3-Hydroxyisoindolin-1-one derivates: Synthesis by palladium-catalyzed C–H activation as BRD4 inhibitors against human acute myeloid leukemia (AML) cells

Article abstract of DOI:10.1016/j.bioorg.2019.01.034

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family, which plays a key role in transcriptional regulation. Recent biological and pharmacological studies have enabled linking of the BET bromodomains wi

Full text of DOI:10.1016/j.bioorg.2019.01.034

BioorganicChemistry86(2019)119–125
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Bioorganic Chemistry
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3-Hydroxyisoindolin-1-one derivates: Synthesis by palladium-catalyzed
CeH activation as BRD4 inhibitors against human acute myeloid leukemia
(AML) cells
⁎
Pan Chena,1, Yifei Yanga,1, Lingyun Yangb, Jiping Tianb, Fangqing Zhangb, Jinpei Zhoua,
,
⁎
Huibin Zhangb,
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein
family, which plays a key role in transcriptional regulation. Recent biological and pharmacological studies have
enabled linking of the BET bromodomains with diseases, including inflammation and cancer, suggesting that
bromodomains are druggable targets. In this study, we made further structural modifications of our previously
reported BRD4 inhibitors, to develop new chemical scaffold 3-Hydroxyisoindolin-1-One. Then a series of com-
pounds (10a-q) were synthesized via palladium-catalyzed CeH activation and BRD4-inhibitory activities and
anti-proliferative effects of these compounds were evaluated. Compound 10e exhibited excellent BRD4-in-
hibitory activity with IC50 value of 80 nM and anti-proliferation potency with IC50 value of 365 nM in HL-60
(human promyelocytic leukemia) cancer cell lines. We have demonstrated compound 10e modulated the in-
trinsic apoptotic pathway. In conclusion, these results suggested that compound 10e could be utilized as a BRD4
inhibitor for further leukemia treatment.
BRD4 inhibitors
Molecular docking
CeH activation
Anticancer
1. Introduction
BRD4 bromodomain protein has developed to be an interesting drug
target for the treatment of cancer [1,15,16], obesity [17], kidney dis-
Epigenetic marks on histones are associated with transcriptional
processes [1]. Proteins of this target class are classified into readers,
writers, and erasers of marks on histones or other nuclear proteins and
DNA [2,3]. Context-specific molecular recognition of lysine residues
(Kac) on histone tails is principally mediated by bromodomains [4].
The bromodomain and extra-terminal domain (BET) family proteins
[5], including BRD2, BRD3, BRD4 and BRDT, shares a common domain
architecture featuring two amino-terminal bromodomains, which in-
teract with acetylated histones [6–8]. Bromodomain protein 4 (BRD4)
is the best studied member of the BET family and is a critical mediator
of transcriptional elongation by recruiting the positive transcription
elongation factor complex (P-TEFb) [1]. Through its direct interaction
with the acetylated cyclin T1 subunit of the positive transcription
elongation factor b, BRD4 furthermore controls transcription elonga-
tion via phosphorylation of RNA polymerase II by CDK9 [9–12]. BRD4
also binds to acetylated RelA, thus stabilizing nuclear NF-B and con-
trolling the expression of downstream target genes [13,14]. Above all,
ease [13], lung fibrosis [18] and other inflammatory diseases [19].
The potential of targeting BET family proteins for cancer therapy
has been substantiated using small-molecule inhibitors, such as JQ1,
Abbv-075, I-BET151 and OTX015 etc. (Fig. 1). JQ1 was the first un-
covered BRD4 inhibitor [7] and I-BET762 was acquired from optimi-
zation of a hit which was designed to enhance ApoA1 expression [20].
Gratifyingly, I-BET762 has entered clinical trials for NUT midline car-
cinoma and other cancers. OTX015 has completed Phase I trials for
hematologic malignancies and NUT midline carcinoma, and the results
exhibited clinically meaningful activity with nontoxic doses [21–23].
ABBV-075 is in phase I clinical trials for the treatment of patients with
solid tumors, acute myeloid leukemia or multiple myeloma on the basis
of the strong biochemical potency for BRD4 (Ki = 15 nM) [24–27].
Upon the basis of the previously disclosed BRD4 inhibitors, we
herein report a rapid synthesis of 3-Hydroxyisoindolin-1-One derivates
by a Palladium-Catalyzed CeH Activation strategy, which enabled the
investigation of their SAR, and the biological activities of BRD4
⁎ Corresponding authors.
E-mail addresses: jpzhou668@163.com (J. Zhou), zhanghb80@163.com (H. Zhang).
1 These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2019.01.034
Received 2 September 2018; Received in revised form 6 January 2019; Accepted 21 January 2019
Availableonline22January2019
0045-2068/©2019PublishedbyElsevierInc.

P. Chen et al.
BioorganicChemistry86(2019)119–125
Fig. 1. Structures of known BET bromodomain inhibitors.
inhibitors for further leukemia treatment.
2. Results and discussion
2.1. Chemistry
The general synthetic routes of 3-hydroxyisoindolin-1-one derivates
(3a-3p) were outlined in Scheme 1. All the target products and key
intermediates were confirmed by 1H NMR and 13C NMR spectrum, mass
spectra. Purifications by column chromatography were carried out over
silica gel (200–300 mesh) and purity was checked by using HPLC before
being tested in biological evaluation (purity was > 98%).
