Discovery and optimization of novel N-benzyl-3,6-dimethylbenzo[d]isoxazol-5-amine derivatives as potent and selective TRIM24 bromodomain inhibitors with potential anti-cancer activities

Article abstract of DOI:10.1016/j.bioorg.2019.103424

Tripartite motif-containing protein 24 (TRIM24), recognized as an epigenetic reader for acetylated H3K23 (H3K23ac) via its bromodomain, has been closely involved in tumorigenesis or tumor progression of several cancers. Developing inhibitors of TRIM24 is significant for functional studies and drug discovery. Herein, we report the identification, optimization and evaluation of N-benzyl-3,6-dimethylbenzo[d]isoxazol-5-amines as TRIM24 bromodomain inhibitors starting from an in house library screening. Structure-based optimization leads to two potent and selective compounds 11d and 11h in an Alphascreen assay with IC₅₀ values of 1.88 μM and 2.53 μM, respectively. The viability assay demonstrates the great potential of this series of compounds as inhibitors of proliferation of prostate cancer (PC) cells LNCaP, C4-2B. A colony formation assay further supports this inhibitory activity. Compounds 11d and 11h inhibit cell proliferation of other cancer types such as non-small cell lung cancer (NSCLC) cells A549 with IC₅₀ values of 1.08 μM and 0.75 μM, respectively. These data suggests that compounds 11d and 11h are promising lead compounds for further research.

Full text of DOI:10.1016/j.bioorg.2019.103424

Journal Pre-proofs
Discovery and optimization of novel N-benzyl-3,6-dimethylbenzo[d]-isoxa-
zol-5-amine derivatives as potent and selective TRIM24 bromodomain inhibi-
tors with potential anti-cancer activities
Qingqing Hu, Chao Wang, Qiuping Xiang, Rui Wang, Cheng Zhang,
Maofeng Zhang, Xiaoqian Xue, Guolong Luo, Xiaomin Liu, Xishan Wu, Yan
Zhang, Donghai Wu, Yong Xu
PII:
S0045-2068(19)30805-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103424
YBIOO 103424
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 June 2019
2 September 2019
5 November 2019
Please cite this article as: Q. Hu, C. Wang, Q. Xiang, R. Wang, C. Zhang, M. Zhang, X. Xue, G. Luo, X. Liu, X.
Wu, Y. Zhang, D. Wu, Y. Xu, Discovery and optimization of novel N-benzyl-3,6-dimethylbenzo[d]-isoxazol-5-
amine derivatives as potent and selective TRIM24 bromodomain inhibitors with potential anti-cancer activities,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103424
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Discovery and optimization of novel N-benzyl-3,6-dimethylbenzo[d]-isoxazol-5-
amine derivatives as potent and selective TRIM24 bromodomain inhibitors with
potential anti-cancer activities
Qingqing Hu,a,b,c Chao Wang,a,b,c Qiuping Xiang,a,b,c Rui Wang,a Cheng Zhang,a
Maofeng Zhang,d Xiaoqian Xue,e Guolong Luo,a Xiaomin Liu,a Xishan Wu,a Yan
Zhang,a,b,c Donghai Wu,f and Yong Xua,c,*
aGuangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences,
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences;
Guangzhou Medical University, Guangzhou 510530, China.
bUniversity of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049,
China.
cGuangzhou Regenerative Medicine and Health Guangdong Laboratory (GRMH-GDL),
Guangzhou 510530, China
dTaizhou Polytechnic College, No. 8 Tianxing Road, Medical High-tech Development
Zone, Taizhou 225300, Jiangsu Province, China
eSchool of Life Science, Huizhou University, Huizhou 516007, China
fGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences;
Guangzhou 510530, China
*Corresponding Author: Yong Xu, PhD, E-mail: xu_yong@gibh.ac.cn

Abstract
Tripartite motif-containing protein 24 (TRIM24), recognized as an epigenetic reader
for acetylated H3K23 (H3K23ac) via its bromodomain, has been closely involved in
tumorigenesis or tumor progression of several cancers. Developing inhibitors of
TRIM24 is significant for functional studies and drug discovery. Herein, we report the
identification, optimization and evaluation of N-benzyl-3,6-dimethylbenzo[d]isoxazol-
5-amines as TRIM24 bromodomain inhibitors starting from an in house library
screening. Structure-based optimization leads to two potent and selective compounds
11d and 11h in an Alphascreen assay with IC50 values of 1.88 M and 2.53 M,
respectively. The viability assay demonstrates the great potential of this series of
compounds as inhibitors of proliferation of prostate cancer (PC) cells LNCaP, C4-2B.
A colony formation assay further supports this inhibitory activity. Compounds 11d and
11h inhibit cell proliferation of other cancer types such as non-small cell lung cancer
(NSCLC) cells A549 with IC50 values of 1.08 M and 0.75 M, respectively. These
data suggests that compounds 11d and 11h are promising lead compounds for further
research.
Keywords: TRIM24 bromodomain, inhibitor, prostate cancer, anti-cancer, structure-
based drug design

1. Introduction
TRIM24 (Tripartite motif-containing protein 24) is a multifunctional protein involved
in different biological processes [1] such as homologous or heterogeneous
polymerization [2] and ubiquitylation [3], including the ubiquitylation of the tumor
suppressor p53 as an E3 ubiquitin ligase due to its tripartite RBCC motif [4-6]. TRIM24
is also known as TIF1α (Transcription Intermediary Factor 1 alpha) because its nuclear
receptor interacting LXXLL domain regulates multiple transcriptional activities [7, 8].
Another important domain of TRIM24 is the bromodomain which defines the whole
BCP (bromodomain containing protein) superfamily which includes 61 members that
are classified into eight groups [9]. TRIM24 belongs to the fifth subfamily and as a
histone epigenetic reader, it can specifically recognize acetylated H3K23 (H3K23ac)
via its bromodomain [10]. Previous studies have indicated that TRIM24 regulates
tumorigenesis or tumor progression of various cancer types including prostate cancer
[11], lung cancer [12], colorectal cancer [13, 14], breast cancer [10] and gliobastoma
[15].
Prostate cancer (PC) is a common cancer in men worldwide. The clinically dominant
treatment is androgen-deprivation approaches that inhibit the androgen synthesis or
androgen receptor (AR) signaling pathways [16, 17]. Unfortunately, most patients
eventually develop resistance to castration therapies and progress to castration-resistant
prostate cancer (CRPC) [17]. CRPC is generally fatal and these patients are in
significant need of more effective treatments such as targeted therapeutic drugs. In
recent years, we have been focused on development of small molecule inhibitors of
BCPs and nuclear receptors which are potential therapeutic targets for CRPC [18-22].
Pioneer work by Brown and others supported that the TRIM24 mediated CRPC growth
through its bromodomain and LXXLL motif and suggested that the bromodomain was
a potential drug target [11, 23-25]. The discovery of new potent and specific TRIM24
inhibitors will greatly enhance our understanding of the therapeutic potential of
bromodomain inhibition for the treatment of CRPC.

