Discovery of potent, orally bioavailable ERK1/2 inhibitors with isoindolin-1-one structure by structure-based drug design

Article abstract of DOI:10.1016/j.ejmech.2018.12.040

Constitutive activation of MAPK (RAS/RAF/MEK/ERK) pathway is frequently observed in many tumors and thus has become an interesting therapeutic target for cancer therapy. Despite the successful development of BRAF and MEK inhibitors in clinic treatment, resistance often appears to re-enhance ERK1/2 signaling. Inspired by the central role of the ERK1/2 signaling cascade in cancer, we describe the scaffold-hopping generation of a series of isoindolin-1-one ERK1/2 inhibitors. Our new compounds could inhibit proliferation of KRAS and BRAF mutant cells lines at low nanomolar concentrations. Compound 22a possesses acceptable pharmacokinetic profiles and showed considerable in vivo antitumor efficacy in a HCT-116 xenograft model, providing a promising basis for further optimization towards clinical ERK1/2 inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.12.040

Accepted Manuscript
Discovery of potent, orally bioavailable ERK1/2 inhibitors with isoindolin-1-one
structure by structure-based drug design
Dezhong Ji, Lingzhi Zhang, Qihua Zhu, Ying Bai, Yaoyao Wu, Yungen Xu
PII:
S0223-5234(18)31077-8
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.040
EJMECH 10973
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 October 2018
Revised Date: 28 November 2018
Accepted Date: 16 December 2018
Please cite this article as: D. Ji, L. Zhang, Q. Zhu, Y. Bai, Y. Wu, Y. Xu, Discovery of potent, orally
bioavailable ERK1/2 inhibitors with isoindolin-1-one structure by structure-based drug design, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.040.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of potent, orally bioavailable ERK1/2 inhibitors with isoindolin-1-one
structure by structure-based drug design
a
, £
b,
£
, a, b
b
b
, a, b
Dezhong Ji , Lingzhi Zhang , Qihua Zhu* , Ying Bai , Yaoyao Wu , Yungen Xu*
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 21009, China
b
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China
£
Both authors contributed equally to this work
ABSTRACT
Constitutive activation of MAPK (RAS/RAF/MEK/ERK) pathway is frequently observed in many
tumors and thus has become an interesting therapeutic target for cancer therapy. Despite the successful
development of BRAF and MEK inhibitors in clinic treatment, resistance often appears to re-enhance
ERK1/2 signaling. Inspired by the central role of the ERK1/2 signaling cascade in cancer, we describe
the scaffold-hopping generation of a series of isoindolin-1-one ERK1/2 inhibitors. Our new compounds
could inhibit proliferation of KRAS and BRAF mutant cells lines at low nanomolar concentrations.
Compound 22a possesses acceptable pharmacokinetic profiles and showed considerable in vivo
antitumor efficacy in a HCT-116 xenograft model, providing a promising basis for further optimization
towards clinical ERK1/2 inhibitors.
1
. INDTODUCTION
The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway
plays a central role in controlling mammalian cellular functions, including adhesion, migration,
1
differentiation, metabolism, and proliferation. This pathway is activated by a variety of extracellular
stimulus, resulted in activation of the RAS (KRAS, NRAS, and HRAS) and then leads to the
recruitment and activation of RAF, a serine-threonine kinase. Subsequently, signal is transmitted
downstream through activated RAF by activates its main substrates MEK1 and MEK2, which are dual-
specificity kinases that activate their only substrates ERK1 and ERK2 via the phosphorylation of
2
conserved threonine and tyrosine residues located within the activation loop. When activated, ERK1/2
3, 4
phosphorylate several downstream substrates involved in both nuclear and cytoplasmic components
,
leading to a wide range of cellular events including cytoskeletal changes, transcriptional activation,
5
which promote cell cycle progression and also regulate negative feedback mechanisms.
The ERK pathway is aberrant activated in more than 30% of human cancers through a high
frequency of genetic alteration. BRAF mutation has been identified in colorectal (10%), thyroid (∼
5
0%) and melanoma (60%) tumors, whereas RAS is mutated in melanoma (20%), colorectal (50%) and
6
pancreatic (90%) tumors , and thus has attracted significant interest as a therapeutic target for cancer.
Specifically inhibit BRAF and its downstream effectors MEK have shown promising activities in the
7
clinical to target BRAF and RAS mutant tumors. In particular, the BRAF inhibitors, vemurafenib and
8
9
10
dabrafenib , and the MEK inhibitor, trametinib and cobimetinib as treatments for BRAF-mutant

