Design, synthesis and biological evaluation of novel isoindolinone derivatives as potent histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.02.032

Histone deacetylases (HDACs) as appealing targets for the treatment of many diseases has been studied extensively and its use in cancer care is the most important. Here, we developed a series of novel derivatives containing isoindolinone skeleton. Twelve compounds demonstrated nanomolar IC₅₀ values against HDAC1, and the best compounds were 5a (65.6 nM), 5b (65.1 nM) and 13a (57.9 nM). In vitro, 5a and 5b also showed potent antiproliferative activities against several cancer cell lines, in particular 5b, which behaved better than approved drug chidamide. Morever, enzyme inhibition and western blot assay established 5b to be a selective inhibitor for HDAC1-3. Molecular docking was performed to rationalize the high potency of isoindolinones. Additionally, 5b had more appropriate drug metabolism in human liver microsome (HLM) compared with chidamide and moderate pharmacokinetics properties. These results indicated that 5b was worthy of further biological studies.

Full text of DOI:10.1016/j.ejmech.2019.02.032

Accepted Manuscript
Design, synthesis and biological evaluation of novel isoindolinone derivatives as
potent histone deacetylase inhibitors
Xin Chen, Shuang Zhao, Hongmei Li, Xin Wang, Aixin Geng, Hao Cui, Tao Lu,
Yadong Chen, Yong Zhu
PII:
S0223-5234(19)30146-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.032
EJMECH 11124
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2018
Revised Date: 10 February 2019
Accepted Date: 10 February 2019
Please cite this article as: X. Chen, S. Zhao, H. Li, X. Wang, A. Geng, H. Cui, T. Lu, Y. Chen, Y.
Zhu, Design, synthesis and biological evaluation of novel isoindolinone derivatives as potent histone
deacetylase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.02.032.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Isoindolinone Derivatives
as Potent Histone Deacetylase Inhibitors
Xin Chena, Shuang Zhaob, Hongmei Lib, Xin Wangc, Aixin Gengb, Hao Cuib, Tao Lub, d, Yadong Chenb, *, Yong
Zhub, *
aShaanxi Key Labotory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University,
Yangling 712100, P. R. China
bSchool of Science, China Pharmaceutical University, Nanjing, 210009, P. R. China
cSchool of Pharmacy, China Pharmaceutical University, Nanjing 210009, P. R. China
dState Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, P. R. China
Corresponding Author
*Yadong Chen
E-mail: ydchen@cpu.edu.cn; Fax: +86-25-86185170
*Yong Zhu
E-mail: zhuyong@cpu.edu.cn; Fax: +86-25-86185182
Notes
The author declare no competing interest.
Abstract
Histone deacetylases (HDACs) as appealing targets for the treatment of many diseases has been studied
extensively and its use in cancer care is the most important. Here, we developed a series of novel derivatives
containing isoindolinone skeleton. Twelve compounds demonstrated nanomolar IC50 values against HDAC1, and
the best compounds were 5a (65.6 nM), 5b (65.1 nM) and 13a (57.9 nM). In vitro, 5a and 5b also showed potent
antiproliferative activities against several cancer cell lines, in particular 5b, which behaved better than approved
drug chidamide. Morever, enzyme inhibition and western blot assay established 5b to be a selective inhibitor for
HDAC1-3. Molecular docking was performed to rationalize the high potency of isoindolinones. Additionally, 5b
had more appropriate drug metabolism in human liver microsome (HLM) compared with chidamide and moderate
pharmacokinetics properties. These results indicated that 5b was worthy of further biological studies.
Keywords
HDAC inhibitor; Antiproliferation; Isoindolinone; Synthesis ; Structural optimization
1. Introduction
Histone lysine acetylation is an epigenetic marker associated with gene transcriptional activation and
repression[1]. Histone deacetylases (HDACs), which remove acetyl groups from lysine residues in histones or
non-histone substrates, are overexpressed in many kinds of cancers. Inhibition of HDACs gives rise to
anticarcinogenic effects through several different mechanisms including reduced cell motility/migration, invasion,
induction of apoptosis, angiogenesis, and blocking of DNA repair. As a result, HDACs have emerged as an
attractive target for the tumor therapy[2-5]. The human HDACs family containing 18 isoforms is divided into class
I (HDACs 1, 2, 3 and 8), class IIa (HDACs 4, 5, 7 and 9), class IIb (HDACs 6 and 10), class III (named sirtuins
1-7) and class IV (HDAC 11). The classical HDACs refer to class I, II and IV isforms which require zinc ion as a
cofactor for deacetylating activity, while that of class III isoforms is nicotine adenine dinucleotide[6-8]. Each
HDAC isotype has different biological function. Specifically, class I HDACs are widespread across all tissues and

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play key role in epigenetic processes [9-12]. Morever, they are highly expressed in a number of cancers and
sustain malignant growth, enabling tumor cells susceptive to HDAC inhibitors (HDACis).
To data, numerous HDACis have been exploited, several of which have been approved for cancer treatment.
Nonselective hydroxamic acid inhibitors, such as vorinostat (SAHA)[13], belinostat (PXD-101)[14], panobinostat
(LBH-589)[15] and natural romidepsin (FK228)[16] have gained U.S. Food and Drug Administration approvals
for the treatment of cutaneous T-cell lymphoma, peripheral T-cell lymphoma and multiple myeloma. Chidamide, a
benzamide inhibitor which was reported to be a class I selective HDACi[17-18], was approved for treating
recurrent and refractory peripheral T-cell lymphoma by the China Food and Drug Administration in 2015 (Fig. 1).
Linker
Cap
ZBG
O
HN
O
O
H
O
N
OH
S
OH
H
N
H
N
N
N
N
H
H
H
OH
O
Vorinostat
O
Belinostat
Panobinostat
O
H
N
O
O
NH
S
N
NH2
H
S
H
HN
N
N
O
O
O
O
F
Chidamide
O
Romidepsin
Fig.1. Approved HDACs inhibitors
Generally, HDAC inhibitors have common structural characteristics: cap, linker and zinc binding group (ZBG)
[19]. Cap moiety matches the rim region of the protein pocket, greatly influencing inhibitory potency and isoforms
selectivity. It usually contains large aromatic group such as aryl or heteroaryl[20-21]. Macrocyclic structures as
cap moiety was also reported[22]. Linker segment embeds into the native channel and makes the ZBG in a proper
position to chelate with the catalytic zinc ion below. Unlike the well-established linker and ZBG group,
modification of cap moiety generates a lot of novel potent inhibitors. Among these, a large and rigid aromatic
capping group is considered to be a key portion for favoring the interaction between the molecular and HDACs.
Moreover, inhibitor bearing a rigid skeleton usually exhibits a favorable metabolism profile[23-24].
O
N
O
O
O
O
N
O
HN
O
O
N
HO
O
O
O
O
vasorelaxant
magallanesine
enterocarpam II
Fig. 2 Known 3-methyleneisoindolin-1-one-based bioactive compounds
Isoindolinones are important scaffolds in medicinal chemistry and organic chemistry for their prevalence in
many natural and synthetic bioactive molecules. In particular, 3-methyleneisoindolin-1-ones have been recognized
as core components in natural products such as enterocarpam II, the secophthalide-isoquinoline ene-lactam
fumaridine[25], magallanesine, an isoindolobenzazocine derived from the South-American plant Berberis
darwinii[26]. 3-Methyleneisoindolin-1-ones with vasorelaxant activity were also reported[27] (Fig. 2). These

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results prompted us to identify novel potential HDAC inhibitors based on the inflexible isoindolinone skeleton. In
the previous study, we described an efficient stereoselective synthesis of (Z) 3-methyleneisoindolin-1-ones via
base-catalyzed intermolecular reaction[28]. With this result in hand, we synthesized a series of novel HDACis
bearing rigid (Z) 3-methyleneisoindolin-1-one as capping group in combination with various linkers and ZBGs
(Fig. 3). Here, we report the design, synthesis, structure−activity relationship (SAR) and metabolism researches of
this new class of HDACis.
Fig. 3. Design of our isoindolinone-based HDACis
2. Results and discussion
2.1 Chemistry
To evaluate the effect on enzymatic activity of the linker between ZBG and the isoindolinone capping group,
compounds 5a-e, 6 with phenyl linkers and 12a-d, 13a-b with chain linkers were synthesized. Among these,
2-aminobenzamides and hydroxamic acid moieties were chosen as ZBG respectively. Compounds 5a-d were
synthesized as shown in Scheme 1. (Z) 3-methyleneisoindolin-1-one derivative 1 was synthesized as the procedure
which we previously reported[21]. Hydrolyzation of 1 with lithium hydroxide (LiOH) in methanol (MeOH)
afforded intermediate 2. Amidation of compound 2 with methyl 4-(aminomethyl)benzoate hydrochloride using
2-(7-Azabenzotri azol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) gave intermediate 3.
Subsequent hydrolysis of 3 yielded compound 4. Coupling of 4 with benzene-1, 2-diamine and various substituded
benzene-1, 2-diamine gave target compounds 5a-d. Under basic conditions, the methyl ester group of 3 was
directly converted to the corresponding hydroxamic acid 6 by hydroxylamine (NH2OH). Compound 5e was
synthesized as shown in Scheme 2. The synthesis of intermediate 8 was performed starting from
4-fluorobenzene-1, 2-diamine (7), the amino was selectively protected in a (Boc)2O/KHCO3 system. Then 8
reacted with 2 by HATU-mediated amide formation to afford 9. The Boc protecting group was subsequently
removed in the presence of trifluoroacetic acid (TFA), which generated the desired compound 5e.
Scheme 1. Synthesis of compounds 5a-d and 6. Reagents and conditions: (a) LiOH, MeOH, 50 °C, 2 h; (b) HATU, N, N-diisopropylethyl
amine (DIPEA), N, N-dimethylformamide (DMF), Methyl 4-(aminomethyl)benzoate hydrochloride, r.t., 6 h; (c) LiOH, MeOH, 60 °C, 2 h;
(d) HATU, DIPEA, DMF, r.t., 6 h; and (e) NH2OH, KOH, MeOH, r.t., overnight.

