Novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety for anaplastic lymphoma kinase (ALK) inhibitor

Article abstract of DOI:10.1016/j.bmcl.2017.03.073

In this study, a series of novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety was described for ALK inhibitor. The pyrazole, imidazole, 1,2,4-triazole, piperazine and phenanthridine moieties were employed at the 2-position of pyrimidine. Amo

Full text of DOI:10.1016/j.bmcl.2017.03.073

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety for
anaplastic lymphoma kinase (ALK) inhibitor
Raghavendra Achary a,c,e, Gangadhar Rao Mathi a,c,e, Dong Ho Lee b,c, Chang Soo Yun d, Chong Ock Lee c,
Hyoung Rae Kim d, Chi Hoon Park a,c, Pilho Kim a,c, , Jong Yeon Hwang a,c,
⇑
⇑
a Department of Medicinal Chemistry and Pharmacology, University of Science & Technology, Daejeon 34113, Republic of Korea
b Department of Chemistry, Sogang University, Seoul 04107, Republic of Korea
c Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
d Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety was described
for ALK inhibitor. The pyrazole, imidazole, 1,2,4-triazole, piperazine and phenanthridine moieties were
employed at the 2-position of pyrimidine. Among the compounds synthesized, 28, 29, 36, and 42 showed
promising anti-ALK activities in enzymatic- and cell-based assays. In vivo H3122 xenograft model study
showed that compound 29 effectively suppressed ALK-driven tumor growth, similar to the extent of cer-
itinib, suggesting that it could be used for a novel ALK inhibitor development.
Received 23 January 2017
Revised 13 March 2017
Accepted 21 March 2017
Available online xxxx
Keywords:
Anaplastic lymphoma kinase
Cancer
Ó 2017 Published by Elsevier Ltd.
2,4-Diaminopyrimidine
Fused tricyclic moiety
Anaplastic lymphoma kinase (ALK) is one of the receptor tyro-
sine kinases and has been implicated in a variety of tumors includ-
ing anaplastic large-cell lymphomas (ALCL), diffuse large B-cell
lymphoma (DLBCL), inflammatory myofibroblastic tumors (IMT),
and solid tumors.1–4 Since ALK gene is fused with various partner
genes, such as NMP, EML4, and KIF5 genes, the resulting fusion pro-
tein is constitutively activated, leading to a state of oncogenic
addiction.5 The blocking of constitutively activated ALK signal with
small molecules can inhibit uncontrolled tumor proliferation, thus
inducing rapid cell death.6,7 Several drugs targeting ALK, such as
crizotinib, ceritinib, and alectinib have been approved for the treat-
ment of ALK-driven non-small cell lung cancer (NSCLC) patients.
Brigatinib and lorlantinib are currently undergoing evaluation in
clinical trials. Crizotinib, the first-in-class drug, was approved in
2011 for the treatment of ALK-positive NSCLC patients (Fig. 1).
Although treatment of NSCLC patients with crizotinib displayed
dramatic suppression of tumor growth, patients eventually suf-
fered from rebounded tumor due to drug resistance predominantly
by ALK mutations including L1196M, F1174L, C1156M, G1202R,
etc.8,9 In addition, crizotinib is not effective against brain metas-
Fig. 1. ALK drugs approved.
tases owing to its poor BBB penetration.10 Thereby, many efforts
to overcome crizotinib-resistant issues and poor brain penetration
have been intensively pursued, leading to second-generation ALK
inhibitors, such as ceritinib, alectinib, and brigatinib. Ceritinib
⇑
Corresponding authors at: Bio
Institute of Chemical Technology, Daejeon 34114, Republic of Korea.
& Drug Discovery Division, Korea Research
E-mail addresses: pkim@krict.re.kr (P. Kim), jyhwang@krict.re.kr (J.Y. Hwang).
These authors contributed equally to this study.
e
http://dx.doi.org/10.1016/j.bmcl.2017.03.073
0960-894X/Ó 2017 Published by Elsevier Ltd.
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R. Achary et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
(LDK378) was approved for the treatment of ALK-positive cancer
following treatment with crizotinib.11,12 Ceritinib is effective
against crizotinib-resistant mutants, L1196M, G1269A, and
S1206Y, but failed to inhibit mutant G1202R and 1151Tins. As
the recent data showed that brigatinib showed remarkable
in vitro activity against both ALK wild type and mutants, it is con-
sidered a potential regimen for the patients with resistance to
crizotinib, ceritinib, and alectinib.13
The ALK-targeted drug discovery program mostly focuses on
the modification of substituents at the 2- and 4-positions of pyrim-
idines, as shown in ceritinib and brigatinib. In particular, the 2-
position of the pyrimidines have been extensively studied to dis-
cover novel ALK inhibitors by introducing terminal secondary or
tertiary amines that play an important role in interacting with
Glu1210 of ALK.14–16 Similarly, our efforts to develop novel ALK
inhibitors have been reported, such as KRCA-0377 and 0713
Fig. 2. Rational design of KRCAs in this study.