As shown in Scheme1, the target compounds 10a-p were prepared
from the commercially starting material 4-bromobenzoic acid (7). Then
4-bromobenzoic acid was treated with MeONH2·HCl or BnONH2·HCl by
acylation to obtain the intermediates 8a-b. As recently reported, a
palladiumcatalyzed oxidative acylation was employed as an efficient
synthesis for hydroxyl isoindolones [28,29]. Therefore, the key inter-
mediates 9a-p could be synthesized via palladium-catalyzed CeH ac-
tivation (Fig. 2) avoiding the tedious multistep processes. A series of
different substituted benzaldehydes were employed under mild reaction
conditions via CeH activation to get 3-hydroxyisoindolin-1-one deri-
vates 9a-p. The target 10a-p were obtained by Suzuki coupling reaction
with 3, 5-dimethylisoxazole-4-boronic acid pinacol ester.
Fig. 2. Synthesis of 3-hydroxyisoindolin-1-one via CeH activation.
2.2. Palladium-Catalyzed CeH activation
Phthalimides were used as starting materials for the synthesis of 3-
hydroxyisoindolin-1-ones in traditional methods. The Grignard reaction
of the carbonyl groups of phthalimides [30] or the photochemical
Scheme 1. Reagents and conditions: (a) (1) ClCOCOCl, DMF, DCM, 0 ˚C to room temperature;(2) MeONH2·HCl or BnONH2·HCl, K2CO3, EtOAc, H2O, 0 ˚C to r.t; (b)
benzaldehyde, Pd(OAc)2, TBHP, Dioxane, 80 ˚C; (c) phenylboronic acids, Pd(P(Ph)3)4, Na2CO3, N2, H2O/EtOH/Toluene, 80 ˚C.
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P. Chen et al.
BioorganicChemistry86(2019)119–125
Table 1
Structures and BRD4 inhibitory effects of compounds 10a-p.
a
Compound No.
R1
R2
BRD4(1) IC50 (μmol/L)
BRD4(2) IC50 (μmol/L)
10a
10b
10c
10d
10e
10f
2,4-di-F-phenyl
4-Me-phenyl
3-F-phenyl
4-F-phenyl
phenyl
Me
0.224
0.245
0.090
0.077
0.080
2.560
0.300
2.100
0.330
0.532
0.780
1.500
0.390
1.800
1.680
0.320
0.078
0.012
0.014
0.012
0.018
0.011
0.023
0.015
0.022
0.014
0.012
0.019
0.021
0.016
0.022
0.025
0.012
0.009
4.260
4.580
2.140
1.700
0.290
NAb
0.024
0.028
0.014
0.027
0.011
Me
Me
Me
Me
3-CF3-phenyl
2- F-phenyl
2-Cl-phenyl
3-Cl-phenyl
3-F-phenyl
2-F-phenyl
3-CN- phenyl
4-F-phenyl
4-Me-phenyl
3-OCH3- phenyl
phenyl
Me
10g
10h
10i
Me
1.300
NA
0.018
0.024
Me
Me
1.400
NA
10j
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
–
10k
10l
NA
NA
10m
10n
10o
10p
JQ1c
1.560
NA
0.023
NA
1.240
0.093
0.026
0.012
–
a
b
c
IC50 values for BRD4-BD1 activities presented are the mean
Mean of the BRD4 inhibitory activity IC50 > 5 μM.
Employed as positive control.
SD values of three independent determinations
reaction of phthalimides to afford 3-benzyl-3-hydroxyisoindol-1-ones
commonly require harsh reaction conditions and complicated reagents.
Kim et al. first reported secondary benzamides and aryl aldehydes via
the rhodium-catalyzed CeH bond activation with an intramolecular
cyclization [31]. Then, palladiumcatalyzed oxidative acylation was
demonstrated as an efficient synthesis for hydroxyl isoindolones
(Fig. 2). Recently, Yang et al. disclosed the synthesis of 3-hydro-
xyisoindolin-1-ones through the palladium-catalyzed CeH bond ortho
acylation/annulation with toluene derivatives [32].
BRD4BD1 protein (PDB id: 3P5O) via Glide docking protocol of Maestro
10.2 and overlapped with compound 5i (Fig. 3). The docking con-
formations showed the isoxazole group of compound 10e behaving as
the KAc mimetic. The amine formed hydrogen bonds to Asn140 and a
through-water interaction to Tyr97. The phenyl group is placed on the
WPF stack (W81, P82, and F83) in a similar manner to compound 5i.
This result showed that 3-hydroxyisoindolin-1-one module was feasible
as a core of BRD4 inhibitors.
Then various substituents phenyl were introduced to explore
structure-activity relationship for the improvement of binding potency
and druggable profile (Table 1). Initially, it was pleasing to observe
increase selectivity in BD1 potency with the addition of single F sub-
stituent to the shelf group (10c, d vs 10e) without significant increase
in BD1 potency but decreasing BD2 potency. The di-F-phenyl group
(10a) caused both decrease in BD1 and BD2 potency. The compound
10b displayed similar potency and selectivity as 10a. Disappointingly,
compound 10f showed poor selectivity and reduced potency, indicating
that strong electron-withdrawing group changes the electronic cloud
density in phenyl leading to evident decrease in potency. The m-posi-
tion and p-position substituents showed higher potency in BRD4BD1
than o-position (compound 10c, d vs 10 g). Compound 10j-p con-
taining benzyl replacement with methoxy group at the 2-position
2.3. Biological activity and SAR study
In our previous research, dihydroquinoxalinone compound 5i ex-
hibited IC50 value of 73 nM of binding activity in BRD4 (1) and 258 nM
of cellular activity in MV4-11 cancer cell lines. Rational scaffold hop-
ping was employed to increase the type of BRD4 inhibitors and the
protein binding potency. Then, a series of compounds (10a-q) con-
taining 3-hydroxyisoindolin-1-one skeleton were designed and synthe-
sized for biological and pharmacodynamics evaluation (Table 1). The
initially dihydroquinoxalinone core of compound 5i was replaced by 3-
hydroxyisoindolin-1-one to obtain compound 10e with binding potency
(IC50 = 80 nM). Subsequently, compound 10e was docked into
Fig. 3. (A) Docking conformation of compound 5i in BRD4 protein (PDB id: 3P5O). (B) Docking conformation of compound 10e in BRD4 protein (PDB id: 3P5O). (C)
Superimposition docking conformation of compound 5i (Pink) and compound 10e (Blue) in BRD4 protein (PDB id: 3P5O).