LXXLL motif is a conserved short sequence [26] which interacts with the AF-2 domain
of nuclear receptors [8, 27, 28]. From a general perspective of druggable targets,
LXXLL motif is too small to provide sufficient protein surface to which drug molecules
can bind, although it is large enough to bind with nuclear receptors [26]. Unlike the
LXXLL motif, the bromodomain contains approximately 110 amino acids [9] which
are folded into a conserved four helical bundle that forms an adequate protein surface
for drug molecules’ occupation [9, 10]. A separate evaluation of the druggability of
TRIM24 bromodomain without involving the interaction between the bromodomain
and the plant homeodomain (PHD) led to a low score, suggesting the difficulty for drugs
to bind with this bromodomain [29]. Encouragingly, two groups initially disclosed a
class of inhibitors targeting the TRIM24 bromodomain [30, 31], which casted a new
light on the druggability of the bromodomain of TRIM24. To date, the best inhibitor,
IACS-9571, has nanomolar activity in a protein-level assay [30], but the activity data
of TRIM24 bromodomain inhibitors tested on various cancer cells is still limited and
deficient [31, 32].
In view of the significance of TRIM24 in cancer development and the limited structure
types, it is worthwhile to discover novel molecules as tool molecules or lead compounds
for drug discovery. Herein, we reported identification of a series of novel TRIM24
bromodomain inhibitors through a sequential process including in-house library
screening, structure-based optimization and molecular docking studies. Two potent and
selective leads also inhibited the growth of PC cells and other cancer cells including
non-small cell lung cancer cells, suggesting the clinical anti-cancer potential of these
compounds.
2. Results and discussion
2.1. Discovery of hit compounds through in-house library screening
In this work, we performed an Alphascreen assay [19, 21] to screen our in-house
compound library against a recombinant human TRIM24 bromodomain produced in-
house. As shown in Figure 1A, we identified three compounds with inhibition rates

above 90% by conducting the screening at a concentration of 20 μM. These compounds
(1, 2 and 3) have dose-dependent inhibitory activities against the TRIM24
bromodomain with an IC50 value of 11.18 μM, 14.22 μM, and 13.76 μM, respectively
(Figure 1B, Table 1). These compounds exhibit fair binding capability and their
inhibition rates were comparable with that of IACS-9571, making them perfect hits in
the traditional medicinal chemistry sense. We identified these three compounds as hit
compounds for subsequent structure optimization.
Figure 1. The discovery of hit compounds. (A) The inhibitory rate of three hit compounds at a
concentration of 20 μM in an in-house library screening. (B) The dose-dependent inhibitory activities of
three hit compounds. All experiments were carried out in an Alphascreen assay.
2.2. Synthesis
Based on three hit compounds mentioned above, a series of derivatives were designed
and synthesized. Two important intermediates 8 (for synthesizing compounds 11a’,
11b’, 11d’ and 11x’) and 9 (for synthesizing compounds 10, 11a-11z) were prepared
as depicted in scheme 1 [20].

O
O
O
O
NH2
NH
NH
NH
NH
d
c
b
a
O
HO
O
O
HO
O
N
N
OH
Br
8
6
4
7
5
e
O
HO
R4
O
NH
h
i
O
g
f
NH2
NH
NH
NH
N
10
O
O
O
R4
O
O
N
N
N
N
9
11k-11n, 11v, 11y-11z
11a
11b
l
NH
O
HO
O
R4
R4
k
j
O
N
11c-11j, 11o-11u, 11w-11x
OH
14c-14j, 14o-14u, 14w-14x 13c-13j, 13o-13u, 13w-13x
OH
Br
12
R4 =
c:
d:
l:
e:
g:
o:
h:
p:
i:
j:
f:
k:
m:
u:
n:
q:
r:
O
O
Cl
O
F
F
O
F
x:
v:
s:
t:
w:
y:
z:
Br
O
Cl
Br
Cl
O
F
F F
Scheme 1. Synthesis of compounds 10, 11a-11z.
Reagents and conditions: (a) Acetyl chloride, Et3N, DCM, 0 C, 15 min, 78%; (b) Acetyl chloride, AlCl3,
DCM, 0-44 C, 2 h, 68%; (c) H2NOH-HCl, AcONa, EtOH/H2O (3:1), 80 C, reflux, 70 min, 100%; (d)
DMF-DMA, 1,4-dioxane, 100 C, 10 min, 78%; (e) HCl/H2O, 100 C, reflux, 2.5 h, 100%; (f) 1-bromo-
3-(bromomethyl)benzene, Cs2CO3, DMF, 60 C, overnight, 40-65%; (g) 1-(bromomethyl)- 3-
methoxybenzene, Cs2CO3, DMF, 60 C, overnight, 40-65%; (h) BBr3, DCM, 0 C; RT, 2.5 h, 57%; (i)
R4Br, K2CO3, 80 C, 40 min-overnight, 2-47% (for 11k-11l, 11v, 11z) or KOH, RT, DMF, 1h, 28-43%
(for 11n and 11y); (tetrahydrofuran-3-yl)methyl 4-methylbenzenesulfonate (for 11m), K2CO3, NMP, 95
C, 4 h, 97%; (j) R4Br or R4I, K2CO3, DMF, 80 C, 0.5-1.0 h, 49-100%; (k) PBr3, DCM, RT, 0.5-2.0 h,
47-98%; (l) Cs2CO3, DMF, 60 C, overnight, 13-57%.

To obtain the final products 10 and 11a-11z, shown in Scheme 1, we adopted two
slightly different strategies, mostly through a chain of substitution reactions. The raw
material R4Br or R4I can be directly purchased or synthesized in-house from the
corresponding alcohols that also are commercially available.
After obtaining compound 11d by the steps shown in Scheme 1, its simple methylation
led to the synthesis of the compound 11d’ (Scheme 2). The synthetic procedure of 11x’
used a strategy similar to the step i shown in Scheme 1 and additionally, we obtained
two intermediates 11a’ and 11b’ (Scheme 2).
O
O
a
NH
N
O
O
N
N
O
11d
11d'
O
O
HO
O
NH
b
c
d
N
N
N
O
O
O
O
N
O
O
O
N
N
N
8
11b'
11x'
11a'
Scheme 2. Synthesis of compounds 11a’, 11b’, 11d’, 11x’.
Reagents and conditions: (a) MeI, NaH, 0 C, 15 min; rt, 10.5 h, 63%; (b) 1-(bromo- methyl)-3-
methoxybenzene, Cs2CO3, DMF, 60 C, 10.5 h, 91%; (c) BBr3, DCM, 0 C, rt, 0.5-2.5 h, 92%; (d) 1-
(bromomethyl)-3-methoxybenzene, K2CO3, 80 C, 3 h, 61%.
2.3. Structure-activity relationship (SAR) studies
We identified three hit compounds (1, 2 and 3) with equivalent inhibitory activities
against the TRIM24 bromodomain. Based on their similar inhibitory activities and
structures, it was difficult for us to initiate a structure optimization process (Table 1).
When we attempted to replace the carboxyl group of compound 3 with a halogen atom,

the newly obtained compound 10 (IC50 = 6.46 μM) exhibited an approximately 2-fold
increase in inhibitory activity versus compounds 1-3 (Table 1). Comparing the structure
and activities of compounds 1, 2, and 10 might be helpful to determine the optimal
position of substituents, thus we chose compound 10 as the initial compound and R2 of
compound 10 as the next optimization direction.
Table 1. The discovery of hit compounds
R3
H
N
R2
N
O
R1
Alphascreen
IC50 (µM)
No.
R1
R2
R3
IACS-9571
0.24
1
2
Cl
H
H
H
H
H
H
Cl
H
11.18
14.22
13.76
6.46
3
COOMe
Br
10
H
Based on the docking results, we supposed that the bromine atom of compound 10 was
directed towards the hydrophobic “upper pocket” (Figure 2A, 3A, see the next section).
We synthesized compounds 11a-11j to explore the optimal space available to the R4
group (Table 2). The inhibitory activity was improved from 11a to 11d and then
decreased from 11d to 11e, suggesting that three-carbon atom length was most suitable
for this “upper pocket” (Table 2) [30]. To further explore the optimal size of the R4
group, we synthesized 11f-11j in which the R4 group has a relatively fixed length of 3
or 4 carbon atoms (Table 2). An R4 group larger than n-propyl (11d) was found to be
disadvantageous for improving the activity. Introduction of a hydrophilic oxygen atom
into the R4 group (11k-11n, Table 2) failed to improve the activity, which suggested
hydrophilic R4 group was also disadvantageous for improving the activity. Based on
compounds 11a-11n, we could verify the hydrophobic requirement of the R4 group for
better occupation of the hydrophobic “upper pocket”.

