 (nM)
4.0
11
-OCD3
-OCD2CD3
12
45.5
6.0
13
-OCD(CD3)2
-OCH(CH3)2
-OCH(CH3)2
-OCH(CH3)2
111.3
97.0
28.4
14
15
28.5
98.5
24.7
Ceritinib (16)
97.0
27.2
The deuterated alkoxy compounds 11, 12, 13 were tested against three enzymes ALK
wild type (ALKwt) and mutant type (ALKF1196M and ALKF1174L) and compared with standard
drug ceritinib (Table 2). In vitro enzyme activity of compound 11, 12 were better in wild type
enzyme and mutant variations. Compound 11 is potent to ALK wt. and mutants in
biochemical enzyme assay (ALKwt IC50 = 71 nM, ALKL1196M IC50 = 18.72 nM, ALK F1174L
IC50 = 36.81 nM).
The compound 11 showed a better tumor growth inhibition in vivo activity in H2228 human
lung cancer bearing mice model comparable to ceritinib without significant body weight
change (Figure 2). In vivo efficacy evaluation of 11 was conducted on a 2-week NCI-H2228
(EML4-ALK NSCLC) xenografts model.22 A dose-dependent tumor growth inhibition was
observed when the animals were dosed 11 orally once daily for two weeks. Our compound 11
exhibited better antitumor inhibition at middle dose (the relative tumor growth inhibition rate
= 53% at 10 mg/kg per day for 14 days) and showed better efficacy than standard drug
ceritinib (the relative tumor growth inhibition rate =26%) at same dose. When the animals
were dosed at 30 mg/kg for 14 days with 11, the relative tumor growth inhibition rate = 77%

on day 14 without obvious body weight loss. The compound 11 displayed very potent in vivo
target inhibition in mice xenograft model. The compound 11 showed suitable
pharmacokinetic parameters, good bioavailability = 60%. Tmax = 6-8h, Cmax = 508.42±
63.73mg/L, AUC0-t 6979 ± 1220.18ng/mL*hr.
Table 2. In vitro biochemical activity
Compound ID
ALK Wild
IC50 (nM)
71.0
ALK F1196M
IC50 (nM)
18.72
ALK F1174L
IC50 (nM)
36.81
11
12
13
106.3
36.98
51.62
169.8
63.42
109.3
Ceritinib (16)
198
47.65
114.3

Figure 2. H2228 xenograft in mice
In summary, the deuterated alkoxy-piperazinesubstituted-2,4-dianilinopyrimidines is
described and their activity study to an aplastic lymphoma kinase (ALK) is discussed. The
compound 11 is selective and potent to ALK. Compound 11 is potent inhibitor to ALK wt.
and mutants in biochemical enzyme assay. Its molecular weight is less than ceritinib. It is
more active than ceritinib in vitro in both enzyme and cell assay. It also demonstrated better
efficacy than standard drug ceritinib in vivo lung cancer xenograft mice model. As per the
results, compound 11 is a suitable drug candidate for further development.
Acknowledgments
The authors are thankful to Shanghai Medicilon Inc. for providing full biological tests
services.
References
1. Mosse YP, Wood A, Maris JM. Inhibition of ALK Signaling for Cancer Therapy.
Clinical Cancer Research. 2009;15(18):5609-5614. doi:10.1158/1078-0432.CCR-08-
2762
2. Shaw AT, Engelman JA. ALK in lung cancer: past, present, and future. J Clin Oncol.
2013;31(8):1105-1111. doi:10.1200/JCO.2012.44.5353

3. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4–ALK
fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153):561-566.
doi:10.1038/nature05945
4. Cui JJ, Tran-Dubé M, Shen H, et al. Structure Based Drug Design of Crizotinib (PF-
02341066), a Potent and Selective Dual Inhibitor of Mesenchymal–Epithelial Transition
Factor (c-MET) Kinase and Anaplastic Lymphoma Kinase (ALK). Journal of Medicinal
Chemistry. 2011;54(18):6342-6363. doi:10.1021/jm2007613
5. Cui JJ, McTigue M, Kania R, Edwards M. Case History. In: Annual Reports in Medicinal
Chemistry. Vol 48. Elsevier; 2013:421-434. doi:10.1016/B978-0-12-417150-3.00025-9
6. Perez CA, Velez M, Raez LE, Santos ES. Overcoming the resistance to Crizotinib in
patients with Non-Small Cell Lung Cancer harboring EML4/ALK translocation. Lung
Cancer. 2014;84(2):110-115. doi:10.1016/j.lungcan.2014.02.001
7. Doebele RC, Pilling AB, Aisner DL, et al. Mechanisms of resistance to crizotinib in
patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res.
2012;18(5):1472-1482. doi:10.1158/1078-0432.CCR-11-2906
8. Sasaki T, Koivunen J, Ogino A, et al. A novel ALK secondary mutation and EGFR
signaling cause resistance to ALK kinase inhibitors. Cancer Res. 2011;71(18):6051-6060.
doi:10.1158/0008-5472.CAN-11-1340
9. Kim S, Kim TM, Kim D-W, et al. Heterogeneity of genetic changes associated with
acquired crizotinib resistance in ALK-rearranged lung cancer. J Thorac Oncol.
2013;8(4):415-422. doi:10.1097/JTO.0b013e318283dcc0
10. Gainor JF, Shaw AT. Emerging paradigms in the development of resistance to tyrosine
kinase inhibitors in lung cancer.
doi:10.1200/JCO.2012.45.2029
J
Clin Oncol. 2013;31(31):3987-3996.
11. Kinoshita K, Asoh K, Furuichi N, et al. Design and synthesis of a highly selective, orally
active and potent anaplastic lymphoma kinase inhibitor (CH5424802). Bioorganic &
Medicinal Chemistry. 2012;20(3):1271-1280. doi:10.1016/j.bmc.2011.12.021
12. Huang W-S, Liu S, Zou D, et al. Discovery of Brigatinib (AP26113), a Phosphine Oxide-
Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. Journal of
Medicinal Chemistry. 2016;59(10):4948-4964. doi:10.1021/acs.jmedchem.6b00306
13. Marsilje TH, Pei W, Chen B, et al. Synthesis, Structure–Activity Relationships, and in
Vivo Efficacy of the Novel Potent and Selective Anaplastic Lymphoma Kinase (ALK)
Inhibitor 5-Chloro- N 2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)- N 4-(2-
(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (LDK378) Currently in Phase 1 and
Phase 2 Clinical Trials. Journal of Medicinal Chemistry. 2013;56(14):5675-5690.
doi:10.1021/jm400402q
14. Sakamoto H, Tsukaguchi T, Hiroshima S, et al. CH5424802, a Selective ALK Inhibitor
Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell. 2011;19(5):679-690.
doi:10.1016/j.ccr.2011.04.004

