Design, synthesis and biological evaluation of novel 2,3-indolinedione derivatives against mantle cell lymphoma

Article abstract of DOI:10.1016/j.bmc.2019.06.009

2,3-Indolinedione derivatives have been identified as a novel class of promising agents for cancer treatment. In this study, eighteen 2,3-indolinedione derivatives were designed and synthesized, and their anticancer activities against mantle cell lymphoma (MCL) cells were evaluated. Most of them exhibited significant antiproliferative activity against the tested cell lines, and compound K5 was the most potent (MCL cellular IC₅₀ = 0.4–0.7 μM). Further, compound K5 could induce cell apoptosis and cell cycle arrest in G2/M phase. Additionally, the results of drug-likeness analysis demonstrated that these novel 2,3-indolinedione derivatives could have potential as novel treatment strategies for MCL.

Full text of DOI:10.1016/j.bmc.2019.06.009

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel 2,3-indolinedione
derivatives against mantle cell lymphoma
⁎
Shengping Yua,1, Yang Liub,1, Zhen Zhanga, Jingya Zhanga, Guisen Zhaoa,
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan,
Shandong 250012, PR China
b Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
2,3-Indolinedione derivatives have been identified as a novel class of promising agents for cancer treatment. In
this study, eighteen 2,3-indolinedione derivatives were designed and synthesized, and their anticancer activities
against mantle cell lymphoma (MCL) cells were evaluated. Most of them exhibited significant antiproliferative
activity against the tested cell lines, and compound K5 was the most potent (MCL cellular IC50 = 0.4–0.7 μM).
Further, compound K5 could induce cell apoptosis and cell cycle arrest in G2/M phase. Additionally, the results
of drug-likeness analysis demonstrated that these novel 2,3-indolinedione derivatives could have potential as
novel treatment strategies for MCL.
2,3-Indolinedione
Mantle cell lymphoma
Derivatives
Biological activities
1. Introduction
It was reported that compound 1 (Fig. 1), a 2,3-indolinedione de-
rivative bearing an amide moiety, could predominantly inhibit human
Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma
with poor prognosis.1 Unlike other non-Hodgkin's lymphomas, MCL is
insidious, and most patients are in clinical stage III or stage IV at the
time of diagnosis and often relapse after first-line treatment.2 Median
overall survival for MCL patients is approximately 3–4 years.3 The pa-
thogenesis of MCL is complex and involves molecular changes at all
levels, including cell cycle mechanisms (CDKN2C/RB1/TP53/BMI1),
cellular survival signaling pathways (FAF1/BCL2L11/CDK4/MDM2),
and DNA damage response pathways (ATM/SP100/SP140/TP53).4,5
Currently, drugs used as targeted therapy for MCL include proteasome
inhibitors,6 BTK inhibitors,7 PI3K/AKT/mTOR pathway inhibitors,8,9
CDK inhibitors,10 HDAC inhibitors,11 targeted death proteins12 and so
on. In spite of the diverse therapeutic strategies available for MCL
treatment, chemo-resistance and increasing mortality rate remain
challenging. This finding emphasizes that more effective treatment
plans for MCL are urgently needed.
myeloid leukemia HL-60, U-937 and human lymphoid leukemia MOLT-
3 cells.25 The previous work of our laboratory once indicated that
compound 2 (Fig. 1), another 2,3-indolinedione analog with an in-
corporated amide group, effectively inhibited the proliferation of tumor
cells.26 Taking the above into consideration, herein, we retained the
2,3-indolinedione amide functionality and concentrated on chemical
modifications of substituents on the N-1 and C-5 positions, which led to
the discovery of new 2,3-indolinedione derivatives as shown in Fig. 1.
All of them were evaluated with respect to antiproliferative activity
against MCL cells. Moreover, cell apoptosis and cell cycle were as-
sessed. Last, we predicted their drug-likeness with the SwissADME web
tool.
2. Results and discussion
2.1. Chemistry
Isatin (2,3-indolinedione, Fig. 1) is an endogenous active compo-
nent in mammalian tissue and body fluids that has led to a series of
derivatives that display a wide range of biological properties including
antiproliferative, anticonvulsant, antifungal, anti-mycobacterial, anti-
protozoal, anti-HIV and anti-inflammatory activities.13–19 In the last
several years, new isatin-based analogs have exerted significant activ-
ities against different human malignancies.20–24
The syntheses of these 2,3-indolinedione derivatives are depicted in
Scheme 1. Commercially available isatin (1) was nitrified to obtain 5-
nitroisatin (2). Intermediate (3) was prepared by treating intermediate
(2) with 2,2-dimethylpropane-1,3-diol, with catalysis by p-toluene-
sulfonic acid. The intermediate (4) was obtained from reaction of in-
termediate (3) and 1,2-dichloro-4-(chloromethyl)benzene under
⁎ Corresponding author.
E-mail address: guisenzhao@sdu.edu.cn (G. Zhao).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2019.06.009
Received 5 April 2019; Received in revised form 3 June 2019; Accepted 5 June 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:ShengpingYu,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.06.009

S. Yu, et al.
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Fig. 1. Design of the target compounds.
alkaline conditions via electrophilic substitution. With Pd/C (10%, w/
w) as the catalyst, the nitro group in intermediate (4) was converted to
an amino group by hydrogenation to prepare intermediate (5). The
resulting compound was allowed to react with acyl chloride in the
presence of anhydrous potassium carbonate to obtain acylated products
(6a-6h), followed by deprotection under acidic conditions to achieve
the target compound K.
higher inhibitory potency to MCL cell lines than that with benzamide
methylene moiety (K2, Q6 vs. D2). Notably, series K and Q behaved
better (IC50 = 0.5–4.9 μM) than IBN (IC50 = 20.0 μM) for Jeko-R cells,
an IBN-resistant MCL cell line. K5 was the most potent inhibitor, with
IC50 values ranging between 0.4 and 0.7 μM against tested MCL cell
lines.
