Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic lymphoma kinase (ALK): Synthesis, in vitro, ex vivo, and in vivo efficacy studies

Article abstract of DOI:10.1016/j.bmcl.2016.02.052

A series of novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety were synthesized and evaluated for their anti-anaplastic lymphoma kinase (ALK) activities using enzymatic and cell-based assays. Among the compounds synthesized, compound 17b showed promising pharmacological results in in vitro, ex vivo, and pharmacokinetic studies. An in vivo efficacy study with compound 17b demonstrated highly potent inhibitory activity in H3122 tumor xenograft model mice. A series of kinase assays showed that compound 17b inhibited various kinases including FAK, ACK1, FGFR, RSK1, IGF-1R, among others, thus demonstrating its potential for synergistic anti-tumor activity and development as a multi-targeted non-small cell lung cancer (NSCLC) therapy.

Full text of DOI:10.1016/j.bmcl.2016.02.052

Accepted Manuscript
Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against
anaplastic lymphoma kinase (ALK): synthesis, in vitro, ex vivo, and in vivo
efficacy studies
Dawn Song, Minji Lee, Chi Hoon Park, Sunjoo Ahn, Chang Soo Yun, Chong
Ock Lee, Hyoung Rae Kim, Jong Yeon Hwang
PII:
S0960-894X(16)30164-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.052
BMCL 23606
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 October 2015
5 February 2016
19 February 2016
Please cite this article as: Song, D., Lee, M., Park, C.H., Ahn, S., Yun, C.S., Lee, C.O., Kim, H.R., Hwang, J.Y.,
Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic lymphoma kinase (ALK):
synthesis, in vitro, ex vivo, and in vivo efficacy studies, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.02.052
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Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic
lymphoma kinase (ALK):synthesis, in vitro, ex vivo, and in vivo efficacy studies
b
c
Dawn Songa, Minji Lee , Chi Hoon Park c, d, Sunjoo Ahnc, d, Chang Soo Yun c, d, Chong Ock Lee ,
Hyoung Rae Kim c*, and Jong Yeon Hwang c, d*
c College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28644, Korea
b Department of Chemistry, Korea University, Seoul 02841, Korea
c Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114,
Korea
d
Department of Medicinal Chemistry and Pharmacology, University of Science & Technology,
Daejeon 34113, Korea
* Corresponding author. Tel: +82-42-860-7045; Email: hyngrk@krict.re.kr; jyhwang@krict.re.kr
Abstract
A series of novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety were synthesized
and evaluated for their anti-anaplastic lymphoma kinase (ALK) activities using enzymatic and cell-
based assays. Amongst the compounds synthesized, compound 17b showed promising
pharmacological results in in vitro, ex vivo, and pharmacokinetic studies. An in vivo efficacy study
with compound 17b demonstrated highly potent inhibitory activity in H3122 tumor xenograft model
mice. A series of kinase assays showed that compound 17b inhibited various kinases including FAK,
ACK1, FGFR, RSK1, IGF-1R, among others, thus demonstrating its potential for synergistic anti-
tumor activity and development as a multi-targeted non-small cell lung cancer (NSCLC) therapy.
Keywords
Anaplastic lymphoma kinase, cancer, 2,4-diaminopyrimidine, tetrahydronaphthalenyl

Anaplastic lymphoma kinase (ALK) belongs to the insulin receptor family of receptor tyrosine
kinases (RTK) and is responsible for many cancers including anaplastic large-cell lymphomas
(ALCL), diffuse large B-cell lymphoma (DLBCL), inflammatory myofibroblastic tumors (IMT), and
a variety of solid tumors [1-4]. ALK gene is fused to various partner genes, such as NPM-ALK,
EML4-ALK, KIF5-ALK, and the fusion genes are detected in approximately 3-7 % of non-small cell
lung cancer (NSCLC) in humans [4]. The constitutive kinase activity associated with ALK fusions
seems to play an essential role in the growth and survival of cancer cells [5]. Crizotinib bearing 2-
aminopyridine structure is the first-in-class drug approved in 2011 for the treatment of ALK-positive
lung cancer patients (Figure 1). Although crizotinib displayed impressive anti-ALK efficacy, patients
eventually developed resistance owing to ALK mutations including L1196M and C1156M [6, 7].
Ceritinib (LDK378), bearing 2,4-diaminopyrimidine, was approved in 2014 for the treatment of ALK-
positive cancer following treatment with crizotinib [8, 9]. Ceritinib could inhibit crizotinib-resistant
mutants, L1196M, G1269A, and S1206Y, but failed to inhibit mutant G1202R and 1151Tins.
Alectinib having a novel benzo[b]carbazole moiety is also active against the crizotinib-resistant ALK
mutations L1196M, C1156Y, and F1174L in preclinical studies [10]. A Phase 3 clinical trial study of
alectinib is currently ongoing for ALK-positive NSCLC patients [11]. Other inhibitors including
AP26113 (Phase 2), [12] PF-06463922 (Phase 1/2), [13] and CEP-37440 (Phase 1) [14] are currently
undergoing evaluation in clinical trials.
In our previous effort to discover potent ALK inhibitors [15-17], we synthesized LDK378 and
CEP-37440 with hybridized structures, thereby leading to a novel structure, KRCA-0445, which is a
2,4-diaminopyrimidine bearing 4-(isopropylsulfonyl)anilino and bicyclic benzazepine moiety as
shown in Figure 2 [18]. In further optimization to improve the anti-ALK potency of KRCAs and to
increase structural diversity, we devised novel compounds bearing tetrahydronaphthalenyl (THN)
moiety (Figure 2). Particularly, we incorporated a germinal dimethyl substituent in the benzylic
methylene of THN since it was found to be easily oxidized to hydroxyl group by Cytochrome P450
[19]. Herein, we report the synthesis of THN analogs and their anti-ALK activities in biochemical and
cellular assays as well as pharmacokinetic (PK) and in vivo xenograft data of the selected compounds.

