Acrylamide Functional Group Incorporation Improves Drug-like Properties: An Example with EGFR Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.8b00270

We demonstrate that the acrylamide group can be used to improve the drug-like properties of potential drug candidates. In the EGFR inhibitor development, both the solubility and membrane permeability properties of compounds 6a and 7, each containing an acrylamide group, were substantially better than those of gefitinib (1) and AZD3759 (2), respectively. We demonstrated that incorporation of an acrylamide moiety could serve as a good strategy for improving drug-like properties.

Full text of DOI:10.1021/acsmedchemlett.8b00270

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Letter
Acrylamide Functional Group Incorporation Improves
Drug-Like Properties: An Example with EGFR Inhibitors
Kuen-Da Wu, Grace Shiahuy Chen, Jia-Rong Liu, Chen-En Hsieh, and Ji-Wang Chern
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00270 • Publication Date (Web): 06 Dec 2018
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ACS Medicinal Chemistry Letters
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Acrylamide Functional Group Incorporation Improves Drug-Like
Properties: An Example with EGFR Inhibitors
Kuen-Da Wu,† Grace Shiahuy Chen,‡ Jia-Rong Liu,† Chen-En Hsieh,‡ Ji-Wang Chern†,*
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†School of Pharmacy and Center for Innovative Therapeutics Discovery, National Taiwan University, Taipei 10055, Taiwan,
‡Department of Applied Chemistry, Providence University, Taichung 43301, Taiwan
KEYWORDS: acrylamide, EGFR inhibitors, hydrophilicity, lipophilicity.
ABSTRACT: We demonstrate that the acrylamide group can be used to improve the drug-like properties of potential drug
candidates. In the EGFR inhibitor development, both the solubility and membrane permeability properties of compounds 6a and 7,
each containing an acrylamide group, were substantially better than those of gefitinib (1) and AZD3759 (2), respectively. We
demonstrated that incorporation of an acrylamide moiety could serve as a good strategy for improving drug-like properties.
functional group modification to penetrate the BBB remains a
Molecules containing an acryloyl functional group
major challenge in medicinal chemistry.
constitute an important class of biologically active
compounds, characterized by the formation of a covalent bond
with the target enzyme through Michael addition. Over the
past decades, this approach has had limited success in clinical
applications since it will induce unwanted side effects by
reacting with various off-targets.1,2 Recently, the advancement
of targeted therapy has led to a better understanding of the
balances between unavoidable side effects and efficacy in the
treatment oncology diseases.3,4 Kinase inhibitors afatinib and
ibrutinib, recently approved by the U.S. Food & Drug
Administration (FDA), belong to the class of acryloyl
compounds. This type of drug has advantages in terms of
lengthening the residence time through the formation of a
covalent bond with the targeted enzymes (Figure 1).5
Poor aqueous solubility and high lipophilicity are both
obstacles to drug absorption, increasing the risk of the patient
experiencing microsomal clearance, toxicity, and other side
effects.10 To increase either cell permeability or solubility of
lead compounds without detriment to the potency is a
challenging task in drug development. Previous approaches to
overcome solubility and permeability problems have been the
introduction of functional groups such as methyl, phenyl,
piperazine, morpholine, sugar, and amino acid.11,12
Log P is a tool to evaluate lipophilicity of the compound.