15. Timmins GS. Deuterated drugs: where are we now? Expert Opinion on Therapeutic
Patents. 2014;24(10):1067-1075. doi:10.1517/13543776.2014.943184
16. Harbeson SL, Tung RD. Deuterium in Drug Discovery and Development. In: Annual
Reports in Medicinal Chemistry. Vol 46. Elsevier; 2011:403-417. doi:10.1016/B978-0-
12-386009-5.00003-5
17. Chen P, Ren S, Song H, et al. Synthesis and biological evaluation of BMS-986120 and its
deuterated derivatives as PAR4 antagonists. Bioorganic & Medicinal Chemistry.
2019;27(1):116-124. doi:10.1016/j.bmc.2018.11.024
18. Raffa RB, Pergolizzi JV, Taylor R. The First Approved “Deuterated” Drug: A Short
Review of the Concept. Pharmacology & Pharmacy. 2018;09(10):440-446.
doi:10.4236/pp.2018.910033
19. Das D, Xie L, Wang J, et al. Discovery of new quinazoline derivatives as irreversible
dual EGFR/HER2 inhibitors and their anticancer activities – Part 1. Bioorganic &
Medicinal Chemistry Letters. 2019;29(4):591-596. doi:10.1016/j.bmcl.2018.12.056
20. Hong J., Liu G, Wang J, Diphenylaminopyrimidine and triazine compound, and
pharmaceutical composition and use thereof. WO 2018 018986
21. Synthesis of compound 11: Compound 10 (2.1 g, 3.3 mmol) was added to a solution of
dichloromethane (10 mL) and trifluoroacetic acid (10 mL), and stirred at room
temperature for 2 hrs. After the reaction was complete as monitored by TLC, the organic
solvent was removed by evaporation, and the residue was dissolved in dichloromethane
(100 mL), washed with a saturated sodium carbonate solution, dried, filtered and
concentrated, to obtain a free amine. The free amine product was dissolved in
dichloromethane (20 mL), a hydrogen chloride/ethanol solution (4 M, 10 mL) was added,
and then the resulting solution was stirred at room temperature for 2 hrs. A white solid
was precipitated, filtered under suction, and dried to obtain compound 11 (1.8 g, 95.2%).
1H NMR (400 MHz, DMSO-d6) δ = 8.32 (s, 1H), 8.27 (d, J = 7.2Hz, 1H), 7.88 (d, J = 7.2
Hz, 1H), 7.67 (dd, J = 7.2 Hz, 7.0 Hz, 1H), 7.45 (dd, J = 7.2 Hz, 7.0 Hz, 1H), 7.42 (s,
1H), 6.79 (s, 1H), 3.49−3.32 (m, 3H), 3.10−2.91 (m, 3H), 2.09 (s, 3H), 1.89−1.77 (m,
4H), 1.13 (d, 6H); 13C NMR (100MHz, CDCl3)  157.5, 155.2, 146.8, 138.5, 138.4,
134.7, 131.1, 126.5, 126.1, 124.6, 123.5, 123.0, 120.9, 107.8, 105.7, 55.4, 54.9 (Sep, J =
21.7 Hz), 47.3, 38.6, 33.7, 18.8, 15.3 LC-MS: m/z= 533.3 (M+1).
22. In vivo efficacy was conducted as following procedure: SCID Beige mice inoculated with
H2228 were randomized into five groups (8 mice per group) with an average tumor
volume of around 200 mm3. The day of grouping was recorded as Day 0, and the animals
were administered immediately after grouping. Compound 11 and ceritinib were orally
administered once a day for consecutive 14 days, and then observed for 7 days. The
weight and tumor size of the animals were measured twice a week during the experiment.
The clinical symptoms were observed and recorded daily. After the animals were
weighed for the last time on Day 21, they were sacrificed by euthanization with CO2.
The tumor volume and relative tumor volume were calculated based on the measured
data. The tumor volume (TV) was calculated by a formula below: TV=0.5 a x b2, where
a and b represent the major and minor diameters of the tumor, respectively. According to
the measurement results, the relative tumor volume (RTV) was calculated, by a formula
below: RTV=Vt/V0, where V0 is the tumor volume at the start of the experiment, and Vt

is the tumor volume measured each time. The evaluation indices for the anti-tumor
activity were the relative tumor growth rate T/C (%), which was calculated by a formula
below: T/C(%) = TRTV/ CRTV×100%, where TRTV is the RTV of the treatment group
and CRTV is the RTV of the negative control group; and the relative tumour growth
inhibition rate (tumour inhibition rate) (%) =(1-T/C)×100%.

Table 1. In vitro cell assay
Cl
Cl
Cl
N
N
N
HN
N
NH
O
S
HN
N
D
NH
O
HN
N
NH
O
S
O
O
O
R
R
S
R
D
15
14
11 -13, 16
N
H
N
H
N
H

Compounds
R
NCI-H2228
IC50 (nM)
41.3
NCI-H3122
IC50 (nM)
4.0
11
-OCD3
12
-OCD2CD3
-OCD(CD3)2
-OCH(CH3)2
-OCH(CH3)2
-OCH(CH3)2
45.5
6.0
13
111.3
97.0
28.4
14
15
28.5
98.5
24.7
Ceritinib (16)
97.0
27.2
Table 2. In vitro biochemical activity
Compound ID
ALK Wild
ALK F1196M ALK F1174L
IC50 (nM)
71.0
IC50 (nM)
18.72
IC50 (nM)
36.81
11
12
13
106.3
169.8
198
36.98
51.62
63.42
109.3
Ceritinib (16)
47.65
114.3
Highlights
 Synthesis of novel deuterated diphenylaminopyrimidine analogs as ALK inhibitors.
 In vitro evaluation of anti-proliferation activity on NCI-H2228 NCI-H3122 .
 Identified ALK inhibitors in nanomolar (nM) concentration.
 In vivo efficacy of 11 was better than ceritinib on mice NCI H2228 xenograft tumor
model.

Graphical Abstract
Cl
N
Cl
N
HN
N
NH
O
HN
N
NH
O
S
O
O
O
S
O
D3C
N
H
N
H
Ceritinib
11
IC50 = 4.0 nM (NCI-H3122)
IC50 = 41.3 nM (NCI-H2228)
IC50 = 27.2 nM (NCI-H3122)
IC50 = 97.0 nM (NCI-H2228)