Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor

Article abstract of DOI:10.1016/j.bmcl.2015.07.004

Abstract A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were synthesized and evaluated for their anti-ALK activities. The activities of these compounds were confirmed in both enzyme- and cell-based ALK assays. Amongst

Full text of DOI:10.1016/j.bmcl.2015.07.004

Accepted Manuscript
Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic
aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor
Ga Ae Kang, Minji Lee, Dawn Song, Heung Kyoung Lee, Sunjoo Ahn, Chi
Hoon Park, Chong Ock Lee, Chang Soo Yun, Heejung Jung, Pilho Kim, Jae Du
Ha, Sung Yun Cho, Hyoung Rae Kim, Jong Yeon Hwang
PII:
S0960-894X(15)00708-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.004
BMCL 22893
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 April 2015
2 July 2015
4 July 2015
Please cite this article as: Kang, G.A., Lee, M., Song, D., Lee, H.K., Ahn, S., Park, C.H., Lee, C.O., Yun, C.S., Jung,
H., Kim, P., Ha, J.D., Cho, S.Y., Kim, H.R., Hwang, J.Y., Synthesis and evaluation of novel 2,4-diaminopyrimidines
bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.07.004
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Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for
anaplastic lymphoma kinase (ALK) inhibitor
Ga Ae Kanga,#, Minji Leeb,#, Dawn Song c, Heung Kyoung Leea, Sunjoo Ahna,d, Chi Hoon Parka,d,
Chong Ock Leea, Chang Soo Yuna,d, Heejung Junga,d, Pilho Kima,d, Jae Du Ha a, Sung Yun Cho a,d,
Hyoung Rae Kima,*, and Jong Yeon Hwanga,d,*
aBio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-
600, Korea
bDepartment of Chemistry, Korea University, Seoul 136-701, Korea
cCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 362-763, Korea
dDepartment of Medicinal Chemistry and Pharmacology, University of Science & Technology,
Daejeon 305-550, Korea
*Corresponding author. Tel: +82-42-860-7045; Fax: +82-42-860-7160; Email:
hyngrk@krict.re.kr; jyhwang@krict.re.kr
# These authors contributed equally to this study.
Abstract
A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were
synthesized and evaluated for their anti-ALK activities. The activities of these compounds were
confirmed in both enzyme- and cell-based ALK assays. Amongst compounds synthesized, KRCA-
0445 showed very promising results in pharmacokinetic study and in vivo efficacy study with H3122
xenograft mouse model.

Keywords: Cancer, ALK, 2,4-diaminopyrimidine
Introduction
Anaplastic lymphoma kinase (ALK) belongs to the insulin receptor family of receptor tyrosine kinase
(RTK). ALK-fused genes, such as NPM-ALK, EML4-ALK, KIF5-ALK, are putative oncogenes and
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]
Crizotinib, Xalkori developed by Pfizer, is the first approved drug by FDA in 2011 for the treatment
of ALK-positive lung cancer patients (Figure 1). Although crizotinib demonstrateed initial impressive
efficacy, patients eventually developed resistance with ALK mutations including L1196M and
C1156M.[5, 6] In 2014, LDK378, Ceritinib developed by Novartis, was approved for the treatment of
ALK-positive cancer following treatment with crizotinib.[7, 8] LDK378 retains potency towards
mutants, L1196M, G1269A, and S1206Y, but loses potency towards G1202R and 1151Tins. Alectinib
having a benzo[b]carbazole moiety is also an ALK-selective second generation inhibitor developed by
Roche. In terms of ALK selectivity, alcectinib is better than crizotinib.[6, 9] Alectinib 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], Others, AP26113 (Ariad Pharmaceuticals, Phase 2)[12], PF-06463922 (Pfizer, Phase
1/2),[13] and CEP-37440 (Teva, Phase 1),[14] are currently undergoing evaluation in clinical trials.
Among those ALK inhibitors, LDK378 bearing a methyl group para to isopropoxy moiety was
designed to circumvent the formation of glutathione adduct from NVP-TAE684, which was originally
reported as a highly potent ALK inhibitor.[1, 15] The CEP-37440 contains a novel 1,4-
diaminopyrimidine structure bearing distinguishing methylacetamidoaniline and bicyclic amine
moieties, which are quite differentfrom LDK378. In our effort to discover potent ALK inhibitors,[16-
18] we came up with hybridized structures of LDK378 and CEP-37440, thereby leading to a novel
structure, KRCAs, which is 2,4-diaminopyrimidine bearing 4-(isopropylsulfonyl)anilino and bicyclic
benzazepine moiety as shown in Figure 2. Although Liu et al. recently reported 2,4-
diarylaminopyrimidine analog bearing bicyclic benzazepine moiety and methylacetamidoaniline as a
c-Met/ALK multikinase inhibitor,[19] its chemical features differ from KRCAs. Herein, we report the
synthesis of KRCAs and their anti-ALK activities in biochemical and cellular assays as well as PK
and in vivo xenograft data of the selected compounds.
Figure 1. Structure of the potent ALK inhibitors.