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P. Chen et al.
BioorganicChemistry86(2019)119–125
Fig. 4. (A) Docking conformation of compound 10p in BRD4 protein (PDB id: 3P5O). (B) Superimposition docking conformation of compound 5i (Pink) and
compound 10p (Yellow) in BRD4 protein (PDB id: 3P5O). (C) Superimposition docking conformation of compound 10p (Yellow) and compound 10e (Blue) in BRD4
protein (PDB id: 3P5O).
exhibited poor selectivity and reduced potency, suggesting that the 2-
position toward a small lipophilic pocket, as occupied by the methoxy
group of 10e (Fig. 3). The docking conformation of compound 10p
demonstrated that the small pocket was not taken up by big benzyl
substituent (Fig. 4).
Fig. 5. BRD4 inhibitor compound 10e induces apoptosis in HL-60 cancer cells.
2.4. Anti-proliferative activities across cancer cell lines
10e induced apoptosis via the mitochondrial pathways in leukemia
Subsequently, the compounds (10a-e, 10 g, 10i, 10 m and 10p)
with excellent BRD4 inhibitory potency were further evaluated in
LNcap-1(human prostatic cells), MV4-11(Human acute monocytic leu-
kemia cells) and HL-60 (human promyelocytic leukemia) for their anti-
proliferation activities (Table 2). Most compounds exhibited favorable
anti-proliferation activities in HL-60 and MV4-11 cell lines. Gratify-
ingly, compound 10e showed remarkable biological activities with
IC50 value of 365 nM in HL-60 and 420 nM in MV4-11 cell lines. These
results indicated that compound 10e was worthy for further in-
vestigation in leukemia therapy. In addition, activities of compounds on
BRD4-independent LNcap-1 cells was weaker than on BRD4-sensitive
lines and this result is consistent with our previous reports [24].
cells.
3. Conclusion
In summary, we report our structural modification chemical
synthesis and evaluation of a new series of BRD4 inhibitors. Upon the
basis of the previously disclosed BRD4 inhibitors, a novel chemical
scaffold 3-hydroxyisoindolin-1-one was synthesized by Palladium-
Catalyzed CeH Activation. The BRD4-inhibitory activities and anti-
proliferative effects of these compounds were evaluated. Meanwhile,
we found compound 10e has remarkable anti-proliferative activities in
HL-60 cancer cells and could induce apoptosis via mitochondrial
pathways. All of these results demonstrated compound 10e could be
utilized as a BRD4 inhibitor for further investigation in leukemia
therapy.
2.5. Induce apoptosis via the mitochondrial pathways in leukemia cells
Western blot was employed to acquire more insight on how com-
pound 10e trigger apoptosis in HL-60 cancer cells. Then varied con-
centrations of compound 10e were utilized to evaluate the expression
of several key proteins in intrinsic apoptotic pathway (Fig. 5). These
results showed that compound 10e downregulated c-Myc at 24 h,
consistent with JQ1. Early apoptotic events such as caspase activation
(caspase 3) and PARP cleavage were already apparent at the low con-
centration of compound 10e (Fig. 5). These data suggest that compound
4. Experiment section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sinopharm
Chemical Reagent Co., Ltd. (China) and treated with standard methods.
Melting points were measured on capillary tube and were uncorrected.
1H and 13C NMR spectra (DMSO-d6) were measured on Bruker AV-300
spectrometer at 25 ˚C and referenced to TMS. Chemical shifts were
reported in ppm (d) using the residual solvent line as internal standard.
High-resolution mass spectra were recorded using an Agilent QTOF
6520 (Beijing, China)
Table 2
Anti-proliferation evaluation against cancer cell lines LNcap-1, HL-60 and MV4-
11.
a
Compound No.
LNcap-1 IC50 (μM)
HL-60 IC50 (μM)
MV4-11 IC50 (μM)
10a
10b
10c
10d
10e
10g
10i
6.850
NA
0.023
6.537
7.340
1.223
NA
0.021
0.016
0.013
5.970
5.134
0.895
0.021
0.022
0.012
4.1.1. General procedure for the preparation of 8a-b
NA
To a solution of the 4-bromobenzoic acid (2 g, 9.95 mmol) and DMF
(cat.) in DCM (20 mL), oxalyl chloride (1.27 mL, 1.89 mmol) was added
dropwise at 0 °C and stirred at room temperature for another 3 h. The
solution was concentrated under reduced pressure to give yellow oil.