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Scheme 2. Synthesis of compound 5e. Reagents and conditions: (a) (Boc)2O, KHCO3, CH3CN, 0 °C ~30 °C, 4 h; (b) HATU, DIPEA,
andydrous DMF, r.t., 10 h; and (c) DCM/TFA (v:v = 1:1), 0 °C, 10 h.
Then we changed phenyl linker with common chain linkers which had different lengths. A series of
isoindolinone analogues (12a-d, 13a-b) were synthesized as shown in Scheme 3. Amidation of 2 with commercial
methyl 6-aminohexanoate or methyl 7-aminohexanoate gave intermediates 10a or 10b, respectively. The
intermediates 10a-b were then converted to hydroxamic acids 13a-b in good yields under conditions similar to
those shown in Scheme 1. The coupling of different benzene-1, 2-diamines with the hydrolysates of compounds
10a-b generated the target compounds 12a-d.
Scheme 3. Synthesis of compounds 12a-d and 13a-b. Reagents and conditions: (a) HATU, DIPEA, DMF, 6-aminohexanoate or methyl
7-aminohexanoate hydrochloride, r.t., 6 h; (b) NH2OH, KOH, MeOH, r.t., overnight; (c) LiOH, MeOH, 50 °C, 2 h; and (d) HATU, DIPEA,
DMF, r.t., 6 h;
To explore the structure and activity relationship of cap motif, different substituents were introduced on the
phenyl of isoindolinone. Compounds 20a-c were synthesized as shown in Scheme 4. Cyclizations of substituded
benzonitriles 14a-c and ethyl acrylate in the presence of RuCl2 as catalyst gave the key isoindolinones 15a-c[29],
which then were hydrolyzed with lithium hydroxide to give 16a-c. Compound 19 was prepared through two steps:
the first synthetic step involved in the amidation of 4-cyanobenzoic acid 17 with 4-fluorobenzene-1,2-diamine to
afford 18, and then 18 was reduced by hydrogen in the presence of Pd/C. Target compounds 20a-c were obtained
by coupling of intermediates 16a-c with 19 in the presence of HATU. In addition, dichloro-substituted
isoindolinone derivative 20d was synthesized beginning with the (Z)-ethyl 2-(5, 6-dichloro-3-oxoisoindolin-1-ylid
ene)acetate (21, Scheme 5) under conditions similar to those shown in Scheme 1.
Scheme 4. Synthesis of compounds 20a-c. Reagents and conditions: (a)Dichloro(p-cymene)ruthenium(II) dimer, AgSbF6, Cu(OAc)2 H2O,
ethyl acrylate, AcOH, 120 °C, 24 h; (b) LiOH, MeOH, 60 °C, 6 h; (c) HATU, DIPEA, DMF, r.t., 12 h; (d) H2, Pd/C, MeOH, r.t., 4 h; and
(e) HATU, DIPEA, DMF, r.t., 6 h.

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Cl
O
F
O
Cl
NH
O
d
H
a
b
c
N
N
H
O
Cl
H2N
NH
O
21
20d
O
Cl
Scheme 5. Synthesis of compound 20d. Reagents and conditions: (a) LiOH, MeOH, 60 °C, 2 h; (b) HATU, DIPEA, DMF, Methyl
4-(aminomethyl)benzoate hydrochloride, r.t., 6 h; (c) LiOH, MeOH, 50 °C, 2 h; and (d) 4-fluorobenzene-1,2-diamine, HATU, DIPEA,
DMF, r.t., 6 h;
Considering that the surface of the active pocket where the capping group binds is adjacent to the solvent
accessible region, increasing the polarity of cap moiety was a logical choice. Hence, we introduced nitrogen atom
to the phenyl of isoindolinone scaffold yielding two novel azaisoindolinone scaffolds 23a and 23b. Up to now,
there is no literatures have revealed the structures and synthesis route of 23a and 23b, thus we tried to obtain them
follow the same procedure as in isoindolinones. 6-hydroxy-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione (22) was
obtained by ammonolysis reaction of furo[3,4-b]pyridine-5,7-dione with hydroxylamine hydrochloride using
acetic acid as solvent. As we anticipated, reaction of (22) with ethyl propionate generated cyclization
azaisoindolinones 23a and 23b simultaneously in equal yields, and the structures of which were further
characterized by X-ray crystallography (see supporting information). With the key intermediates 23a and 23b in
hand, target compounds 27a-c were synthesized (Scheme 6) under conditions similar to those shown in Scheme 1.
Scheme 6. Synthesis of compounds 27a-c. Reagents and conditions: (a) hydroxylamine hydrochloride, AcOH, 119 °C, 4 h; (b)
tributylphosphine (Bu3P), ethyl propionate, anhydrous DMF, 150 °C, 2 h; (c) LiOH, MeOH, 50 °C, 2 h; (d) HATU, DIPEA, DMF, Methyl
4-(aminomethyl)benzoate hydrochloride, r.t., 6 h; (e) LiOH, MeOH, 50 °C, 2 h; and (f) substituted benzene-1,2-diamine, HATU, DIPEA,
DMF, r.t., 6 h;
2.2 HDAC inhibition and SAR study of target compounds
We tested all acquired compounds for inhibitory ability against HDAC1, using chidamide and SAHA as
references. We selected the HDAC1 enzyme because it was widely involved in transcriptional repression and was
intimately linked to the tumors. Compounds 5a-e, 6, 12a-d and 13a-b were synthesized to evaluate the influence
of the modifications of linker and ZBG on HDAC1 activity. As shown in Table 1, the inhibition rates (IRs) against
HDAC1 of most tested compounds were above 70% at 50 µM concentration except 6 and 13b. IC50 results
illustrated that the benzamide compounds 5a, 5b, and 5e, with IC50 values of 65.6, 65.1 and 70.3 nM, exhibited
better activities than chidamide (IC50 = 296 nM). Hydroxamic acid analogue 13a also showed a potent HDAC1
inhibition with IC50 value of 57.9 nM, although this was obviously weaker than SAHA (IC50 = 4 nM).
When we chose 2-aminobenzamide moiety as ZBG, the HDAC1 inhibitory activities were markedly
influenced by different substituents on the phenyl of ZBG group. Fluorine (F) atom was similar in size and bulk
with hydrogen (H) atom, therefore introducing F substituent on the para-position of the amide (R1 position) had
marginal influence on the HDAC1 potency, i.e. 5b vs 5a. However, replacement of F with larger chlorine (5c, IC50
= 466 nM) or methyl (5d, IC50 = 5.72 µM) substituents decreased the HDAC1 potency substantially which might

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be attributed to the resulting hindrance at R1 position. While changing the F substituent from R1 to R2 position,
compound 5e still exhibited a comparable HDAC1 activity. Compounds 5a, 5b with phenyl as linker showed
better HDAC1 inhibitory activity than compounds 12a-d with aliphatic chain as linker whose IC50 values
distributed between 320 nM and 6.42 µM. When we chose hydroximic acid as ZBG, compound 6 with phenyl as
linker only had a HDAC1 IR of 49.91% at 50 µM concentration. Compound 13a with a five carbon length chain as
linker (n = 5) displayed an excellent activity (IC50 = 57.9 nM), whereas increasing the length of the aliphatic chain
to six carbon as in 13b resulted in a lost of HDAC1 inhibition. The inappropriate linker lengths of hydroximic
acids 6 (IR = 49.91%) and 13b (IR = 15.49%) might be responsible for the decrease of HDAC1 activity.
Table 1
The HDAC1 inhibitory activity of compounds 5a-e, 6, 12a-d and 13a-b.
HDAC1
IR%a (50 µM)
HDAC1
IR%a (50 µM)
Cpd.
R1
R2
Cpd.
n
R1
IC50b(µM)
IC50b(µM)
6.4200± 0.3253
0.8720± 0.0037
1.6000± 0.1573
0.3200± 0.0299
0.0579± 0.0061
-
5a
5b
H
F
H
H
H
H
F
99.28
99.31
90.63
81.60
99.23
49.91
99.87
99.36
0.0656± 0.0092
0.0651± 0.0073
0.4660± 0.0524
5.7200± 0.8476
0.0703± 0.0051
12a
12b
5
5
6
6
5
6
F
H
F
H
/
75.60
89.96
81.57
93.98
97.30
15.49
90.92
5c
Cl
CH3
H
12c
5d
12d
5e
13a
c
6
/
/
-
13b
/
SAHA
TSA
0.0040± 0.0007
0.0125± 0.0012
Chidamide
0.296± 0.0417
aIR%: Enzymatic inhibition rate of HDAC1 was tested at 50 µM. bIC50 values for enzymatic inhibition of HDAC1. c- : not tested. We ran
experiments in duplicate. Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).
The preliminary enzymatic results outlined in Table 1 demonstrated that the strategy of introducing
isoindolinone as the capping group was rational and feasible. In consideration of chemical instability, potential
toxicity[30] of hydroximic acid HDACis, and lower potency against HDAC1 of 13a compared with SAHA, further
modification on isoindolinone was performed based on benzamide analogue 5b with 2-aminobenzamide moiety as
ZBG and phenyl as linker. As shown in Table 2, the introduction of different substituents on capping group had a
detrimental impact on HDAC1 activity. Electron withdrawing groups like F (20a) or large trifluoromethyl (20b) at
R3 position led to a 3-fold and 8-fold decrease on potency than that of 5b, with IC50 values of 167 nM and 491 nM,
respectively. Dichloro-substituted (R3 and R4 positions) analogue 20d, were also over seven times less potent
compared to 5b, with an IC50 value of 489 nM. Moreover, a slightly reduced enzymatic activity was also observed
for compound 20c (IC50 = 96.8 nM) with an electron donor methoxy group at R3 position. The general trend
established was that an electron donor substituent on isoindolinone was more favorable for HDAC1 activity than
electron withdrawing group.
When the phenyl of isoindolinone was replaced with pyridine, we observed a sharp fall on HDAC1 inhibitory
potency for compound 27a and 27c compared with 5b. Azaisoindolinone 27b without a fluoro substituent on ZBG
also had a nearly 12-fold decrease in comparison with 5a. These results indicated that a more hydrophobic
structure might be suitable for binding with the surface area of HDAC1.
Table 2
HDAC1 inhibitory activities of compounds 20a-d and 27a-c.