Figure 2. Rational design of KRCAs.
The representative synthesis of KRCA analogs is shown in Scheme 1. Commercially available 4-
amino-2,5-dichloropyrimidine 1 was coupled with aminobenzazepines 2 under acidic conditions to
afford compounds 3,[20, 21] which were converted to the final compounds 4 by following hydrolysis
of trifluoroacetamide group with K2CO3 in MeOH. Compounds 4 were reacted with various
electrophiles such as alkyl halides, acids, and ketones for further diversification.
Scheme 1. General synthesis of KRCAs.
Reagents and conditions: (a) 4 N HCl in dioxane, 2-ethoxyethanol, 80 ℃, overnight; (b) K2CO3,
MeOH, 90 ℃; (c) electrophiles (alkyl halides, acids, and ketones).

The bicyclic aminobenzazepines 2 were synthesized from the corresponding starting material as
shown in Scheme 2. Nitrobenzazepinone 7 was prepared by nitration with KNO3 from benzazepinone
6, which was synthesized from the corresponding 1-tetralone by applying Schmidt reaction with
NaN3/HCl.[22] Reduction of amide group in compound 7 by treatment of BH3/THF provided
benzoazepine 8, which was treated with trifluoroacetic anhydride (TFAA) to afford compound 9.
Reduction of nitro group in assistance of Pd/C gave the aminobenzazepine 10. Compound 13 was
prepared from intermediate 11 by same synthetic manner described above (nitration  amide
reduction  protection  nitro reduction). Compound 17 was prepared from compound 14 which
was synthesized by known procedure.[23] Compound 14 was hydrogenated over 10% Pd/C in AcOH
to give amide 15. A following series of reactions (nitration  amide reduction  protection  nitro
reduction) provided compound 17. Benzoxazepinone 18, which was synthesized from resorcinol by
known procedure in the literature,[24] was subjected to a series of reactions as described above to
afford the benzoxazepine 19. Reduction of nitro compounds 7, 11, and 15 gave aminobenzazepinones
20-22, respectively.

Scheme 2. Synthesis of aminobenzazepine analogs.
Reagents and conditions: (a) KNO3, TFAA, MeCN, 0 oC to rt; (b) BH3, THF, reflux; (c) TFAA, TEA,
DCM, rt; (d) H2, 10% Pd/C, MeOH, rt; (e) H2, 10% Pd/C, AcOH, MeOH, rt.
Table 1. Activity profiles of compound 23-29.

The IC50 values are for the inhibition of ALK wild type, ALK L1196M mutant, and Insulin Receptor
using homogenous time resolved fluorescence (HTRF) KinEASE-TK assay in 384-well plate. The
CC50 values are for cellular proliferation inhibition. Data were fitted to a four-parameter sigmoidal
dose response for determination of IC50 and CC50 values. The errors were reported as the 95%
confidence interval.
All final compounds synthesized were evaluated for their anti-ALK activity in biochemical and cell-
based assays. For kinase inhibition assay, ALK wild type (wt.), ALK L1196M, and insulin receptor
(IR) homogeneous time resolved fluorescence (HTRF) KinEASE-TK assays were used in 384-well
plate. The cell cytotoxicity assay was conducted with H3122 (wt.) cells which are EML4-ALK
addicted non-small cell lung cancer cells. For ALK mutant cells, Ba/F3 cells transfected with ALK

L1196M were used. The KRCAs bearing various aminobenzazepine moieties were evaluated for anti-
ALK potency as summarized in Table 1. While no selectivity against insulin receptor was observed,
all compounds (23-29) exhibited very good ALK inhibitory activity with low nanomolar of IC50
values in both wild type and mutant ALK enzymatic assays. Most of compounds in this series
displayed better enzymatic activities than those of LDK378. In cell-based assay with H3122 cells,
benzazepines 26-29 bearing basic nitrogen atom showed low double-digit nanomolar of IC50 values,
whereas benzazepinones 23-25 bearing relatively acidic amide group turned out to be less potent.
Compounds 26-29 exhibited similar anti-proliferative activities against H3122 cells compared to
LDK378 although those were at least 7-fold more active than LDK378 in ALK wt. enzymatic assay.
These patterns were also observed in Ba/F3 L1196 mutant cells.
Table 2. Activity profiles of compounds 30-46.