Figure 1. Chemical structures of currently available ALK inhibitors.
Figure 2. Rational design of KRCAs in this study.
To synthesize novel KRCAs with THN moiety, key THN intermediates 4, 8, 10, and 11 were
generated as shown in Scheme 1. 7-Methoxy-2-tetralone (1) was treated with MeI in the presence of
tetrabutylammonium sulfate (TBAS) and KOH to yield 1,1’-dimethyltetralone (2) [19]. Nitration of 2
with KNO3 afforded 6-nitrotetralone (3). Reduction of the nitro group in the assistance of Pd/C gave
6-aminotetralone (4). Dimethyltetralone (2) was converted to oxime (5) by treatment of
hydroxylamine [20]. Reduction of 5 by treatment of BH3/THF and subsequent addition of
trifluoroacetic anhydride (TFAA) afforded THN (6) protected with trifluoroacetyl group. Nitration
and reduction of 6 provided the desired amino-THN (8). Compounds 10 and 11 were also prepared

from the 6-methoxy-2-tetralone (9) by same synthetic manner described above.
Scheme 1. Synthesis of compound 4, 8, 10, and 11. Reagents and conditions: (a) MeI, TBAS, KOH,
THF, rt, 2 h; (b) KNO3, TFA, 0 oC to rt, 3 h; (c) H2, 10% Pd/C, MeOH, rt, 2 h; (d) NH2OH∙HCl,
AcONa, H2O, MeOH, 90 oC, reflux, overnight; (e) BH3, THF, reflux, 3 h; (f) TFAA, TEA, DCM, rt, 4
h; (g) KNO3, TFAA, MeCN, 0 oC to rt, 4 h.
Scheme 2. General synthesis of KRCAs. Reagents and conditions: (a) 4 N HCl in dioxane, 2-
ethoxyethanol, 100 oC, overnight; (b) NaBH4, MeOH, rt, 2 h; (c) Nucleophiles, conc. H2SO4, EtOH,
80 oC, overnight; (d) LiOH, MeOH, THF, H2O, rt, overnight; (e) acids, EDCI, HOBt, TEA, DCM, rt,
overnight.

The synthesis of KRCAanalogs is shown in Scheme 2. Commercially available 4-amino-2,5-
dichloropyrimidine 12 was coupled with amino-THNs 4, 8, 10, and 11 under acidic conditions to
afford compounds 13 and 16 [21, 22]. The ketone of 13 was reduced by NaBH4 to afford hydroxyl
compound 14. Condensation with various nucleophiles such as hydroxylamine or hydrazines
including acetyl hydrazide, semicarbazide, aminoguanidine, 1-amino-4-methylpiperazine, and 1-
aminomorpholine gave hydrazone compound 15. Compound 16 were converted to compound 17 by
hydrolysis of trifluoroacetamide group with K2CO3 in MeOH. Compound 17 were reacted with
glycolic acid or N,N-dimethylglycine in the presence of 1-ethyl-3-3-dimethylaminopropyl
carbodiimide (EDC) and hydroxybenzotriazole (HOBt) to yield compound 18.
Figure 3. List of KRCAs synthesized in this study.
Table 1. Activity profiles of compounds 13-18.
BaF3
L1196M
CC50 (nM)
ALK (wt.)
IC50 (nM)
L1196M
IC50 (nM)
H3122
CC50 (nM)
No.