Intermediate (7) was obtained from intermediate (3), which was
converted by hydrogenation. Intermediate (7) was reacted with acyl
chloride in the presence of anhydrous potassium carbonate to obtain
acylated products (8a-8h). Treatment of phenyl nitrile (9) with hy-
droxylamine hydrochloride under alkaline conditions afforded inter-
mediate (10), and subsequent reaction with chloroacetyl chloride in a
methanol solution provided intermediate (11). Different intermediates
(12a-12b) were reacted with bromoacetyl bromide to prepare inter-
mediates (13a-13b). Then, intermediates (8a-8f) reacted with inter-
mediate (11) and intermediates (8f-8h) reacted with intermediates
(13a-13b) by electrophilic substitution to obtain intermediates (14a-
14f) and intermediates (15a-15d), respectively. Finally, products 14
and 15 were converted to target compounds Q and D by deprotection
under acidic conditions.
2.3. Cell apoptosis and cell cycle assay
Many factors mediate apoptosis and the cell cycle to maintain a
stable internal environment. Accordingly, to shed light on the under-
lying antitumor mechanism of compound K5, an apoptosis assay was
performed in Rec-1 cells and Z138 cells after K5 treatment, as well as
the cell cycle assay in Rec-1 cells. Annexin V-FITC/PI staining showed
that K5 induced cell apoptosis in Rec-1 cells and Z138 cells in a dose-
dependent manner, and the rate of cell apoptosis was over 90% after
24 h of treatment at a concentration of 2.5 μM (Fig. 2A–D). The cell
cycle assay demonstrated that treatment with K5 dose-dependently
induced cell cycle arrest in G2/M phase after a 24-h incubation, ef-
fectively preventing cell cycle progression and promoting cell death
(Fig. 2E).
2.2. Cell antiproliferative activity
2.4. Prediction of drug-likeness
All of the newly synthesized compounds were assessed in a panel of
MCL cell lines (Mino, Granta-519, Z138, Jeko-R, Maver-1) to explore
the antiproliferative activities. Ibrutinib (IBN), which was approved by
the FDA to treat MCL, served as a positive control.27 As shown in
Table 1, most of the compounds increased antiproliferative activities
compared with IBN. Compounds with 3,4-dichlorobenzyl group and 3-
phenyl-1,2,4-oxadiazol-5-methylene group at the N1 position displayed
To reduce time and resource consumption in drug discovery phases,
molecules should be evaluated for absorption, distribution, metabolism
and excretion (ADME). With the SwissADME web tool (http://www.
swissadme.ch),28 we calculated the physicochemical properties, lipo-
philicity, water solubility, pharmacokinetics and drug-likeness of
compound K5. The results are summarized in Table 2: we could see that
the drug-likeness of K5 was predicted by five diverse filters (Lipinski
2

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Scheme 1. Reagents and conditions: (a) fuming nitric acid, concentrated H2SO4, 0 °C, 1 h; (b) neopentyl glycol, p-toluenesulfonic acid, cyclohexane, 85 °C, 16 h; (c)
K2CO3, N,N-dimethylformamide, 85 °C, 1.5 h; (d) Pd/C, H2, ethyl acetate, r.t., 12 h; (e) substituted acyl chloride, K2CO3, ethyl acetate, 0 °C, 12 h; (f) glacial acetic
acid, concentrated hydrochloric acid, r.t., 12 h; (g) hydroxylamine hydrochloride, Et3N, CH3OH, 60 °C, 0.5 h; (h) chloroacetyl chloride, K2CO3, tetrahydrofuran, 0 °C,
2 h; (i) bromoacetyl bromide, NaHCO3, ethyl acetate/H2O = 1:1, 0 °C, 1 h; (j) K2CO3, N,N-dimethylformamide, r.t., 12 h.
filter, Ghose filter, Veber filter, Egan filter and Muegge filter), and it
passed all the rules of filter evaluation, which indicated that K5 was
promising for further in-depth research. From the prediction, we could
ascertain that early prediction contributed to designing and synthe-
sizing excellent compounds.
400 MHz and 100 MHz, respectively, on a Bruker Avance DRX-400
Spectrometer (Bruker, Germany) in deuterated dimethyl sulfoxide
(DMSO‑d6) with tetramethylsilane (TMS) as the internal standard. Peak
multiplicities were expressed as follows: singlet (s), doublet (d), triplet
(t), quartet (q), multiplet (m), broad singlet (br s), doublet of doublets
(dd), doublet of triplets (dt), and quartet of doublets (qd). The mass
spectra (MS) were measured with LCQ FLEET (ThermoFisher, USA).
The purity of the compounds was determined by HPLC performed on a
Shimadzu LC-20ATVP Liquid Chromatograph equipped with an SPD-
M20A UV VIS Detector using a C18 column (size: 250 mm × 4.6 mm).
Elution solvent: 75% methanol and 25% water. The elution rate was
1.00 mL/min, and the injection volumes were 10 μL at 25 °C and de-
tection at 253 nm.
3. Conclusion
In summary, our study outlined the design, synthesis and biological
evaluation of novel 2,3-indolinedione derivatives as potential agents for
the treatment of MCL. Most compounds showed increased anti-
proliferative activity against MCL cell lines compared with IBN. Among
these compounds, K5 remarkably exhibited considerable efficiency in
MCL cells, with IC50 values lower than 1 μM. Meanwhile, it induced cell
apoptosis and G2/M cell cycle arrest in a dose-dependent manner. The
prediction of drug-likeness proved that K5 was worthy of thorough
research. The results suggested that the 2,3-indolinedione structure did
offer potential for the discovery of novel potent inhibitors for MCL.
4.1.1. Synthesis of 5-nitroindoline-2,3-dione (2)
To a solution of isatin (1, 200 mg, 1.36 mmol) in concentrated
H2SO4 (3 mL) was added fuming nitric acid (124 mg, 1.77 mmol) at
0 °C. The resulting reaction mixture was stirred at 0 °C for 1 h. After
completion of the reaction, the mixture was slowly poured into ice-cold
water (50 mL). The precipitate was filtered, washed with water
(15 mL × 3), and then dried to obtain intermediate 2.
4. Experimental section
Yellow solid; yield 85%; mp: 257–259 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 11.66 (s, 1H), 8.45 (dd, J = 8.7, 2.4 Hz, 1H), 8.22 (d,
J = 2.4 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H).
4.1. Chemistry
All of the chemical reagents and solvents were purchased from
commercial sources and were used without further purification.