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HDAC1
IR% a (50 µM)
Cpd.
R3
R4
IC50b(µM)
20a
20b
20c
20d
F
CF3
OCH3
Cl
H
H
94.43
93.49
94.26
92.38
0.1670± 0.0189
0.4910± 0.0713
0.0968± 0.0117
0.4890± 0.0614
H
Cl
27a
27b
62.93
88.53
50.38
90.9
NAc
0.7580± 0.1094
8.3600± 0.8365
0.2960± 0.0433
27c
Chidamide
c
aIR%: Enzymatic inhibition rate of HDAC1 was tested at 50 µM. bIC50 values for enzymatic inhibition of HDAC1. NA: no inhibition
activity. We ran experiments in duplicate. Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).
To investigate the selectivity of benzamide isoindolinones versus other HDAC isoforms, the representative 5b
was tested against all of the 11 class I, II and IV HDACs with chidamide as reference compound. As described in
Table 3, 5b preferentially inhibited HDAC1-3 (IC50 values of 65.1, 75 and 112 nM, respectively) and almost lost
activity against class II HDACs, indicating a similar selectivity profile to that of chidamide. Moreover, a 17-fold
and 10-fold selectivity toward HDAC8 and HDAC11 versus HDAC1 were also observed for 5b. These data
displayed that 5b was a potent class I selective HDACs inhibitor.
Table 3
Complete Characterization of 5b at all 11 class I, II, and IV HDACs (IC50a, µM).
Zn2+-dependent HDAC isoforms
5b
Chidamide
0.2960± 0.0117
0.4500± 0.0216
0.2650± 0.0005
0.8330± 0.0073
NA
Class I
HDAC1
0.0651± 0.0049
0.0750± 0.0009
0.1120± 0.0082
1.0800± 0.0305
NAb
HDAC2
HDAC3
HDAC8
Class IIa
Class IIb
HDAC4/5/7/9
HDAC6
NA
NA
HDAC10
HDAC11
0.9500± 0.0913
0.6320± 0.0315
0.2350± 0.0227
0.7600± 0.0771
Class IV
aIC50 values for enzymatic inhibition of HDACs. bNA: no inhibition activity. We ran experiments in duplicate. Assays were performed by
Reaction Biology Corporation (Malvern, PA, USA).

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2.3 In vitro antiproliferative activity
On the basis of the enzymatic results above, the most promising compounds 5a and 5b was determined to
evaluate antiproliferative activities of isoindolinones against human leukemia cell lines HL-60 and K562, colon
cancer cell line HCT116 and breast cancer cell line MCF-7 (the results are summarized in Table 4). As we
expected, both 5a and 5b exhibited excellent inhibitory activities against HL-60 and K562 cell lines with IC50
values ranging from 193 to 450 nM. In addition, nanomolar antiproliferative activities against solid tumor cell line
HCT116 were also observed. Notably, 5a, 5b and chidamide were less potent in inducing MCF-7 cell death with
IC50 values in double-digit micromolar range. Compound 5b, with a lipophilic F substituent on the terminal phenyl,
showed better antiproliferative activity than 5a despite of their equipotent HDAC1 inhibition. This could be
attributed to the increased lipophilicity which made 5b penetrate cell membrane more easily. Compared with
chidamide, 5b displayed overall superior antiproliferative activities including HL-60 (5-fold), K562 (4-fold),
HCT116 (2.5-fold) and MCF-7 (2-fold).
Table 4
Antiproliferative activity of 5a, 5b and reference inhibitor chidamide against HL-60, K562, HCT116 and MCF-7 cell lines
IC50, µM
Compound
HL-60
K562
HCT116
MCF-7
5a
5b
0.450± 0.067
0.373±0.046
1.970± 0.208
0.286± 0.039
0.193± 0.020
0.747± 0.116
0.483± 0.071
0.432± 0.055
1.090± 0.136
20.080± 1.641
14.560± 2.133
29.070± 4.289
Chidamide
Western blot assay of 5b was performed to detect the expression of histone 3 (H3) acetylation which was an
important biomarker related to intracellular HDAC1-3 inhibition. MCF-7 cells were treated with compound 5b and
chidamide at 0.25, 2.5, 25 µM for 24 h to measure the acetylation status of total H3 (Fig. 4). As expected, we
observed a dose-dependent improvement in the level of Ac-H3 when the cells were incubated with compound 5b.
Not until concentration of 25 µM was used was an apparent increase in histone H3 acetylation visible for
chidamide. This might partly be contributing to a relatively higher biochemical IC50 value of chidamide against
class I HDACs.
Fig 4. Effect of histone H3 acetylation in breast cancer cell line (MCF-7) by chidamide and 5b respectively. Cells were treated with 5b
and chidamide for 24 h and followed by western blot analysis.
Further screening for antiproliferative activity of 5b against a set of 59 solid or hematological tumor cell lines
was carried out in NCI (termed NCI-60). The human tumor cell lines of the NCI-60 are some of the most
representative cell lines used in laboratory which come from different tissue origins including blood, lung, colon,

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central nervous system (CNS), melanoma, ovary, kidney, prostate and breast. Concretely, 5b was screened at 10
µM, and cell viability was assessed after 2 days of treatment. As shown in Table 5, compound 5b displayed
notable antiproliferative potential against human leukemia cell lines, and all the IRs of which were above 50%, in
particular CCRF-CEM and HL60 (TB) with IRs of 84.48% and 84.01%. However, a bad antiproliferation profile
was observed for the most solid tumor cell lines, although several exceptions like NCI-H522 (lung cancer),
HCT116 (colon cancer) and TK-10 (renal cancer) also exhibited moderate cellular activities with IRs of 70.66%,
73.27% and 72.33% respectively. It appeared that the current HDACs inhibitors including 5b are more inclined to
treat hematological tumors compared with solid tumors.
Table 5.
In vitro antiproliferative screening of compound 5b (Inhibition rate% at 10 µM concentration)
Origin of cancer
Leukemia
Cell line
CCRF-CEM
HL60 (TB)
K562
IR %a
84.48
84.01
61.56
63.05
59.78
57.61
12.19
11.68
29.93
58.58
30.21
26.46
14.19
26.75
70.66
27.8
Origin of cancer
Cell line
M14
IR %
29.39
28.84
52.05
33.76
34.74
36.56
57.78
22.82
41.95
21.34
36.41
55.90
18.02
29.54
7.02
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
MOLT-4
RPMI-8226
SR
Non-Small
Cell
A549/ATCC
EKVX
Lung Cancer
Ovarian Cancer
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
Renal Cancer
Colon Cancer
A498
47.14
15.04
17.53
30.75
72.33
50.08
39.35
13.04
35.53
22.42
29.27
6.06
20.41
73.27
0.12
ACHN
RXF 393
SN12C
HT29
40.35
26.91
40.14
35.98
16.23
44.80
13.55
30.64
35.89
15.71
52.58
TK-10
KM12
UO-31
SW-620
Prostate Cancer
Breast Cancer
PC-3
CNS Cancer
SF-268
DU-145
SF-295
MCF7
SF-539
MDA-MB-231/ATCC
HS 578T
BT-549
SNB-19
SNB-75
U251
T-47D
58.17
9.71
Melanoma
LOX IMVI
MALME-3M
MDA-MB-468
aIR% was calculated by 100%-growth %, the values of cell growth percent were provided by NCI-60 program.
2.4 Docking study

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The molecular docking study was performed with compound 5b in the catalytic pocket of human HDAC1
crystallographic structure (PDB code: 5ICN) to further rationalize the SAR of isoindolinones. As illustrated in
Fig.5(A), the phenyl linker of 5b could insert into the narrow channel of the binding site and form a π−π stacking
interaction with the residues of amino acids Phe150 and Phe205. The amino of the 2-aminobenzamide ZBG group
formed a key H-bond with Asp176, and the H atom of the amide between the phenyl linker and ZBG also
generated an H-bond interaction with carbonyl of Gly149. These H-bond forces positioned 5b in a suitable
space-conformation to efficiently contact with the zinc atom. Additionally, although isoindolinone core occupied
the surface groove which was adjacent to the solvent region, it was surrounded by hydrophobic residues including
Ile305, Tyr303, Leu271 and Pro29. This might be responsible for the poor HDAC1 inhibition of
azaisoindolinones.
Fig. 5 Docking models of isoindolinone analogues in complex with the HDAC1 catalytic domain. (A) Docking model of 5b (yellow) in
the binding pocket, with key residues in the hydrophobic channel labeled in cyan. The H-bonding interactions with residues were labeled
in blue. Zinc ion was labeled in brown. (B) Docked positions obtained for 5b (yellow) and 5d (green) in HDAC1 pocket. Methyl
substituent was marked with a cyan label and F substituent was marked with a black label. The HDAC1 protein is displayed as a surface
representation in gray for clarity.
It should be noted that the distance between H atom of the amide in isoindolinone scaffold and the oxygen
atom of the carbonyl linked to the methylene was only 2.381 Å which indicated a potential H-bond formation. This
conclusion might also be confirmed by the X-ray crystallography of (Z)3-methyleneisoindolin-1-one in our
previous article[28]. The approximately tricyclic capping group of compound 5b further enhanced the interaction
with the large surface of pocket which resulted in the excellent HDAC1 activity and isoforms selectivity of
isoindolinones. Additionally, comparing the binding models of 5b and 5d (Fig. 5B), it appeared that the sterical
hinderence between the methyl substituent on ZBG and nearby residues made 5d in a adverse position close
chelate with the zinc ion. This poor combination was probably responsible for the terrible HDAC1 inhibition as
exemplified by 5c and 5d.
2.5 In vitro metabolic stability
In view of the promising results gained from the biological activity assays in vitro, a preliminary investigation
for metabolic stability of compound 5b was conducted to determinate half-life (T1/2) and CLint in liver microsomes
(LMs) from male SD rats, dogs, and human. As shown in Table 6, the elimination half-life (T1/2) value of 5b was
292 min in human LM, a bit better than that of chidamide (276 min), whereas shorter T1/2 values in dog and rat
LMs were observed for 5b (201 and 121 min) than chidamide (302 min and 190 min). The CLints of compound 5b
were calculated as 4.80, 6.92 and 11.7 µL×min-1×mg-1 in human, dog, rat LMs correspondingly. This result
inspired us to perform further pharmacokinetic studies in vivo for 5b.