Generally speaking, the cLog P value for a potential drug
candidate should range between 0 to 5. When this value is
over 5, however, it is considered a high-risk compound for
development because of anticipated poor pharmacokinetics
profile for its difficulty in crossing cell membrane. The ideal
value for a drug candidate is ~2 to 3 (BBB-penetrating drugs
have values close to 2).13 A previous study has shown that
more than 50% of high-potency compounds possess cLog P
values of over 4.25.14 A widely used approach for decreasing
the cLog P value of a compound involves the addition of a
basic group, but this also increases the number of hydrogen
bond acceptors which is not beneficial for BBB penetration.15
O
N
O
N
H2N
N
O
N
N
N
H
N
N
HN
O
N
O
F
Cl
Afatinib
Ibrutinib
Figure 1. FDA-Approved Irreversible Inhibitors
As a general rule, a balance between potency and
lipophilicity in molecules results in reasonable efficacy and
pharmacokinetics clinical drug candidates. The enzymatic
interactions between afatinib and EGFR have been
described.16 The acryloyl group at the 6-position demonstrated
a covalent bond with Cys797.5 Replacement of the N-hydrogen
atom at the 4-position with a methyl group decreased the
binding activity. Thus, the NH group at the 4-position has
been considered to form critical hydrogen bond in the active
site of enzyme. In this study, we chose EGFR inhibitors
gefitinib (1) and AZD3759 (2) as lead compounds because of
their well-documented interactions with the active sites of the
Gefitinib (1) came to the market as the first-generation
epidermal growth factor receptor (EGFR) inhibitor for the
treatment of non-small cell lung cancer (NSCLC).6 Due to
drug resistance, scientists have developed the fourth-
generation EGFR tyrosine kinase inhibitors (TKIs) that enable
comparatively prolonged survival rates in patients.7 However,
metastasis to the CNS is observed in about 50% of treated
lung cancer patients. Unfortunately, no drug has yet been
developed to overcome this issue.8 AZD3759 (2), as an
alternative to EGFR-TKIs, is in Phase 1 clinical trials and it
may have a potential use advantage since it penetrates the
blood brain barrier (BBB).9 Designing drug candidates by
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enzymes. Compounds 6 and 7 were designed by introducing
Two types of acryloyl quinazoline compounds 6a-l and 7
were subjected to EGFR enzyme kinetic assay (Table 1).
Gefitinib derivative 6a exhibited an IC50 value of 65.2 nM,
1
2
3
4
5
6
7
8
an acryloyl group to the 4-anilinoquinazoline to study the
effects of the physicochemical properties of acrylamide
derivatives on the lead compounds gefitinib (1) and AZD3759
(2), respectively (Figure 2). We assumed that the N-acryloyl
group at the 4-position of the quinazoline core would not form
covalent bond with the target enzyme, acryloyl group situated
away from residue Cys797, but it might provide better
physicochemical properties. The solubility and lipophilicity of
the designed target compounds are also investigated in this
study.
less potent by over a 100-fold than that of gefitinib (1) (IC50
0.5 nM). When the 3ʹ-chlorine atom of 6a (3ʹ-chloro-4ʹ-fluoro
derivative) was replaced with either methyl or
˂
a
trifluoromethyl group, the resulting compounds 6b and 6c lost
activity with IC50 values of 499 nM and > 1.0 μM,
respectively. We assumed that the decreased inhibitory
activity might be due to the steric effect.
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Next, 3ʹ,4ʹ-difluoro (6e) and 3ʹ,4ʹ-dichloro (6f) derivatives
were prepared, and they exhibited enzyme inhibitory activity
with IC50 values of 368 and 31.7 nM, respectively.
Replacement of the 3ʹ-trifluoromethyl of 6c by a fluorine atom
(6e, IC50 = 368 nM) enhanced the potency against EGFR
kinase. Surprisingly, 3ʹ,4ʹ-dichloro derivative 6f exhibited an
increased enzyme inhibitory activity with an IC50 value of 31.7
nM.
Cl
Cl
3'
F
F
4'
MeN
HN
O
HN
4
N
O
N
N
O
N
6
7
O
MeO
N
MeO
N
Gefitinib (1)
AZD3759 (2)
It should be pointed out that there is slightly different
from 4ʹ-fluoro to 4ʹ-chloro between compound 1 and 5f, the
activity of 1 (IC50 ˂ 0.5 nM) against the enzyme is better than
that of 5f (IC50 = 1.0 nM). However, when the acryloyl group
was onto the 4-N-position of 1 and 5f, the enzyme inhibitory
activity of 6f is 2-fold better than that of 6a. This indicates
acryloyl moiety might play an important role in changing the
physicochemical properties related to biological activity.