Reactions were monitored using thin-layer chromatography (TLC)
performed on SGF254 plates. Chromatographic separations were per-
formed using column chromatography on silica gel (60 Å,
200–300 mesh). Melting points were determined on a Büchi capillary
melting point apparatus (Buchi Labortechnik AG, Switzerland) without
correction. The 1H NMR and 13C NMR spectra were recorded at
4.1.2. Synthesis of 5′,5′-dimethyl-5-nitrospiro[indoline-3,2′-[1,3]dioxan]-
2-one (3)
Intermediate 2 (200 mg, 1.04 mmol), neopentyl glycol (108 mg,
1.04 mmol) and p-toluenesulfonic acid (25 mg, 145 μmol) were suc-
cessively added and dissolved in cyclohexane (10 mL). The resulting
reaction mixture was refluxed for 16 h at 85 °C. After completion of the
3

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Table 1
Cell proliferation assay assessing the effects of novel 2,3-indolinedione derivatives on MCL cell lines.
Code
X
Y
Cell viability assay, IC50/μM
Mino
Granta-519
Z138
Jeko-R
Maver-1
IBN
K1
—
—
2.0
0.4
9.7
1.4
14.4
0.4
20.0
0.8
14.4
0.6
K2
K3
K4
K5
K6
K7
K8
0.4
0.4
0.4
0.4
0.4
0.4
1.8
1.2
1.1
1.1
0.6
0.9
0.8
4.2
0.4
0.4
0.4
0.4
0.4
0.4
0.9
0.8
0.5
0.6
0.7
0.7
0.5
3.3
0.5
0.5
0.5
0.4
0.5
0.5
2.3
Q1
Q2
Q3
Q4
Q5
Q6
D1
D2
D3
D4
2.3
6.5
1.3
4.1
2.9
1.2
3.0
0.6
2.0
1.0
0.4
1.7
0.5
0.9
0.7
3.5
7.3
2.2
4.9
3.9
0.4
1.5
0.4
0.7
0.5
0.6
2.1
0.5
1.3
0.7
7.3
aND
aND
aND
aND
29.6
28.0
> 60
> 60
49.4
52.5
> 60
58.0
25.3
43.8
> 60
39.6
12.3
> 60
15.7
aND, Not Detected.
reaction, the mixture was cooled to room temperature. Then, the pre-
cipitate was filtered, washed with water and dried. Purification by
chromatography on silica gel (petroleum ether/ethyl acetate = 5/1)
afforded intermediate 3.
K2CO3 (199 mg, 1.44 mmol) in dried N,N-dimethylformamide (2 mL)
was added 1,2-dichloro-4-(chloromethyl)benzene (169 mg, 0.86 mmol).
The resulting reaction mixture was stirred for 1.5 h at 85 °C. The re-
action mixture was cooled to room temperature and then poured into
ice water (50 mL), and a precipitate was produced. The precipitate was
filtered, washed with water, and then recrystallized with ethyl acetate/
petroleum ether to obtain intermediate 4.
White solid; yield 84%; mp: 201–203 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 11.20 (s, 1H), 8.28 (dd, J = 8.7, 2.4 Hz, 1H), 8.08 (d,
J = 2.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.49 (d, J = 11.0 Hz, 2H),
3.55 (d, J = 11.2 Hz, 2H), 1.34 (s, 3H), 0.84 (s, 3H).
White solid; yield 80%; mp: 137–141 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 8.34 (dd, J = 8.7, 1.6 Hz, 1H), 8.14(d, J = 2.2 Hz, 1H),
7.64 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 8.8 Hz, 1H), 7.22 (dd, J = 8.3,
2.0 Hz, 1H), 4.98 (s, 2H), 4.50 (d, J = 11.0 Hz, 2H), 3.61 (d,
J = 10.9 Hz, 2H), 1.37 (s, 3H), 0.87 (s, 3H).
4.1.3. Synthesis of 1-(3,4-dichlorobenzyl)-5′,5′-dimethyl-5-nitrospiro
[indoline-3,2′-[1,3]dioxan]-2-one (4)
To a solution of intermediate 3 (200 mg, 0.72 mmol) and anhydrous
4

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Fig 2. Cell apoptosis with K5 treatment in Rec-1 cells and Z138 cells, and cell cycle assay with K5 treatment in Rec-1 cells. A/C) Apoptosis assay of K5 at the
indicated concentrations in Rec-1 cells for 24 h; B/D) Apoptosis assay of K5 at the indicated concentrations in Z138 cells for 24 h; E) Cell cycle analysis of K5 at the
indicated concentrations in Rec-1 cells for 24 h.
4.1.4. Synthesis of 5-amino-1-(3,4-dichlorobenzyl)-5′,5′-dimethylspiro
[indoline-3,2′-[1,3]dioxan]-2-one (5)
filtered, and the filtrate was concentrated under reduced pressure. The
crude material was recrystallized with ethyl acetate/petroleum ether to
obtain intermediate 5.
To a solution of intermediate 4 (280 mg, 640 μmol) in ethyl acetate
(25 mL) was added 10% Pd/C (140 mg), and the mixture was subjected
to hydrogen for 12 h at room temperature. The reaction mixture was
Light yellow solid; yield 77%; mp: 171–174 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 7.63 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.21 (dd,
5

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Table 2
was filtered and purified by recrystallization in methanol to obtain
Several important properties of compound K5 as determined using SwissADME
compounds K1-K8.
web tools.
4.1.6.1. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)propionamide
Properties
K5
(K1). Red solid; yield 81%; mp: 227–230 °C; 1H NMR (400 MHz,
Physicochemical Properties
Molecular weight
Num. heavy atoms
Num. arom. heavy atoms
Fraction Csp3
DMSO‑d6)
δ 9.97 (s, 1H), 7.90 (s, 1H), 7.76 (s, 1H), 7.61 (d,
397.64 g/mol
J = 8.3 Hz, 2H), 7.44 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H),
4.89 (s, 2H), 2.30 (dd, J = 14.9, 7.5 Hz, 2H), 1.07 (t, J = 7.5 Hz, 3H);
13C NMR (100 MHz, DMSO‑d6) δ 183.43 (s), 172.53 (s), 159.11 (s),
145.56 (s), 137.34 (s), 135.76 (s), 131.74 (s), 131.16 (s), 130.57 (s),
129.81 (s), 128.23 (s), 128.21 (s), 118.39 (s), 115.66 (s), 111.50 (s),
42.28 (s), 29.84 (s), 10.05 (s); MS (ESI): calcd for C18H14Cl2N2O3
[M−H]− 375.04, found 375.33; HPLC: tR 5.566 min, purity 95.0%.
