Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives as novel selective Axl inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.128247

Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

Full text of DOI:10.1016/j.bmcl.2021.128247

Bioorg. Med. Chem. Lett. 48 (2021) 128247
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives as
novel selective Axl inhibitors
,
Satoshi Inouea *, Yoshinobu Yamanea, Shuntaro Tsukamotob, Norio Muraia, Hiroshi Azumaa,
Satoshi Nagaoa, Kyoko Nishibatab, Sayo Fukushimab, Kenji Ichikawab, Takayuki Nakagawab,
Naoko Hata Sugib, Daisuke Itob, Yu Katob, Aya Gotoc, Dai Kakiuchic, Takashi Uenod,
Junji Matsuib, Tomohiro Matsushimaa
a Medicinal Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
b Biopharmacology, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
c Drug Safety, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
d Drug Metabolism and Pharmacokinetics, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we re-
ported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice,
whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane
permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine
derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000
as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.
Axl
Mer
Structure-activity relationships
Cancer
Inhibitor
Small molecule
Drug resistance to conventional, targeted therapy and immune
checkpoint inhibitors is a major cause of failure of anticancer treatment.
Understanding of resistance mechanisms and identification of the mol-
ecules driving resistance will allow development of targeted agents that
can destroy the drug resistant cancer. Axl is a member of the TAM
(Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when acti-
vated, can promote tumor cell survival, proliferation, migration and
invasion, angiogenesis, and tumor host interactions.1 Axl is often highly
expressed in resistant tumor cells that show mesenchymal
phenotypes.2–3 Furthermore, Axl is involved in acquired drug resistance
to multiple chemotherapies and molecular targeted-, radio- and immuno
therapies.4–9 Inhibition of Axl signaling suppresses such resistance in a
variety of cancer types.10 Therefore, we consider Axl to be a crucial drug
target to eradicate therapy-resistant cancer cells to improve treatment
outcomes.
In mice, chronic inhibition of Mer leads to retinal toxicity.11 Recently
we reported that enzyme-mediated inhibition of Mer by Axl/Mer dual
inhibitor leads to retinal toxicity in mice, on the other hand, the com-
pound 1, which selectively inhibited the activity of Axl over Mer, didn’t
show the retinal toxicity at a dose of 100 mg/kg in mice.12Fig 1.
The issue with compound 1 was its low membrane permeability and
Pe value was not detected. Low membrane permeable compounds are
expected to be a major issue in clinical development. The cause of low
membrane permeability was considered to be due to the polarity of the
ureidopyrimidine moiety, and it was necessary to discover a new tem-
plate. To obtain more selective compounds with well-balanced physi-
cochemical and pharmacokinetic profiles, subsequent derivatization of
1 led to the discovery of a series of novel Axl inhibitors. Here, we report
Abbreviations: Boc, tert-butoxycarbonyl; EtONa, sodium ethoxide; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate; iPr2NEt, N,N-diisopropylethylamine; NaBH(OAc)3, sodium triacetoxyborohydride; PPh3, triphenylphosphine; RT, room temperature; TAM, Tyro3-
Axl-Mer; TDI, time-dependent inhibition; tBuOK, potassium tert-butoxide.
* Corresponding author.
E-mail address: s4-inoue@hhc.eisai.co.jp (S. Inoue).
https://doi.org/10.1016/j.bmcl.2021.128247
Received 24 May 2021; Received in revised form 18 June 2021; Accepted 1 July 2021
Available online 13 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S. Inoue et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Fig. 1. Chemical structure of previously reported Axl selective inhibitors.
the design and synthesis of a novel series of 5,6,7,8-tetrahydropyrido
[3,4-d]pyrimidine derivatives.
Table 1
Effects of the R substituent on the in vitro inhibitory activities of the four tem-
plate compounds.
First, we designed and synthesized four new compounds as templates
(6, 11, 15, 19) listed in Table 1. The syntheses of these compounds are
shown in Schemes 1–4, respectively. In short, to obtain compound 6,
commercially obtained 2 was treated with 3 in the presence of Cs2CO3 to
obtain aniline derivative 4 via a SNAr reaction, then condensation of 4
with 512 afforded 6. To obtain compound 11, reported compound 713
was treated under Appel reaction conditions to obtain chloride 8. Next,
8 was treated with 3 to give aniline derivative 9 via a SNAr reaction.
Condensation of 9 with 512 afforded 10, and subsequent removal of the
tert-butoxycarbonyl group of 10 under acidic conditions, followed by
reductive amination with formaldehyde, afforded 11. Compounds 15
and 19 were synthesized from reported compounds 1213 and 1613 via
SNAr reaction, condensation, removal of protective group and reductive
amination, respectively.
Table 1 lists the results of the inhibitory activities of the four tem-
plates against Axl or Mer, as assessed by cell-free assay or cell-based
assay. In the cell-free assays, all of the templates showed Axl-specific
inhibitory activity. In particular, Axl inhibitory activities of 11 and 15
were more than 45-fold and 80-fold higher, respectively, than the cor-
responding Mer inhibitory activities. In the cell-based assays, all of the
Scheme 1. Reagents and conditions: (a) Cs2CO3, DMSO, 110 ◦C; (b) HATU, iPr2NEt, DMF, RT.
Scheme 2. Reagents and conditions: (a) PPh3, CCl4, 1,2-dichloroethane, 70 ◦C; (b) 3, tBuOK, DMF, 0 ◦C; (c) 5, HATU, iPr2NEt, DMF, RT; (d) TFA, DCM, RT; (e)
formaldehyde, NaBH(OAc)3, THF, RT.
2