R1
O
R2
N
N
R3O
N
MeO
6
7
&
Figure 2. Design of EGFR Inhibitors
It is known that a fluorine atom at the 2ʹ-position of
aniline was beneficial to form an intramolecular halogen bond
with the amide group.18,19 We introduced a fluorine at the 2ʹ-
position to investigate the effect of the anilino orientation,
Hence, variously substituted 2ʹ-fluoroanilines were used to
synthesize the corresponding acrylamide derivatives 6g-l, and
they were subjected to enzyme assays. Compound 6g with 3ʹ-
chloro-2ʹ-fluoro substituent showed an IC50 value of 52.6 nM
meanwhile 4ʹ-chloro-2ʹ-fluoro (6h, IC50 = 234 nM) and 5ʹ-
chloro-2ʹ-fluoro (6i, IC50 = 133 nM) derivatives were less
potent. When the 4ʹ-chlorine atom of 6h was replaced by 4ʹ-
methoxy (6j, IC50 ˃ 1.0 μM), markedly reduced activity was
observed, indicating that an electron donating group at the 4ʹ-
position reduces potency in this series of compounds. Neither
replacement of 4ʹ-chlorine with a fluorine atom (difluoro 6k,
IC50 = 302 nM) nor trifluoro compound 6l (with one more
fluorine atom at the 5ʹ-position, IC50 = 500 nM) improved
activity.
At the outset, commercially available 7-methoxy-6-(3-
morpholinopropoxy)-3,4-dihydroquinazolin-4-one (3) was
treated with phosphorus oxychloride at 80 °C to provide key
intermediate 4 according to the reported method17 (Scheme 1).
Then, compound 4 was reacted with a variety of anilines in the
presence of acetic acid to obtain 4-anilinoquinazoline
derivatives 5b-l. Subsequent treatment with acryloyl chloride
furnished the acrylamide derivatives 6b-l. Similarly,
commercially available gefitinib (1) and AZD3759 (2) were
reacted with acryloyl chloride to give compounds 6a and 7,
respectively.
Scheme 1. Synthetic Route to Compounds 6a-l and 7a
O
O
O
Cl
N
O
N
O
a
NH
N
MeO
N
MeO
N
3
4
R
R
O
Based on the results described in supra, we observed that
the active compounds 6a, 6f, and 6g all have the 3ʹ-
chloroaniline substitution in common, indicating that the
chlorine atom at the 3ʹ-position is important for efficient
binding. An SAR analysis of this series of compounds allowed
us to assume that the pronounced enzyme inhibition activity
might arise from the 3ʹ-chloroaniline. In addition, we were
motivated to investigate the effect of the acryloyl group at the
4-position of AZD3759 (2). Notably, compound 7 showed the
greatest activity with an IC50 value of 8.7 nM. and this
pronounced activity might be due to the fluorine atom at the
2ʹ-position allowing co-planarity through internal halogen
bonding.
O
HN
N
O
N
N
b
c
N
O
N
O
N
N
MeO
MeO
6a
,R = 3-Cl, 4-F
,R = 3-Me, 4-F
6g
6h
6i
6j
,R = 2-F, 3-Cl
,R = 2-F, 4-Cl
,R = 3-CF3, 4-F , R = 2-F, 5-Cl
1
5b
5c
5d
5e
5f
5g
5h
5i
5j
,
R = 3-Cl, 4-F
,R = 3-Me, 4-F
,R = 2-F, 3-Cl
,R = 2-F, 4-Cl
,R = 3-CF3, 4-F , R = 2-F, 5-Cl
6b
6c
6d
6e
,R = 3-CF3
,R = 3-F, 4-F
, R = 3-Cl, 4-Cl
, R = 2-F, 4-OMe
,R = 2-F, 4-F
6l
,R = 2-F, 4-F, 5-F
,R = 3-CF3
,R = 3-F, 4-F
, R = 3-Cl, 4-Cl
, R = 2-F, 4-OMe
,R = 2-F, 4-F
5l
,R = 2-F, 4-F, 5-F
6k
5k
6f
Cl
Cl
F
F
O
MeN
HN
N
MeN
N
N
N
O
N
O
c
N
N
O
O
MeO
MeO
2
7
aReagents and conditions: (a) POCl3, Et3N, 80 °C, 6 h;
(b) substituted aniline, AcOH, 80 °C, 7 h; (c) acryloyl
chloride, Et3N, CH2Cl2, 8 h.