Then the residue was quenched with EtOAc (30 mL) and K2CO3 (2.75 g,
19.9 mmol), MeONH2·HCl or BnOBH2·HCl (11.94 mmol), water (15 mL)
were added subsequently. The reaction mixture was stirred at room
temperature until the reaction was completed. The organic layer was
separated and washed with saturated sodium bicarbonate solution
(3 × 20 mL) and brine (20 mL) and the organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure to give 8a-
b
NA
NA
NA
0.365
1.399
NA
0.018
0.019
0.420
1.250
6.530
NA
0.011
0.014
0.024
NA
NA
10m
10p
NA
NA
NA
2.983
0.686
0.022
0.013
2.460
1.380
0.026
0.023
c
JQ1
4.720
0.024
a
IC50 values for BRD4-BD1 activities presented are the mean
three independent determinations
SD values of
b
c
Mean of the BRD4 inhibitory activity IC50 > 5 μM.
Employed as positive control.
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P. Chen et al.
BioorganicChemistry86(2019)119–125
b without further purification.
J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 5.12 (d, J = 10.0 Hz, 1H),
4.99 (d, J = 10.0 Hz, 1H), 2.27 (s, 3H). MS (ESI, m/z): 424.3[M+H]+
.
4.1.1.1. 4-Bromo-N-methoxybenzamide (8a). White solid, yield 78%. 1H
NMR (300 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.02 – 7.32 (m, 4H), 3.71 (s,
4.1.2.10. 2-(Benzyloxy)-5-bromo-3-hydroxy-3-phenylisoindolin-1-one
3H). MS (ESI, m/z): 230.3[M+H]+
.
(9p). White solid, yield 79%. 1H NMR (300 MHz, DMSO-d6) δ 7.83 –
7.68 (m, 3H), 7.55 (d, J = 1.0 Hz, 1H), 7.46 – 7.28 (m, 10H), 5.17 –
4.1.1.2. N-Benzyl-O-(4-bromobenzoyl)hydroxylamine (8b). White solid,
yield 89%. 1H NMR (300 MHz, DMSO-d6) δ 7.75 – 7.64 (m, 4H), 7.39
(ddd, J = 22.2, 14.0, 7.4 Hz, 6H), 4.93 (s, 2H). MS (ESI, m/z): 306.3[M
5.09 (m, 1H), 4.99 (d, J = 10.0 Hz, 1H). MS (ESI, m/z): 411.1[M+H]+
4.1.3. General procedure for the preparation of 10a-p
.
+H]+
.
A mixture of 9a-p (0.36 mmol), 3,5-dimethyl-4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl) isoxazole (0.08 g, 0.36 mmol), sodium
carbonate (0.14 g, 1.08 mmol), Tetrakis (triphenylphosphine) palla-
dium (0.04 g, 0.036 mmol) in a mixed solvent of water (3 mL), ethanol
(1 mL) and toluene (3 mL) was refluxed under N2 for 12 h. After the
reaction was completed, the mixture was diluted with water (20 mL)
and filtered with diatomite. Then the filtrate was extracted with ethyl
acetate (10 mL × 3). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure to give
brown oil. The resulting crude oil was purified by silica gel column
chromatography (petroleum ether/ethyl acetate, 3:1) to afford target
product 10a-p.
4.1.2. General procedure for the preparation of 9a-p
To a solution of compound 8a, b (0.87 mmol), varied substituent
benzaldehyde (1.3 mmol) in toluene, Pd(OAc)2 (19.52 mg, 0.08 mmol)
and TBHP (1 mL, 10.43 mmol) were added respectively in sealed tube.
Then the reaction mixture was stirred at 80 °C under air for 12 h. After
cooling to the room temperature, the reaction mixture was diluted with
saturated sodium bicarbonate solution (10 mL) and extracted with
EtOAc (3 × 15 mL). The combined organic layers were washed with
and brine (30 mL), dried over Na2SO4 and concentrated. The residue
was purified by column chromatography (petroleum ether/ethyl
acetate, 5/1 to 3/1) to give 9a-p.
4.1.3.1. 3-(2,
4-Difluorophenyl)-5-(3,
5-dimethylisoxazol-4-yl)-3-
4.1.2.1. 5-Bromo-3-(2, 4-difluorophenyl)-3-hydroxy-2-methoxyisoindolin-
1-one (9a). Offwhite solid, yield 81%. 1H NMR (300 MHz, DMSO-d6) δ
8.13 – 7.99 (m, 1H), 7.90 (s, 1H), 7.76 (ddd, J = 20.6, 11.0, 4.7 Hz,
2H), 7.52 (d, J = 1.2 Hz, 1H), 7.27 – 7.06 (m, 2H), 3.81 (d, J = 1.2 Hz,
hydroxy-2-methoxyisoindolin-1-one (10a). White solid, yield 78%.
m.p.: 128–130 °C; 1H NMR (300 MHz, DMSO-d6)
δ 8.05 (d,
J = 6.9 Hz, 1H), 7.93 – 7.76 (m, 2H), 7.58 (d, J = 7.7 Hz, 1H), 7.29
(s, 1H), 7.14 (dd, J = 20.7, 8.7 Hz, 2H), 3.82 (s, 3H), 2.35 (s, 3H), 2.16
(s, 3H). 13C NMR (75 MHz, DMSO) δ 166.38, 162.96, 158.28, 146.41,
135.40, 131.55, 131.40, 130.80, 127.93, 123.63, 123.05, 115.77,
111.95, 111.71, 105.13, 104.79, 104.44, 87.80, 65.48, 40.89, 40.62,
40.35, 40.07, 39.80, 39.52, 39.24, 11.81, 10.80. MS (ESI, m/z):
387.4[M+H]+; Anal. calcd. for C20H16F2N2O4: C, 62.18; H, 4.17; N,
7.25. Found: C, 62.48; H, 4.21; N, 7.38.
3H). MS (ESI, m/z): 370.4[M+H]+
.