25
12
0.17
Num. rotatable bonds
Num. H-bond acceptors
Num. H-bond donors
Molar Refractivity
TPSAa
5
3
1
100.67
66.48 Å2
Lipophilicity
4.1.6.2. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)butyramide
Log P (average of iLOGP, XLOGP3, WLOGP,
MLOGP, SILICOS-IT)
Water Solubility
3.06
(K2). Red solid; yield 77%; mp: 248–250 °C; 1H NMR (400 MHz,
DMSO‑d6)
δ 9.98 (s, 1H), 7.91 (s, 1H), 7.76 (s, 1H), 7.61 (d,
Log S (ESOL)b
−4.50
J = 8.3 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H),
4.89 (s, 2H), 2.26 (t, J = 7.3 Hz, 2H), 1.65–1.53 (m, 2H), 0.88 (t, 3H);
13C NMR (100 MHz, DMSO‑d6) δ 183.42 (s), 171.68 (s), 159.10 (s),
145.59 (s), 137.33 (s), 135.73 (s),131.74 (s), 131.16 (s), 130.57 (s),
129.81 (s), 128.26 (s), 128.20 (s), 118.37 (s), 115.70 (s), 111.50 (s),
42.29 (s), 38.65 (s), 18.97 (s), 14.04 (s); MS (ESI): calcd for
Solubility
1.25e-02 mg/ml; 3.13e-
05 mol/l
Class
Moderately soluble
Pharmacokinetics
GI absorption
High
Yes
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Log Kp (skin permeation)c
19H16Cl2N2O3 [M−H]− 389.05, found 389.07; HPLC: tR 6.710 min,
Yes
C
Yes
purity 92.3%.
No
Yes
4.1.6.3. (E)-N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)but-2-
enamide (K3). Red solid; yield 72%; mp: 257–259 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.06 (s, 1H), 7.97 (d, J = 2.1 Hz, 1H), 7.77
(d, J = 2.0 Hz, 1H), 7.65 (dd, J = 8.5, 2.3 Hz, 1H), 7.61 (d, J = 8.3 Hz,
1H), 7.44 (dd, J = 8.4, 2.0 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.80 (dq,
J = 13.8, 6.9 Hz, 1H), 6.06 (dd, J = 15.2, 1.7 Hz, 1H), 4.89 (s, 3H),
1.86 (dd, J = 6.9, 1.4 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 183.38
(s), 163.97 (s), 159.10 (s), 145.73 (s), 140.78 (s), 137.32 (s), 135.69 (s),
131.75 (s), 131.16 (s), 130.58 (s), 129.81 (s), 128.38 (s), 128.20 (s),
126.06 (s), 118.43 (s), 115.80 (s), 111.56 (s), 42.30 (s), 18.01 (s); MS
(ESI): calcd for C19H14Cl2N2O3 [M−H]− 387.04, found 387.18; HPLC:
tR 7.598 min, purity 87.1%.
−6.28 cm/s
Drug-likeness
Lipinski (Pfizer) filter
Ghose filter
Yes; 0 violation
Yes
Yes
Yes
Yes
0.55
Veber (GSK) filter
Egan (Pharmacia) filter
Muegge (Bayer) filter
Bioavailability Scored
a
TPSA: Topological Polar Surface Area. Molecular polar surface area (PSA)
calculated as sum of tabulated surface contributions of polar fragments.29
b
ESOL – Estimated SOLubility: Simple method for estimating the aqueous so-
lubility of a compound directly from its structure.30 Solubility Class: Log S scale:
−Insuluble < −10 < Poorly < −6 < Moderately < −4 < Soluble < −2
< Very < 0 < Highly.
4.1.6.4. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)acrylamide
c
LogKp: Permeability coefficient from the Quantitative Structure
(K4). Red solid; yield 74%; mp: 220–222 °C; 1H NMR (400 MHz,
Permeability Relationships (QSPR) model based upon permeant size [molecular
volume (MV) or molecular weight (MW)] and octanol/water partition coeffi-
DMSO‑d6)
δ 10.29 (s, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.77 (d,
cient (Koct).31
J = 2.0 Hz, 1H), 7.68 (dd, J = 8.5, 2.2 Hz, 1H), 7.61 (d, J = 8.3 Hz,
1H), 7.45 (dd, J = 8.3, 2.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.39 (dd,
J = 17.0, 10.0 Hz, 1H), 6.26 (dd, J = 17.0, 2.0 Hz, 1H), 5.77 (dd,
J = 10.0, 2.1 Hz, 1H), 4.90 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ
183.33 (s), 163.67 (s), 159.11 (s), 145.97 (s), 137.31 (s), 135.39 (s),
131.97 (s), 131.75 (s), 131.17 (s), 130.58 (s), 129.82 (s), 128.52 (s),
128.21 (s), 127.68 (s), 118.48 (s), 115.89 (s), 111.61 (s), 42.31 (s); MS
(ESI): calcd for C18H12Cl2N2O3 [M−H]− 373.02, found 373.11; HPLC:
tR 5.523 min, purity 88.9%.
d
Abbott Bioavailability Score: Probability that
F > 10% in the rat.32
a compound will have
J = 8.3, 2.0 Hz, 1H), 6.76 (d, J = 2.2 Hz, 1H), 6.64 (d, J = 8.3 Hz,
1H),6.48 (dd, J = 8.3, 2.3 Hz, 1H), 4.97 (s, 2H), 4.75 (s, 2H), 4.50 (d,
J = 10.9 Hz, 2H),3.51 (d, J = 11.0 Hz, 2H), 1.30 (s, 3H), 0.84 (s, 3H).
4.1.5. Synthesis of intermediates (6a-6h)
To a solution of intermediate 5 (200 mg, 491 μmol) and anhydrous
K2CO3 (81 mg, 589 μmol) in ethyl acetate (30 mL) was added differ-
entially substituted acyl chloride (589 μmol) at 0 °C. The resulting so-
lution was stirred overnight at room temperature. After completion of
the reaction, the mixture was filtered. The organic phase was succes-
sively washed with water (15 mL × 3) and saturated brine (15 mL × 3)
and then evaporated under reduced pressure to obtain intermediates
6a-6h, which were directly used for the next step without further
purification.