Scheme 1. Synthesis of compounds 7 and 8. Reagents and conditions: (a) N-acetylimidazole, NaH, THF, 30%; (b) hydrazine monohydrate, EtOH, reflux, 92%; (c) HNO3, TFA, rt,
69%; (d) H2, 10% Pd/C, MeOH, rt, 18 h, 57%; (e) 0.08 M HCl in ethoxyethanol, 110 °C, 33%; (h) glycolic acid, EDCI, HOBt, TEA, DCM, rt, 67%.
Scheme 2. Synthesis of compound 18. Reagents and conditions: (a) 2-chloroacetyl chloride, TEA, DCM, rt, 4 h, 76%; (b) potassium phthalimide, DMF, 90 °C, 12 h, 80%; (c)
POCl3, EtOH, 110 °C, 12 h, 60%; (d) cat. HCl, NaBH3CN, MeOH, rt, 1 h, 99%; (e) hydrazine monohydrate, EtOH, 90 °C, 1 h, then CNBr, EtOH, rt, 1 h, 54%; (f) TFAA, TEA, DCM, rt,
1 h, 45%; (g) HNO3, TFA, rt, 3 h, 55%; (h) i) H2, 10% Pd/C, EtOAc, rt, 4 h; ii) 6, 0.08 M HCl in ethoxyethanol, 80 °C, 12 h, 53% (two-step); (i) K2CO3, EtOH/H2O, 90 °C, 1 h, 67%.
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R. Achary et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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(Fig. 2), bearing bicyclic amines at the 2-position of the pyrimidine
in ceritinib.17–19
out that tricyclic kinase inhibitors could improve activities as well
as selectivities.24–26 Thus, novel 2,4-diaminopyrimidine derivatives
as ALK inhibitors were designed by replacing the 2-position sub-
stituent of pyrimidine in ceritinib with fused tricyclic scaffolds
incorporating pyrazole, imidazole, piperazine, 1,2,4-triazine, and
phenanthridine moieties (Fig. 2). Herein, we report the synthesis
During the course of our search for novel ALK inhibitors,17–23
the encouraging biological activities of the bicyclic amines led us
to further investigate tricyclic amines, since several precedent
reports in other kinases, such as JAK3, PDGFR, and Pim, pointed
Scheme 3. Synthesis of compounds 18 and 19. Reagents and conditions: (a) Ethyl bromoacetate, NaH, DMF, rt, 79%; (b) Lawesson’s reagent, toluene, reflux, 73%; (c) hydrazine
monohydrate, EtOH, rt, 1 d, 84%; (d) LAH, THF, reflux, 3 h, 83%; (e) TFAA, TEA, DCM, rt, 64%; (f) HNO3, TFA, rt, 47%; (g) H2, 10% Pd/C, MeOH, rt, 18 h, 92%; (h) 0.08 M HCl in
ethoxyethanol, 110 °C; (i) LiOH, MeOH/THF, H2O, rt, 3 h, 68%; (j) formaldehyde, NaBH3CN, AcOH (cat.), MeOH, rt, 24 h, 98%.
Scheme 4. Synthesis of 36 and 41. Reagents and conditions: (a) ClCOCH2Cl, TEA, DCM, rt, 4 h, 94%; (b) 2,2-diethoxyethan-1-amine, toluene, reflux, 2 h, 77%; (c) H2SO4, rt, 4 h,
28%; (d) TFAA, TEA, DCM, rt, 2 h, 72%; (e) HNO3, TFA, 0 °C to rt, 2 h, 40%; (f) 10% Pd/C, H2, EtOAc, rt, 12 h, 82%; (g) 6, 0.08 M HCl in ethoxyethanol, 80 °C, 12 h, 43%; (h)
LiOHÁH2O, THF/MeOH/H2O, rt, 2 h, 40%; (j) i) LiAlH4, THF, 50 °C, 2 h; ii) TFAA, TEA, DCM, rt, 2 h, 30%; (k) HNO3, TFA, 0 °C to rt, 2 h, 40%; (l) 10% Pd/C, H2, EtOAc, rt, 12 h, 89%; (m)
6, 0.08 M HCl in ethoxyethanol, 80 °C, 12 h, 27%; (n) LiOHÁH2O, THF/MeOH/H2O, rt, 2 h, 59%.
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R. Achary et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
of designed compounds and their in vitro and in vivo biological
evaluation results to provide more options for the discovery of
ALK inhibitors.
