G Model
CCLET 5250 No. of Pages 7
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X. Hu et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
Fig. 1. The reported fourth generation EGFR inhibitors overcoming EGFRC797S mutation.
EGFRL858R/T790M/C797S. When exocyclic N atom in the linker was
incorporated into the cycloalkyl group, the resulting compounds
(
6c–6h)
showed
greatly
decreased
potency
on
L858R/T790M/C797S
EGFR
. Compound 6i with cis-cyclohexanamine
instead of the trans-cyclohexanamine exhibited an IC50 value of
L858R/T790M/C797S
3
.1 nmol/L against EGFR
, which is comparable to
that of JND3229. Moreover, this compound is 7-fold more potent
L858R/T790M
than JND3229with the inhibitoryactivityof EGFR
double
mutant. These results indicated that the cyclohexanamine group in
the linker fraction was preferable for the high inhibitory activity.
After determined that the cyclohexanamine was the optimal
substituent for the L part, we then turned to optimize the R1
substitution (Table 1). It was revealed that the ethyl in JND3229
could be replaced with a variety of relative small substituents (e.g.,
methyl (6j), isopropyl (6k), tert-butyl (6l)), without obviously
affecting the EGFR inhibitory potency. However, when the ethyl was
replaced with larger hydrophobic groups, including cyclopentyl
(6m), or cyclohexyl (6n), and phenyl (6o), the resulting molecules
displayed about 3- to 7-folds potency loss. For instance, 6o with the
Fig. 2. The designed 2-oxo-3,4-dihydropyrimido[4, 5-d] pyrimidines privileged
scaffold based on JND3229.
water molecule. Herein, we would like to describe extensive
structure-activity relationships (SARs) of this 2-oxo-3,4-dihydro-
pyrimido[4,5-d]pyrimidine privileged scaffold based compounds
phenyl substituent exhibited an IC50 value of 14.4 nmol/L against
L858R/T790M/C797S
(
Fig. 2).
The synthetic routes of the designed2-oxo-3, 4-dihydropyrimido
4, 5-d]pyrimidinyl analogues were outlined in Scheme 1. Briefly,
EGFR
. Interestingly, introducing an N atom into this
phenyl group in 6o further reduced the compound’s activity (6p,
IC50 = 35.2 nmol/L). It was worthy to note that when the propionyl
moiety in JND3229 was replaced with a propyl group, the resulting
compound (6x) dramatically decreased the kinase inhibitory
[
reduction of 4-chloro-2-(methylthio)-pyrimidine-5-carboxylate
(
(
7) with diisobutyl aluminium hydride (DIBAL) gave (4-chloro-2-
methyl thio)pyrimidin-5-yl)methanol, which were subsequently
potency with an IC50 value of 139.7 nmol/L against
L858R/T790M/C797S
oxidized byMnO
2
toyieldaldehyde 9. Forcompounds6a-6uand6x,
EGFR
. The investigation suggested that a critical
9
then reacted with BOC-protected aliphatic amines, followed by
interaction mightexist betweenthe carbonylgroup andthe protein.
T790M/C797S
Borch reductive amination, cyclization and oxidation to yield key
pyrimidopyrimidinone 14. For compounds 6v and 6w, 9 went
through Grignard reaction to give alcohol 15, which followed by
chlorination, nucleophilic substitution, cyclization and oxidation to
yield key pyrimidopyrimidinone 20. The intermediates 14 and 20
were respectively subjected to nucleophilic, deprotection and
acylation reaction to give the title compounds 6a-6w.
The kinase inhibitory activities of the title compounds were
evaluated by the enzyme-linked-immunosorbent assay (ELISA)
assay (Table 1). Osimertinib, staurosporine and JND3229 were used
The X-ray structure analysis of EGFR
and JND3229
complex revealed that the methyl substituted phenylpiperazine
moiety of JND3229 was exposed to a solvent accessible surface
0
[16]. We then explored the preliminary SARs of 3 -methyl
0
substituent. When the 3 -methyl group in JND3229 was replaced
0
0
with 3 -CF
3
(6r) or changed to 2 -position (6u), the resulting
compounds exhibited comparable activities, while replacement of
0
0
3 -methyl group with the other substituents, e.g., 3 -F (6r), 3ꢀOCH
3
(6s), or removal of it (6t), yielded compounds with potency loss
ranging from 2- to 3-folds.
as positive controls to validate the screening conditions. As shown
3
At last, we investigated the potential steric impact of R position
L858R/T790M
in Table 1, osimertinib potently inhibits EGFR
, while is
by substitution with methyl (6v) and ethyl (6w). The compound 6v
exhibited the equal activity compared to the parental compound
JND3229, while the ethyl substituted compound 6w displayed
about 4-folds potency loss, suggesting that the bulky moieties
were not tolerated at this site.
L858R/T790M/C797S
less potent against EGFR
. However, staurosporine,
a broad spectrum of multi-kinase inhibitor, suppresses the
L858R/T790M
L858R/T790M/C797S
enzymatic activity of the EGFR
and EGFR
mutants with low nanomolar IC50 values.
We firstlyinvestigatedthe SARs on the linker L(Fig. 2)andtheresults
were summarized in Table 1. Change of trans-cyclohexanamine in
JND3229withacis-cyclopyrrilidinamine(6a)andlinearethylamine
In order to get insight into the binding mode of cys-cyclo-
T790M/C797S
hexanamine with EGFR
, a co-crystal structure of 6i
T790M/C797S
complexed with EGFR
was determined (Fig. 3A and
(
6b) led to 14-fold and complete potency loss against
Table S1 in Supporting information). It was indicated that 6i bound
(