S. Inoue et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Scheme 3. Reagents and conditions: (a) 3, K2CO3, DMF, 60 ◦C; (b) 5, HATU, iPr2NEt, DMF, RT; (c) TFA, DCM, RT; (d) formaldehyde, NaBH(OAc)3, THF, RT.
Scheme 4. Reagents and conditions: (a) 3, tBuOK, DMF, 0 ◦C; (b) 5, HATU, iPr2NEt, DMF, RT; (c) TFA, DCM, RT; (d) formaldehyde, NaBH(OAc)3, THF, RT.
templates again showed Axl-specific inhibitory activity. In particular,
the Axl inhibitory activity of 11 was more than 10-fold higher than the
Mer inhibitory activity. Therefore, in the remainder of the experiments,
we examined derivatives only of template 11 (5,7-dihydro-pyrrolo[3,4-
d]pyrimidine) and 15 (5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine).
Next, we designed and synthesized 5,7-dihydro-pyrrolo[3,4-d]py-
rimidine derivatives 20–26 listed in Table 2. The synthesis of these
compounds are shown in Scheme 5. In short, these compounds were
obtained by deprotection of 10 under acidic conditions, followed by
condensation with an acid (20–23), by amidation with cyclo-
propanecarbonyl chloride (24), or by reductive amination with alde-
hyde (25–26).
Table 2
Effects of the R substituent on the in vitro inhibitory activities of 5,7-dihydro-
pyrrolo[3,4-d]pyrimidine derivatives.
Table 2 lists the inhibitory activities of template 11 and of de-
rivatives 20–26. All of the derivatives except 20 showed higher Axl
inhibitory activities than 11 in both the cell-free and the cell-based as-
says. However, similar increases were also seen for Mer inhibitory ac-
tivity. Therefore, despite its weak inhibitory activity, 11 remained as the
compound with the highest Axl selectivity.
We then designed and synthesized 5,6,7,8-tetrahydropyrido[3,4-d]
pyrimidine derivatives 27–31, 33–35, and 39–42 listed in Table 3. The
synthesis of these compounds are shown in Scheme 6. In short, these
compounds were obtained by deprotection of 14 under acidic condi-
tions, followed by condensation (27–31), alkylation then deprotection
of 32 and condensation (33–34), or reductive amination (35–42).
Table 3 lists the inhibitory activities of template 15 and derivatives
27–31, 33–35, and 39–42. Converting the R moiety increased the Axl
inhibitory activities of some of the derivative, particularly those of 35,
40, and 42 in cell-free assay. However, all of the derivatives, except 27,
also showed increased Mer inhibitory activity. Derivatives 27, 28, 30,
31, 33 and 35 showed more than 20-fold greater Axl selectivity over
Mer. In the cell-based assays, similar results were obtained for 28, 29,
31, 34 and 35, which showed more than 5-fold greater Axl selectivity
over Mar. Derivatives 35, 39, and 42 showed the greatest Axl inhibitory
activity. Taking into account the Axl selectivity data obtained from the
cell-free and cell-based assays and data obtained from in vivo efficacy
screening (data not shown), 35 was selected for further optimization.
Derivative 35 was further optimized by varying the substituents on
the uracil moiety listed in Table 4. The synthesis of these compounds are
shown in Scheme 7. In short, to obtain these compounds, 43 was treated
with corresponding alkyl halide in the presence of K2CO3, followed by
hydroxylation of 44 to obtain carboxylic acid 45, then condensation of
45 with 13, deprotection of 46, followed by reductive amination
afforded 47–52. The syntheses of 60 and ER-001259851–000 are
shown in Scheme 8. Commercially obtained 53 was treated with the
corresponding amine to give 54. Cyclization of 54 with 56 in the pres-
ence of K2CO3 afforded 57. Hydroxylation of 57, then condensation with
3