Table 2. Structure−activity relationships of compounds 11a-11z
H
R4
N
O
N
O
Alphascreen
IC50 (µM)
No.
R4
IACS-9571
11a
0.24
7.30
7.37
3.84
1.88
CH3
H
11b
11c
11d
11e
11f
4.19
3.34
11g
11h
3.47
2.53
11i
12.22
11j
11k
11l
2.86
9.01
4.3
O
O
O
11m
11n
2.54
2.72
O
F
11o
70.76
F
11p
11q
78.36
65.05
F

Cl
11r
11s
11t
76.09
76.41
24.15
Cl
Cl
Br
11u
87.31
Br
11v
11w
11x
54.49
64.42
8.42
F
F F
O
O
11y
11z
2.57
2.33
It was found that the activity was promoted more than 4-fold when the cyclohexyl
methyl group (11i) was replaced by the benzyl group (11j). This suggested a potential
opportunity to improve the activity by optimizing the structure of 11j. Since benzene
ring of the R4 group in compound 11j could be easily substituted, we introduced
electron-donating or electron-accepting groups in the o-, m- and p- positions of the
phenyl ring to explore the optimization space (Table 2). The activities of compounds
11o-11w were drastically decreased comparing with the reference compound (11j),
which implied the disadvantage of electron-attracting substituents. While an electron-
donating methyl or methoxyl group was added in the o-position of the phenyl ring, the
inhibitory activities of 11y and 11z were only slightly improved over that of the

reference compound (11j), revealing the weak and insignificant advantage of electron-
donating substituents (Tables 2). Eventhough optimization of the phenyl ring of the
starting compound (11j) failed to distinctly improve activity, the unimpressive success
of the hydrophobic ortho-methyl substituted 11z further supported the hydrophobicity
requirement of the R4 group. Taking these findings together, we concluded that
introducing hydrophobic groups with appropriate length and shape into the R4 group is
an efficient strategy to improve activity. The n-propyl group (11d) was identified as the
best substituent [30]. A cyclopentyl methyl group (11h) and an o-tolylmethyl group
(11z) were suboptimal substituents.
Based on the common N-benzyl-3,6-dimethylbenzo-[d]isoxazol-5-amine skeleton, we
synthesized four methyl or acetyl derivatives 11a’, 11b’, 11d’ and 11x’ to further
investigate the importance of the hydrogen atom in the backbone structure. However,
replacement of the hydrogen atom led to significant loss of activity, indicating the
essential role of the hydrogen atom of these derivatives (Table 3).
Table 3. Exploration of Substitutability in R5
R5
N
R4
O
N
O
Alphascreen
IC50 (µM)
No.
11a
R4
R5
H
H
H
7.30
32.79
7.37
11a’
11b
11b’
O
H
H
34.19
O
11d
1.88
11d’
34.97
O
11x
H
8.42
O
11x’
39.61
O

2.4. Binding mode analysis
To further reveal the molecular mechanism of inhibitory activity of these compounds,
we analyzed the binding mode from several perspectives. As shown in Figure 2A, the
“upper pocket” is defined by hydrophobic residues Ala989, Met920 and a hydrophobic
LAF/V shelf (Leu922, Ala923, Phe924/Val986) as well as hydrophilic residues Glu919
and Glu985 with both carboxyl groups extending into the solvent area, making the
“upper pocket” highly hydrophobic. As previously reported by Anderson et al., there
was one conserved water molecule in the “upper pocket” (see Figure 2B water
molecules depicted in red) [30]. After aligning all crystal structures of the TRIM24
bromodomain revealed by Anderson et al., it was found that there actually was a second
water molecule relatively conserved in the “upper pocket” (see Figure 2B water
molecules depicted in magenta) for two reasons: Firstly, almost every available crystal
structures of TRIM24-ligand retain two water molecules in relatively fixed positions in
the “upper pocket”, and secondly, in most aligned PDBs, two water molecules and
residue Glu919 could form a hydrogen-bond network (Figure 2A, 2B).
Figure 2. Structure of the “upper pocket”. (A) Constitutive residues of the “upper pocket”. PDB 4YBT
was used as an example. (B) Conserved water molecules in the “upper pocket” based on alignment of
released PDBs (4YAB, 4YAD, 4YAT, 4YAX, 4YBM, 4YBS, 4YBT, 4YC9; all PDBs were aligned with
4YBT) [30]. The reported conserved water molecules are depicted as red spheres and the second
conserved water molecules are depicted as magenta spheres. All residues are shown as lines. The main

chain of 4YBT was generated as an electrostatic potential surface. The ligand of PDB 4YBT was shown
as sticks. All aligned PDBs were shown as transparent cartoons under surface for clarity.
We investigated the binding mode after docking compound 10 into the TRIM24
bromodomain (PDB: 4YBT, with two conserved water molecules in the “upper pocket”)
with the Glide Standard Precision module [33]. The bromine atom of compound 10
extended into the “upper pocket” (Figure 3A). Due to hydrophobicity of the “upper
pocket”, introduction of hydrophobic ether groups which could occupy this
hydrophobic pocket should benefit activity. Among a series of ether derivatives 11a-
11j, the activity increased from 11a to 11d but subsequently declined from 11d to 11e
as the R4 group became longer, suggesting that a three-carbon-atom length is the
optimal choice of the R4 group to occupy the “upper pocket” (Figure 3B). Keeping the
length fixed and enlarging the shape of the R4 group to increase the hydrophobic
interaction from 11e to 11j led to a “rise-fall-rise” trend in activity, revealing that larger
R4 group led to improvement of activity because the activity of 11h increased versus
that of 11f or 11g. But the clash in the “upper pocket” caused by increasing the volume
of the R4 group was also important. As shown in Figures 3B, 3C and 3D, 11d docked
its R4 group precisely into the “upper pocket” without any clash while the phenyl ring
of 11j clashed with the first conserved water in the pocket and 11i entirely failed to
enter into the pocket because of its relatively massive cyclohexyl group. Taken together,
11d displayed the best balance between scale and hydrophobicity of the R4 group, and
showed optimal inhibitory activity against the TRIM24 bromodomain. Compounds 11h
and 11z were the suboptimal compounds also achieving that balance.