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Table 6
In vitro stabilities of compound 5b and Chidamide toward human, dog, rat liver microsomes.
human
dog
rat
Compound
T1/2 (min)
292
CLint
4.80
4.98
T1/2 (min)
201
CLint
6.92
5.76
T1/2 (min)
121
CLint
11.7
5b
Chidamide
276
302
190
10.35
2.6 Pharmacokinetic studies
The pharmacokinetic studies of 5b were evaluated in SD rats following intravenous administration at 1mg/kg
body weight and oral administration at 10 mg/kg body weight. Blood samples were taken, and the plasma was
detected for the concentrations of 5b by an LC-MS/MS system. As illustrated in Table 7, 5b showed a plasma
clearance of 21.3 mL·kg-1·min-1 with terminal phase elimination half-life (T1/2) of 0.39 h administered iv in rats,
whereas oral administration in rats gave half-time (T1/2) of 1.30 h. The oral bioavailability of 16% suggested that
5b could be further investigated and optimized as a novel class I HDACs inhibitor.
Table 7
Pharmacokinetic parameters of 5b tested in SD rats.
Compound
route
5b
iv
1
po
10
Dose (mg/kg)
CL (mL·kg-1·min-1)
Vdss (L·kg-1)
AUC0-inf (ng·h·mL-1)
AUC0-t (ng·h·mL-1)
T1/2 (h)
21.3
0.493
784
781
0.39
/
/
/
1742.7
1710.5
1.30
16
F (%)
3. Conclusion
In summary, we have developed a series of novel isoindolinone-based HDACs inhibitor, and evaluated their
in vitro activity. Twelve compounds exhibited nanomolar IC50 values on HDAC1 inhibition, and the best
compounds were 5a (65.6 nM), 5b (65.1 nM) and 13a (57.9 nM). Among these, compound 5b as a class I HDACs
inhibitor displayed suprior enzymatic activity than chidamide and an excellent isoforms selectivity profile. 5a and
5b also demonstrated nanomolar antiproliferative activities against human Leukemia cell lines HL-60, K562 and
colon cell line HCT116, whereas a micromolar effect was observed in breast cancer cell line MCF-7. In
comparison of the antiproliferative results of 5a, 5b and chidamide, compound 5b had an overall superior activity
against the four cell lines.
Through the NCI antiproliferative screening toward 59 cell lines involving nine tumor types, compound 5b
inhibited the growth of leukemia cell lines, but showed weak antiproliferative activities against most of the solid
tumors. The following stability evaluation in LMs (human, dog and rat) indicated that 5b possessed a better
stability in HLM than chidamide. In the case of pharmacokinetic, 5b showed moderate oral bioavailability of 16%
in rats. The in vivo pharmacological assay is currently under investigation in our group.
4. Experimental section
4.1 Chemistry
All reagents and solvents were reagent grade or were purified by standard methords in advance. Isolation and

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purification of the compounds were performed by flash column chromatography on silica gel 60 (230-400 mesh).
Analytical thin-layer chromatography (TLC) was conducted on fluka TLC plates (silica gel 60 F254, aluminum
foil). The structures of synthesized compounds were characterized by 1H NMR, 13C NMR and MS. The structural
information and detailed synthetic process for the intermediates and target compounds are shown in this section.
Melting points were measured using an X-4 melting-point apparatus with a microscope (Beijing Tech Instrument)
and were not corrected. 1H and 13C NMR spectra were recorded in DMSO-d6 by a 300 MHz spectrometer:
chemical shifts (δ) are given in parts per million, coupling constants (J) in Hz. MS were determined by a Nicolet
2000 FT-IR mass spectrometer and MAT-212 mass spectrometer. All of the target compounds were examined by
HPLC, and the purity of every case was ≥ 95%. Reverse-phase HPLC was performed on an Agilent Technologies
1260 Infinity, which was equipped with a C18 column (Agilent Zorbax SB-C18, 5 µM, 4.6 mM × 150 mM).
Mobile phase A was water, and mobile phase B was methanol. A gradient of 20−80% B was run at a flow rate of
0.8 mL/min over 30 min.
4.1.1 General procedure for the preparation of isoindolin scaffolds 1, 21 and 23a-b.
To the solution of ethyl propiolate (35.3 mg, 0.36 mmol) and N-hydroxyphthalimide (0.3 mmol) in dry DMF (3
mL) was added Bu
atmosphere for the required period of time. After completion of the reaction as monitored by TLC, the reaction
mixture was quenched with CH Cl (15 mL), which was washed with water and brine successively, dried over
MgSO , filtered, and concentrated in vacuo. Purification by flash chromatography (SiO
1:10−1:3) yielded the desired products.
3P (15.7 mg, 0.06 mmol). The resulting mixture was heated to 150 °C under a nitrogen
2
2
4
2
; ethyl acetate/PE,
1
4.1.1.1 Ethyl (Z)-2-(3-oxoisoindolin-1-ylidene)acetate (1). 95% isolated yield; white solid. Mp: 167-169 °C. H
NMR (300 MHz, DMSO-d6) δ: 10.95 (s, 1H), 8.98 (d, J = 7.6 Hz, 1H), 7.91-7.60 (m, 3H), 5.80 (s, 1H), 4.21 (q, J
= 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ:167.4, 165.5, 148.0, 133.6, 133.0, 131.7,
131.2, 127.6, 122.7, 98.2, 59.8, 14.1. ESIHRMS: Calcd for C12H12NO3: (M+H)+ 218.0812. Found: m/z 218.0815.
4.1.1.2 Ethyl (Z)-2-(5,6-dichloro-3-oxoisoindolin-1-ylidene)acetate (21).90% isolated yield; yellow solid. Mp:
194−196 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.53 (s, 1H), 8.49 (s, 1H), 8.01 (s, 1H), 6.20 (s, 1H), 4.21 (q, J =
6.9 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 167.9, 167.7, 146.4, 138.3, 137.9, 136.3,
130.8, 127.0, 126.3, 95.9, 62.0, 16.0. ESIHRMS: Calcd for C12H9Cl2N1NaO3: (M+Na)+ 307.9852. Found: m/z
307.9857.
4.1.1.3 Ethyl (Z)-2-(5-oxo-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-ylidene)acetate (23a). 45% isolated yield;
black solid. Mp: 128−130 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.79 (s, 1H), 8.90 (dd, J1 = 4.9 Hz, J2 = 1.5 Hz,
1H), 8.26 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.70 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 5.99 (s, 1H), 4.24 (q, J = 7.1
Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); ESI-MS m/z: 219.1 [M+H]+.
4.1.1.4 Ethyl (Z)-2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetate (23b). 45% isolated yield;
black solid. Mp: 125−127 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.79 (s, 1H), 8.88 (dd, J1 = 4.8 Hz, J2 = 1.4 Hz,
1H), 8.57 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz, 1H), 7.73 (dd, J1 = 7.9 Hz, J2 = 4.8 Hz, 1H), 6.22 (s, 1H), 4.23 (q, J = 7.1
Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H); ESI-MS m/z: 241.1 [M+Na]+.
4.1.2 General procedure for the preparation of isoindolin scaffolds 15a-c
.
Substituted nitriles (0.5 mmol, 1.0 equiv), [{RuCl2(p-cymene)}2] (0.025 mmol, 5 mol %), Cu(OAc)2 H2O (1.0

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mmol, 2.0 equiv), AgSbF6 (0.10 mmol, 20 mol %) were taken in a 15 mL pressure tube equipped with a magnetic
stirrer and septum. To the tube were then added liquid alkene (2.0 mmol, 4.0 equiv) and acetic acid solvent (3 mL)
via syringes, allowed the reaction mixture to stir at room temperature for few seconds. Then, the septum was taken
out and immediately a screw cap was used to cover the tube. The reaction mixture was allowed to stir at 120 °C.
Then, the reaction was monitored by TLC until the maximum conversion. The reaction mixture was diluted with
CH2Cl2, filtered through Celite and silica gel, and the filtrate was concentrated. The crude residue was purified
through a silica gel column using hexanes and ethyl acetate as eluent to give pure 15a-c.
4.1.2.1 Ethyl (Z)-2-(6-fluoro-3-oxoisoindolin-1-ylidene)acetate (15a). 23% isolated yield; white solid. Mp:
169-171 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.47 (s, 1H), 8.08 (dd, J1 = 2.1 Hz, J2 = 8.8 Hz, 1H), 7.86 (m, 1H),
7.52 (m, 1H), 6.18 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); ESI-MS m/z: 236.1 [M+H]+.
4.1.2.2 Ethyl (Z)-2-(3-oxo-6-(trifluoromethyl)isoindolin-1-ylidene)acetate (15b). 20% isolated yield; white solid.
Mp: 170-172 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.75 (s, 1H), 8.66 (s, 1H), 8.03 (s, 2H), 6.41 (s, 1H), 4.23 (q,
J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H); ESI-MS m/z: 308.1 [M+Na]+.
4.1.2.3 Ethyl (Z)-2-(6-methoxy-3-oxoisoindolin-1-ylidene)acetate (15c). 25% isolated yield; white solid. Mp:
175-177 °C.1H NMR (300 MHz, DMSO-d6) δ: 10.17 (s, 1H), 7.77 (m, 2H), 7.23 (m, 1H), 6.20 (s, 1H), 4.26 (q, J =
7.0 Hz, 2H), 3.93 (s, 1H), 1.31 (t, J = 7.1 Hz, 3H); ESI-MS m/z: 248.2 [M+Na]+.
4.1.3 Tert-butyl (2-amino-4-fluorophenyl)carbamate (8).
A round-bottom flask charged with 4-fluorobenzene-1, 2-diamine 7 (126 mg, 1 mmol) and KHCO3 (200 mg, 2
mmol) was taken up in CH3CN at 0 °C. After 5 min, (Boc)2O (0.23 mL, 1mmol) was added in portions and the
resulting mixture was stirred overnight. Then, the mixture was concentrated and extracted with EtOAc. The
combined organic extracts were washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The
1
product obtained by chromatography on a silica gel column. 90% isolated yield; white solid; Mp: 95-97 °C. H
NMR (300 MHz, DMSO-d6) δ: 8.18 (s, 1H), 7.22-7.07 (m, 1H), 6.45 (dd, J1 = 2.85 Hz, J2 = 11.13 Hz, 1H), 6.28
(td, J1 = 2.88 Hz, J2 = 8.55 Hz, 1H), 5.10 (s, 2H), 1.44 (s, 9H); ESI-MS m/z: 227.1 [M+H]+.
4.1.4 General procedure for the hydrolysis of esters to yield various acids
To a solution of various esters (1 mmol) in MeOH and H2O (10 mL, v/v = 1: 1) was added Lithium hydroxide
(95.8 mg, 4 mmol) at 0 °C. Then, mixture was allowed to stir at room temperature. The reaction was monitored by
TLC until the maximum conversion. After neutralized with acetic acid, the formed precipitate was collected by
filtration, washed with water, and dried in vacuo to give the title compounds
1
4.1.4.1 (Z)-2-(3-oxoisoindolin-1-ylidene)acetic acid (2). 90.2% isolated yield; white solid. Mp: 218-220 °C. H
NMR (300 MHz, DMSO-d6) δ: 12.06 (brs, 1H), 10.90 (s, 1H), 8.99 (d, J = 7.6 Hz, 1H), 7.92-7.63 (m, 3H), 5.82 (s,
1H). ESI-MS m/z: 190.2 [M+H]+.
4.1.4.2 (Z)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzoic acid (4). 95% isolated yield; white solid.
Mp: 205-207 °C. 1H NMR (300 MHz, DMSO-d6) δ: 12.83 (brs, 1H), 10.39 (s, 1H), 8.89 (s, 1H), 7.92 (d, J = 7.77
Hz, 2H), 7.87-7.71 (m, 4H), 7.43 (d, J = 7.77 Hz, 2H), 6.10 (s, 1H), 4.49 (d, J = 5.58 Hz, 2H); ESI-MS m/z: 321.1
[M+H]+.