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metastatic melanoma, validate the strategy of targeting the ERK pathway as an effective way of
treating cancer. However, acquired resistance to BRAF inhibitors is often occurs in some patients due
11-14
to genetic alteration resulting in pathway reactivation
, and limited efficacy as single-agent therapies
1
5
has been reported with MEK inhibitors . Additionally, negative feedback loops within the ERK
pathway lead to pathway reactivation when inhibits single node, driving innate resistance of some RAS
16
and BRAF mutant tumors to BRAF or MEK inhibitors . The emerged resistance to BRAF and MEK
inhibitors has led to greater interest in directly targeting ERK, a downstream key signaling node of the
MAPK pathway, to provide alternative therapeutic option in tumors with mutations in RAS or BRAF
genes and also may have clinical utility in overcoming acquired resistance to BRAF and MEK
1
7
inhibitors.
In contrast to the advanced development of BRAF and MEK inhibitors, the development of small
molecule inhibitors of ERK has fallen behind. Stimulated by the key role of the ERK in MAPK
pathway, multiple groups have developed their ERK inhibitors, including several reaching clinical
18
19
20
21
trials (Figure 1), such as GDC-0994 (1) , BVD-523 (2) , SCH7729984 (3) , CC-9003 (4) , MK-
2
2
23
24
8
353 , LTT462 , KO-947 . However, the clinical trials involving ERK inhibitors are slow and some
21, 23
have recently been terminated or no longer recruiteparticipants.
Therefore, the identification of
high-quality inhibitors of ERK1/2 remains of great interest. Herein, we report the structure-based
design of a novel scaffold derived from 1 and a series of modifications to the scaffold, led to the
discovery of 19i as high-level potent ERK inhibitors.
Figure 1. Structures of published clinical ERK1/2 inhibitors
2
. RESULTS AND DISCUSSION
2
.1. Structure-Based Design of Novel ERK Inhibitors
BVD-523 (Figure 2) is an ATP competitive ERK inhibitor , which was chosen as a lead compound
19
in our new structure design. In the docking structure of BVD-523 bond to ERK2 (PDB code 5K4I), the
nitrogen of pyrimidine ring accepts one hydrogen bond from the hinge NH of Met108 while the 2-
amino group donates one hydrogen bond to the carbonyl oxygen of the same residue. Additionally, the
pyrrole-2-carboxamide oxygen makes H-bonds with water mediated H-bond to Gln105 and catalytic
Lys54. The 3-chlorophenyl group inserts into a hydrophobic pocket under the glycine-rich loop. The
hydroxyl group is forms H-bond from the side chains of Asp167 (Figure 2A). In the aspect of
designing a new scaffold, it was desirable to maintain the critical interactions between BVD-523 and
the ERK2 protein. We speculated that the pyrrole-2-carboxamide moiety in BVD-523 might be
effectively replaced by an isoindolin-1-one linker, as shown in Scheme 1, based on the following
considerations. First, modeling studies (Figure 2B) showed that the isoindolin-1-one carbonyl could
mimic the oxygen of pyrrole-2-carboxamide, form the same hydrogen bond to Lys54 and water
mediated hydrogen bond to Gln105. Second, overlap modeling of the isoindolin-1-one and pyrrole-2-
carboxamide scaffold revealed that the left-part pyrimidine formed critical H-bond with hinge and
right-part benzyl moieties occupied the same cave under glycine-rich loop (Figure 2B). Thus, we
designed and synthesized the isoindolin-1-one derivatives 12a~12l. Considering the hydroxymethyl