Figure 3. Predicted binding modes of 11d, 11h, 11j, and 11i in the “upper pocket”. 4YBT was prepared
for docking each compound: (A) 11d, (B) 11h, (C) 11j, (D) 11i. All compounds are shown as sticks and
all residues as lines. Water molecules in the “upper pocket” are shown as spheres. An electrostatic
potential surface was shown as well. The red dashed line represents a serious conflict.
2.5. Evaluation of bromodomain selectivity
To further investigate the selectivity profile, we used an Alphascreen assay to test three
optimal compounds 11d, 11h and 11z against representative BCPs produced in house.
These compounds showed excellent selectivity for TRIM24 bromodomain over BRD4
(1) and BAZ2B, and moderate selectivity over CREBBP and BRD9 (Figure 4A, 4B)
and compound 11h exhibited the best selectivity profile.
Figure 4. The activities and selectivity profiles of three optimal compounds. (A) Dose-dependent
inhibitory activities of three optimal compounds. (B) Selectivity profiles of three optimal compounds
against four representative BCPs. All experiments were carried out in an Alphascreen assay. Compounds
with lower than 50% inhibition rate at concentration of 100 μM were attached corresponding values in
brackets.
2.6. Cell-based evaluation of inhibitory effects
The bromodomain of TRIM24 has been reported to be highly correlated with
progression of prostate cancers [11]. To generally evaluate the inhibitory effects of our
compounds on prostate cancer cell proliferation, compounds whose IC50 values in the
Alphascreen assay were lower than 10 M were selected to conduct a luminescent assay
in prostate cancer cells LNCaP and C4-2B respectively, with the CellTiter-Glo reagent
serving as a detection reagent. As shown in Table S1, most compounds exhibited higher
inhibitory potency than enzalutamide (an androgen receptor antagonist approved for

CRPC [34-36]) on proliferation of LNCaP and C4-2B cells, demonstrating the overall
inhibition capability of this series of compounds. Three optimal compounds 11d, 11h
and 11z showed low to high micromolar activities on the proliferation of LNCaP and
C4-2B cells. And compound 11h was the optimal compound for both cells (Table S1,
Figure 5A).
Figure 5. The dose-dependent inhibitory activities of three optimal compounds in PC cell lines. (A) The
inhibitory activities on the proliferation of LNCaP cells and C4-2B cells. (B) The inhibitory activities on
the colony formation of C4-2B cells.

To further investigate the long-term cell growth inhibitory effects, we tested the
inhibitory capacity of compounds 11d, 11h and 11z on PC cell colony formation.
Consistent with the effects of the viability assay, treatment of C4-2B cells with any of
these compounds reduced the colony formation in a dose-dependent manner (Figures
5A, 5B). Compounds 11d showed a clearer dose-dependent effect in inhibiting the
colony formation (Figure 5B). Based on effects in the above two assays, compounds
11d and 11h were selected for the subsequent cell-based assays.
Previous studies have reported that TRIM24 is involved in the development of various
other cancer types such as lung cancer [12], colorectal cancer [13, 14] and breast cancer
[10]. Accordingly, we tentatively tested the effects of compounds 11d and 11h in
NSCLC cells A549, colorectal cancer cells HT-29, breast cancer cells MCF-7 and
MDA-MB-231 (Figure 6). Both compounds exhibited inhibitory effects on the growth
of each kind of cancer cells. Moreover, compounds 11d and 11h both showed
encouraging low micromolar inhibitory activities in A549 cells with IC50 values of 1.08
M and 0.75 M, respectively (Figure 6).
Figure 6. Dose-dependent inhibitory activities of compounds 11d and 11h in diverse cell lines as shown
above.
Overall, our compounds had preference for prostate cancer cells LNCaP and C4-2B and
NSCLC cells A549. The general inhibitory effects on these types of cancer cells

highlighted the therapeutic potential of TRIM24 bromodomain inhibitors to treat
cancers clinically in the future.
3. Conclusions
In this study, we have reported a novel class of N-benzyl-3,6-dimethylbenzo-[d]-
isoxazol-5-amine derivatives as TRIM24 bromodomain inhibitors. Through a
sequential process including in-house library screening, structure-based medicinal
chemistry optimization and biological evaluation, we obtained two molecules with low
micromolar activities in both protein and cell viability assays. The results from the
colony formation assay further supported the inhibitory effects shown in the viability
assay. Taken together, we reported a novel series of TRIM24 bromodomain inhibitors
and revealed for the first time the inhibitory effects of our inhibitors on proliferation of
previously reported cancer cells, particularly PC cells and NSCLC cells. We obtained
two compounds 11d and 11h which can be used as probe molecules for studying the
biological function of TRIM24 bromodomain in cell-based assays. These compounds
can also be used as lead compounds for further optimization to develop novel cancer
targeted drug candidates especially for intractable CRPC.
4. Methods
4.1. Plasmid construction, protein expression and purification
All BCPs were expressed as His6-tagged fusion proteins using the pET24a expression
vector (Novagen). cDNA encoding bromodomain of human TRIM24 (residues Gly896-
Glu1014), BRD4 (1) (residues Asn44-Glu168), CREBBP (residues Arg1081-Gly1197),
BRD9 (residues Leu14-Gln134) and BAZ2B (residues Ser1858-Ser1972) were
synthesized by Genscript. BL21 (DE3) cells transformed with these expression
plasmids were grown in LB broth at 25 C to an OD600 of ~1.0 and then induced with
0.1 mM isopropyl- β-D-1-thiogalactopyranoside (IPTG) at 16 C for 16 h. Cells were
harvested by centrifugation (6000g for 15 min at 4 C, JLA 81000 rotor, on a Beckman
Coulter Avanti J-20 XP centrifuge) and were frozen at -20 C for storage as pellets.
Cells were resuspended in extract buffer (50 mM HEPES, pH 7.5 at 25 C, 500 mM

NaCl, 5 mM
imidazole, 5% glycerol and 0.5 mM TCEP (Tris (2-
carboxyethyl)phosphine hydrochloride) and were lysed by high pressure
homogenization using an JN3000 PLUS high pressure homogenizer (JNBIO,
Guangzhou, China) at 4 C. The lysate was collected on ice and centrifuged at 12,000
rpm for 40 min at 4 C. The supernatant was loaded onto a 5 mL NiSO4-loaded HisTrap
HP column (Ni-NTA, GE Healthcare, NJ). The column was washed with 20 mL of
extract buffer (50 mM HEPES, pH 7.5 at 25 C, 500 mM NaCl, 50 mM imidazole).
The protein was eluted with a 50-500 mM imidazole gradient in elute buffer with (50
mM HEPES, pH 7.5 at 25 C, 500 mM NaCl, 500 mM imidazole). The protein was
collected during elution, and concentrated and further purified by a gel filtration column
(HiLoad, Superdex 75, 16/60, GE Healthcare). The sample purity of each fraction was
examined by SDS-PAGE. Purified proteins were concentrated and stored in the gel
filtration buffer (10 mM HEPES pH 7.5 at 25 C, 150 mM NaCl, 0.5 mM TCEP) at -
80 C used for the Alphascreen assay.
4.2. Alphascreen assay
Interactions between bromodomain-containing proteins (BCPs) and ligands were
assessed by luminescence-based Alphascreen technology (PerkinElmer) as previously
described [19-21] using a histidine detection kit from Perkin Elmer (Norwalk, CT). All
of the reactions contained bromodomain-containing proteins bound to nickel acceptor
beads (5 μg/mL) and a biotinylated acetylated histone H4 or H3 peptide bound to
streptavidin donor beads (5 μg/mL) in the presence of the indicated amounts of control
compound IACS-9571 (MCE, hydrochloride form) or candidate compounds. The
sequences of biotinylated acetylated histone H4 or H3 peptide were listed as follows:
bH4Kac4: SGRGK(ac)GGK(ac)GLGK(ac)GGAK(ac)RHRK-biotin, bH3K14ac:
YQTARKSTGGK(ac)APRKQLATKAK-biotin,
bH3K23ac:
GKAPRKQLATK(ac)AARKSAPATYKK-biotin (both produced by Genscript). The
experiments were conducted with protein/peptide ratio as follows for sensitive signal:
BRD4 (1): bH4Kac4 = 50 nM: 50 nM, BRD9: bH4Kac4 = 150 nM: 50 nM, CREBBP:
bH4Kac4 = 150 nM: 50 nM, BAZ2B: bH3K14ac = 150 nM: 100 nM, TRIM24:

bH3K23ac = 500 nM: 100 nM.
All reagents were diluted in the buffer (50 mM MOPS, pH 7.4, 50 mM NaF, 50 μM
CHAPS, and 0.1 mg/mL bovine serum albumin) and allowed to equilibrate at RT prior
to their addition to low-volume 384-well plates (ProxiPlate-384 Plus, PerkinElmer,
USA). Plates were sealed in foil to protect them from light, incubated at RT for 1.5 h
and read on an EnSpire plate reader (PerkinElmer, USA). When excited by a laser beam
of 680 nm, the donor beam emits singlet oxygen that activates thioxene derivatives in
the acceptor beads, which releases photons of 520–620 nm as the binding signal. All
experiments were carried out in triplicate. The IC50 values were calculated using
GraphPad Prism 7 software.
4.3. Molecular docking
The molecular docking was carried out using the Glide module in Maestro software
v11.7. The crystal structure of hTRIM24 (PDB: 4YBT) was used as the receptor for
docking. The protein structure was prepared with the Protein Preparation Wizard in
Maestro. A receptor grid was generated with the Receptor Grid Generation program
leaving all settings on the default parameters. All ligands were prepared with the
LigPrep for generating all ionization states and stereoisomers. A standard precision (SP)
module was used to perform the molecular docking.
4.4. Cell viability and cell colony formation assays
For the viability assay, LNCaP, C4-2B, A549, HT-29 cells were cultured in RPMI-1640
supplemented with 10% FBS; MDA-MB-231 cells were cultured in DMEM
supplemented with 10% FBS. Cells were seeded in white 384-well microplates at their
optimum density for growth (500 or 1000 cells per well) in a total volume of 20 μL of
media. After cultivating cells in an incubator for 12 h, the control compound
enzalutamide (Selleck) and compounds were added in a total volume of 10 μL of media
(triple diluted) to each well with final concentration between 5 nM and 100 μM. Then
the plate was incubated for 72-120 h. Each plate and its contents was equilibrated at RT

for 20-30 min then 25 μL of Cell-Titer GLO reagents (Promega) was added to each
well. The contents were mixed on an orbital shaker for 20 min and incubated at RT for
10 min. Luminescence was measured on an EnSpire plate reader (PerkinElmer, USA),
according to the manufacturer’s instructions. All experiments were carried out in
triplicate. The estimated IC50 values were calculated using GraphPad Prism 7 software.
C4-2B cells were seeded in a well of 6-well plates with 4500 cells per well and cultured
with vehicle or indicated concentrations of candidate compounds for 14 days with the
3 mL medium. When the cell clone grew visible, the medium was removed and the
plates were washed once with 2 mL PBS. The cell colonies were stained with 2.5%
crystal violet in MeOH for 2 h. The numbers of colonies were counted after being
washed 3 times with twice-distilled water.
4.5. Chemistry
All solvents and reagents were purchased from commercially available sources and
unless otherwise specified, were used without purification. Column chromatography
was performed using 300-400 mesh silica gel. All reactions were monitored by TLC,
using silica gel plates with fluorescence F254 and UV light visualization. All products
were characterized by their NMR and MS spectra. 1H NMR and 13C NMR spectra were
obtained on Bruker Avance 400 or 500 instruments at 400 MHz or 500 MHz,
respectively. Chemical shifts are reported in parts per million (ppm, ) relative to
tetramethylsilane (TMS) as the internal standard control and coupling constants (J
values) are reported in Hertz (Hz). Proton coupling patterns are described as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m). ESI-MS were recorded on an Agilent
1200 HPLC-MSD mass spectrometer. Compound purities were determined by reverse-
phase high-performance liquid chromatography (HPLC) with solvent A (H2O) and
solvent B (MeOH) as eluents. The purity tests used a Dionex Summit HPLC column

(Inertsil ODS-SP, 5.0 m, 4.6 mm × 250 mm (GL Sciences Inc.)) with a UVD170U
detector, and a manual injector, a P680 pump with a detection wavelength of 254 nm
and a flow rate of 1.0 mL/min. The purity of all the final compounds was determined
by HPLC to be >95%.
4.5.1. General procedure for synthesis of skeleton structures shown in Scheme 1
4.5.1.1 General procedure for synthesis of skeleton structures 8 and 9
As shown in Scheme 1, commercially available 4 was N-acetylated with acetyl chloride
to give 5, which was converted to 6 via a Friedel-Crafts reaction. The intermediate 6
was used to prepare the oxime precursor 7, which was cyclized by high temperature
intramolecular dehydration to afford the key skeleton structure 8. Further deacylation
of compound 8 can yield another important skeleton structure 9.
4.5.1.1.1. N-(4-Methoxy-2-methylphenyl)acetamide (5).
4-Methoxy-2-methylaniline (4) (11.152 g, 81.29 mmol) and Et3N (13.161 g, 130.06
mmol) were dissolved in DCM (70 mL) in an ice bath. Acetyl chloride (8.934 g, 113.81
mmol) was added to the cold mixture sequentially and slowly. The mixture was stirred
at 0 C for 15 min. Then ice water was used to quench the reaction and the mixture was
extracted with DCM. The organic layer was washed with saturated brine, dried over
Na2SO4 and concentrated under reduced pressure. Adding petroleum ether to the
concentrated solution crystallized N-(4-methoxy-2-methylphenyl) acetamide (5) as
light purple crystals (11.398 g, 78% yield). 1H NMR (500 MHz, DMSO-d6) δ 9.16 (s,
1H), 7.18 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 2.6 Hz, 1H), 6.70 (dd, J = 8.6, 2.8 Hz, 1H),
3.71 (s, 3H), 2.14 (s, 3H), 2.00 (s, 3H). MS (ESI) m/z [M + H]+ calcd. 180.09; found
180.0.
4.5.1.1.2. N-(5-Acetyl-4-hydroxy-2-methylphenyl)acetamide (6).
N-(4-Methoxy-2-methylphenyl)acetamide (5) (11.398 g, 63.6 mmol) was dissolved in
DCM. Acetyl chloride (14.977 g, 190.8 mmol) and aluminum chloride (33.922 g, 254.4