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4.1.4.3 (Z)-2-(5-oxo-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-ylidene)acetic acid (24a). 84% isolated yield; black
solid. Mp: 142−156 °C. 1H NMR (300 MHz, DMSO-d6) δ: 12.83 (brs, 1H), 10.89 (s, 1H), 8.93 (dd, J1 = 4.9 Hz, J2
= 1.5 Hz, 1H), 8.28 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.73 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 5.98 (s, 1H);
ESI-MS m/z: 219.1 [M+H]+.
4.1.4.4 (Z)-2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetic acid (24b). 87% isolated yield; black
solid. Mp: 138−140 °C. 1H NMR (300 MHz, DMSO-d6) δ: 12.83 (brs, 1H), 10.88 (s, 1H), 8.98 (dd, J1 = 4.8 Hz, J2
= 1.4 Hz, 1H), 8.59 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz, 1H), 7.77 (dd, J1 = 7.9 Hz, J2 = 4.8 Hz, 1H), 6.28 (s, 1H);
ESI-MS m/z: 191.1 [M+H]+.
4.1.4.5 (Z)-6-(2-(3-oxoisoindolin-1-ylidene)acetamido)hexanoic acid (11a). 71% isolated yield; white solid. Mp:
178-180 °C; 1H NMR (300 MHz, DMSO-d6) δ: 11.88 (brs, 1H), 10.44 (s, 1H), 8.35 (t, J = 5.5 Hz, 1H), 7.85-7.80
(m, 2H), 7.78-7.73 (m, 1H), 7.69-7.64 (m, 1H), 6.02 (s, 1H), 3.23-3.16 (m, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.60-1.42
(m, 4H), 1.36-1.28 (m, 2H); ESI-MS m/z: 340.1 [M+Na]+.
4.1.4.6 (Z)-7-(2-(3-oxoisoindolin-1-ylidene)acetamido)heptanoic acid (11b). 79% isolated yield; white solid. Mp:
180-182 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.89 (brs, 1H), 10.44 (s, 1H), 8.36 (t, J = 5.5 Hz, 1H), 7.84-7.73
(m, 3H), 7.68 (m, 1H), 6.01 (s, 1H), 3.22-3.16 (m, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.59-1.38 (m, 4H), 1.30 (s, 4H);
ESI-MS m/z: 317.4 [M+H]+.
4.1.4.7 (Z)-2-(6-fluoro-3-oxoisoindolin-1-ylidene)acetic acid (16a). 63% isolated yield; white solid. Mp:
189-191 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.89 (brs, 1H), 10.48 (s, 1H), 8.09 (dd, J1 = 2.1 Hz, J2 = 8.8 Hz,
1H), 7.88 (m, 1H), 7.54 (m, 1H), 6.19 (s, 1H); ESI-MS m/z: 208.2 [M+H]+.
4.1.4.8 (Z)-2-(6-trifluoromethyl-3-oxoisoindolin-1-ylidene)acetic acid (16b). 60% isolated yield; white solid; Mp:
1
178-180 °C. H NMR (300 MHz, DMSO-d6) δ: 11.91 (brs, 1H), 10.76 (s, 1H), 8.67 (s, 1H), 8.04 (s, 2H), 6.42 (s,
1H); ESI-MS m/z: 258.2 [M+H]+.
4.1.4.9 (Z)-2-(6-methoxy-3-oxoisoindolin-1-ylidene)acetic acid (16c). 68% isolated yield; white solid. Mp:
185-187 °C.1H NMR (300 MHz, DMSO-d6) δ: 11.91 (brs, 1H), 10.18 (s, 1H), 7.78 (m, 2H), 7.23 (m, 1H), 6.22 (s,
1H), 3.94 (s, 1H); ESI-MS m/z: 220.2 [M+H]+.
4.1.4.10 (Z)-4-((2-(5-oxo-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-ylidene)acetamido)methyl)benzoic acid (26a).
69% isolated yield; black solid. Mp: 158−160 °C. ESI-MS m/z: 346.3 [M+H]+. 1H NMR (300 MHz, DMSO-d6) δ:
12.71 (s, 1H), 10.79 (s, 1H), 9.07 (s, 1H), 8.91 (dd, J1 = 4.8 Hz, J2 = 1.4 Hz, 1H), 8.25 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz,
1H), 7.77-7.70 (m, 5H), 5.98 (s, 1H), 4.54 (d, J = 5.7 Hz, 2H).
4.1.4.11 (Z)-4-((2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetamido)methyl)benzoic acid (26b).
71% isolated yield; black solid. Mp: 158−160 °C. ESI-MS m/z: 346.3 [M+H]+. 1H NMR (300 MHz, DMSO-d6) δ:
12.70 (s, 1H), 10.79 (s, 1H), 9.08 (s, 1H), 8.89 (dd, J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.57 (dd, J1 = 7.9 Hz, J2 = 1.4
Hz, 1H), 7.81-7.71 (m, 5H), 6.24 (s, 1H), 4.43 (d, J = 5.9 Hz, 2H).
4.1.5 General procedure for the amidation of various acids with various amides
To a solution of acid (1 mmol) was added DIPEA (0.66 mL, 4 mmol), HATU (353.2 mg, 1.1 mmol) and

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DMF (10 mL) at 0 °C. After 30 min, various amides (1mmol) were added. The mixture was stirred for 6 h. After
completion of the reaction as monitored by TLC, the mixture was poured into water (30 mL) and extracted with
EtOAc (3×30 mL). The combined organic extracts were washed with brine (2×30 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo. The product was obtained by chromatography on a silica gel column.
4.1.5.1 Methyl (Z)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzoate (3).74% isolated yield; white solid.
Mp: 156-158 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.40 (s, 1H), 8.92 (s, 1H), 7.95 (d, J = 9.00 Hz, 2H),
7.85-7.70 (m, 4H), 7.46 (d, J = 8.13 Hz, 2H), 6.10 (s, 1H), 4.50 (d, J = 5.64 Hz, 2H), 3.85 (s, 1H); ESI-MS m/z:
358.1 [M+Na]+.
4.1.5.2 (Z)-N-(2-aminophenyl)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (5a).35% isolated
yield; white solid. Mp: 179-180 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.65 (s, 1H), 8.94 (t, J = 5.88
Hz, 1H), 7.96 (d, J = 8.07 Hz, 2H), 7.88-7.74 (m, 3H), 7.70-7.68 (m, 1H), 7.43 (d, J = 7.65 Hz, 2H), 7.16 (d, J =
7.59 Hz, 1H), 6.99-6.94 (m, 1H), 6.79-6.76 (m, 1H), 6.60 (m, 1H), 6.11 (s, 1H), 4.90 (s, 2H), 4.49 (d, J = 5.85 Hz,
2H); 13C NMR (75 MHz, DMSO-d6) δ: 167.26, 166.26, 165.08, 143.14, 143.10, 142.70, 136.48, 133.26, 132.97,
131.22, 128.90, 127.87, 127.80, 126.63, 126.43, 123.33, 123.28, 121.00, 116.25, 116.12, 95.17, 41.93; ESI-MS
m/z: 435.1 [M+Na]+.
4.1.5.3 (Z)-N-(2-amino-4-fluorophenyl)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (5b). 37%
isolated yield; white solid. Mp: 208-209 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.44 (s, 1H), 9.58 (s, 1H,), 8.95 (t,
J = 5.88 Hz, 1H), 7.96 (d, J = 8.07 Hz, 2H), 7.84-7.77 (m, 3H), 7.74-7.68 (m, 1H), 7.43 (d, J = 8.07 Hz, 2H),
7.13-7.08 (m, 1H), 6.54 (dd, J1 = 2.7 Hz, J2 = 11.2 Hz, 1H), 6.35 (dd, J1 = 2.7 Hz, J2 = 8.6Hz, 1H), 6.11 (s, 1H),
5.23 (s, 2H), 4.49 (d, J = 5.85 Hz, 2H); ESI-MS m/z: 429.2 [M+H]+. 13C NMR (75 MHz, DMSO-d6) δ: 167.24,
166.26, 165.15, 143.15, 142.96, 138.83, 136.43, 132.97, 133.00 131.23, 128.90, 127.91, 127.10, 123.28, 121.00,
116.58, 112.57, 112.38, 112.29, 112.06, 95.14, 41.93. ESI-MS m/z: 431.4 [M+H]+
4.1.5.4 (Z)-N-(2-amino-4-chlorophenyl)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (5c). 31%
isolated yield; white solid. Mp: 160-162 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.64 (s, 1H), 8.94 (s,
1H), 7.95 (d, J = 8.16 Hz, 2H), 7.88-7.68 (m, 5H), 7.43 (d, J = 8.16 Hz, 2H), 6.80 (m, 1H), 6.10 (s, 1H), 5.18 (m,
2H), 4.49 (d, J = 5.85 Hz); 13C NMR (75 MHz, DMSO-d6) δ: 167.78, 166.74, 165.79, 145.30, 143.64, 143.35,
136.98, 133.55, 133.49, 129.40, 128.75, 128.45, 127.54, 123.80, 122.47, 121.52, 115.87, 115.26, 95.66, 42.42.
ESI-MS m/z: 469.1 [M+Na]+.
4.1.5.5 (Z)-N-(2-amino-4-methylphenyl)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (5d). 29%
isolated yield; white solid. Mp: 145-146 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.58 (s, 1H), 8.94 (s,
1H), 7.95 (d, J = 8.10 Hz, 2H), 7.88-7.68 (m, 4H), 7.42 (d, J = 8.10 Hz, 2H), 7.01 (d, J = 8.10 Hz, 1H), 6.58 (s,
1H), 6.41 (d, J = 7.92 Hz, 1H), 6.10 (s, 1H), 4.81 (s, 2H), 4.49 (d, J = 5.85 Hz, 2H), 2.17 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ: 167.78, 166.74, 165.54, 143.63, 143.35, 143.14, 136.98, 135.98, 133.79, 133.49, 131.75,
129.39, 128.36, 127.53, 127.08, 123.80, 121.53, 121.40, 117.63, 117.04, 95.68, 42.41. ESI-MS m/z: 449.2
[M+Na]+.
4.1.5.6 Tert-butyl (Z)-(4-fluoro-2-(4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamido)Phenyl)carbam
ate (9). 72% isolated yield; white solid. Mp: 168-170 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.40 (s, 1H), 9.79 (s,
1H), 8.92 (s, 1H), 8.75 (s, 1H), 7.92 (d, J = 8.13 Hz, 2H), 7.89-7.72 (m, 3H), 7.72-7.63 (m, 1H), 7.58-7.41 (m, 4H),