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group could form H-bonds with Asp167 and Asn154 residues, we also synthesized the isoindolin-1-one
derivatives 19a~19l with hydroxymethyl group.
Scheme 1. Structure-Based Design: From 4-pyridone to isoindolin-1-one
Figure 2. (A) BVD-523 docked into X-ray structure of ERK2 (PDB code 5K4I). Hydrogen bond
interactions are illustrated with yellow dashed lines. (B) Overlap of the proposed binding modes of 12a
(
(
green) and BVD-523 (yellow) in ERK2 (PDB code 5K4I). The figure was generated using PyMol
http://www.pymol.org/).
2
.2. Chemistry
The isoindolin-1-one derivatives 12a-12l were synthesized via two different procedures described in
Scheme 2 and Scheme 3. In Scheme 2, Pinacol boronate ester 6 was prepared by palladium mediated
coupling from compound 5, followed by protection of amide obtained compound 7. Then a second
palladium catalyzed Suzuki-coupling to furnish the pyrimidine derivative 8 and deprotection to get
amide 9. The N-substituted isoindolin-1-one derivatives 10a-10e were prepared with sodium hydride
and the corresponding benzyl bromide. Later 10a-10e were oxidized to sulfone 11a-11e, then
displacement of 2-methylsulfonyl group with corresponding amine gave the final compounds 12a, 12b
and 12f-12l. In Scheme 3, Pinacol boronate ester 14 was prepared by palladium mediated coupling
from compound 13. Brominated with 14 gave aryl bromide 15, which was cyclized with corresponding
amine to intermediate 16a-16c. A palladium catalyzed Suzuki-coupling to furnish the pyrimidine
derivative 17a-17c. Then a second palladium catalyzed Buchwald-coupling with corresponding amine
gave compounds 12c-12e.
Hydroxymethylated analogues 22a-22l were prepared as illustrated in Scheme 4. Suzuki coupling of
1
4 and commercially available 4-chloro-2-(methylthio) pyrimidine produced compound 18, which was
converted to the intermediate 19 via oxidation. Brominated with 19 gave aryl bromide 20, which was
cyclized with corresponding benzyl amine to key intermediate 21a-21c. Installation of the
corresponding amine afforded 22a-22l. Hydroxymethylated analogues such as 24a-24e requiring a
slightly different synthesis route as illustrated in Scheme 5. TBS protected 23 installed the
corresponding amine followed by TBS deprotection afforded 24a-24e. The (S) or (R)-22i and (S) or
(
R)-22l were prepared by corresponding (S) or (R) benzyl amine within scheme 4.

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a
Scheme 2. Preparation of Compounds 12a, 12b and 12f-12l
a
o
Reaction conditions: (a) B (pin) , KOAc, Pd(dppf)Cl , dioxane, N , 90 C, 12 h, 71%; (b) (Boc) O,
2
2
2
2
2
o
DMAP, DCM, 25 C, 2 h, 96%; (c) 4-chloro-2-(methylthio)pyrimidine, Pd(PPh ) , Na CO ,
3
4
2
3
o
o
PhMe/MeOH/H O, 70 C, 8 h, 90%; (d) TFA, DCM, 25 C, 1 h, 95%; (e) corresponding arylmethyl
2
o
o
bromide， NaH, dioxane, 80 C, 12 h, 55%; (f) m-CPBA, DCM, 25 C, 5 h, 83%; (g) corresponding
o
amine, 2-butyl alcohol 125 C, 12 h, 31%.
a
Scheme 3. Preparation of Compounds 12c-12e
a
o
Reaction conditions: (a) B (pin) , KOAc, Pd(dppf)Cl , dioxane, N , 90 C, 5 h; (b) NBS, BPO, CCl ,
2
2
2
2
4
o
8
0 C, 4 h; (c) 2-Aminomethyl-6-methyl-pyridine or 2-Aminomethyl pyridine or 3-Aminomethyl
o
o
pyridine, Et N, MeCN, 70 C, 12 h; (d) 2,4-dichloropyrimidine, Pd(PPh ) , K PO , dioxane/H O, 80 C,
3
3 4
3
4
2
o
1
2 h; (e) Pd(OAc) , XantPhos, Cs CO , 1,4- dioxane, N , 100 C, 12 h.
2 2 3 2
a
Scheme 4. Preparation of Compounds 22a-22l