4.1.6.5. 2-Chloro-N-(1-(3,4-dichlorobenzyl)-2,3-dioxoindolin-5-yl)
acetamide (K5). Red solid; yield 83%; mp: 205–206 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.42 (s, 1H), 7.88 (d, J = 2.1 Hz, 1H), 7.77
(d, J = 1.9 Hz, 1H), 7.61 (dd, J = 8.4, 2.2 Hz, 2H), 7.45 (dd, J = 8.3,
2.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 4.90 (s, 2H), 4.25 (s, 2H); 13C
NMR (100 MHz, DMSO‑d6) δ 183.32 (s), 165.26 (s), 159.11 (s), 146.24
(s), 137.26 (s), 134.76 (s), 131.76 (s), 131.16 (s), 130.59 (s), 129.81 (s),
128.72 (s), 128.21 (s), 118.51 (s), 116.00 (s), 111.65 (s), 43.85 (s),
42.32 (s); MS (ESI): calcd for C17H11Cl3N2O3 [M−H]− 394.98, found
395.19; HPLC: tR 5.300 min, purity 94.1%.
4.1.6. Synthesis of target compounds (K1-K8)
To the solvent of glacial acetic acid (20 mL) and concentrated hy-
drochloric acid (2 mL) was added intermediate 6 (421 μmol). The
mixture was stirred overnight at room temperature and then poured
into water (100 mL), and a precipitate was produced. The precipitate
4.1.6.6. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)
cyclopropanecarboxamide (K6). Red solid; yield 75%; mp: 247–250 °C;
1H NMR (400 MHz, DMSO‑d6) δ 10.29 (s, 1H), 7.89 (d, J = 2.1 Hz, 1H),
6

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7.76 (d, J = 2.0 Hz, 1H), 7.65–7.58 (m, 2H), 7.44 (dd, J = 8.4, 2.0 Hz,
1H), 6.89 (d, J = 8.5 Hz, 1H), 4.89 (s, 2H), 1.76–1.67 (m, 1H), 0.80 (d,
J = 6.3 Hz, 4H); 13C NMR (100 MHz, DMSO‑d6) δ 183.41 (s), 172.17
(s), 159.10 (s), 145.54 (s), 137.34 (s), 135.77 (s), 131.75 (s), 131.16 (s),
130.58 (s), 129.81 (s), 128.20 (s), 128.14 (s), 118.41 (s), 115.64 (s),
111.52 (s), 42.29 (s), 14.99 (s), 7.76 (s, 2C); MS (ESI): calcd for
under reduced pressure to obtain intermediate 10 as a white solid.
4.1.10. Synthesis of 5-(chloromethyl)-3-phenyl-1,2,4-oxadiazole (11)
To a solution of intermediate 10 (100 mg, 734 μmol) and anhydrous
K2CO3 (152 mg, 1.1 mmol) in tetrahydrofuran (15 mL) was added
chloroacetyl chloride (166 mg, 1.47 mmol) at 0 °C. The mixture was
stirred for 2 h at room temperature. The reaction mixture was filtered,
and the filtrate was concentrated under reduced pressure. The residue
was dissolved in ethyl acetate (25 mL). The organic phase was succes-
sively washed with water (20 mL × 3) and saturated sodium carbonate
solution (20 mL × 3), dried over Na2SO4 and then filtered and con-
centrated under reduced pressure to obtain intermediate 11 as yellow
oil.
C
19H14Cl2N2O3 [M−H]− 387.04, found 387.48; HPLC: tR 7.195 min,
purity 99.6%.
4.1.6.7. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)benzamide
(K7). Red solid; yield 70%; mp: 238–240 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 10.36 (s, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.98–7.93 (m,
2H), 7.87 (dd, J = 8.6, 2.2 Hz, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.61 (dd,
J = 11.0, 7.8 Hz, 2H), 7.54 (t, J = 7.3 Hz, 2H), 7.47 (dd, J = 8.4,
2.0 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 4.92 (s, 2H); 13C NMR (100 MHz,
DMSO‑d6) δ 183.36 (s), 165.97 (s), 159.19 (s), 146.12 (s), 137.34 (s),
135.50 (s), 134.92 (s), 132.23 (s), 131.75 (s), 131.17 (s), 130.58 (s),
129.83 (s), 129.70 (s), 128.92 (s, 2C), 128.22 (s), 128.10 (s, 2C), 118.40
(s), 117.05 (s), 111.43 (s), 42.32 (s); MS (ESI): calcd for C22H14Cl2N2O3
[M−H]− 423.04, found 423.42; HPLC: tR 9.789 min, purity 97.4%.
4.1.11. Synthesis of intermediates (14a-14f)
To a solution of intermediate 8a-8f (532 μmol) in dried N,N-di-
methylformamide (5 mL) was added anhydrous K2CO3 (147 mg,
1.06 mmol). The mixture was stirred for 30 min at room temperature.
Intermediate 11 (124 mg, 639 μmol) was then added to the mixture.
The mixture was stirred for 12 h at room temperature. The reaction
mixture was poured into ice water (100 mL) and a precipitate was
produced. The precipitate was filtered, washed with water, and dried to
obtain intermediates 14a-14f.
4.1.6.8. N-(1-(3,4-Dichlorobenzyl)-2,3-dioxoindolin-5-yl)-2-
phenylacetamide (K8). Red solid; yield 75%; mp: 257–259 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.30 (s, 1H), 7.90 (d, J = 2.1 Hz, 1H), 7.76 (d,
J = 1.9 Hz, 1H), 7.62 (dd, J = 13.5, 5.2 Hz, 2H), 7.44 (dd, J = 8.3,
2.0 Hz, 1H), 7.32 (d, J = 4.4 Hz, 4H), 7.29–7.20 (m, 1H), 6.89 (d,
J = 8.5 Hz, 1H), 4.89 (s, 2H), 3.62 (s, 2H); 13C NMR (100 MHz,
DMSO‑d6) δ 183.35 (s), 169.69 (s), 159.11 (s),145.76 (s), 137.30 (s),
136.24 (s), 135.60 (s), 131.75 (s), 131.15 (s), 130.58 (s),129.80 (s),
129.56 (s, 2C), 128.79 (s, 2C), 128.37 (s), 128.19 (s), 127.04 (s),
118.41(s), 115.73 (s), 111.55 (s), 43.62 (s), 42.28 (s); MS (ESI): calcd
4.1.12. Synthesis of target compounds (Q1-Q6)
Glacial acetic acid (30 mL) and concentrated hydrochloric acid
(4 mL) were added to intermediate 14 (421 μmol). The mixture was
stirred overnight at room temperature and then poured into distilled
water (100 mL), and a precipitate was produced. The precipitate was
filtered and purified by column chromatography with di-
chloromethane/methanol (20:1) to obtain compounds Q1-Q6.
for
C
23H16Cl2N2O3 [M−H]− 437.05, found 437.22; HPLC: tR
8.042 min, purity 97.2%.