4.1.2.2. 5-Bromo-3-hydroxy-2-methoxy-3-(p-tolyl)isoindolin-1-one
(9b). White solid, yield 73%. 1H NMR (300 MHz, DMSO-d6) δ 7.77 –
7.66 (m, 2H), 7.60 (s, 1H), 7.47 (d, J = 1.5 Hz, 1H), 7.20 (dd, J = 20.8,
8.2 Hz, 4H), 3.82 (s, 3H), 2.28 (s, 3H). MS (ESI, m/z): 348.4[M+H]+
.
4.1.2.3. 5-Bromo-3-(3-fluorophenyl)-3-hydroxy-2-methoxyisoindolin-1-
4.1.3.2. 5-(3, 5-Dimethylisoxazol-4-yl)-3-hydroxy-2-methoxy-3-(p-tolyl)
isoindolin-1-one (10b). Yellow solid, yield 83%. m.p.: 133–135 °C; 1H
NMR (300 MHz, DMSO-d6) δ 7.83 (d, J = 7.8 Hz, 1H), 7.61 – 7.50 (m,
2H), 7.34 – 7.21 (m, 3H), 7.16 (d, J = 8.3 Hz, 2H), 3.85 (s, 3H), 2.36 (s,
3H), 2.27 (s, 3H), 2.18 (s, 3H). 13C NMR (75 MHz, DMSO) δ 166.38,
162.62, 158.30, 147.86, 138.03, 136.50, 135.32, 130.48, 129.36,
127.35, 126.49, 123.67, 123.37, 115.82, 90.83, 65.34, 40.89, 40.63,
40.35, 40.07, 39.79, 39.51, 39.24, 21.09, 11.82, 10.83. MS (ESI, m/z):
365.2[M+H]+; Anal. calcd. for C21H20N2O4: C, 69.22; H, 5.53; N, 7.69.
Found: C, 69.48; H, 5.51; N, 7.71.
one (9c). White solid, yield 68%. MS (ESI, m/z): 352.1[M+H]+
.
4.1.2.4. 5-Bromo-3-(4-fluorophenyl)-3-hydroxy-2-methoxyisoindolin-1-
one (9d). Yellow solid, yield 79%.1H NMR (300 MHz, DMSO-d6) δ 7.81
– 7.66 (m, 3H), 7.54 (s, 1H), 7.46 – 7.34 (m, 2H), 7.20 (t, J = 8.6 Hz,
2H), 3.83 (s, 3H). MS (ESI, m/z): 352.3[M+H]+
.
4.1.2.5. 5-Bromo-3-hydroxy-2-methoxy-3-phenylisoindolin-1-one
(9e). Yellow solid, yield 73%. 1H NMR (300 MHz, DMSO-d6) δ 7.82 –
7.59 (m, 3H), 7.52 (s, 1H), 7.37 (s, 5H), 3.83 (d, J = 4.3 Hz, 3H). MS
(ESI, m/z): 334.3[M+H]+
.
4.1.3.3. 5-(3,5-Dimethylisoxazol-4-yl)-3-(3-fluorophenyl)-3-hydroxy-2-
methoxyisoindolin-1-one (10c). Yellow solid, yield 91%. m.p.:
147–149 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.90 – 7.75 (m, 2H),
7.58 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 10.4 Hz,
1H), 7.21 – 7.05 (m, 2H), 3.88 (s, 3H), 2.36 (s, 3H), 2.18 (s, 3H). 13C
NMR (75 MHz, DMSO) δ 166.45, 164.25, 162.68, 158.33, 147.17,
142.53, 142.44, 135.57, 130.87, 130.79, 127.24, 123.84, 123.48,
122.54, 115.78, 113.91, 113.59, 90.30, 65.53, 40.91, 40.63, 40.35,
40.07, 39.79, 39.51, 39.23, 11.81, 10.80. MS (ESI, m/z): 369.2[M
+H]+; Anal. calcd. for C20H17FN2O4: C, 65.21; H, 6.65; N, 7.60. Found:
C, 65.24; H, 6.51; N, 7.83.
4.1.2.6. 5-Bromo-3-hydroxy-2-methoxy-3-(3-(trifluoromethyl)phenyl)
isoindolin-1-one (9f). White solid, yield 78%. 1H NMR (300 MHz,
DMSO-d6) δ 7.94 (s, 1H), 7.85 – 7.69 (m, 4H), 7.58 (d, J = 24.3 Hz,
3H), 3.86 (d, J = 3.3 Hz, 3H). MS (ESI, m/z): 402.1[M+H]+
.
4.1.2.7. 5-Bromo-3-(2-fluorophenyl)-3-hydroxy-2-methoxyisoindolin-1-
one (9 g). White solid, yield 84%. 1H NMR (300 MHz, DMSO-d6) δ 8.02
(td, J = 8.1, 1.8 Hz, 1H), 7.83 – 7.68 (m, 3H), 7.51 – 7.29 (m, 3H), 7.11
– 7.01 (m, 1H), 3.81 (s, 3H). MS (ESI, m/z): 352.1[M+H]+
.