ACCEPTED MANUSCRIPT
7.07-7.01 (m, 1H), 6.11 (s, 1H), 4.51 (d, J = 5.7 Hz, 2H), 1.44 (s, 9H); ESI-MS m/z: 553.2 [M+Na]+.
4.1.5.7 Methyl (Z)-6-(2-(3-oxoisoindolin-1-ylidene)acetamido)hexanoate (10a). 82% isolated yield; white solid.
1
Mp: 120−122 °C. H NMR (300 MHz, DMSO-d6) δ: 10.43 (s, 1H), 8.35 (t, J = 5.5 Hz, 1H), 7.85-7.80 (m, 2H),
7.78-7.73 (m, 1H), 7.69-7.64 (m, 1H), 6.00 (s, 1H), 3.58 (s, 3H), 3.21-3.14 (m, 2H), 2.31 (t, J = 7.4 Hz, 2H),
1.60-1.42 (m, 4H), 1.35-1.27 (m, 2H); ESI-MS m/z: 339.1 [M+Na]+.
4.1.5.8 Methyl (Z)-7-(2-(3-oxoisoindolin-1-ylidene)acetamido)heptanoate (10b). 91% isolated yield; white solid.
Mp: 125−127 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.42 (s, 1H), 8.36 (t, J = 5.5 Hz, 1H), 7.84-7.73 (m, 3H),
7.67 (m, 1H), 6.01 (s, 1H), 3.57 (s, 3H), 3.21-3.14 (m, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.58-1.35 (m, 4H), 1.29 (s,
4H); ESI-MS m/z: 353.1 [M+Na]+.
4.1.5.9 Methyl (Z)-4-((2-(5-oxo-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-ylidene)acetamido)methyl)benzoate (25a).
71% isolated yield; black solid. Mp: 150−152 °C. ESI-MS m/z: 338.3 [M+H]+. 1H NMR (300 MHz, DMSO-d6) δ:
10.79 (s, 1H), 9.05 (s, 1H), 8.91 (dd, J1 = 4.8 Hz, J2 = 1.3 Hz, 1H), 8.25 (d, J = 7.5 Hz, 1H), 7.77-7.70 (m, 5H),
5.99 (s, 1H), 4.53 (d, J = 5.7 Hz, 2H), 3.89 (s, 3H).
4.1.5.10 Methyl (Z)-4-((2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetamido)methyl)benzoate
(25b). 74% isolated yield; black solid. Mp: 151−153 °C. ESI-MS m/z: 338.3 [M+H]+. 1H NMR (300 MHz,
DMSO-d6) δ: 10.79 (s, 1H), 9.06 (s, 1H), 8.88 (dd, J1 = 5.0 Hz, J2 = 1.4 Hz, 1H), 8.57 (dd, J1 = 7.9 Hz, J2 = 1.4
Hz, 1H), 7.81-7.71 (m, 5H), 6.23 (s, 1H), 4.42 (d, J = 5.8 Hz, 2H), 3.88 (s, 3H).
4.1.5.11 (Z)-N-(2-amino-4-fluorophenyl)-6-(2-(3-oxoisoindolin-1-ylidene)acetamido)hexanamide (12a). 43%
isolated yield; white solid. Mp: 188-189 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.43 (s, 1H), 9.02 (s, 1H), 8.36 (t,
J = 5.5 Hz, 1H), 7.84-7.73 (m, 3H), 7.69-7.64 (m, 1H), 7.11-7.06 (m, 1H), 6.50-6.45 (m, 1H), 6.30-6.23 (m, 1H),
6.01 (s, 1H), 5.14 (s, 2H), 3.23-3.17 (m, 2H), 2.33-2.28 (m, 1H), 1.66-1.59 (m, 2H), 1.55-1.46 (m, 2H), 1.41-1.32
(m, 2H); 13C NMR (75 MHz, DMSO-d6) δ: 171.82, 167.67, 166.57, 144.80, 144.65, 143.04, 137.00, 133.41,
131.61, 129.41, 127.63, 127.50, 123.76, 121.40, 119.99, 96.12, 38.96, 36.05, 29.36, 26.63, 25.43. ESI-MS m/z:
411.2 [M+H]+.
4.1.5.12 (Z)-N-(2-aminophenyl)-6-(2-(3-oxoisoindolin-1-ylidene)acetamido)hexanamide (12b). 45% isolated yield;
Mp: 179-180 °C; white solid. 1H NMR (300 MHz, DMSO-d6) δ: 10.44 (s, 1H), 9.10 (s, 1H), 8.35 (s, 1H),
7.97-7.78 (m, 2H), 7.76-7.72 (m, 1H), 7.69-7.65 (m, 1H), 7.13 (d, J = 13.9 Hz, 1H), 6.87 (t, J = 7.1 Hz, 1H), 6.71
(d, J = 7.9 Hz, 1H), 6.52 (t, J = 7.2 Hz, 1H), 6.01 (s, 1H), 5.10 (s, 2H), 3.23-3.17 (m, 2H), 2.33-2.28 (m, 1H),
1.66-1.59 (m, 2H), 1.55-1.46 (m, 2H), 1.41-1.32 (m, 2H);. 13C NMR (75 MHz, DMSO-d6) δ: 171.56, 167.68,
166.57, 143.05, 142.37, 137.01, 133.42, 131.61, 129.42, 126.16, 125.78, 124.02, 123.76, 121.41, 116.61, 116.34,
96.13, 38.97, 36.16, 29.36, 26.63, 25.51. ESI-MS m/z: 415.2 [M+Na]+.
4.1.5.13 (Z)-N-(2-amino-4-fluorophenyl)-7-(2-(3-oxoisoindolin-1-ylidene)acetamido)heptanamide (12c). 39%
isolated yield; white solid. Mp: 189-191 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.44 (s, 1H), 9.04 (s, 1H), 8.36 (s,
1H), 7.84-7.78 (m, 2H), 7.75-7.70 (m, 1H), 7.69-7.64 (m, 1H), 7.11-7.06 (m, 1H), 6.50-6.45 (m, 1H), 6.38-6.23 (m,
1H), 6.01 (s, 1H), 5.14 (s, 2H), 3.23-3.16 (m, 2H), 2.33-2.23 (m, 2H), 1.61-1.48 (m, 4H), 1.34 (s, 4H); 13C NMR
(75 MHz, DMSO-d6) δ: 171.86, 167.67, 166.55, 144.78, 144.62, 143.03, 137.00, 133.41, 131.61, 129.41, 127.53,
123.76, 121.39, 120.03, 96.14, 39.01, 36.09, 29.43, 28.89, 26.75, 25.65. ESI-MS m/z: 447.2 [M+Na]+.