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a
Reaction conditions: (a) 4-chloro-2-(methylthio)pyrimidine, Pd(PPh ) , Na CO , PhMe/MeOH/H O,
3
4
2
3
2
o
o
o
7
0 C, 8 h, 88%; (b) m-CPBA, DCM, 25 C, 5 h, 95%; (c) NBS, AIBN, Benzene, 80 C, 4 h; (d)
o
o
corresponding amine, Et N, MeCN, 85 C, 8 h; (e) corresponding amine, 2-butyl alcohol, 125 C, 12 h,
3
3
3%.
a
Scheme 5. Preparation of Compounds 24a-24e
a
o
Reaction conditions: (a) TBSCl, imidazole, DCM, 35 C, 8 h, 95%; (b) corresponding amine,
o
LiHMDS, THF, -78 C, 3 h, then HCl (gas), 3 h, 20%.
2
.3. In Vitro ERK Inhibition Assay
To test the hypothesis that isoindolin-1-one scaffold could mimic the pyrrole-2-carboxamide of
BVD-523, we first introduced the isoindolin-1-one without considering the hydroxymethyl group in
BVD-523. It was found that compound 12a showed a promising activity with an enzyme IC = 5.6 nM.
50
This encouraging result confirmed the rationality of our docking experiments and gave us more
confidence in using this scaffold to proceed SAR. Thus 1-methyl-1H-pyrazol-5-yl group was employed
for our initial SAR studies (Table 1). It is gratifying that the 3-chlorobenzyl analogue 12b was more
potent than 12a, but other analogues 12c, 12d, 12e which contain a pyridyl group were almost
completely loss of potency indicated that pyridyl group was not tolerated at this position. Then we
fixed the 3-chlorobenzyl group and studied SAR of R group (Table1). The pyridyl (12f), 1-methyl-1H-
1
pyrazol-4-yl (12g), 4-fluorophenyl (12h) and tetrahydro-2H-pyran-4-yl (12i) were used to mimic the
hydrogen receptor 1-methyl-1H-pyrazol-5-yl group in 12b. The results showed that 1-methyl-1H-
pyrazol-4-yl derivative 12g has the same potent as 12b, however pyridyl derivative 12f and 4-
fluorophenyl derivative 12h were less potent than 12b. Gratifyingly, the tetrahydro-2H-pyran-4-yl
analogue 12i displayed twofold increase of potency over 12b. Following SAR showed the position of

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Cl substituent is critical to the potency (12j, 12k, 12l), 3-Chlorophenyl (12i, 12j) was favored over 4-
chlorophenyl (12k, 12l) in enzyme activity.
1
2
Table 1. Initial SAR at Right-Part (R ) and Left-Part Ring (R )
1
2
R
R
ERK2 IC (nM)
50
1
2a
5.6
3.5
12b
12c
12d
12e
> 1000
> 1000
> 1000
1
2f
36.3
3.6
1
1
2g
2h
> 1000
1.6
1
1
2i
2j
2.9
1
2k
2l
BVD-523
17.8
1
35.6
0.7
Encouraged by the high inhibitory effect on ERK2 of the novel isoindolin-1-one derivatives with a
-chlorophenyl at right-part, we then introduced hydroxymethyl group and focused on substituents at
3
left-part (Table 2). First, the hydroxymethyl analogue 22a resulted in twofold increase in potency over
2i, indicating the hydroxymethyl group, which could form hydrogen bond with Asp167 and Asn154,
1
is essential to the activity. Second, halogens at the 4- or 3, 4-position (22b, 22c) led to a decrease in
potency, which further verified 3-Chlorophenyl was favored over 4-chlorophenyl. Replaced tetrahydro-
2
H-pyran-4-yl to tetrahydrofuran-3-yl (22d) and 4-hydroxycyclohexyl (22e) preserve ERK inhibitory
potency while replaced oxygen to nitrogen eliminate the enzyme potency (22f, 22g). In addition, the
enzyme potency decreased dramatically when the tetrahydro-2H-pyran-4-yl was opened (22h, 22i).
The analogues with alkyl substituents such as cyclohexyl and isopropyl groups (22j, 22k) at left-part
suffered loss of the enzyme potency. Finally, we explored aromatic groups at left-part. The potency of
2
2l and 24a was maintained over 22a, while the potency of 24b-24e decreased, presumably due to
absence of the interaction with Lys114.
Table 2. Expanded SAR of the Left-Part Ring of Hydroxylated Analogues

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1
3
R
R
ERK2 IC (nM)
50
22a
22b
22c
22d
3-Cl
3-Cl, 4-Cl
4-Cl
0.7
3.1
30.4
5.4
3-Cl
2
2e
3-Cl
3-Cl
2.7
2
2f
> 1000
2
2
2g
2h
3-Cl
3-Cl
3-Cl
> 1000
17.6
2
2
2i
2j
38.4
3-Cl
3-Cl
7.2
2
2k
2l
11.2
2
3-Cl
1.2
2
4a
4b
3-Cl
3-Cl
1.9
2
25.6
24c
24d
24e
3-Cl
3-Cl
3-Cl
19.8
22.3
93.3
As ERK have two isoforms, in order to completely suppress ERK pathway, we also evaluated part of
compounds’ ERK1 inhibition activities (table 3). The results showed that our compounds also have
good inhibitory effects on ERK1.
Table 3. ERK1 Inhibition Activities of Selected Compounds
ERK1 IC (nM)
50
1
1
2
2
2g
2i
2a
2l
4a
21
3.3
1.5
4.1
3.6
2
2
.4. Structure-activity relationship (SAR) summary
Based on the results of ERK inhibition screening, we summarized the structure-activity relationship
1
of isoindolin-1-one derivatives (Figure 3). Firstly, the effect of substituent R showed that hydrogen