4.1.12.1. N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)indolin-
5-yl)cyclopropanecarboxamide (Q1). Red solid; yield 61%; mp:
216–219 °C; 1H NMR (400 MHz, DMSO‑d6) δ 10.31 (s, 1H), 7.88 (d,
J = 2.1 Hz, 1H), 7.62 (dd, J = 8.5, 2.2 Hz, 1H), 7.42 (d, J = 7.0 Hz,
2H), 7.34 (t, J = 7.3 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 6.92 (d,
J = 8.5 Hz, 1H), 4.88 (s, 2H), 1.71 (s, 1H), 0.80 (s, 4H); 13C NMR
(100 MHz, DMSO‑d6) δ 182.70 (s), 175.25 (s), 172.33 (s), 168.25 (s),
158.69 (s), 145.33 (s), 136.23 (s), 132.26 (s), 129.79 (s, 2C), 128.68 (s),
127.54 (s, 2C), 126.15 (s), 118.16 (s), 115.67 (s), 111.89 (s), 36.22 (s),
14.99 (s), 7.80 (s, 2C); MS (ESI): calcd for C21H16N4O4 [M−H]−
387.12, found 387.72; HPLC: tR 4.209 min, purity 97.9%.
4.1.7. Synthesis
of
5-amino-5′,5′-dimethylspiro[indoline-3,2′-[1,3]
dioxan]-2-one (7)
To a solution of intermediate 3 (395 mg, 1.42 mmol) in ethyl acetate
(40 mL) was added 10% Pd/C (240 mg). The mixture was subjected to
hydrogen for 12 h at room temperature. The reaction mixture was fil-
tered, and the filtrate was concentrated under reduced pressure. The
crude material was recrystallized with ethyl acetate/petroleum ether to
obtain intermediate 7.
White solid; yield 81%; mp: 212–214 °C; 1H NMR (400 MHz,
DMSO‑d6) δ 9.97 (s, 1H), 6.68 (s, 1H), 6.47 (s, 2H), 4.82 (s, 2H), 4.49
(d, J = 10.8 Hz, 2H), 3.43 (d, J = 11.0 Hz, 2H), 1.27 (s, 3H), 0.81 (s,
3H).
4.1.12.2. N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)indolin-
5-yl)propionamide (Q2). Red solid; yield 55%; mp: 207–209 °C; 1H
NMR (400 MHz, DMSO‑d6) δ 10.06 (s, 1H), 8.01–7.91 (m, 3H), 7.76
(dd, J = 8.6, 2.2 Hz, 1H), 7.61–7.52 (m, 3H), 7.26 (d, J = 8.6 Hz, 1H),
5.38 (s, 2H), 2.32 (q, J = 7.5 Hz, 2H), 1.08 (t, J = 7.5 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6) δ 182.71 (s), 175.26 (s), 172.64 (s), 168.25 (s),
158.69 (s), 145.36 (s), 136.18 (s), 132.26 (s), 129.80 (s, 2C), 128.77 (s),
127.54 (s, 2C), 126.15 (s), 118.16 (s), 115.70 (s), 111.87 (s), 36.22 (s),
29.86 (s), 10.03 (s); MS (ESI): calcd for C20H16N4O4 [M−H]− 375.12,
found 375.78; HPLC: tR 4.008 min, purity 98.5%.
4.1.8. Synthesis of intermediates (8a-8h)
To a solution of intermediate 7 (240 mg, 967 μmol) and anhydrous
K2CO3 (160 mg, 1.16 mmol) in ethyl acetate (20 mL) was added dif-
ferentially substituted acyl chloride (1.16 μmol) at 0 °C. The resulting
solution was stirred overnight at room temperature. After completion of
the reaction, the mixture was filtered. The organic phase was succes-
sively washed with water (15 mL × 3) and saturated brine (15 mL × 3)
and then evaporated under reduced pressure to obtain intermediates
8a-8h, which were directly used for the next step without further
purification.
4.1.12.3. N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)indolin-
5-yl)-2-phenylacetamide (Q3). Red solid; yield 66%; mp: 189–192 °C;
1H NMR (400 MHz, DMSO‑d6) δ 10.37 (s, 1H), 7.99–7.91 (m, 3H), 7.77
(dd, J = 8.6, 2.2 Hz, 1H), 7.63–7.52 (m, 3H), 7.33 (d, J = 4.4 Hz, 4H),
7.27 (d, J = 8.7 Hz, 2H), 5.38 (s, 2H), 3.63 (d, J = 7.9 Hz, 2H); 13C
NMR (100 MHz, DMSO‑d6) δ 182.62 (s), 175.24 (s), 169.78 (s), 168.24
(s), 158.68 (s), 145.60 (s), 136.17 (s), 135.93 (s), 132.27 (s), 129.80 (s,
2C), 129.57 (s, 2C), 128.92 (s), 128.81 (s, 2C), 127.54 (s, 2C), 127.08
(s), 126.15 (s), 118.21 (s), 115.80 (s), 111.94 (s), 43.63 (s), 36.21 (s);
MS (ESI): calcd for C25H18N4O4 [M−H]− 437.13, found 437.21; HPLC:
tR 5.342 min, purity 96.7%.