LDK378
13a
13b
14a
14b
15a
15b
15c
14 ± 4
342 ± 158
43 ± 23
34 ± 17
21 ± 12
424 ± 339
52 ± 32
16 ± 14
16 ± 9
29 ± 10
1376 ± 1111
207 ± 164
109 ± 47
56 ± 16
38 ± 7
1310 ± 140
100 ± 7
75 ± 9
1995 ± 49
432 ± 3
726 ± 72
99 ± 7
1600 ± 87
299 ± 11
1363 ± 358
640 ± 32
400 ± 20
2092 ± 190
2601 ± 400
1643 ± 47
1528 ± 104
314 ± 2
600 ± 30
201 ± 121
97 ± 70
1220 ± 100
146 ± 17
146 ± 17
2404 ± 343
2828 ± 478
732 ± 123
276 ± 24
128 ± 37
117 ± 10
235 ± 69
14 ± 2
100 ± 25
200 ± 98
222 ± 100
N.D.
15d
15e
44 ± 22
122 ± 57
212 ± 154
21 ± 11
29 ± 7
15f
15g
15h
15i
40 ± 15
42 ± 11
334 ± 10
1584 ± 23
66 ± 6
18 ± 6
37 ± 10
15j
0.5 ± 0.2
40 ± 22
27 ± 10
8.4 ± 0.9
40 ± 22
15k
15l
30 ± 6
416 ± 165
1748 ± 47
94 ± 40
2094 ± 379
17a
0.7 ± 0.2
6.1 ± 2.5
4.2 ± 1.1
1.5 ± 0.3
9.9 ± 2.6
3.6 ± 0.9
16 ± 4
44 ± 6
39 ± 4
62 ± 2
86 ± 5
71 ± 2
17b
18a
18b
The IC50 values are for the inhibition of ALK wild type and ALK L1196M mutant using homogenous
time resolved fluorescence (HTRF) KinEASE-TK assay in a 384-well plate. The CC50 values are for
cellular proliferation inhibition. Data are fitted to a four-parameter sigmoidal dose response for
determination of IC50 and CC50 values. The errors are reported as the 95% confidence interval. N.D.:
not determined.
All final compounds synthesized were evaluated for their anti-ALK activities in biochemical
and cell-based assays and summarized in Table 1. For kinase inhibition studies, ALK wild type (wt.)
and ALK L1196M homogenous time resolved fluorescence (HTRF) KinEASE-TK assays were
performed in a 384-well plate. The cytotoxicity assay was conducted with H3122 (wt.) cells, which
are EML4-ALK addicted non-small cell lung cancer cells [23]. For ALK mutant cells, Ba/F3 cells
transfected with ALK L1196M gene were used [24]. Most of the compounds in this study exhibited
excellent ALK inhibitory activities with IC50 values in low double-digit nanomolar ranges in wild type
ALK enzymatic assays, except for compounds 13a, 15a, 15f, and 15g. Interestingly, compounds 15k

and 17b were highly potent (IC50 = 0.5 nM and 0.7 nM, respectively), and showed 88-fold and 39-fold
more potency than their structural isomers 15e and 17a, respectively. Although most of the
compounds showed good inhibitory activity against mutant L1196M ALK in a similar pattern
observed in wild type, the mutant enzyme was relatively less sensitive, about 2~6-fold against
inhibitors in comparison with wild type. Among the compounds synthesized, 15k and 17b were the
most promising in both wild and mutant ALK enzyme assays.
In the cell-based assay with H3122 cells, five compounds 15k, 15l, 17b, and 18a-18b
showed excellent anti-ALK activities with CC50 values in low double-digit nanomolar ranges (14-44
nM). These compounds also exhibited moderate inhibitory potencies against L1196M BaF3 mutant
cell lines. Remarkably, 15d, 15e, and 17a, which showed excellent activity in the enzyme assays,
exhibited weak cytotoxicity with IC50 values in micromolar ranges. It is worthwhile to note that
compounds 13b, 14b, 15g-15l, and 17b derived from THN intermediates 4 and 8 are generally more
potent than compounds 13a, 14a, 15a-15f, and 17a derived from THNs 10 and 11. It is reported that
the terminal piperidine in LDK378 fits closely to the protein surface and plays an important role in the
interaction with E1210 in ALK [24]. The activity differences observed might be related to the
interaction with E1210. Amongst the compounds tested in this study, 15k and 17b were the most
potent in both enzymatic and cell-based assays.
Two highly active compounds (15k and 17b) were tested in the human and mouse liver
microsomal models to aid in the prediction of metabolic stability (Table 2). The hERG inhibition with
patch clamp experiment and CYP inhibition assay were also conducted for predicting in vivo toxicity.
Compound 17b displayed moderate stability (56 % remaining at 30 min) in human microsomal
models but relatively short half-life in mouse microsomal models (23 % remaining at 30 min).
Compound 15k was rapidly metabolized in both mouse and human microsomal models (14 % in
mouse and 20 % in human). In hERG assay, compounds 15k and 17b exhibited weak hERG
inhibition with an IC50 value of 24 and 44 µM, respectively, thus posing weak or no cardiac toxicity in
relation to potassium channel blockade. In case of CYP inhibition, compound 15k showed no or poor