S. Inoue et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Scheme 5. Reagents and conditions: (a) TFA, DCM, RT; (b) corresponding acid, HATU, iPr2NEt, DMF, RT; (c) cyclopropanecarbonyl chloride, iPr2NEt, DCM, RT; (d)
corresponding aldehyde, NaBH(OAc)3, THF, RT.
13 afforded 59. Deprotection of 59, followed by reductive amination
afforded 60 and ER-001259851–000.
affected Axl and Mer inhibitory activities. Among 47–52 (various R1
groups; R2 = 4-fluorophenyl), 47, 48, and 49 (less bulky substituents
compared with those in 50–52) showed lower inhibitory activities than
those of 35 in all four assays, suggesting that a bulky R1 substituent
might be preferable for increased inhibitory activity. Indeed, 50–52
showed higher inhibitory activities than those of 35, together with
marked Axl selectivity over Mer. When R2 was changed from 4-fluoro-
phenyl to 5-methylpyridine (60, ER-001259851-000), Axl inhibitory
activity was slightly diminished in the cell-free and cell-based assays,
but Axl selectivity over Mer was improved. Based on in vivo safety
screening for retinal toxicity and in vivo effect screening (data not
shown), ER-001259851–000 was chosen as the best compound. To
clarify the kinase selectivity of ER-001259851–000, cell-free kinase
assays using a panel of 52 kinases were conducted (Table S1). ER-
001259851–000 showed highly selective Axl inhibition activity.
Finally, the physicochemical and pharmacokinetic properties of ER-
001259851-000 were examined. ER-001259851-000 showed moder-
ate solubility in Dulbecco’s phosphate-buffered saline and moderate
membrane permeability, moderate stability in mouse liver microsomes,
but high stability in human liver microsomes (Table 5), and minimal
reversible inhibition on six cytochrome P450 isoforms (CYP1A2,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A) with IC50 values of
>20 µM. ER-001259851–000 showed moderate time-dependent inhi-
Table 4 shows how varying the R1 and R2 groups on the uracil moiety
Table 3
Effects of the R substituent on the in vitro inhibitory activities of 5.6.7.8-tetra-
hydropyrido[3,4-d]pyrimidine derivatives.
bition of CYP3A at 50 μM, no or minimal time-dependent inhibition of
the other isoforms were observed (Table 5). After a single oral admin-
istration of ER-001259851-000 at 25, 50, or 100 mg/kg to female
CAnN.Cg-Foxn1nu/CrlCrlj mice, the plasma concentration of ER-
001259851-000 peaked at 1 to 2 h postdose, and Cmax and AUC(0-24h)
values increased dose-proportionally within the dose range examined
(Table 6).
In summary, 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives
were designed as novel selective Axl inhibitors. Several derivatives with
high Axl inhibitory activity and high Axl selectivity over Mer were ob-
tained by using a combination of cell-free and cell-based in vitro assays
(e.g., 31, 35, 50, 51, 52, 60, ER-001259851-000). ER-001259851-000
was selected as the best derivative, and in vitro and in vivo assessments
showed that it has well-balanced physicochemical and pharmacokinetic
profiles. When compared with a previously reported pyrimidine deriv-
ative (1),12 ER-001259851-000 showed improved Axl selectivity and
membrane permeability in in vitro assays. Further biological evaluations
will be reported in due course.
Declaration of Competing Interest
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
All authors are employees of Eisai Co., Ltd., Japan.
4