4.1.9. Synthesis of (E)-N'-hydroxybenzimidamide (10)
Benzonitrile 9 (2 g, 19.39 mmol), hydroxylamine hydrochloride
(2.7 g, 38.79 mmol) and Et3N (3.93 g, 38.79 mmol) were successively
added and dissolved in methanol (100 mL). The mixture was stirred for
30 min at 60 °C. The solvent was removed under reduced pressure, and
the residue was dissolved in ethyl acetate (50 mL). The organic phase
was successively washed with water (30 mL × 3) and saturated brine
(30 mL × 3), dried over MgSO4 and then filtered and concentrated
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4.1.12.4. N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)indolin-
5-yl)benzamide (Q4). Red solid; yield 67%; mp: 264–266 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.45 (s, 1H), 8.10 (s, 1H), 8.06–7.90 (m, 5H),
7.66–7.49 (m, 6H), 7.34 (d, J = 8.5 Hz, 1H), 5.42 (s, 2H); 13C NMR
(100 MHz, DMSO‑d6) δ 182.65 (s), 175.26 (s), 168.27 (s), 166.06 (s),
158.76 (s), 145.92 (s), 135.93 (s), 134.86 (s), 132.27 (s), 130.29 (s),
129.80 (s, 2C), 128.92 (s, 2C), 128.89 (s), 128.16 (s, 2C), 127.55 (s,
2C), 126.16 (s), 118.15 (s), 117.16 (s), 111.80 (s), 36.26 (s); MS (ESI):
calcd for C24H16N4O4 [M−H]− 423.12, found 423.28; HPLC: tR
5.233 min, purity 85.5%.
4.1.15. Synthesis of target compounds (D1-D4)
Glacial acetic acid (15 mL) and concentrated hydrochloric acid
(3 mL) were added to intermediate 15 (354.22 μmol). The mixture was
stirred overnight at room temperature and then poured into ice water
(100 mL), and a precipitate was produced. The precipitate was filtered,
washed with water, and dried. The crude material was recrystallized
with methanol to obtain compounds D1-D4.
4.1.15.1. N-(1-(2-((3-Cyanophenyl)amino)-2-oxoethyl)-2,3-dioxoindolin-
5-yl)acrylamide (D1). Red solid; yield 75%; mp: 283–285 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.57 (s, 1H), 10.34 (s, 1H), 8.03 (d, J = 4.3 Hz,
2H), 7.80 (s, 2H), 7.57 (s, 2H), 7.16 (d, J = 7.6 Hz, 1H), 6.40 (m,
6.44–6.37, 1H), 6.28 (d, J = 16.7 Hz, 1H), 5.79 (d, J = 10.3 Hz, 1H),
4.59 (s, 2H); 13C NMR (100 MHz, DMSO‑d6) δ 183.39 (s), 166.10 (s),
163.73 (s), 159.04 (s), 146.84 (s), 139.57 (s), 135.52 (s), 131.95 (s),
130.86 (s), 129.24 (s), 127.89 (s), 127.77 (s), 124.61 (s), 122.75 (s),
118.99 (s), 117.94 (s), 115.82 (s), 112.14 (s), 111.90 (s), 43.71 (s); MS
(ESI): calcd for C20H14N4O4 [M−H]− 373.10, found 373.22; HPLC: tR
2.943 min, purity 98.8%.
4.1.12.5. (E)-N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)
indolin-5-yl)but-2-enamide (Q5). Red solid; yield 72%; mp: 200–203 °C;
1H NMR (400 MHz, DMSO‑d6) δ 10.19 (s, 1H), 8.02 (d, J = 1.8 Hz, 1H),
7.97 (d, J = 6.7 Hz, 2H), 7.81 (dd, J = 8.6, 2.0 Hz, 1H), 7.65–7.52 (m,
3H), 7.28 (d, J = 8.6 Hz, 1H), 6.82 (dq, J = 13.9, 6.8 Hz, 1H), 6.10 (d,
J = 15.2 Hz, 1H), 5.39 (s, 2H), 1.87 (d, J = 6.1 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6) δ 182.67 (s), 175.24 (s), 168.27 (s), 164.10 (s),
158.68 (s), 145.54 (s), 140.81 (s), 136.15 (s), 132.25 (s), 129.79 (s, 2C),
128.97 (s), 127.55 (s, 2C), 126.17 (s), 126.10 (s), 118.20 (s), 115.89 (s),
111.92 (s), 36.24 (s), 28.56 (s); MS (ESI): calcd for C21H16N4O4
[M−H]− 387.12, found 387.29; HPLC: tR 4.416 min, purity 97.0%.
4.1.15.2. N-(1-(2-((4-Cyanophenyl)amino)-2-oxoethyl)-2,3-dioxoindolin-
5-yl)butyramide (D2). Red solid; yield 77%; mp: 290–293 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.70 (s, 1H), 10.06 (s, 1H), 7.94 (d, J = 1.5 Hz,
1H), 7.81 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 2H), 7.71 (dd,
J = 8.6, 1.6 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 4.61 (s, 2H), 2.28 (t,
J = 7.3 Hz, 2H), 1.68–1.55 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6) δ 183.47 (s), 171.76 (s), 166.25 (s), 159.03 (s),
146.51 (s), 143.02 (s), 135.87 (s), 133.85 (s, 2C), 129.01 (s), 119.99 (s,
2C), 119.38 (s), 117.79 (s), 115.60 (s), 111.76 (s), 106.05 (s), 43.76 (s),
38.65 (s), 18.96 (s), 14.07 (s); MS (ESI): calcd for C21H18N4O4 [M−H]−
389.13, found 389.30; HPLC: tR 3.146 min, purity 95.2%.
4.1.12.6. N-(2,3-Dioxo-1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)indolin-
5-yl)butyramide (Q6). Red solid; yield 78%; mp: 205–208 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.07 (s, 1H), 8.03–7.91 (m, 3H), 7.76 (dd,
J = 8.6, 2.2 Hz, 1H), 7.64–7.54 (m, 3H), 7.26 (d, J = 8.6 Hz, 1H), 5.38
(s, 2H), 2.28 (t, J = 7.3 Hz, 2H), 1.67–1.54 (m, 2H), 0.91 (t, J = 7.4 Hz,
3H); 13C NMR (100 MHz, DMSO‑d6) δ 182.70 (s), 175.25 (s), 171.82 (s),
168.25 (s), 158.70 (s), 145.39 (s), 136.14 (s), 132.26 (s), 129.80 (s, 2C),
128.80 (s), 127.54 (s, 2C), 126.15 (s), 118.15 (s), 115.73 (s), 111.86 (s),
38.65 (s), 36.21 (s), 18.97 (s), 14.06 (s); MS (ESI): calcd for C21H18N4O4
[M−H]− 389.13, found 389.42; HPLC: tR 4.614 min, purity 98.7%.