4.1.2.8. 5-Bromo-3-(3-chlorophenyl)-3-hydroxy-2-methoxyisoindolin-1-
one (9i). White solid, yield 88%. 1H NMR (300 MHz, CDCl3) δ 7.63 –
7.53 (m, 3H), 7.43 (d, J = 1.0 Hz, 1H), 7.38 – 7.23 (m, 3H), 5.26 (s,
4.1.3.4. 5-(3,5-Dimethylisoxazol-4-yl)-3-(4-fluorophenyl)-3-hydroxy-2-
methoxyisoindolin-1-one (10d). Yellow solid, yield 81%. m.p.:
143–145 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J = 7.8 Hz, 1H),
7.65 (d, J = 6.0 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.44 (dd, J = 8.7,
5.5 Hz, 2H), 7.34 (s, 1H), 7.19 (t, J = 8.9 Hz, 2H), 3.88 (s, 3H), 2.37 (s,
3H), 2.19 (s, 3H). 13C NMR (75 MHz, DMSO) δ 166.42, 162.58, 158.31,
147.49, 135.66, 135.50, 130.67, 128.90, 128.78, 127.28, 123.79,
123.43, 115.79, 115.50, 90.45, 65.47, 40.90, 40.62, 40.34, 40.06,
1H), 3.87 (d, J = 13.8 Hz, 3H). MS (ESI, m/z): 368.2[M+H]+
.
4.1.2.9. 2-(Benzyloxy)-5-bromo-3-hydroxy-3-(p-tolyl)isoindolin-1-one
(9n). White solid, yield 81%. 1H NMR (300 MHz, DMSO-d6) δ 7.80 –
7.67 (m, 3H), 7.52 (d, J = 1.2 Hz, 1H), 7.41 – 7.31 (m, 5H), 7.28 (d,
123

P. Chen et al.
BioorganicChemistry86(2019)119–125
39.78, 39.50, 39.23, 11.81, 10.81. MS (ESI, m/z): 369.2[M+H]+; Anal.
calcd. for C20H17FN2O4: C, 65.21; H, 6.65; N, 7.60. Found: C, 65.14; H,
6.61; N, 7.63.
2.18 (s, 3H). 13C NMR (75 MHz, DMSO) δ 166.47, 162.95, 158.34,
147.20, 142.58, 142.48, 135.67, 135.63, 130.98, 130.87, 129.47,
129.02, 128.76, 127.31, 123.89, 123.59, 122.62, 115.80, 113.99,
113.68, 90.50, 79.61, 40.91, 40.63, 40.35, 40.07, 39.79, 39.52,
39.24, 11.82, 10.81. MS (ESI, m/z): 445.4[M+H]+; Anal. calcd. for
C26H21FN2O4: C, 70.26; H, 4.76; N, 6.30. Found: C, 70.31; H, 4.81; N,
6.24.
4.1.3.5. 5-(3, 5-Dimethylisoxazol-4-yl)-3-hydroxy-2-methoxy-3-phenyli-
soindolin-1-one (10e). Yellow solid, yield 71%. m.p.: 139–141 °C; 1H
NMR (300 MHz, DMSO-d6) δ 7.84 (d, J = 7.8 Hz, 1H), 7.66 (s, 1H), 7.56
(d, J = 7.7 Hz, 1H), 7.49 – 7.24 (m, 6H), 3.86 (s, 3H), 2.35 (s, 3H), 2.17
(s, 3H). 13C NMR (75 MHz, DMSO) δ 166.40, 162.65, 158.30, 147.73,
139.46, 135.38, 130.56, 128.83, 128.73, 127.34, 126.54, 123.72,
123.42, 115.81, 90.85, 65.40, 40.91, 40.63, 40.35, 40.08, 39.80,
39.52, 39.24, 11.80, 10.81. MS (ESI, m/z): 351.2[M+H]+; Anal.
calcd. for C20H18N2O4: C, 68.56; H, 5.18; N, 8.00. Found: C, 65.44;
H, 5.31; N, 7.93.
2-(Benzyloxy)-5-(3,5-dimethylisoxazol-4-yl)-3-(2-fluorophenyl)-3-
hydroxyisoindolin-1-one (10 k)
Yellow solid, yield 79%. m.p.: 146–148 °C; 1H NMR (300 MHz,
DMSO-d6) δ 8.09 (s, 1H), 7.84 (s, 2H), 7.59 (s, 1H), 7.34 (s, 8H), 7.05 (s,
1H), 5.23 – 4.90 (m, 2H), 2.37 (s, 3H), 2.19 (s, 3H). 13C NMR (75 MHz,
DMSO) δ 165.89, 162.87, 157.80, 146.20, 135.15, 134.81, 131.05,
131.01, 130.95, 130.93, 130.21, 129.53, 128.89, 128.47, 128.21,
127.53, 124.39, 123.14, 122.59, 115.92, 115.63, 115.27, 87.85, 78.92,
40.32, 40.04, 39.77, 39.49, 39.21, 38.93, 38.65, 11.32, 10.33. MS (ESI,
m/z): 445.5[M+H]+; Anal. calcd. for C26H21FN2O4: C, 70.26; H, 4.76;
N, 6.30. Found: C, 70.21; H, 4.74; N, 6.33.