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Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Scheme 6. Reagents and conditions: (a) TFA, DCM, RT; (b) corresponding acid, HATU, iPr2NEt, DMF, RT; (c) tert-butyl 2-bromoacetate, DMF, RT; (d) corresponding
amine, HATU, iPr2NEt, DMF, RT; (e) corresponding aldehyde or ketone, NaBH(OAc)3, THF, RT; (f) corresponding aldehyde, NaBH(OAc)3, THF, RT.
Table 4
Effects of R1 and R2 substituents on the in vitro inhibitory activities of 5.6.7.8-tetrahydropyrido[3,4-d]pyrimidine
derivatives.
5

S. Inoue et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Scheme 7. Reagents and conditions: (a) corresponding alkyl halide, K2CO3, DMF, 60 ◦C; (b) HCl in 1,4-dioxane, H2O, 70 ◦C; (c) 13, HATU, iPr2NEt, DMF, RT; (d)
TFA, DCM, RT; (e) 1-methylpiperidine-4-carbaldehyde, NaBH(OAc)3, THF, RT.
Scheme 8. Reagents and conditions: (a) corresponding amine, EtOH, 0 ◦C ~ RT; (b) phenyl chloroformate, iPr2NEt, THF, 0 ◦C ~ RT; (c) K2CO3, DMF, 80 ◦C; (d) HCl
in 1,4-dioxane, H2O, 80 ◦C; (e) 13, HATU, iPr2NEt, DMF, RT; (f) TFA, DCM, RT; (g) 1-methylpiperidine-4-carbaldehyde, NaBH(OAc)3, THF, RT.
Table 5
Physicochemical and pharmacokinetic properties of ER-001259851–000.
1
*
Solubility in Dulbecco’s phosphate-buffered saline. *2 Parallel artificial membrane
permeability assay. *3 Residual ratio was determined after incubation at 0.1 µM for 30 min
with human or mouse liver microsomes. *4 Time-dependent inhibition was evaluated at 50 µM
using a cocktail of probe CYP substrates with human liver microsomes.
6

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Bioorganic & Medicinal Chemistry Letters 48 (2021) 128247
Table 6
Pharmacokinetic parameters* of ER-001259851–000 in mice.
*
After a single oral administration of ER-001259851–000 at 25, 50, or 100 mg/kg to female CAnN.Cg-Foxn1nu/
CrlCrlj mice, blood was collected from the retinal vein at 0.5, 1, 2, 4, 8, and 24 h postdose and centrifuged to obtain
plasma samples. Plasma concentrations of ER-001259851–000 were measured by liquid chromatography tandem
mass spectrometry.
Activation of the AXL Kinase Causes Resistance to EGFR-Targeted Therapy in Lung
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