ACCEPTED MANUSCRIPT
4.1.5.14 (Z)-N-(2-aminophenyl)-7-(2-(3-oxoisoindolin-1-ylidene)acetamido)heptanamide (12d). 42% isolated yield;
1
Mp: 150-151 °C; white solid. H NMR (300 MHz, DMSO-d6) δ: 10.42 (s, 1H), 9.10 (s, 1H), 8.34 (s, 1H), 7.96 –
7.78 (m, 2H), 7.75 (t, J = 7.3 Hz, 1H), 7.66 (t, J = 7.1 Hz, 1H), 7.13 (d, J = 13.9 Hz, 1H), 6.88 (t, J = 7.1 Hz, 1H),
6.71 (d, J = 7.9 Hz, 1H), 6.53 (t, J = 7.2 Hz, 1H), 6.00 (s, 1H), 3.19 (dd, J = 12.1, 6.1 Hz, 2H), 2.31 (t, J = 7.2 Hz,
2H), 1.60 (s, 2H), 1.48 (s, 2H), 1.34 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ: 171.12, 167.19, 166.05, 142.52,
141.74, 136.51, 132.91, 131.09, 128.91, 125.64, 125.24, 123.62, 123.23, 120.88, 116.22, 115.92, 95.64, 38.52,
35.70, 28.91, 28.36, 26.23, 25.21. ESI-MS m/z: 429.3 [M+Na]+.
4.1.5.15(Z)-N-(2-amino-4-fluorophenyl)-4-((2-(6-fluoro-3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide(2
1
0a). 13% isolated yield; Mp: 158-160 °C; white solid. H NMR (300 MHz, DMSO-d6) δ: 10.50 (s, 1H), 9.60 (s,
1H), 8.99 (t, J = 5.3 Hz, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.94-7.89 (m, 1H), 7.83-7.80 (m, 1H), 7.62-7.51 (m, 1H),
7.47 (d, J = 8.2 Hz, 2H), 7.20-7.04 (m, 1H), 6.58 (dd, J = 11.3, 2.7 Hz, 1H), 6.40 (m, 1H), 6.16 (s, 1H), 5.25 (s,
2H), 4.53 (d, J = 5.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ: 167.80, 166.76, 165.81, 145.36, 143.66, 143.33,
136.99, 133.57, 133.50, 131.77, 129.41, 128.77, 128.54, 127.55, 123.90, 122.49, 121.54, 115.89, 115.23, 95.80,
42.43. ESI-MS m/z: 449.2 [M+H]+.
4.1.5.16 (Z)-N-(2-amino-4-fluorophenyl)-4-((2-(6- trifluoromethyl-3-oxoisoindolin-1-ylidene)acetamido)methyl)be
-nzamide (20b). 15% isolated yield; Mp: 156-157 °C; white solid. 1H NMR (300 MHz, DMSO-d6) δ: 10.78 (s, 1H),
9.64 (s, 1H), 8.97 (s, 1H), 8.37 (s, 1H), 8.25 (s, 2H), 8.08-8.00 (m, 2H), 7.49-7.39 (m, 2H), 7.18-7.14 (m, 1H), 6.60
(m, J = 11.6 Hz, 1H), 6.43-6.31 (m, 1H), 6.37 (s, 1H), 5.29 (s, 2H), 4.55-4.42 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) δ: 168.67, 167.68, 166.57, 147.73, 144.58, 144.38, 140.26, 137.98, 134.40, 132.66, 130.33, 129.33,
128.53, 124.71, 122.43, 117.95, 115.66, 113.81, 113.48, 96.56, 42.47. ESI-MS m/z: 521.1 [M+Na]+.
4.1.5.17(Z)-N-(2-amino-4-fluorophenyl)-4-((2-(6-methoxy-3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide
(20c). 16% isolated yield; white solid. Mp: 159-160 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.24 (s, 1H), 9.58 (s,
1H), 9.01-8.76 (m, J = 5.8 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.57-7.33 (m, 3H),
7.26-7.16 (m, 1H), 7.16-7.03 (m, 1H), 6.67-6.47 (m, 1H), 6.44-6.30 (m, 1H), 6.10 (s, 1H), 5.23 (s, 2H), 4.49 (d, J =
5.9 Hz, 2H), 3.91 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ: 167.60, 166.80, 165.85, 163.80, 146.01, 143.67,
143.31, 139.43, 133.58, 128.39, 127.45, 125.34, 121.88, 119.74, 118.35, 106.03, 102.66, 102.36, 102.10, 101.77,
95.49, 42.42. ESI-MS m/z: 483.2 [M+Na]+.
4.1.5.18(Z)-N-(2-amino-4-fluorophenyl)-4-((2-(5,6-dichloro-3-oxoisoindolin-1-ylidene)acetamido)methyl)benzami
1
de (20d). 30% isolated yield; white solid. Mp: 140-142 °C; H NMR (300 MHz, DMSO-d6) δ: 10.67 (s, 1H), 9.59
(s, 1H), 8.98 (s, 1H), 8.30 (s, 1H), 8.11 (s, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.18-7.11 (m,
1H), 6.61-6.56 (m, 1H), 6.43-6.36 (m, 1H), 6.22 (s, 1H), 5.24 (s, 1H), 4.53 (d, J = 5.6 Hz, 2H); 13C NMR (75 MHz,
DMSO-d6) δ: 169.93, 168.88, 167.68, 148.39, 147.42, 143.64, 139.12, 138.12, 135.93, 135.64, 133.90, 131.54,
130.50, 129.68, 129.23, 125.95, 123.67, 123.55, 119.78, 119.18, 97.82, 42.56. ESI-MS m/z: 497.1 [M-H]-.
4.1.5.19(Z)-N-(2-amino-4-fluorophenyl)-4-((2-(5-oxo-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-ylidene)acetamido)
1
methyl)benzamide (27a). 41% isolated yield; white solid. Mp: 232-233 °C; H NMR (300 MHz, DMSO-d6) δ:
10.79 (s, 1H), 9.57(s, 1H), 9.05 (s, 1H), 8.79 (dd, J = 4.9, 1.5 Hz, 1H), 8.13 (dd, J1 = 1.5 Hz, J2 = 7.6 Hz, 1H), 7.92
(d, J = 8.1 Hz, 2H), 7.58 (m, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.09 (m, 1H), 6.62 (s, 1H), 6.53 (dd, J1 = 2.8 Hz, J2 =
11.2 Hz, 1H), 6.35 (td, J1 = 8.5 Hz, J2 = 2.7 Hz, 1H), 5.22 (s, 2H), 4.86 (d, J = 16.2 Hz, 1H), 4.58 (d, J = 16.3 Hz,

ACCEPTED MANUSCRIPT
1H); 13C NMR (75 MHz, DMSO-d6) δ: 166.63, 165.34, 164.86, 162.58, 159.41, 152.79, 145.52, 145.37, 142.22,
133.03, 131.17, 128.58, 128.45, 127.79, 127.10, 124.51, 123.78, 119.26, 88.05, 40.85. ESI-MS m/z: 430.2 [M-H]-.
4.1.5.20 (Z)-N-(2-aminophenyl)-4-((2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetamido)methyl)
1
benzamide (27b). 40% isolated yield; white solid. Mp: 152-153 °C; H NMR (300 MHz, DMSO-d6) δ: 9.65 (s,
1H), 8.77 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 7.2 Hz, 2H), 7.62-7.67 (m, 1H), 7.49 (d, J = 7.2 Hz, 2H),
7.15 (d, J = 5.6 Hz, 1H), 7.06-6.91 (m, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.61 (s, 1H), 6.56 (s, 3H, -NH2), 4.86 (d, J =
16.0 Hz, 1H), 4.60 (d, J = 18.1 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ: 165.61, 165.05, 162.98, 161.66, 159.81,
151.60, 149.13, 145.54, 143.56, 142.52, 133.45, 130.94, 129.70, 128.84, 128.19, 127.82, 127.52, 126.76, 119.72,
86.64, 41.39. ESI-MS m/z: 412.1 [M-H]-.
4.1.5.21(Z)-N-(2-amino-4-fluorophenyl)-4-((2-(7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-ylidene)acetamido)
1
methyl)benzamide (27c). 38% isolated yield; white solid. Mp: 205-207 °C; H NMR (300 MHz, DMSO-d6) δ:
9.58(s, 1H), 8.76 (dd, J1 = 1.3 Hz, J2 = 4.8 Hz, 1H), 8.13 (dd, J1 = 1.4 Hz, J2 = 7.7 Hz, 1H), 7.93 (d, J = 8.2 Hz,
2H), 7.65 (dd, J1 = 4.8 Hz, J2 = 7.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 2H), 7.10 (dd, J1 = 6.5 Hz, J2 = 8.6 Hz, 1H), 6.60
(s, 1H), 6.53 (dd, J1 = 2.8 Hz, J2 = 11.2 Hz, 1H), 6.35 (td, J1 = 2.9 Hz, J2 = 8.5 Hz, 1H), 5.22 (s, 2H), 4.86 (d, J =
16.2 Hz, 1H), 4.59 (d, J = 16.3 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ: 165.34, 164.81, 162.57, 151.41, 148.73,
145.51, 145.36, 143.15, 133.05, 130.53, 129.29, 128.57, 128.43, 127.78, 127.41, 127.12, 126.36, 119.31, 86.23,
40.98. ESI-MS m/z: 430.2 [M-H]-.
4.1.5.22 N-(2-amino-4-fluorophenyl)-4-cyanobenzamide (18). 72% isolated yield; white solid. 1H NMR (300 MHz,
DMSO-d6) δ: 9.87 (s, 1H), 8.13 (d, J = 8.2 Hz, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.21-6.98 (m, 1H), 6.62-6.44 (m, 1H),
6.44-6.25 (m, 1H), 5.34 (s, 2H); ESI-MS m/z: 256.3 [M+Na]+.
4.1.6 General procedure for the reaction of esters and hydroxylamine to afford hydroxamic acids 6 and 13a-b
KOH (28 g, 509 mmol) and NH2OH (23.35 g, 343 mmol) were dissolved in methanol (70 mL). After filtering
the precipitate (KCl), a mix solution of NH2OK and NH2OH was obtained. Various esters (1 mmol) was dissolved
in 10 mL NH2OK solution and stirred overnight. After the reaction was complete, it was evaporated under vacuum.
The residue was acidified with 1 N HCl to a pH 3-4 and then extracted with EtOAc (3×20 mL). The organic layer
was washed with brine (2×30 mL) and dried over Na2SO4 overnight. The crude material was purified via flash
chromatography to afford the product.
4.1.6.1 (Z)-N-hydroxy-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (6). 72% isolated yield;
1
white solid. Mp: 133-135 °C; H NMR (300 MHz, DMSO-d6) δ: 12.92 (s, 1H), 10.42 (s, 1H), 8.94 (t, J = 5.9 Hz,
1H), 7.92 (d, J = 8.2 Hz, 2H), 7.89-7.73 (m, 3H), 7.70-7.66 (m, 1H), 7.42 (d, J = 8.2 Hz, 2H), 6.10 (s, 1H), 4.49 (d,
J = 5.8 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ: 167.78, 167.63, 166.75, 144.86, 143.64, 136.97, 133.48, 131.74,
129.94, 129.88, 129.38, 127.81, 123.79, 121.51, 95.64, 42.42. ESI-MS m/z: 360.2 [M+Na]+.
4.1.6.2 (Z)-N-hydroxy-6-(2-(3-oxoisoindolin-1-ylidene)acetamido)hexanamide(13a). 71% isolated yield; white
solid. Mp: 96-98 °C; 1H NMR (300 MHz, DMSO-d6) δ: 12.00 (s, 1H), 10.42 (s, 1H), 8.33 (t, J = 5.3 Hz, 1H), 7.94
– 7.55 (m, 4H), 6.01 (s, 1H), 3.19 (dd, J1 = 7.0 Hz, J2 = 12.1 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.50 – 1.53 (m, 4H),
1.38 – 1.27 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ: 174.91, 167.71, 166.53, 143.02, 136.96, 133.41, 131.56,
129.36, 123.75, 121.39, 96.17, 38.90, 34.07, 29.27, 26.50, 24.69. ESI-MS m/z: 318.4 [M+Na]+.