4.1.3.6. 5-(3,5-Dimethylisoxazol-4-yl)-3-hydroxy-2-methoxy-3-(3-
(trifluoromethyl)phenyl)isoindolin −1-one (10f). White solid, yield 78%.
m.p.: 143–145 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.92 (s, 1H), 7.88 (d,
J = 7.6 Hz, 1H), 7.82 (s, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.62 – 7.56 (m,
3H), 7.42 (s, 1H), 3.89 (s, 3H), 2.36 (s, 3H), 2.17 (s, 3H). 13C NMR
(75 MHz, DMSO) δ 166.47, 162.69, 158.33, 146.90, 143.63, 141.04,
135.71, 134.32, 132.40, 130.97, 130.77, 130.23, 127.23, 123.95,
123.57, 123.09, 115.76, 90.31, 65.64, 40.92, 40.64, 40.36, 40.08,
39.80, 39.52, 39.24, 11.78, 10.76. MS (ESI, m/z): 419.2[M+H]+; Anal.
calcd. for C21H17F3N2O4: C, 60.29; H, 4.10; N, 6.70. Found: C, 60.24; H,
4.21; N, 6.73.
4.1.3.11. 3-(2-(Benzyloxy)-6-(3,5-dimethylisoxazol-4-yl)-1-hydroxy-3-
oxoisoindolin-1-yl)benzonitrile (10 l). Yellow solid, yield 81%. m.p.:
151–152 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.98 (d, J = 8.5 Hz, 2H),
7.89 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.0 Hz, 1H), 7.59 (dd, J = 11.7,
9.2 Hz, 3H), 7.46 (s, 1H), 7.43 – 7.32 (m, 5H), 5.12 (dd, J = 31.6,
10.1 Hz, 2H), 2.38 (s, 3H), 2.20 (s, 3H). 13C NMR (75 MHz, DMSO) δ
170.02, 166.02, 162.52, 146.27, 140.67, 135.24, 135.06, 132.27,
132.02, 131.99, 131.51, 131.38, 130.91, 130.50, 130.06, 129.77,
129.02, 128.80, 128.64, 128.58, 128.27, 126.73, 123.51, 123.20,
79.24, 40.31, 39.76, 39.47, 39.20, 38.92, 38.63, 11.34, 10.33. MS
(ESI, m/z): 452.5[M+H]+; Anal. calcd. for C27H21N3O4: C, 71.83; H,
4.69; N, 9.31. Found: C, 71.23; H, 4.63; N, 9.35.
4.1.3.7. 5-(3,5-Dimethylisoxazol-4-yl)-3-(2-fluorophenyl)-3-hydroxy-2-
methoxyisoindolin-1-one (10 g). White solid, yield 88%. m.p.:
149–151 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (t, J = 8.0 Hz, 1H),
7.84 (d, J = 7.5 Hz, 1H), 7.76 (s, 1H), 7.60 – 7.55 (m, 1H), 7.42 (d,
J = 5.5 Hz, 1H), 7.35 – 7.25 (m, 2H), 7.05 (dd, J = 11.8, 8.5 Hz, 1H),
3.83 (s, 3H), 2.35 (s, 3H), 2.17 (s, 3H). 13C NMR (75 MHz, DMSO) δ
166.35, 158.27, 146.66, 135.27, 131.37, 130.69, 130.06, 128.06,
126.06, 125.93, 124.86, 123.58, 123.02, 116.39, 116.09, 115.79,
88.04, 65.40, 40.91, 40.63, 40.35, 40.08, 39.80, 39.52, 39.24, 11.80,
10.80. MS (ESI, m/z): 369.4[M+H]+; Anal. calcd. for C20H17FN2O4: C,
65.21; H, 4.65; N, 7.60. Found: C, 65.27; H, 4.61; N, 7.57.
4.1.3.12. 2-(Benzyloxy)-5-(3,5-dimethylisoxazol-4-yl)-3-(4-
fluorophenyl)-3-hydroxyisoindolin-1-one (10 m). Yellow solid, yield
79%. m.p.: 149–151 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.87 (d,
J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.50 – 7.32
(m, 8H), 7.19 (t, J = 8.8 Hz, 2H), 5.10 (dd, J = 38.5, 10.1 Hz, 2H), 2.38
(s, 3H), 2.20 (s, 3H). 13C NMR (75 MHz, DMSO) δ 166.45, 162.84,
160.83, 158.33, 147.52, 135.68, 135.55, 130.74, 129.46, 129.00,
128.97, 128.85, 128.75, 127.33, 123.83, 123.53, 115.81, 115.51,
90.65, 79.55, 40.91, 40.63, 40.35, 40.08, 39.80, 39.52, 39.24, 11.83,
10.83. MS (ESI, m/z): 445.5[M+H]+; Anal. calcd. for C26H21FN2O4: C,
70.26; H, 4.76; N, 6.30. Found: C, 70.23; H, 4.73; N, 6.31.
4.1.3.8. 3-(2-Chlorophenyl)-5-(3,5-dimethylisoxazol-4-yl)-3-hydroxy-2-
methoxyisoindolin-1-one (10 h). White solid, yield 84%. m.p.:
148–150 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.28 (d, J = 8.3 Hz, 1H),
7.83 (d, J = 7.6 Hz, 1H), 7.69 (s, 1H), 7.61 – 7.30 (m, 4H), 7.13 (s, 1H),
3.76 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). 13C NMR (75 MHz, DMSO) δ
166.27, 162.99, 158.27, 146.12, 135.09, 134.94, 131.31, 131.13,
131.08, 130.73, 129.32, 127.73, 123.55, 122.96, 115.84, 88.67,
64.94, 40.81, 40.53, 40.26, 39.98, 39.70, 39.42, 39.14, 11.75, 10.78.
MS (ESI, m/z): 385.3[M+H]+; Anal. calcd. for C20H17ClN2O4: C, 62.42;
H, 4.45; N, 7.28. Found: C, 62.41; H, 4.41; N, 7.27.