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ACS Medicinal Chemistry Letters
Table 1. Structure–Activity Relationships for EGFR Inhibitors
Obtained by Enzyme Assay
Table 2. In Vitro Cytotoxicity Assay against A549 cancer cell
lines
1
2
3
4
5
6
7
8
9
Compound R1
R2
IC50 (μM)
Cl
F
R2
1
6a
5f
6f
5g
6g
2
H
3-Cl,4-F
3-Cl,4-F
3-Cl,4-Cl
3-Cl,4-Cl
2-F,3-Cl
2-F,3-Cl
2-F,3-Cl
2-F,3-Cl
5.74 ± 0.40
0.15 ± 0.01
24.67 ± 2.08
5.72 ± 0.16
7.51 ± 0.28
0.45 ± 0.02
28.67 ± 5.69
6.10 ± 0.13
R1
R1
MeN
N
O
N
N
N
O
acryloyl
H
N
N
O
N
O
MeO
N
MeO
1, 5
6
2
7
&
&
acryloyl
H
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EGFR
Compound R1
R2
IC50 (nM)
˂ 0.5
65.2
499
acryloyl
H
1
H
3-Cl, 4-F
3-Cl, 4-F
3-Me, 4-F
3-CF3, 4-F
3-CF3
6a
6b
6c
6d
6e
5f
6f
5g
6g
6h
6i
acryloyl
acryloyl
acryloyl
acryloyl
acryloyl
H
7
acryloyl
˃ 1000
844
To investigate the underlying cause of the non-correlation
between cellular cytotoxicity and enzymatic inhibition, the
physicochemical properties of the studied compounds were
determined (Table 3). It should be noted that AZD3759 (2) has
better solubility in 1-octanol than gefitinib (1), indicating that
it crosses the BBB more easily (Sol1-octanol = 4.54 mM and 1.40
mM, respectively).9 Interestingly, an introduction of the
acryloyl group to 1, leading to 6a, significantly enhanced the
solubility in both aqueous solution (from ˂0.01 mM to 1.65
mM) and 1-octanol (~3.5-fold; from 1.40 mM to 4.94 mM).
The enhanced solubility was also observed in acrylamide-
containing compounds 6f, 6g, and 7. However, compound 7
showed only slightly improved solubility relative to 2 (~1.7-
fold in aqueous solution; 0.02 mM for 2, 0.03 mM for 7 and
1.1-fold in 1-octanol; 4.54 mM for 2, 5.02 mM for 7). This
observation might be probably due to the intramolecular
interaction between the 2ʹ-fluorine atom and the oxygen atom
of amide group leading to either a reduced acrylamide
hydration21 or an aggregation of planar structure.
3-F, 4-F
368
3-Cl, 4-Cl
3-Cl, 4-Cl
2-F, 3-Cl
2-F, 3-Cl
2-F, 4-Cl
2-F, 5-Cl
1.0
acryloyl
H
31.7
˂ 0.5
52.6
234
acryloyl
acryloyl
acryloyl
acryloyl
acryloyl
acryloyl
H
133
6j
6k
6l
2-F, 4-OMe
2-F, 4-F
˃ 1000
302
2-F, 4-F, 5-F
500
The thermodynamic solubility assay demonstrated that
the acrylamide derivative was more soluble than its precursor
in both aqueous solution and 1-octanol. In general, enhancing
the aqueous solubility of one molecule would lead to reduce
lipophilicity. Surprisingly, we found out that acrylamide
modification not only significantly enhanced aqueous
solubility but also slightly promoted 1-octanol solubility. This
might lead to improved intracellular drug concentrations as
well.