mmol) were added in succession to the reaction in an ice bath with continuous stirring.
Then the mixture was heated at 45 C and refluxed for 2 h. The mixture was cooled to
RT and the reaction was quenched with ice water. The mixture was extracted with DCM
and the organic layer was washed with water, then with saturated brine, and dried over
Na2SO4. Decolorizing the organic solution by suction filtration with silica gel powder
to give light yellow filtrate. The residue was washed with DCM twice and the filtrate
was concentrated to produce the product which was collected as white crystals by
suction filtration again to yield N-(5-acetyl- 4-hydroxy-2-methylphenyl) acetamide (6)
(9.03 g, 68% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 9.31 (s, 1H), 7.77
(s, 1H), 6.83 (s, 1H), 2.57 (s, 3H), 2.18 (s, 3H), 2.03 (s, 3H). MS (ESI) m/z [M + H]+
calcd. 208.09; found 208.0.
4.5.1.1.3. N-(4-Hydroxy-5-(1-(hydroxyiminoethyl)-2-methylphenyl)acetamide (7).
N-(5-acetyl-4-hydroxy-2-methylphenyl)acetamide (6) (9.03 g, 43.6 mmol) was
dissolved in an EtOH-H2O mixture (3:1, 100 mL). Hydroxylamine hydrochloride
(4.84g, 69.7 mmol) and NaOAc (5.72 g, 69.7 mmol) were added successively and the
mixture was refluxed at 80 C for 70 min. Then the mixture was cooled to RT and the
EtOH was removed under reduced pressure to produce crystals. Water (~ 60 mL) was
added to dissolve ionic impurities and then the crystals were filtered and the filter cake
was washed with water. The cake was dried to afford N-(4-hydroxy- 5-(1-
(hydroxyimo)ethyl)-2-methylphenyl)acetamide (7) as a white powder containing solid
NaCl (12.3 g, 100% yield,). 1H NMR (400 MHz, DMSO-d6) δ 11.48 (s, 1H), 11.37 (s,
1H), 9.21 (s, 1H), 7.35 (s, 1H), 6.72 (s, 1H), 2.19 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H). MS
(ESI) m/z [M - H]- calcd. 221.09; found 221.0.
4.5.1.1.4. N-(3,6-Dimethylbenzo[d]isoxazol-5-yl)acetamide (8).
To a solution of N-(4-hydroxy-5-(1-(hydroxyimino)ethyl)-2-methylphenyl)acetamide
(7) (12.3 g, 55.35 mmol, theoretically 43.6 mmol) dissolved in 1,4-dioxane (46 mL)
was added DMF-DMA (33.8 mL) under vigorous stirring. The mixture was stirred at
100 C for 37 min. Then the reaction mixture was cooled to RT. The product was

extracted between the water layer and EtOAc layer. The organic layer was washed with
once with water and saturated brine, then dried over Na2SO4. The organic solution was
concentrated under reduced pressure and the concentrated solution was filtered to give
N-(3,6-dimethylbenzo-[d]isoxazol-5-yl)acetamide (8) as a brown solid (6.955 g, 78%
yield). 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.35 (s, 1H), 7.13 (s, 1H), 2.52 (s,
3H), 2.39 (s, 3H), 2.24 (s, 3H). MS (ESI) m/z [M + H]+ calcd. 205.09; found 205.0.
4.5.1.1.5. 3,6-Dimethylbenzo[d]isoxazol-5-amine (9).
To the compound 8 (6.955 g, 34.05 mmol) was added 3N solution of hydrochloric acid
(204 mL). The mixture was stirred and refluxed at 100 C for 2.5 h. Then the reaction
was terminated and the mixture was cooled to RT. Unreacted compound 8 was removed
by extraction from the water layer with EtOAc. In an ice bath, the pH of the aqueous
solution was adjusted to pH 7~8 with sodium hydroxide solution to precipitate the
product. The solid was collected by filtration, washed with water and dried to give 3,6-
dimethylbenzo-[d]isoxazol-5-amine (9) as a brown powder (6.54 g, 100% yield,
1
containing NaCl solid). H NMR (400 MHz, DMSO-d6) δ 7.29 (s, 1H), 6.83 (s, 1H),
4.89 (s, 2H), 2.41 (s, 3H), 2.21 (s, 3H). MS (ESI) m/z [M + H]+ calcd. 163.08; found
163.0.
4.5.1.2. General procedure for synthesis of compounds 11c-11j, 11o-11u, 11w-11x
4.5.1.2.1. General procedure for synthesis of intermediates 14c-14j, 14o-14u, 14w-14x
As shown in Scheme 1, commercially available 12 was etherified on the phenolic
hydroxyl group to give compounds 13c-13j, 13o-13u, 13w-13x in 49-100% yield with
corresponding R4Br or R4I purchased from reagent companies or synthesized in-house
from corresponding commercially available alcohols with the same reaction reagents
and conditions as were used in step k. The synthesized R4Br was used for next reactions
without further purification and characterization. Compounds 13c-13j, 13o-13u, 13w-
13x were then transformed to the corresponding bromides 14c-14j, 14o-14u, 14w-14x
(47-98% yield) with PBr3. The following monosubstitution of 9 with 14c-14j, 14o-14u,
14w-14x finally yielded the target products 11c-11j, 11o-11u, 11w-11x (13-57% yield).

For steps j and k, we used synthetic procedures of intermediates 13c and 14c as
examples.
4.5.1.2.1.1. (3-Ethoxyphenyl)methanol (13c).
To a solution of compound 12 (200 mg, 1.61 mmol, 1.0 eq) dissolved in DMF were
added K2CO3 (668 mg, 4.83 mmol, 3.0 eq), bromoethane (193 mg, 1.77 mmol, 1.1 eq)
(for most other intermediates, R4Br or R4I could be 1.1 eq or 1.5 eq). The mixture was
stirred at 60 C overnight. For most other intermediates, the stirring could be 80 C for
0.5-1.0 h. Then the mixture was cooled, poured into water and extracted with EtOAc.
The organic layer was washed with saturated brine, dried over NaSO4 and concentrated
under reduced pressure to give the crude (3-ethoxyphenyl)methanol (13c) as a colorless
or light yellow liquid (210 mg, 86% yield). For most other intermediates, the crude
product was further purified by silica gel chromatography with petroleum ether/EtOAc
(1/5~1/2) to obtain products which were monitored by TLC. The intermediates 13c-
13j, 13o-13u, 13w-13x were not characterized but were used directly in the next steps.
4.5.1.2.1.2. 1-(Bromomethyl)-3-ethoxybenzene (14c).
To a solution of compound 13c (210 mg, 1.38 mmol, 1.0 eq) dissolved in DCM was
added PBr3 (374 mg, 1.38 mmol, 1.0 eq). The mixture was stirred at RT for 2 h. For
most other intermediates, the stirring time could be 0.5~2.0 h. Then water was added
to quench the reaction and the DCM was removed under reduced pressure. The water
solution was extracted with EtOAc, washed with saturated brine, dried over Na2SO4,
and concentrated to give the crude product 1-(bromomethyl)-3-ethoxybenzene (14c) as
a light yellow oil (150 mg, 51% yield). All intermediates 14c-14j, 14o-14u, 14w-14x
were not characterized but used directly in the next steps.
4.5.1.2.2. General procedure for synthesis of compounds 11c-11j, 11o-11u, 11w-11x
To a solution of compound 9 (1.0 eq) dissolved in DMF (3 mL) were added Cs2CO3
(1.5 eq) and 1.0 eq of intermediates 14c-14j, 14o-14u, 14w-14x. The mixture was
stirred at 60 C overnight. Then the mixture was cooled, poured into water and extracted