2-(Benzyloxy)-5-(3,5-dimethylisoxazol-4-yl)-3-hydroxy-3-(p-tolyl)
isoindolin-1-one (10n)
White solid, yield 78%. m.p.: 149–151 °C; 1H NMR (300 MHz,
DMSO-d6) δ 7.85 (d, J = 7.7 Hz, 1H), 7.64 (s, 1H), 7.58 (dd, J = 7.8,
1.4 Hz, 1H), 7.46 – 7.27 (m, 8H), 7.16 (d, J = 8.0 Hz, 2H), 2.37 (s, 3H),
2.26 (s, 3H), 2.19 (s, 3H). 13C NMR (75 MHz, DMSO) δ 165.92, 162.33,
157.82, 147.37, 137.56, 136.04, 135.21, 134.87, 131.99, 131.51,
131.38, 130.04, 129.00, 128.89, 128.79, 128.63, 128.49, 128.24,
126.87, 126.05, 123.25, 122.97, 115.32, 90.55, 78.97, 40.32, 40.03,
39.76, 39.48, 39.21, 38.93, 38.66, 20.60, 11.33, 10.36. MS (ESI, m/z):
441.3[M+H]+; Anal. calcd. for C27H24N2O4: C, 73.62; H, 5.49; N, 6.36.
Found: C, 73.53; H, 5.51; N, 6.32.
4.1.3.9. 3-(3-Chlorophenyl)-5-(3,5-dimethylisoxazol-4-yl)-3-hydroxy-2-
methoxyisoindolin-1-one (10i). White solid, yield 78%. m.p.:
151–153 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.93 – 7.75 (m, 2H),
7.52 (dd, J = 41.3, 17.7 Hz, 5H), 7.25 (s, 1H), 3.89 (s, 3H), 2.37 (s, 3H),
2.19 (s, 3H). 13C NMR (75 MHz, DMSO) δ 166.00, 162.16, 157.86,
146.53, 141.45, 135.11, 133.11, 130.40, 130.35, 128.33, 126.65,
126.01, 124.80, 123.39, 122.98, 115.24, 113.14, 89.79, 65.12, 40.30,
40.04, 39.75, 39.48, 39.20, 38.92, 38.65, 11.33, 10.32. MS (ESI, m/z):
385.4[M+H]+; Anal. calcd. for C20H17ClN2O4: C, 62.42; H, 4.45; N,
7.28. Found: C, 62.31; H, 4.51; N, 7.34.
4.1.3.13. 2-(Benzyloxy)-5-(3, 5-dimethylisoxazol-4-yl)-3-hydroxy-3-(3-
methoxyphenyl)isoindolin-1-o ne (10o). White solid, yield 85%. m.p.:
138–140 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J = 8.1 Hz, 1H),
7.72 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 – 7.21 (m, 7H), 7.08 (s, 1H),
6.97 – 6.79 (m, 2H), 5.10 (dd, J = 38.3, 10.1 Hz, 2H), 3.73 (s, 3H), 2.38
(s, 3H), 2.20 (s, 3H). 13C NMR (75 MHz, DMSO) δ 165.91, 162.39,
159.23, 157.85, 147.11, 140.63, 135.18, 134.91, 130.14, 129.51,
129.01, 128.52, 128.25, 126.81, 123.29, 123.01, 118.15, 115.31,
113.42, 112.22, 90.39, 79.01, 55.04, 40.28, 39.98, 39.73, 39.45,
4.1.3.10. 2-(Benzyloxy)-5-(3,5-dimethylisoxazol-4-yl)-3-(3-
fluorophenyl)-3-hydroxyisoindolin-1-one (10j). Yellow solid, yield 81%.
m.p.: 156–158 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J = 11.0 Hz,
2H), 7.58 (d, J = 7.9 Hz, 1H), 7.37 (dd, J = 16.4, 6.3 Hz, 8H), 7.13 (dd,
J = 22.3, 8.0 Hz, 2H), 5.09 (dd, J = 34.0, 10.0 Hz, 2H), 2.36 (s, 3H),
39.17, 38.90, 38.60, 11.33, 10.35. MS (ESI, m/z): 457.3[M+H]+
;
124

P. Chen et al.
BioorganicChemistry86(2019)119–125
Anal. calcd. for C27H24N2O5: C, 71.04; H, 5.30; N, 6.14. Found: C,
71.03; H, 5.41; N, 6.12.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.01.034.
4.1.3.14. 2-(Benzyloxy)-5-(3,
5-dimethylisoxazol-4-yl)-3-hydroxy-3-
phenylisoindolin-1-one (10p). White solid, yield 88%. m.p.:
142–143 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.87 (d, J = 7.6 Hz, 1H),
7.73 (s, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.53 – 7.23 (m, 11H), 5.11 (dd,
J = 41.3, 10.1 Hz, 2H), 2.38 (s, 3H), 2.20 (s, 3H). 13C NMR (75 MHz,
DMSO) δ 166.42, 162.85, 158.31, 147.73, 139.51, 135.72, 135.43,
130.62, 129.45, 128.99, 128.84, 128.78, 128.74, 127.41, 126.61,
123.77, 123.53, 91.04, 79.47, 40.91, 40.64, 40.36, 40.08, 39.80,
39.52, 39.25, 11.82, 10.82. MS (ESI, m/z): 427.3[M+H]+; Anal.
calcd. for C26H22N2O4: C, 73.23; H, 5.20; N, 6.57. Found: C, 73.26;
H, 5.21; N, 6.62.
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