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bond accepter is better than hydrogen bond donor in which tetrahydropyran-4-yl, 1-methyl-1H-pyrazol-
5
-yl and 1-methyl-1H-pyrazol-4-yl (12b, 12g, 12i) showed best ERK2 inhibition activities. Secondly,
2
analyzing the effect of R substituents, we found that phenyl > pyridyl and the substitution position at
phenyl ring is important for the activity, in which 3-position > 3, 4-position > 4-position (12i, 12j, 12k,
3
1
2l). Finally, the introduction of hydroxymethyl group at R (12i vs 22a, 12b vs 24a, 12g vs 22l)
further enhanced the activities. Therefore, compounds 12b, 12g, 12i, 22a, 22l, 24a were selected for
further studies.
Figure 3. Structure-activity relationship analysis (SAR) of isoindolin-1-one derivatives.
2
.5. In Vitro Anti-proliferation Assay
Given sufficient potency in inhibiting the intracellular signaling activity of ERK kinase, we then
evaluated 12g, 12i, 22a, 22l and 24a for their anti-proliferative activities against four tumor cell lines
carrying BRAF and K-Ras activating mutations. According to the results in Table 4, all the inhibitors
showed moderate activities against Colo205 (BRAFV600E), WM-266-4 (BRAFV600D), SW-626
(
KRASG12V) and HCT-116 (K-RasG12D). It was revealed that the IC50 of 12g, 12i and 22a against
HCT-116 were 148.00±16.27 nM, 150.44±14.16 nM and 154.97±14.16 nM respectively, much higher
than other IC values against tumor cell lines. We also evaluated the anti-proliferation effect of these
5
0
compounds on K562 cell (Ras & BRAF wild-type). The anti-proliferation activities decreased by more
than 10 times which indicated that the effects of these compounds are achieved by affecting the ERK
pathway (Table 1 in SI). Western blot analysis was conducted to confirm the intracellular efficacies of
2
2a. Result in Figure 4A showed that 22a could dose-dependent decrease the intracellular
phosphorylation levels of ERK1/2 and RSK in HCT116 cells. To determine if apoptosis contributed to
the reduced cell proliferation. HCT116 cells were stained with Hoechst 33258 after incubating with
2
2a and BVD-523 (Figure 4B). The condensed bright apoptotic nuclei were readily observed. The
presence of apoptotic cells was further confirmed by stained with PI and analyzed by flow cytometry
Figure 4C&4D). Hypodiploid DNA appeared in the sub-G0/G1 region, which was represented dead
(
cells.
Table 4. In Vitro Anti-Proliferation Activities of Selected Compounds
IC (nM)
50
Colo205
WM-266-4
173.64±17.80
219.89±23.74
259.94±22.34
SW-626
HCT-116
1
2g
2i
2a
203.75±19.27
170.49±15.79
193.53±15.46
208.84±16.84
254.71±28.47
246.72±26.87
148.00±16.27
150.44±14.16
154.97±14.16
1
2