ACCEPTED MANUSCRIPT
4.1.6.3 (Z)-N-hydroxy-7-(2-(3-oxoisoindolin-1-ylidene)acetamido)heptanamide (13b). 83% isolated yield; white
1
solid. Mp: 164-165 °C; H NMR (300 MHz, DMSO-d6) δ: 12.03 (s, 1H), 10.42 (s, 1H), 8.36 (t, J = 5.5 Hz, 1H),
7.84-7.73 (m, 3H), 7.67 (m, 1H), 6.01 (s, 1H), 3.21-3.14 (m, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.58-1.35 (m, 4H), 1.29
(s, 4H); 13C NMR (75 MHz, DMSO-d6) δ: 174.99, 167.67, 166.54, 143.01, 137.01, 133.41, 131.60, 129.41, 123.75,
121.40, 96.15, 39.00, 34.14, 29.38, 28.75, 26.68, 24.94. ESI-MS m/z: 354.1 [M+Na]+.
4.1.6.4 (Z)-N-(2-amino-5-fluorophenyl)-4-((2-(3-oxoisoindolin-1-ylidene)acetamido)methyl)benzamide (5e).
To a solution of trifluoroacetic acid and CH2Cl2 (24 mL, v/v = 1:1) was added compound 9 (53.1mg,
0.1mmol). The mixture was stirred for 10 h at 0 °C. The reaction was quenched with Na2CO3 and the aqueous
layer extracted with DCM (3×10 mL). The combined organic extracts were washed with brine, dried over sodium
sulfate, concentrated in vacuo, and purified via flash chromatography, affording the product as a waxy solid. 61%
isolated yield; Mp: 212-214 °C; 1H NMR (300 MHz, DMSO-d6) δ: 10.41 (s, 1H), 9.62 (s, 1H), 8.91 (t, J = 6.2 Hz,
1H), 7.95 (d, J = 8.2 Hz, 2H), 7.89-7.71 (m, 3H), 7.73-7.63 (m, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.23-7.11 (m, 1H),
6.89-6.73 (m, 2H), 6.11 (s, 2H), 4.83 (s, 2H), 4.50 (d, J = 6.0 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ: 167.24,
166.26, 165.15, 143.15, 142.95, 138.83, 136.48, 133.03, 132.97, 131.23, 128.90, 127.91, 127.10, 123.28, 121.00,
116.56, 112.57, 112.38, 112.29, 112.06, 95.14, 41.93. ESI-MS m/z: 453.1 [M+Na]+.
4.1.7 N-(2-amino-4-fluorophenyl)-4-(aminomethyl)benzamide (19)
To a solution of compound 18 (255.3 mg, 1 mmol) in methanol (10 mL) was added Pd/C (21.3 mg, 0.2 mmol).
The mixture was heated to reflux for 5 h. After maximum conversion observed by TLC, the reaction mixture
cooling to ambient temperature, the stock liquid was collected by filtration. Then the reaction mixture was diluted
with 1 N HCl (0.5 mL). When the crystallizing was finished, the precipitate was collected as crude product by
filtration. 61% isolated yield; white solid. Mp: 135-137 °C. 1H NMR (300 MHz, DMSO-d6) δ: 9.87 (s, 1H), 8.91 (s,
2H), 8.10 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 7.16-6.92 (m, 1H), 6.60-6.42 (m, 1H), 6.41-6.23 (m, 1H),
5.34 (s, 2H), 4.52 (s, 2H); ESI-MS m/z: 260.3 [M+Na]+.
4.1.8 6-hydroxy-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione (22)
To a solution of furo[3,4-b]pyridine-5,7-dione (5 g, 33.56 mmol) in acetic acid (100 mL) was added
hydroxylamine hydrochloride (2.80 g, 40.27 mmol). The reaction mixture was heated to reflux for 4 h. After
maximum conversion observed by TLC, the reaction mixture was cooled to ambient temperature, concentrated in
vacuo，then poured into water (40 mL), the formed precipitate was collected by filtration, washed with water, and
dried in vacuo. 34% isolated yield; white solid. Mp: 163-165 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.03 (s, 1H),
8.95 (dd, J1 = 1.4 Hz, J2 = 5.0 Hz, 1H), 8.25 (dd, J1 = 1.4 Hz, J2 =7.6 Hz, 1H), 7.79-7.75 (m, 1H); ESI-MS m/z:
163.0 [M-H]-
4.2 In vitro HDAC enzyme assay
Enzyme inhibition activity were conducted by the Reaction Biology Corporation, Malvern , PA using HDAC
fluorescent activity assay based on the unique Fluor de Lys™ substrate and developer combination. Compounds
were dissolved in DMSO and tested in at least 10-dose IC50 mode with 3-fold serial dilution starting at 50 µM.
GraphPad Prism 5.0 software was used to calculate the IC50 values for each compound.
4.3 In vitro cell growth inhibitory activity.
Culture medium and culture condition of cell lines. Cell growth inhibitory assay was conducted by 3D
BioOptima, Co., Ltd. 1338 Wuzhong Blvd., Suzhou 215104, China using the Alamar blue cell growth inhibition

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test module. HCT116 and MCF-7 human cancer cells were cultured in McCoy's 5A medium with 10% FBS, 100
U/mL penicillin and 100 ug/mL streptomycin. HL-60 cell was cultured in IMDM medium with 20% FBS, 100
U/mL penicillin and 100 ug/mL streptomycin. K562 was cultured in IMDM medium with 10% FBS, 100 U/mL
penicillin and 100 ug/mL streptomycin. All cells were maintained at 37 °C in a humidified atmosphere of 5% CO2
in air.
Cell Growth Inhibitory Assay. The general procedure using Alamar blue reagent for antiproliferative
activitives was as follows: 1. plated 100 µL cell suspension or completed medium into 96-well plate using a
Matrix 12-channel pipettor. And filled residual wells with 200 µL PBS per well; 2. drugs were added to each well
of 96-well plate; 3. placed plate into an incubator with corresponding culture condition for 72 h; 4. pipetted 22 µL
Alamar blue solution (1 mM) into each well of 96-well plate; 5. returned plate to incubator and leave for 5~6 h; 6.
shook plate for 10 seconds and recorded fluorescence at 530/590 nm.
4.4 Western Blotting
MCF-7 cells (1 × 106) were seeded overnight and then incubated with indicated concentrations of compounds
for 24 h. Cell extract was prepared by lysing cultured cells with a mammalian protein extraction reagent
supplemented with EDTA-free protease inhibitor for 15 min. Supernatants were collected following centrifugation
of lysed cells at 15000 g for 10 min at 4 °C. To analyze the cell lysate, 30 µg of total protein per sample was
resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes with immobilized proteins
were probed with antibodies for H3 acetylation. The reactive protein bands were visualized using an ECL
detection system.
4.5 Growth Percent on 60 Cell Lines by NCI.
All detailed test method is described on the NCI Web site (https://dtp.cancer.gov/discovery_developm
ent/nci-60/methodology.htm).
4.6 Computational Methods
All computational work was performed in Discovery Studio 3.0 software (BIOVIA, 5005 Wateridge Vista
Drive, San Diego, CA92121 USA). Docking was conducted using Cdocker module based on the cocrystal of
HDAC1 (PDB: 5ICN). HDAC1 was used as the receptor. The cavity occupied by a peptide inhibitor was selected
as binding site. The default values of docking sphere radius based on the peptide inhibitor was kept. Water
molecules outside the binding pocket were excluded. The energy minimization for compounds was performed by
Powell's method for 1000 iterations using tripos force field and with Gasteiger-Hückel charge. The other docking
parameters were kept as default.
4.7 Microsomal stability assay
Each incubated mixture contained 0.5 mg/mL liver microsome (human or dog or rat), 100 mM potassium
phosphate buffer (pH 7.4), 1 mM NADPH, and 1 µM test compound in a total volume of 200 µL. After
prewarming at 37 °C for 10 min, the NADPH was added to initiate the reaction. The reaction was terminated after
0, 5, 10, 20, 40, or 60 min by adding 0.2 mg/mL glibenclamide (internal standard) in ice-cold methanol into 200
µL of incubation mixture. The sample was then centrifuged at 4000 rpm for 10 min at 4 °C. The supernatant was
then analyzed by LC−MS/MS.
4.8 Pharmacokinetic analysis
All animal experiments were carried out in accordance with the U.K. Animals Act (1986) and associated

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guidelines. The evaluation of compound was carried out using two separate sets of three male SD rats (199-214 g)
after single iv (1 mg/kg) and po (10 mg/kg) administration. The animal was restrained manually at the designated
time points, approximately 200 µL of blood sample was collected via jugular vein into dried heparinized (coated
with 0.25 % heparin in saline) tubes. Blood samples were centrifuged at 5500 rpm for 10 min to obtain plasma
samples. An aliquot of 30 µL of sample was added with 150 µL CH3CN which contains of Verapamil, 5 ng·mL-1
and Glibenclamide, 50 ng·mL-1 for protein precipitation. The mixture was vortexed for 10 min, and then
centrifuged at 3700 rpm for 18 min. After that, 70 µL of supernatant was added with 70 µL of water, then vortexed
for 10 min. An aliquot of 10 µL of the mixture was injected into the LC-MS/MS system.
Acknowledgment
We thank the NCI Division of Cancer Treatment & Diagnosis for the NCI-60 screening of our compounds.
This work was supported by Chinese Universities Scientific Fund (grant No. 2452017174), the Scientific Startup
Foundation for Doctors of Northwest A&F University (grant No. 2452016146) and Natural Science Foundation of
Jiangsu Province (grant No. BK20180573), China.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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HIGHLIGHTS
ò Potent class I HDAC inhibitors based on the natural isoindolinone scaffolds.
ò Two azaisoindolinone scaffolds were first synthesized and characterized by X-ray crystallography.
ò Molecular docking simulations suggested the key protein-ligand interactions for structural optimization.