Compounds 7 and 8 bearing pyrazole moiety were synthesized
as shown in Scheme 1. 7-Methoxy-2-tetralone (1) was treated with
N-acetylimidazole and NaH to afford compound 2. Cyclization of 2
Scheme 5. Synthesis of 50 and 51. Reagents and conditions: (a) ClCOCH2Cl, TEA, DCM, rt, 2 h, 93%; (b) 2,2-diethoxyethan-1-amine, toluene, reflux, 2 h, 82%; (c) CH3SO3H,
DCM, rt, 12 h, 51%; (d) TFAA, TEA, DCM, rt, 2 h, 94%; (e) HNO3, TFA, rt, 2 h, 59%; (f) 10% Pd/C, H2, MeOH, rt, 3 h, 80%; (g) 6, 0.08 M HCl in ethoxyethanol, 80 °C, 12 h, 79%; (h)
LiOHÁH2O, THF/MeOH/H2O, rt, 2 h, 80%; (j) LiAlH4, THF, 50 °C, 2 h, 51%.
Scheme 6. Synthesis of compounds 56, 57, and 58. Reagents and conditions: (a) i) oxalyl chloride, DMF (cat.), DCM, rt; ii) MeONH2ÁHCl, TEA, DCM, rt, 44%; (b) iodobenzene, Pd
(OAc)2, Ag2O, AcOH, 120 °C, 36 h, 42%; (c) H2, Pd/C, MeOH, rt, 3 h, 58%; (d) 6, 0.08 M HCl in ethoxyethanol, 80 °C, 20 h; (e) LAH, THF, rt, 6 h, 63%; (f) i) BH3ÁTHF, THF, 75 °C, 12 h;
ii) 1.5 N HCl, 80 °C, 2 h, 40%.
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R. Achary et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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with hydrazine provided fused tricyclic pyrazole 3 in high yield.27
Nitration of 3 gave nitro compound 4 as a major product. Reduc-
tion of nitro group in the presence of Pd/C afforded tricyclic pyra-
Reduction of amide group in 22 by treatment of LAH provided
dihydro-1,2,4-triazine 23, which was treated with TFAA to afford
24. Typical nitration, reduction, and coupling with 6 provided com-
pounds 25, 26, and a mixture of 27 and 28, respectively. Compound
27 was hydrolyzed by LiOH to afford 28. Compound 28 was treated
with formaldehyde in the presence of NaBH3CN and a catalytic
amount of AcOH to facilitate compound 29.
zoloaniline 5. Compound
5 was coupled with commercially
available 4-amino-2,5-dichlorpyrimidine 6 under acidic conditions
to afford compound 7. Compound 7 was treated with glycolic acid,
EDCI, and HOBt to afford amide 8.
In case of 2-aminodihydroimidazolo compound 18 (Scheme 2),
at first 4-methoxyphenethylamine 9 was treated with 2-chloroa-
cethyl chloride in the presence of TEA to afford amide 10, which
was transformed to phthalimidide 11 by treatment of potassium
phthalimide for the Gabriel synthesis.28 Intramolecular cyclization
of 11 in the assistance of POCl3 provided dihydroquinoline 12,
which was converted to tetrahydroisoquinoline 13 by the follow-
ing NaBHCN3 reduction. Hydrolysis of 13 using hydrazine followed
by cyclization in the presence of CNBr gave 2-aminodihydroimida-
zole 14.29 With amine 14 in hand, sequential amine protection,
nitration, nitro reduction, and coupling with 6 afforded 15, 16,
17, and 18, respectively.19
Piperazinoisoquinolines 36 and 41 were synthesized as shown
in Scheme 4. Piperazinodihydroisoquinoline 31 was prepared from
acid-mediated intramolecular cyclization of aminodimethylacetal
30, which was generated from 10 through amination with 2,2-
diethoxyethan-1-amine. With 31 in hand, sequential protection,
nitration, nitro reduction, coupling with 6, and deprotection deliv-
ered compounds 32–36, respectively. For the preparation of tri-
cyclic piperazine 41, amide of oxopiperazine 31 was reduced by
treatment of LAH and subsequent protection with TFAA afforded
compound 37. Tricyclic piperazine 41 was eventually obtained
using compound 37 through similar reaction conditions and
sequences for the preparation of 36.
Compounds 28 and 29 bearing 1,2,4-triazine moiety were syn-
thesized as described in Scheme 3. 7-Methoxy-3,4-dihydroquino-
lin-2-one (19) was treated with ethyl bromoacetate and NaH to
afford N-alkyl compound 20. The amide of 20 was converted to
thioamide 21 by treatment of Lawesson’s reagent. Cyclization of
21 with hydrazine provided fused tricyclic 1,2,4-triazinone 22.