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2
2l
4a
BVD-523
226.91±25.46
211.34±17.59
216.57±18.56
230.24±21.39
206.12±26.87
200.68±18.48
250.57±24.16
201.74±22.34
2
251.94±25.46
388.81±40.17
252.15±23.97
291.72±31.21
Figure 4. (A) Effects of 22a on the phosphorylation levels of ERK1/2 and RSK in HCT116 cells. The
phosphorylation levels of ERK1/2 and RSK were measured with western blot assay. Results were mean
s± SD for three individual experiments which, for each condition, were performed in triplicate. (B)
Effects of 22a on the apoptosis in HCT116 cells. Hochest staining from HCT116 cells in different
groups (n=3) (Scale bar: 10 µm). (C) Effects of different 22a on the apoptosis in HCT116 cells. Cell
apoptosis were measured using flow cytometry. Results were means ± SD for three individual
experiments which, for each condition, were performed in triplicate. (D) Effects of 22a on the cell
cycle distribution in HCT116 cells. Cell cycle distribution was measured using flow cytometry.
2
.6. In Vitro Cytotoxic Effects to Normal Cells
It is important to avoid side-effect of new compounds before evaluating in vivo activities. Human
colonic epithelial cell NCM-460 was chosen to evaluate our compounds’ (22a, 22l) cytotoxic effects of
normal cells. The result showed that, compounds 22a and 22l have no cytotoxic effects at the
concentration of 10000 nM, proved our compounds have good safety.
2
.7. In Vivo Pharmacokinetic (PK) Profile
Compound 12i and 22a were selected to investigate in vivo pharmacokinetic characteristics. Two
compounds were administrated intravenously (iv) at 5 mg/kg or orally (po) at 50 mg/kg in Sprague-
Dawley (SD) rats. The clearance of 12i (CL = 43.6 mL/min/kg) was higher than 22a (CL = 23.5
mL/min/kg) at 5 mg/kg (iv). The pharmacokinetic parameters of 12i and 22a were respectively shown
in Table 5, which indicated that the pharmacokinetic properties of 22a is superior to that of 12i.
Importantly, 22a possessed the moderate oral bioavailability (F) of 22.5%, insuring further evaluation
of its oral antitumor activity.
Table 5. PK Parameters of 12i and 19a in SD Rats (N = 3)
dose
mk/kg)
Cmax
(ng/mL)
AUC0-t
(ng·h/mL)
compd
t1/2 (h)
MRT (h) F (%)
0-t
(

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1
2i hydrochloride
50
50
0.37
3.6
747
1920
1283
8380
1.2
3.4
6.9
22.5
2
2a hydrochloride
2
.8. In Vivo Antitumor Activity Evaluation
A HCT-116 xenograft model in nude mice was established to evaluate the in vivo antitumor potency
of compound 22a hydrochloride. 22a hydrochloride was administered at a dose of 50 mg/kg (po, q.d.)
for 30 days, and which could result in 71% tumor growth inhibition (TGI). In comparison, the ERK
inhibitor, BVD-523, at a dose of 50 mg/kg (po, q.d.) only resulted in 54% TGI. The tumor growth
delay induced by 22a was visualized by the tumor growth curve in Figure 5A and the final tumor tissue
size in Figure 5B. Considering of the potential toxicity of 22a, we evaluated the nude mice’s body
weight every 3 days. There was no observed body weight loss with treatment by 30 days in this study
(
figure 1 in SI), which proved good safety of 22a.
Figure 5. Antitumor efficacy of 22a in the HCT-116 mouse xenograft model. (A) HCT-116 mouse
xenograft data with compound 22a. (B) The resulting tumors were excised from the indicated groups.
Although racemic 22a and 22l possessed good inhibitory effect on ERK2, in order to confirm
whether there is difference in ERK2 inhibitory activities between optical isomers, (S)-enantiomer and
(
R)-enantiomer of 22a and 22l were synthesized using corresponding enantiomer amine (Figure 2 in
SI). As anticipated, (S)-22a and (S)-22l were 250- and 570-fold more active in the enzyme assay in
comparison to (R)-22a and (R)-22l (Table 6).
Table 6. SAR of (S)-22i, (R)-22i, (S)-22l, (R)-22l
R1
Chirality
ERK2 IC (nM)
50
(
S)-22a
S
0.8
(
R)-22a
R
S
201
(
S)-22l
1.1
(
R)-22l
R
633
3
. CONCLUSION
In summary, a novel series of isoindolin-1-one derivatives targeting ERK was rationally designed,
synthesized, and identified. Some of these compounds have excellent biochemical and cellular ERK
potencies. Comprehensive and systematic SAR investigation led to the identification of 22a, a potent
and highly efficacious ERK inhibitor. 22a exhibited excellent ERK inhibitory activities and anti-
proliferation potencies. Although 22a only possessed acceptable pharmacokinetic profiles with the oral
bioavailability of 22.5 % in SD rats, but showed considerable activity in vivo antitumor efficacy in a

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HCT-116 xenograft model. In addition, 22a possessed a chiral hydroxymethyl group and further
investigation of enantiomer of 22a showed (S)-enantiomer was 250 fold more active in the enzyme
assay in comparison to (R)-enantiomer (0.8 nM vs 201 nM). Currently, related in vitro and in vivo
studies of 22a enantiomer are in progress.
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
ò 31 novel isoindolin-1-one derivatives were synthesized as ERK inhibitors.
ò Antitumor activities of part compounds were evaluated in four cancer cell lines.
ò Compound 22a possesses acceptable pharmacokinetic profiles in SD rat.
ò Compound 22a showed considerable in vivo antitumor efficacy in a HCT-116
xenograft model