4.1.15.3. 2-(5-Acetamido-2,3-dioxoindolin-1-yl)-N-(4-cyanophenyl)
acetamide (D3). Red solid; yield 73%; mp: 282–285 °C; 1H NMR
(400 MHz, DMSO‑d6)
δ 10.67 (s, 1H), 10.10 (s, 1H), 7.91 (d,
4.1.13. Synthesis of intermediates (13a-13b)
J = 2.0 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H),
7.69 (dd, J = 8.5, 2.1 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 4.60 (s, 2H),
2.04 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 183.46 (s), 168.91 (s),
166.26 (s), 159.02 (s), 146.53 (s), 143.01 (s), 135.87 (s), 133.85 (s, 2C),
128.94 (s), 120.00 (s, 2C), 119.38 (s), 117.80 (s), 115.53 (s), 111.78 (s),
106.06 (s), 43.78 (s), 24.32 (s); MS (ESI): calcd for C19H14N4O4
[M−H]− 361.10, found 361.22; HPLC: tR 2.790 min, purity 98.1%.
To differentially substituted aniline 12 (5 mmol) and NaHCO3
(1.26 g, 15 mmol) in ethyl acetate/water (10 mL/10 mL) was added
bromoacetyl bromide (2.02 g, 10 mmol) at 0 °C. The mixture was stirred
for 1 h at 0 °C. After completion of the reaction, the mixture was dis-
solved in ethyl acetate/water (1:1, 100 mL). The aqueous phase was
washed with ethyl acetate (30 mL × 2). The organic phase was then
washed with saturated brine (30 mL × 1) and dried over Na2SO4, fil-
tered and concentrated under reduced pressure. The crude material was
recrystallized with ethyl acetate to obtain intermediates 13a-13b.
4.1.15.4. N-(1-(2-((3-Cyanophenyl)amino)-2-oxoethyl)-2,3-dioxoindolin-
5-yl)butyramide (D4). Red solid; yield 77%; mp: 243–245 °C; 1H NMR
(400 MHz, DMSO‑d6) δ 10.55 (s, 1H), 10.04 (s, 1H), 8.03 (s, 1H), 7.94
(d, J = 2.0 Hz, 1H), 7.83–7.76 (m, 1H), 7.71 (dd, J = 8.5, 2.0 Hz, 1H),
7.56 (dd, J = 3.6, 2.0 Hz, 2H), 7.12 (d, J = 8.5 Hz, 1H), 4.58 (s, 2H),
2.28 (t, J = 7.3 Hz, 2H), 1.70–1.54 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H);
13C NMR (100 MHz, DMSO‑d6) δ 183.48 (s), 171.75 (s), 166.11 (s),
159.05 (s), 146.46 (s), 139.57 (s), 135.87 (s), 130.85 (s), 128.98 (s),
127.88 (s), 124.61 (s), 122.75 (s), 118.99 (s), 117.85 (s), 115.59 (s),
112.13 (s), 111.75 (s), 43.69 (s), 38.66 (s), 18.96 (s), 14.07 (s); MS
(ESI): calcd for C21H18N4O4 [M−H]− 389.13, found 389.30; HPLC: tR
3.170 min, purity 98.0%.
4.1.13.1. 2-Bromo-N-(4-cyanophenyl)acetamide (13a). White solid;
yield 84%; mp: 123–125 °C; 1H NMR (400 MHz, DMSO‑d6) δ (ppm)
10.84 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 4.09
(s, 2H).
4.1.13.2. 2-Bromo-N-(3-cyanophenyl)acetamide (13b). White solid;
yield 87%; mp: 153–155 °C; 1H NMR (400 MHz, DMSO‑d6) δ (ppm)
10.75 (s, 1H), 8.07 (s, 1H), 7.81–7.78 (m, 1H), 7.57–7.56 (m, 2H), 4.08
(s, 2H).
4.1.14. General synthesis of intermediates (15a-15d)
4.2. Cell proliferation activity
To a solution of intermediate 8f-8h (1.03 mmol) in dried N,N-di-
methylformamide (20 mL) was added anhydrous K2CO3 (1.55 mmol).
The mixture was stirred for 30 min at room temperature. Then, the
intermediates 13a-13b (1.24 mmol) were added to the mixture. The
mixture was stirred overnight at room temperature and poured into ice
water (100 mL), and a precipitate was produced. The precipitate was
filtered, washed with water, and dried and purified by column chro-
matography with petroleum ether/ethyl acetate (5:1) to obtain inter-
mediates 15a-15d.
The cell proliferation assay was evaluated on a panel of MCL cell
lines using the CellTiter-Glo Luminescent cell viability assay kit
(Promega) following the manufacturer's protocol. MCL cell lines were
purchased from American Type Culture Collection (ATCC, USA) and
cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum
(FBS, Gibco), 10,000 units/mL penicillin, and 10 mg/mL streptomycin
at 37 °C in a humidified incubator with 5% CO2. In short, the cells were
plated in 96-well plates at a density of 10,000 cells/well. After
8

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
attachment of the cells to the bottom, compounds to be tested were
dissolved to concentrations of 0.93 μM to 60 μM in DMSO and then
added to the plates with no more than a 1‰ final percentage of DMSO.
The treatment lasted for 72 h, and then CellTiter-Glo (30 μL) was added.
The absorbance at 570 nm was measured using an ELX800 Microplate
Reader (BioTek, USA) three times for each plate, and IC50 values were
calculated by Prism 6.0 Software (GraphPad Software, USA).
derivatives as potent BTK inhibitors against B cell lymphoma cell lines. Bioorg Med
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Cell cycle arrest was measured using PI staining by flow cytometry.
Cells were seeded in 6-well plates at a density of 200,000 cells/well,
with 0.5 μM and 1 μM of K5 for 24 h. Then, cells were harvested, wa-
shed twice with cold PBS, and subsequently fixed in cold 70% ethanol
overnight at 4 °C. Samples were washed twice with PBS, followed by
further treatment with 50 mL of 100 μg/mL ribonuclease and 100 μL of
100 μg/mL PI, and finally analyzed by flow cytometry. The result was
determined using the NovoExpress software.
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Acknowledgement
This work was supported by the key research and development
project of Shandong Province (No. 2017CXGC1401).
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Appendix A. Supplementary data
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Synthesis of novel class of N-alkyl-isatin-3-iminobenzoic acid derivatives and their
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Supplementary data to this article can be found online at https://
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