Compounds 50 and 51 were also prepared from the correspond-
ing 3-methoxyphenethylamine 42 by similar synthetic manners as
36 and 41 (Scheme 5). Particularly, oxopiperazine 50 was reduced
by LAH to afford tricyclic piperazine 51.
For phenanthridinones 56–57 and phenanthridine 58
(Scheme 6), the synthesis started from treatment of 52 with oxalyl
Table 1
ALK inhibitory activities of the final compounds.
No.
ALK (wt.)
IC50 (nM)
L1196M
IC50 (nM)
IR
H3122
CC50 (nM)
BaF3
L1196M
CC50 (nM)
IC50 (nM)
Ceritinib
7
8
18
28
29
36
41
14
29
21
5.3
0.33
1.7
3.7
2
29
24
130
7.5
21
11
6.0
2.8
2.1
38
75
700
500
1300
260
120
93
75
170
130
11
8.3
8.1
140
100
380
38
36
30
5.2
1.9
36
50
51
56
57
4.3
12
190
310
>10,000
5.3
4.8
380
530
>10,000
2.2
14
300
260
>10,000
770
240
160
760
410
310
790
2600
>10,000
>10,000
58
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.
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R. Achary et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 3. Antitumor activity of ceritinib and 29 in H3122 xenograft model. Compounds were administered to SCID mice orally at a dose of 50 mg/kg q.d. for 14 days after the
tumor volume reached 200 mm.3 Each group consisted of 8 mice. The results are shown as the mean standard error.
chloride and catalytic DMF followed by treatment with methoxya-
mine hydrochloride in the presence of TEA to afford amide 53.
Compound 53 was reacted with iodobenzene by the assistance of
Pd(OAc)2 and Ag2O to give tricyclic N-methoxyphenanthridinone
54.30 Reduction of the nitro group in 54 and coupling reaction with
6 provided 55 and 56, respectively. Reduction of 56 with LAH pro-
vided phenanthridine 57, whereas reduction of 56 with BH3 and
HCl gave phenanthridine 58
pound 29 exhibited similar antitumor potency compared to ceri-
tinib. No changes in body weight or side effects were observed
during the study (data not shown).
In conclusion, the design, synthesis, and anti-ALK activity of
novel 2,4-diaminopyrimidines bearing fused tricyclic ring moieties
were described. The fused tricyclic structures incorporating pyra-
zole, imidazole, 1,2,4-triazole, piperazine, and phenanthridine
were applied at the 2-position of pyrimidine in ceritinib. Com-
pound 28, 29, 36, and 41 showed promising activities in both enzy-
matic- and cell-based assays, while others were less active in cell-
based assays. An in vivo efficacy study with compound 29 against
H3122 tumor xenograft model in mice demonstrated highly potent
inhibitory activity, similar to ceritinib, suggesting that it might be
used for a novel ALK inhibitor development.
All final compounds synthesized were evaluated for their anti-
ALK activities in biochemical assays (ALK wild type, L1196M, and
insulin receptor kinase) and proliferation assays (H3122, BaF3 with
L1196M) and summarized in Table 1. For kinase inhibition studies,
homogenous time resolved fluorescence (HTRF) KinEASE-TK assays
were performed in a 384-well plate. The proliferation assay was
performed with H3122 (wt.) cells, which are EML4-ALK addicted
non-small cell lung cancer cells. For ALK mutant cells, ALK
L1196M gene was transfected in Ba/F3 cells. Pyrazoles (7 and 8),
imidazole (18), 1,2,4-triazine (28 and 29), piperazine derivatives
(36, 41, 50, and 51) exhibited good ALK inhibitory activities with
IC50 values in single- to low double-digit nanomolar ranges in wild
type ALK enzymatic assays, while phenanthridine derivatives (56,
57, and 58) showed weak or no ALK inhibitory activities. In case
of L1196M ALK mutant, this series of compounds displayed as sim-
ilar activity trends as above. However, no drastic selectivity
between ALK and IR was observed in this series. In cell based-
assay, compound 28, 29, 36, and 41 showed good anti-proliferative
activities with IC50 values of 30–38 nM ranges in H3122 cells and
this tendency was also observed in mutant cell lines (BAF3/
L1196 M). It is worthy to note that compounds 36 and 41 showed
better anti-ALK activities in cell-based assay than 50 and 51, while
these compounds are almost equal in enzymatic assays. Overall,
four promising compounds (28, 29, 36, and 41) displayed similar
anti-ALK activities in comparison with ceritinib.
Acknowledgements
This work was funded by the National Research Foundation of
Korea (NRF-2012M3A9A9054902) and the National Research
Council of Science & Technology (NST) grant by the Korea Govern-
ment (MSIP) (No. CRC-16-01-KRICT).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.03.
073.
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