2
X. Qin et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Reversible EGFR inhibitors
O
F
HN
Cl
HN
HN
N
O
O
O
O
O
O
O
N
O
O
N
N
O
N
N
N
Gefitinib
Erlotinib
Icotinib
Irreversible EGFR inhibitors
F
F
F
O
O
N
HN
N
Cl
HN
Cl
HN
N
Cl
H
HN
O
N
HN
N
N
N
N
O
O
O
N
N
O
O
Canertinib
Afatinib
Figure 1. Chemical structures of EGFR-TKIs.
Dacomitinib
this strategy.25,26 Accordingly, we have designed and synthesized a
series of novel morpholin-3-one fused quinazoline derivatives
through intramolecular cyclization as new EGFR inhibitors (Fig. 2).
The synthetic route of compounds a1–a15 is illustrated in
Scheme 1. Commercially available 4-hydroxy-3-nitrobenzoic acid
(a1–a15), the inhibitory activities against EGFRwt ranged from
53.1 nM to 829.2 nM except a5 and a14 in which IC50 > 1000 nM.
1
2
When R was halogen (F, Cl or Br) and R was hydrogen (a1, a2,
a3), compounds substituted by Cl or Br (a2, IC50 = 63.1 nM; a3,
IC50 = 89.6 nM) displayed 2-fold increase in potency than substi-
tuted by F (a1, IC50 = 245.1 nM). While R was hydrogen and R2
was halogen atom (a9, a10, a11), the results of inhibition against
EGFR was unsatisfactory. This may be that meta-position com-
pounds (a1, a2, a3) could form a stronger interaction with EGFR
1
1
was esterified with methanol using concentrated sulfuric acid
27
as a catalyst to give compound 2. Alkylation of methyl 4-
hydroxy-3-nitrobenzoate 2 with ethyl bromoacetate and reduction
of the nitro group with Fe/acetic acid followed by in situ cyclization
yielded intermediate 4. Subsequent nitration and alkylation pro-
vided 6. Then reduction the nitro group of 6 using Fe/acetic acid
gave 7, which was subjected to cyclization to generate intermedi-
ate 8. Following, 8 was chloridized by phosphorus oxychloride to
generate intermediate 9. Finally, condensation of 9 with substi-
tuted anilines afforded 4-anilinoquinazoline derivatives a1–a15.
Synthesis of compounds b1–b6 is shown in Scheme 2. Interme-
diate 5, which could be synthesized according to Scheme 1, was
reacted with 1-bromo-3-chloropropane and piperidine in sequence
to achieve 11. The target compounds were synthesized in good
yields by the next four step reactions according to Scheme 1.
To evaluate the EGFR inhibitory potency of the new compounds,
Kinase-Glo luminescent assay is used to test their ability to block
EGFR tyrosine kinase. Compounds were initially texted at final con-
centrations of 1000, 200, 40, 8, 1.6, 0.32, and 0.06 nM and per-
formed in duplicate at each concentration with gefitinib as
positive control. Compound inhibition curve was fitted using
Graphpad Prism 5.0 software based on the inhibition rate of all
compounds at different concentrations. The testing results are
summarized in Table 1. Almost all the compounds displayed signif-
1
kinase than para-position compounds (a9, a10, a11). When R
2
was Cl and R was F, compound a8 displayed the most inhibitory
wt
1
2
activity toward EGFR (IC50 = 53.1 nM). When R or R was meth-
oxy or trifluoromethyl group, compounds (a5, a6, a14,) displayed
weaker potency against EGFR kinase. One explanation might be
1
that a larger volume of R group could not effectively fill up well
1
the hydrophobic pocket of EGFR kinase. In summary, when R
2
was Cl or Br, and R was H or F, compounds (a2, a3, a8) displayed
wt
excellent inhibitory activities against EGFR
(IC50 < 89.6 nM).
Among them, compound a8 displayed the most inhibitory activity
wt
toward EGFR (IC50 = 53.1 nM).
To find a more potent EGFR inhibitor, R was substituted by 3-
(piperidin-1-yl)propyl in the subsequent synthesis (b1–b6).
Among them, b3 was the most active compound against EGFR
1
(IC50 = 99 nM). Compounds b2, b3, b6 (R = Cl or Br) showed much
1
more potency than b1 (R = F). This was the same phenomena as
1
the former discussion (a1–a3, R = halogen). The reason may be
that small fluorine atom could be too small to fill up well the
hydrophobic pocket of EGFR kinase pocket. However, when R1
was a relatively larger volume group methyl or ethynyl, com-
pounds (b4, b5) did not showed good inhibitory activities towards
icant EGFR inhibitory activities (IC50 < 1 lM). Among them, com-
wt
1
pound a8 showed the most potent inhibitory activity against
EGFR . As a whole, R was Cl or Br would be beneficial for improv-
wt
wt
EGFR kinase (IC50 = 53.1 nM). When R was 3-morpholinopropyl
ing the inhibitory activities against EGFR .
O
N
F
R
O
R2
R1
HN
N
Cl
HN
N
O
O
Design
N
O
N
N
Cycliczation
Gefitinib
Figure 2. Design strategy and modification of novel EGFR inhibitors.