with EtOAc. The organic layer was washed with saturated brine, dried over NaSO4 and
concentrated under reduced pressure. The residue was purified by silica gel
chromatography with petroleum ether/ EtOAc (1/10~1/5) to give the target
products11c-11j, 11o-11u, 11w-11x.
4.5.1.2.2.1. N-(3-Ethoxybenzyl)-3,6-dimethylbenzo[d]isoxazol-5-amine (11c).
Compound 11c was prepared from 9 and 1-(bromomethyl)-3-ethoxybenzene (14c)
according to the general procedure and isolated as a solid, in 30% yield. 1H NMR (500
MHz, CDCl3) δ 7.32 – 7.27 (m, 2H), 7.01 – 6.96 (m, 2H), 6.84 (dd, J = 8.2, 2.1 Hz,
1H), 6.63 (s, 1H), 4.36 (s, 2H), 4.04 (q, J = 7. 0 Hz, 2H), 3.84 (s, 1H), 2.49 (s, 3H), 2.31
13
(s, 3H), 1.42 (t, J = 7.0 Hz, 3H). C NMR (126 MHz, DMSO-d6) δ 158.61, 155.92,
154.02, 143.51, 141.64, 129.31, 128.61, 120.22, 119.25, 113.27, 112.39, 110.02, 97.89,
62.77, 46.89, 18.83, 14.63, 9.60. MS (ESI) m/z [M + Na]+ calcd. 319.14; found 319.3.
HPLC analysis: MeOH − H2O (85:15), 10.26 min, 95.21% purity.
4.5.1.2.2.2. 3,6-Dimethyl-N-(3-propoxybenzyl)benzo[d]isoxazol-5-amine (11d).
Compound 11d was prepared from 9 and 1-(bromomethyl)-3-propoxybenzene (14d)
according to the general procedure and isolated as a liquid in 28% yield. 1H NMR (500
MHz, CDCl3) δ 7.32 – 7.27 (m, 2H), 7.01 – 6.95 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.63
(s, 1H), 4.36 (s, 2H), 3.93 (t, J = 6.5 Hz, 2H), 3.84 (s, 1H), 2.49 (s, 3H), 2.31 (s, 3H),
1.80 (dt, J = 13.9, 7.0 Hz, 2H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-
d6) δ 159.22, 156.37, 154.46, 143.98, 142.10, 129.76, 129.06, 120.67, 119.71, 113.81,
112.94, 110.47, 98.37, 69.19, 47.37, 22.46, 19.27, 10.84, 10.04. MS (ESI) m/z [M +
Na]+ calcd. 333.39; found.333.4. HPLC analysis: MeOH − H2O (85:15), 10.95 min,
99.03% purity.
4.5.1.2.2.3. N-(3-Butoxybenzyl)-3,6-dimethylbenzo[d]isoxazol-5-amine (11e).
Compound 11e was prepared from 9 and 1-(bromomethyl)-3-butoxybenzene (14e)
according to the general procedure. 11e was isolated as a liquid in 57% yield. 1H NMR
(500 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.03 – 6.94 (m, 2H), 6.83

– 6.74 (m, 1H), 6.60 (s, 1H), 5.61 (t, J = 6.0 Hz, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.93 (t,
J = 6.5 Hz, 2H), 2.35 (s, 3H), 2.32 (s, 3H), 1.71 – 1.60 (m, 2H), 1.40 (dq, J = 14.7, 7.4
Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 158.79, 155.93,
154.02, 143.53, 141.65, 129.31, 128.61, 120.23, 119.25, 113.33, 112.49, 110.02, 97.91,
66.92, 46.91, 30.74, 18.84, 18.74, 13.67, 9.60. MS (ESI) m/z [M + Na]+ calcd. 347.17;
found 347.4. HPLC analysis: MeOH − H2O (85:15), 11.22 min, 97.93% purity.
4.5.1.2.2.4. N-(3-Isobutoxybenzyl)-3,6-dimethylbenzo[d]isoxazol-5-amine (11f).
Compound 11f was prepared from 9 and 1-(bromomethyl)-3-isobutoxybenzene (14f)
according to the general procedure and isolated as a liquid in 32% yield. 1H NMR (400
MHz, DMSO-d6) δ 7.37 (s, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.01 – 6.95 (m, 2H), 6.77 (dd,
J = 8.1, 1.6 Hz, 1H), 6.60 (s, 1H), 5.61 (t, J = 6.0 Hz, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.70
(d, J = 6.5 Hz, 2H), 2.36 (s, 3H), 2.32 (s, 3H), 2.05 – 1.90 (m, 1H), 0.96 (s, 3H), 0.94
13
(s, 3H). C NMR (126 MHz, DMSO-d6) δ 158.88, 155.92, 154.02, 143.54, 141.67,
129.32, 128.62, 120.23, 119.29, 113.44, 112.57, 110.03, 97.93, 73.60, 46.93, 27.67,
19.06 (2 × C), 18.84, 9.59. MS (ESI) m/z [M + Na]+ calcd. 347.17; found 347.4. HPLC
analysis: MeOH − H2O (85:15), 11.06 min, 96.14% purity.
4.5.1.2.2.5.
amine (11g).
Compound
N-(3-(Cyclopropylmethoxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
11g
was
prepared
from
9
and
1-(bromomethyl)-3-
(cyclopropylmethoxy)benzene (14g) according to the general procedure. Product 11g
1
was isolated as a liquid in 24% yield. H NMR (400 MHz, CDCl3) δ 7.32 – 7.27 (m,
2H), 7.01 – 6.97 (m, 2H), 6.86 – 6.83 (m, 1H), 6.62 (s, 1H), 4.36 (s, 2H), 3.85 (s, 1H),
3.81 (d, J = 6.9 Hz, 2H), 2.49 (s, 3H), 2.31 (s, 3H), 1.29 – 1.25 (m, 1H), 0.67 – 0.61 (m,
2H), 0.35 (q, J = 4.8 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 158.73, 155.92, 154.02,
143.52, 141.62, 129.29, 128.62, 120.22, 119.26, 113.41, 112.48, 110.02, 97.90, 71.81,
46.90, 18.84, 10.15, 9.60, 3.06 (2 × C). MS (ESI) m/z [M + Na]+ calcd. 345.16; found
345.9. HPLC analysis: MeOH − H2O (85:15), 11.18 min, 96.02% purity.

4.5.1.2.2.6.
N-(3-(Cyclopentylmethoxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11h).
Compound 11h was prepared from
9
and 1-(bromomethyl)-3-(cyclo-
pentylmethoxy)benzene (14h) according to the general procedure. The product 11h was
isolated as a liquid in 35% yield. 1H NMR (400 MHz, CDCl3) δ 7.33 – 7.27 (m, 2H),
7.01 – 6.96 (m, 2H), 6.88 – 6.83 (m, 1H), 6.63 (s, 1H), 4.35 (s, 2H), 3.87 – 3.80 (m,
3H), 2.49 (s, 3H), 2.41 – 2.33 (m, 1H), 2.31 (s, 3H), 1.88 – 1.79 (m, 2H), 1.70 – 1.58
(m, 4H), 1.41 – 1.32 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 158.91, 155.92, 154.02,
143.53, 141.64, 129.31, 128.62, 120.23, 119.28, 113.44, 112.55, 110.03, 97.93, 71.46,
46.92, 38.54, 29.01 (2 × C), 24.93 (2 × C), 18.85, 9.60. MS (ESI) m/z [M + Na]+ calcd.
373.19; found 373.4. HPLC analysis: MeOH − H2O (85:15), 11.70 min, 97.54% purity.
4.5.1.2.2.7.
N-(3-(Cyclohexylmethoxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11i).
Compound 11i was prepared from 9 and 1-(bromomethyl)-3-(cyclohexyl-
methoxy)benzene (14i) according to the general procedure. The product 11i was
isolated as a liquid in 53% yield. 1H NMR (500 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.20
(t, J = 8.1 Hz, 1H), 6.99 – 6.95 (m, 2H), 6.76 (d, J = 7.4 Hz, 1H), 6.60 (s, 1H), 5.61 (t,
J = 5.9 Hz, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.73 (d, J = 6.4 Hz, 2H), 2.35 (s, 3H), 2.32 (s,
3H), 1.80 – 1.73 (m, 2H), 1.72 – 1.65 (m, 3H), 1.66 – 1.60 (m, 1H), 1.25 – 1.13 (m,
3H), 1.04 – 0.94 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 158.92, 155.93, 154.02,
143.53, 141.65, 129.31, 128.62, 120.23, 119.25, 113.32, 112.58, 110.03, 97.93, 72.57,
46.90, 37.04, 29.28 (2 × C), 26.04, 25.26 (2 × C), 18.85, 9.61. MS (ESI) m/z [M + Na]+
calcd. 387.20; found 387.5. HPLC analysis: MeOH − H2O (85:15), 12.52 min, 95.38%
purity.
4.5.1.2.2.8. N-(3-(Benzyloxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-amine (11j).
Compound 11j was prepared from 9 and 1-(benzyloxy)-3-(bromomethyl)benzene (14j)
according to the general procedure and isolated as a liquid in 51% yield. 1H NMR (400
MHz, DMSO-d6) δ 7.43 – 7.39 (m, 2H), 7.38 – 7.28 (m, 4H), 7.23 (t, J = 7.9 Hz, 1H),