2
˂ 0.5
8.7
7
acryloyl
Potent EGFR inhibitors were subjected to anti-
proliferative assays against EGFR inhibitors sensitive A549
non-small lung carcinoma cells.20 The anti-proliferative effects
of compounds containing the acryloyl moiety, such as 6a, 6f,
6g, and 7, had lower IC50 values than their corresponding
precursors 1, 5f, 5g, and 2, respectively (Table 2). Notably,
compounds 6a and 6g were the most potent with IC50 values of
0.15 and 0.45 μM, respectively. However, 6a and 6g were
100-fold less potent than their corresponding aniline
derivatives 1 and 5g in the enzymatic assay. Surprisingly, we
did not find correlation between the anti-proliferative effect
and EGFR enzyme inhibitory activity. The acryloy containing
compounds 6a, 6f, 6g, and 7 exhibited better anti-proliferative
activity than their precursors 1, 5f, 5g, and 2.
The Log P values (where P is the partition coefficient)
were then predicted using the ALOGPS 2.1 of each compound
listed in Table 3; the Log D (where D is the distribution
coefficient) values were obtained by the shake flask assay to
measure the two-phase distributions.22,23 Both of these values
suggest that the inclusion of an acrylamide group decreased
the lipophilicity of the compounds (such as 6a, 6f, 6g, and 7),
resulting in an ideal balance between solubility and
permeability. Our results demonstrate that acrylamide
modification can be applied for reducing the Log P (partition-
coefficient) or Log D (distribution-coefficient) values.
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Table 3. Assay Results for Physicochemical Properties
Solubility (mM)
1
2
3
4
5
6
7
8
9
d
Cpd
1
pH7.4 aqueous solutiona
1-octanolb
1.40
aLog Pc
4.02
3.23
4.63
3.80
4.08
3.32
3.66
3.83
Log D7.4
2.41
2.01
NDf
t1/2e (min)
NAg
˂ 0.01
1.65
NDf
6a
5f
6f
5g
6g
2
4.94
85.1
4.57
NAg
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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55
56
57
58
59
60
0.63
0.07
1.20
0.02
0.03
4.47
3.57
NDf
17.0
4.34
NAg
5.60
2.64
2.84
2.46
49.0
4.54
NAg
7
5.02
27.5
aThermodynamic solubility in pH7.4 buffer solution. Thermodynamic solubility in 1-octanol. ALOGPS 2.1, molecular property
prediction.22 dShake flask assay (solubility in 1-octanol/pH 7.4 aqueous solution = 1/2, v/v).23 eHalf-life (t1/2) with glutathione in
pH 7.4 and 37 °C (compound/glutathione = 1/10, c/c).24 fND, not detectable. gNA, not applicable.
b
c
It is of our interest to study the stability of this type of
compounds in aqueous solution. Glutathione should add to the
acryloyl moiety easily by Michael addition.24 Thus,
glutathione was used to test stability. Among four acryloyl
compounds (Table 3), the most anti-proliferative compound 6a
demonstrated a half-life value of 85.1 min, even in the
presence of high concentration of glutathione.
Information). Indeed, molecular modeling demonstrated that
compound 6a would have hydrogen bond to the hinge residue
Met793 as that in the crystal structure. This result provided
further evidence for the potency of compound 6a. The residue
Cys797 was far away from the acryloyl moiety of 6a, and thus
there is no possibility to form covalent bond in the binding
model (Supporting Information).
The toxicity assays to FHS and Vero normal cell lines
and hERG were carried out to exclude off-target effect with
acryloyl group (Table 4). Further, compound 6a was screened
against a panel of 22 kinases, and the results showed that the
compound 6a was a highly selective target therapy agent
(Supporting Information).
In summary, acrylamide compound 6a were synthesized
by introducing an acryloyl functional group to the EGFR
inhibitor 1. The acrylamide group significantly improved both
water and 1-octanol solubility. The improved hydrophilicity
should arise from the oxygen atom of the acryloyl group
hydrogen bonded to water, and the improved lipophilicity
came from the change of secondary amine to a tertiary amide.