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 384-well plate. The CC50 values are for
cellular proliferation inhibition. Data were fitted to a four-parameter sigmoidal dose response for
determination of IC50 and CC50 values. The errors were reported as the 95% confidence interval.
Next, several N-substituted benzazepine analogs (30-46) were evaluated and the data were
summarized in Table 2. Interestingly, all compounds in this series exhibited very potent and similar

anti-ALK (wt.) activity ranges regardless of substituents. This trend was also observed in L1196M
mutant enzyme assay, except for compounds 36 and 39. However, substitution on nitrogen generally
led to reduced anti-proliferative activity in both wild type (H3122) and mutant (Ba/F3, L1196M) cell-
based assays. Overall, encouraging effect by recruiting substituents on nitrogen was not observed in
this series.
Table 3. Metabolic stability of the selected compounds
Metabolic Stability
Compound
Mouse / Human
(% remaining at 30 min)
25 / 72
26 (KRCA-0462)
27 (KRCA-0444)
28 (KRCA-0445)
29 (KRCA-0503)
59 / 53
67 / 92
7 / 10
Amongst compounds tested in this study, four active compounds (26-27) in both enzymatic and cell-
based assay were tested in human and mouse liver microsomal models to aid in prediction of
metabolic stability (Table 3). Compounds 27 and 28 have reasonable stability in both human and
mouse microsomal models but compound 26 has relatively short half-life. Compound 29 quickly
metabolized in both mouse and human microsomal models. Based on this results, compounds 27
(KRCA-0444) and 28 (KRCA-0445) were selected for pharmacokinetic study. Serum concentrations
were determined using LC-MS/MS after oral administration (PO) of compounds (10 mg/kg). Both
compounds showed promising pharmacokinetic parameters as good drug candidates summarized in
Figure 3. It is worthwhile to note that the selected compounds showed very long half-life (T1/2) with at
least 12 h, thus expecting long exposure time in in vivo study.

Figure 3. Pharmacokinetic parameters of KRCA-0444 and KRCA-0445 in male rats.
Although both KRCA-0444 and KRCA-0445 exhibited very similar anti-ALK activity ranges,
KRCA-0445 was selected for in vivo study due to slightly better pharmacokinetic parameters,
compared with KRCA-0444. The in vivo study for the antitumor efficacy of compound KRCA-0445
was assessed in H3122 tumor xenograft model in SCID mouse. Compound KRCA-0445 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 q.d. for the 14-day duration of the study (Figure 4). Although complete
tumor regression was not observed, compound KRCA-0445 showed similar tumor growth inhibition,
compared with LDK378. No changes in body weight or side effects were observed during the study.

Figure 4. Antitumor activity of compound KRCA-0445 in H3122 xenograft model. Compounds were
administered to SCID mice orally at doses of 50 mg/kg q.d. for 14-day after the tumor volumn
reached around 200 mm3. Each group consisted of 8 mice. The results were shown as the mean ±
standard error. **p < 0.01 and *p <0.05 vs control group on final day using Student’s t-test.
Table 3. Kinase selectivity of KRCA-0445
Inhibition
Kinases
at 100 nM (%)
100
97
90
ALK
FGFR1
ROS1
78
IGF-1R
EGFR
(T790M, L858R)
c-Met
73
49
43
31
KDR
EGFR
The data was collected from Millipore’s KinaseProfiler panel assays.
Kinase selectivity of KRCA-0445 was performed with several kinases as shown in Table 3.
Interestingly, FGFR1, ROS1, IGF-1R, and EGFR (T790, L858R) were strongly inhibited by KRCA-

0445 at 100 nM concentration, while c-Met, KDR, and EGFR were slightly or weakly inhibited.
Currently, many ALK inhibitors including crizotinib, ceritinib, and PF-06463922 are also under
development for ROS1 inhibitor.[25-28] Therefore, KRCA-0445 could be further optimized to
develop for potential ROS1 intervention. Moreover, it was reported that ceritinib inhibited both ALK
and IGF-1R, thereby showing better potency in in vivo study, compared to crizotinib.[19] Likewise,
KRCA-0445 targeting both ALK and IGF-1R might give clinical benefits in the future development.
In summary, the design and synthesis of novel 2,4-diaminopyrimidines bearing bicyclic
aminobenzazepines and their anti-ALK activity were discussed. Amongst the compounds synthesized,
compound 28 (KRCA-0445) showed promising assay results including anti-ALK activities and
pharmacological properties. Kinase selectivity assay showed that KRCA-0445 is a multi-targeted
inhibitor including FGFR1, ROS1 and IGF-1R, thus expecting synergy effect in anti-tumor activity.
An in vivo efficacy study with compound KRCA-0445 demonstrated potent inhibitory activity against
H3122 tumor xenograft model in mice, suggesting that it could be used as a platform for further
optimization to develop novel ALK inhibitors.
Acknowledgments
This work was financially supported by the National Research Foundation of Korea (NRF-
2012M3A9A9054902), the Korea Research Institute of Chemical Technology (KK-1403), and the
Korea Chemical Bank (SI-1405).
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Graphical abstract