7.07 (s, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 8.1, 1.8 Hz, 1H), 6.59 (s, 1H), 5.60
(t, J = 5.9 Hz, 1H), 5.07 (s, 2H), 4.36 (d, J = 5.8 Hz, 2H), 2.35 (s, 3H), 2.32 (s, 3H). 13C
NMR (126 MHz, DMSO-d6) δ 158.50, 155.95, 154.06, 143.51, 141.77, 137.12, 129.37,
128.64, 128.39 (2 × C), 127.78, 127.66 (2 × C), 120.24, 119.62, 113.69, 112.85, 110.04,
97.89, 69.09, 46.89, 18.85, 9.63. MS (ESI) m/z [M + Na]+ calcd. 381.16; found 381.4.
HPLC analysis: MeOH − H2O (85:15), 11.69 min, 95.02% purity.
4.5.1.2.2.9.
N-(3-((3-Fluorobenzyl)oxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11o).
Compound 11o was prepared from
9
and 1-(bromomethyl)-3- ((3-
fluorobenzyl)oxy)benzene (14o) according to general procedure and isolated as a liquid
in 33% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.43 – 7.35 (m, 2H), 7.27 – 7.21 (m,
3H), 7.15 – 7.09 (m, 1H), 7.07 (s, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 8.0, 1.8
Hz, 1H), 6.57 (s, 1H), 5.61 (t, J = 5.9 Hz, 1H), 5.10 (s, 2H), 4.36 (d, J = 5.8 Hz, 2H),
2.35 (s, 3H), 2.31 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 163.14, 161.20, 158.26,
155.95, 154.04, 143.48, 141.83, 140.15, 140.09, 130.45, 130.38, 129.40, 128.63,
123.42, 123.40, 120.22, 119.78, 114.57, 114.40, 114.20, 114.03, 113.68, 112.88,
110.04, 97.88, 68.21, 46.87, 18.83, 9.61. MS (ESI) m/z [M + Na]+ calcd. 399.15; found
399.3. HPLC analysis: MeOH − H2O (95:5), 11.98 min, 95.89% purity.
4.5.1.2.2.10.
N-(3-((4-Fluorobenzyl)oxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11p).
Compound 11p was prepared from
9
and 1-(bromomethyl)-3-((4-
fluorobenzyl)oxy)benzene (14p) according to the general procedure. The product was
1
isolated as a white solid in 15% yield. H NMR (400 MHz, DMSO-d6) δ 7.49 – 7.41
(m, 2H), 7.37 (s, 1H), 7.26 – 7.21 (m, 1H), 7.20 – 7.13 (m, 2H), 7.06 (s, 1H), 7.01 (d, J
= 7.2 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.58 (s, 1H), 5.67 – 5.56 (m, 1H), 5.05 (s, 2H),
4.36 (d, J = 4.9 Hz, 2H), 2.35 (s, 3H), 2.32 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
162.66, 160.73, 158.37, 155.93, 154.01, 143.48, 141.76, 133.33, 133.31, 129.90,
129.83, 129.36, 128.62, 120.22, 119.66, 115.26, 115.09, 113.65, 112.84, 110.02, 97.86,

68.33, 46.86, 18.82, 9.60. MS (ESI) m/z [M + Na]+ calcd. 399.15; found 399.3. HPLC
analysis: MeOH − H2O (95:5), 11.98 min, 95.44% purity.
4.5.1.2.2.11.
N-(3-((2-Fluorobenzyl)oxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11q).
Compound 11q was prepared from 9 and 1-((3-(bromomethyl)- phenoxy)methyl)-2-
fluorobenzene (14q) according to the general procedure. The product was isolated as a
liquid in 47% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.54 – 7.48 (m, 1H), 7.42 – 7.35
(m, 2H), 7.28 – 7.16 (m, 3H), 7.08 (s, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.89 (dd, J = 8.0,
1.8 Hz, 1H), 6.59 (s, 1H), 5.61 (t, J = 5.8 Hz, 1H), 5.11 (s, 2H), 4.37 (d, J = 5.7 Hz,
13
2H), 2.35 (s, 3H), 2.32 (s, 3H). C NMR (126 MHz, DMSO-d6) δ 161.33, 159.37,
158.32, 155.95, 154.05, 143.49, 141.86, 130.62, 130.59, 130.35, 130.28, 129.43,
128.64, 124.48, 124.45, 120.23, 119.84, 115.44, 115.27, 113.56, 112.80, 110.04, 97.89,
63.36, 63.34, 46.85, 18.84, 9.61. MS (ESI) m/z [M + Na]+ calcd. 399.15; found 399.3.
HPLC analysis: MeOH − H2O (95:5), 10.78 min, 98.60% purity.
4.5.1.2.2.12.
N-(3-((3-Chlorobenzyl)oxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-
amine (11r).
Compound 11r was prepared from 9 and 1-(bromomethyl)-3-((3-chloro-
benzyl)oxy)benzene (14r) according to the general procedure. The product was isolated
1
as a liquid in 13% yield. H NMR (400 MHz, DMSO-d6) δ 7.47 (s, 1H), 7.40 – 7.33
(m, 4H), 7.24 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 7.01 (d, J = 7.6 Hz, 1H), 6.87 (dd, J =
8.0, 2.1 Hz, 1H), 6.57 (s, 1H), 5.60 (t, J = 5.9 Hz, 1H), 5.09 (s, 2H), 4.36 (d, J = 5.8 Hz,
13
2H), 2.35 (s, 3H), 2.31 (s, 3H). C NMR (126 MHz, DMSO-d6) δ 158.21, 155.93,
153.99, 143.45, 141.81, 139.74, 133.06, 130.27, 129.38, 128.60, 127.62, 127.13,
126.01, 120.20, 119.79, 113.68, 112.86, 110.01, 97.88, 68.14, 46.88, 18.79, 9.58. MS
(ESI) m/z [M + Na]+ calcd. 415.12; found 414.9. HPLC analysis: MeOH − H2O (95:5),
10.19 min, 95.47% purity.
4.5.1.2.2.13.
N-(3-((2-Chlorobenzyl)oxy)benzyl)-3,6-dimethylbenzo[d]isoxazol-5-