Both improved physicochemical properties contributed to the
improved cell permeability. Our results suggest that
acrylamide could serve as a potential functional group for the
optimization of desirable physicochemical properties in drug
design.
Table 4. Evaluation of drug toxicity
IC50 (μM)
Cpd
FHSa
Verob
hERG
NDc
1
˃ 10 (34%)
9.17 ± 0.09
˃ 10 (30%)
˃ 10 (47%)
ASSOCIATED CONTENT
6a
˃ 10 (20%)
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
aFHS: normal cell of human small intestine. Vero: normal
cell of Cercopithecus aethiops kidney. cND: not
determined.
b
Experimental details for preparation of compound 6a-l and 7,
including characterization data (PDF)
AUTHOR INFORMATION
Corresponding Author
To investigate whether a covalent bond exists or not,
computation and molecular modeling were performed. Firstly,
the structure of 6a was optimized by DFT theory of
computation, and the optimized structure showed a dihedral
angle of 40 degree between aniline and quinazoline rings
which was in close to that of 1 (45°) in cocrystal structure to
EGFR kinase. Further, the optimized 6a was docked into
EGFR kinase (pdb ID: 2ITY), and the results illustrated no
covalent bond formation between 6a and kinase (Supporting
*Phone: +886 2 33668697. E-mail: jwchern@ntu.edu.tw
Author Contributions
KD Wu and JW Chern conceived the project and designed the
experiments. KD Wu synthesized the compounds and analyzed
the physicochemical properties. JR Liu performed the in vitro cell
assay. CE Hsieh and GS Chen carried out the molecular modeling
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Gilles, R. W.; Greene, N.; Huang, E.; Krieger-Burke, T.; Loesel,
J.; Wager, T.; Whiteley, L.; Zhang, Y. Physiochemical drug
properties associated with in vivo toxicological
and computational calculation, respectively. KD Wu and JW
Chern analyzed the data and wrote the manuscript.
1
2
3
4
5
6
7
8
Funding Sources
outcomes. Bioorg. Med. Chem. Lett. 2008, 18, 4872–4875.
11. Gorham, R. D., Jr.; Forest, D. L.; Khoury, G. A.; Smadbeck, J.;
Beecher, C. N.; Healy, E. D.; Tamamis, P.; Archontis, G.;
Larive, C. K.; Floudas, C. A.; Radeke, M. J.; Johnson, L. V.;
Morikis, D. New compstatin peptides containing N-terminal
extensions and non-natural amino acids exhibit potent
complement inhibition and improved solubility characteristics. J.
Med. Chem. 2015, 58, 814–826.
This study was supported by grants from the Aiming for Top
University Project, Ministry of Education, Taiwan.
Notes
The authors declare no competing financial interest.
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crystal lattice. J. Med. Chem. 2013, 56, 2690–2694.
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Y. J.; Zilberstein, A.; Johnson, S. E.; Hook, L. E.; Jacoski, M. V.
The preparation and sar of 4-(anilino), 4-(phenoxy), and 4-
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ACKNOWLEDGMENT
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This study was supported by grants from the Aiming for Top
University Project, Ministry of Education, Taiwan.
ABBREVIATIONS
EGFR, epidermal growth factor receptor; NSCLC, non-small cell
lung cancer; TKI, tyrosine kinase inhibitor; CNS, central nervous
system; SAR, structure−activity relationship; BBB, blood brain
barrier.
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Cl
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MeO
MeO
Gefitinib (1)
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EGFR IC50 = 65.2 nM
A549 IC50 = 0.15 M
Solubility pH7.4 = 1.65 mM
Solubility 1-Octanol = 4.94 mM
EGFR IC50  0.5 nM
A549 IC50 = 5.74 M
Solubility pH7.4  0.01 mM
Solubility 1-Octanol = 1.40 mM
Log D7.4 = 2.41
Log D7.4 = 2.01
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