inhibition against a panel of five CYP isozymes at 10 µM, while compound 17b inhibited 97 % and
72 % of CYP3A4 and CYP2C19 isozymes, respectively.
Table 2. Metabolic stability, hERG, and CYP inhibition of 15k and 17b
Metabolic stability
Mouse / Human
(% remaining at 30 min)
14 / 20
hERG
patch clamp
(IC50, µM)
24
CYP inhibition at 10 µM (%)
Compound
1A2
2C9
2D6
3A4
2C19
7.2
9.5
17.1
38.0
11.4
31.0
47.5
97.0
43.4
72.0
15k
17b
23 / 56
44
Next, pharmacokinetic studies were performed with compounds 15k and 17b. Serum
concentrations were determined using LC-MS/MS after oral administration (PO) of compounds (10
mg/kg) and the data is summarized in Figure 4. Interestingly, compounds 15k and 17b showed
promising pharmacokinetic parameters in rats although these had a short half-life in the ex vivo mouse
microsomal model. Particularly, compound 17b showed very a long half-life (T1/2) with 9.5 h and
large AUC values, thus could exhibit long drug exposure time in the in vivo study.

Figure 4. Pharmacokinetic parameters of 15k and 17b in male rats.
Both 15k and 17b exhibited excellent anti-ALK activities and moderate pharmacological
properties; hence, their in vivo antitumor efficacy was assessed in H3122 tumor xenograft-bearing
SCID mouse. Compounds and controls (DMSO and LDK378) were administered to SCID mice orally
in 20 % PEG400 and 3 % Tween 80 in DDW at doses of 50 mg/kg or 25 mg/kg q.d. for the 14-day
duration of the study (Figure 5). Tumor growth was inhibited and regression was observed in all
conditions during drug treatment for 14 days. Although slight rebound of tumor growth was observed
after discontinuation of dose as observed in LDK378, compound 17b exhibited better tumor growth
inhibition than LDK378, while compound 15k was less potent. No changes in body weight or side
effects were observed during the study (data not shown).

Figure 5. Antitumor activity of compounds 15k and 17b in H3122 xenograft model. Compounds
were administered to SCID mice orally at doses of 50 mg/kg q.d. for 14 days after the tumor volume
reached 200 mm3. Each group consisted of 8 mice. The results are shown as the mean ± standard error.

Figure 6. Kinase selectivity of compound 17b A, Pie chart for kinase inhibition by compound 17b; B,
list of kinases with >90% inhibition.
Kinase selectivity of compound 17b was evaluated with a set of 96 kinases and is
summarized in Figure 6 and Supplementary data. Interestingly, 26 out of 96 kinases were strongly
inhibited by compound 17b at 1 µM (>90% inhibition) and 19 kinases were moderately inhibited
(90%-70%), while 45 kinases were weakly inhibited (<70%). Although IC50 values of compound 17b
against those kinases showing >70% inhibition were not determined, it seems to be a multi-targeting
inhibitor. It was reported that ceritinib (LDK378) inhibited both ALK and IGF-1R, thereby showing
better potency in in vivo study than crizotinib [24]. Many ALK inhibitors including crizotinib,
ceritinib, and PF-06463922 are also currently under development as ROS1 inhibitor [25-30]. In this
context, compound 17b targeting ALK and other kinases may provide clinical benefits for the
treatment of NSCLC in the future.
In conclusion, the design, synthesis, and anti-ALK activity of novel 2,4-diaminopyrimidines
bearing THN moiety were investigated. Compound 17b showed the most potent anti-ALK activity in
enzymatic and cell-based assays. In addition, compound 17b showed reasonable pharmacological

properties such as PK, hERG, and CYP inhibition. An in vivo efficacy study with compound 17b
demonstrated highly potent inhibitory activity against H3122 tumor xenograft model in mice. Kinase
selectivity assay showed that compound 17b is a multi-targeting inhibitor; hence, it could possess
synergistic effect in anti-tumor activity and possibility for multi-targeted NSCLC drug development.
The compound 17b is currently undergoing further optimization to develop novelALK inhibitors.
Acknowledgements
This work was funded by the National Research Foundation of Korea (NRF-2012M3A9A9054902),
the Korea Research Institute of Chemical Technology (KK-1503), and the Korea Chemical Bank
(SI1512-01).
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Graphical Abstract