Design and synthesis of tranylcypromine derivatives as novel LSD1/HDACs dual inhibitors for cancer treatment

Article abstract of DOI:10.1016/j.ejmech.2017.09.038

Lysine specific demethylase 1 (LSD1) and Histone deacetylases (HDACs) are promising drug targets for cancers. Recent studies reveal an important functional interplay between LSD1 and HDACs, and there is evidence for the synergistic effect of combined LSD1 and HDAC inhibitors on cancers. Therefore, development of inhibitors targeting both LSD1 and HDACs might be a promising strategy for epigenetic therapy of cancers. We report herein the synthesis of a series of tranylcypromine derivatives as LSD1/HDACs dual inhibitors. Most compounds showed potent LSD1 and HDACs inhibitory activity, especially compound 7 displayed the most potent inhibitory activity against HDAC1 and HDAC2 with IC₅₀ of 15 nM and 23 nM, as well as potent inhibition against LSD1 with IC₅₀ of 1.20 μM. Compound 7 demonstrated stronger anti-proliferative activities than SAHA with IC₅₀ values ranging from 0.81 to 4.28 μM against MGC-803, MCF-7, SW-620 and A-549 human cancer cell lines. Further mechanistic studies showed that compound 7 treatment in MGC-803 cells dose-dependently increased cellular H3K4 and H3K9 methylation, as well as H3 acetylation, decreased the mitochondrial membrane potential and induced remarkable apoptosis. Docking studies showed that compound 7 can be well docked into the active binding sites of LSD1 and HDAC2. This finding highlights the potential for the development of LSD1/HDACs dual inhibitors as novel anticancer drugs.

Full text of DOI:10.1016/j.ejmech.2017.09.038

Accepted Manuscript
Design and synthesis of tranylcypromine derivatives as novel LSD1/HDACs dual
inhibitors for cancer treatment
Ying-Chao Duan, Yong-Cheng Ma, Wen-Ping Qin, Li-Na Ding, Yi-Chao Zheng, Ying-
Li Zhu, Xiao-Yu Zhai, Jing Yang, Chao-Ya Ma, Yuan-Yuan Guan
PII:
S0223-5234(17)30752-3
10.1016/j.ejmech.2017.09.038
EJMECH 9753
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2017
Revised Date: 18 September 2017
Accepted Date: 19 September 2017
Please cite this article as: Y.-C. Duan, Y.-C. Ma, W.-P. Qin, L.-N. Ding, Y.-C. Zheng, Y.-L. Zhu, X.-Y.
Zhai, J. Yang, C.-Y. Ma, Y.-Y. Guan, Design and synthesis of tranylcypromine derivatives as novel
LSD1/HDACs dual inhibitors for cancer treatment, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and synthesis of tranylcypromine derivatives as novel
LSD1/HDACs dual inhibitors for cancer treatment
Ying-Chao Duana*, Yong-Cheng Mab, Wen-Ping Qina, Li-Na Dingc, Yi-Chao Zhengc
Ying-Li Zhub, Xiao-Yu Zhaia, Jing Yangc, Chao-Ya Mac, Yuan-Yuan Guana**
a School of Pharmacy, Xinxiang Medical University, Xinxiang, Henan 453003, China.
bDepartment of Pharmacy, Zhengzhou University People’s Hospital, No. 7, Wei Wu Road,
Zhengzhou, Henan,China
cKey Laboratory of Advanced Drug Preparation Technologies(Zhengzhou University ) , Ministry
of Education; Collaborative Innovation Center of New Drug Research and Safety Evaluation,
Henan Province; Key Laboratory of Henan Province for Drug Quality and Evaluation; School of
Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001,
China.
* Corresponding authors
E-mail address:duanyingchao1986@163.com (Y.C. Duan)
E-mail address:131011@xxmu.edu.cn (Y.Y. Guan)
Abstract
Lysine specific demethylase 1 (LSD1) and Histone deacetylases (HDACs) are
promising drug targets for cancers. Recent studies reveal an important functional
interplay between LSD1 and HDACs, and there is evidence for the synergistic effect
of combined LSD1 and HDAC inhibitors on cancers. Therefore, development of
inhibitors targeting both LSD1 and HDACs might be a promising strategy for
epigenetic therapy of cancers. We report herein the synthesis of a series of
tranylcypromine derivatives as LSD1/HDACs dual inhibitors. Most compounds
showed potent LSD1 and HDACs inhibitory activity, especially compound 7
displayed the most potent inhibitory activity against HDAC1 and HDAC2 with IC50
of 15 nM and 23 nM, as well as potent inhibition against LSD1 with IC50 of 1.20 µM.
Compound 7 demonstrated stronger anti-proliferative activities than SAHA with IC50
values ranging from 0.81 to 4.28 µM against MGC-803, MCF-7, SW-620 and A-549
human cancer cell lines. Further mechanistic studies showed that compound 7
treatment in MGC-803 cells dose-dependently increased cellular H3K4 and H3K9
methylation, as well as H3 acetylation, decreased the mitochondrial membrane
potential and induced remarkable apoptosis. Docking studies showed that compound
7 can be well docked into the active binding sites of LSD1 and HDAC2. This finding
highlights the potential for the development of LSD1/HDACs dual inhibitors as
novel anticancer drugs.
Key words: Lysine-specific demethylase 1; Histone deacetylases; dual inhibitor;
synthesis
1. Introduction
Diverse epigenetic abnormalities have been directly implicated in the origin,
development and metastasis of cancers [1]. Over the past decade, epigenetic therapies
for cancer treatment have attracted extensive attention of researchers and clinicians as
they provide alternative therapeutic options [2-4]. Epigenetic regulators like lysine
specific demethylase1(LSD1) and histone deacetylases (HDACs) are being
increasingly used as targets for chemotherapeutic intervention in cancers [5].

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LSD1 was the first identified histone demethylase in 2004 [6]. As a component
of the CoREST corepressor complex, LSD1 specifically catalyses the demethylation
of mono- and di-methylated histone H3 lysine 4 (H3K4) and represses gene
transcription [7]. While in the presence of the androgen receptor, it specifically
removes mono- or di-methylated H3 lysine 9 (H3K9) and activates gene transcription
[8]. LSD1 also demethylates non-histone protein, such as p53 [9], DNA
methyltransferases [10], E2F transcription factor 1 (E2F1) [11]and regulates their
cellular functions. High level expression of LSD1 was frequently found in
malignancies [12]and is associated with development, progression and poor clinical
outcome in cancers of the lung [13], acute myeloid leukemia [14, 15], gallbladder [16],
colon [17] and breast cancers [18]. Downregulation or inhibition of LSD1 was shown
to re-express the epigenetically silenced tumor suppressor genes and inhibit cancer
cell proliferation and metastasis [19, 20]. Therefore, LSD1 is regarded as a promising
drug target for cancer intervention [21, 22]. A number of LSD1 inhibitors (LSD1i)
with various scaffolds, including cyclopropylamine (1, 2), amidoxime(3) [23],
pyridine(4) [24], benzohydrazide(5) [25], thieno[3,2-b]pyrrole(6) [26]and others have
been developed [27-30](Fig. 1). We have also contributed to the field with the
identification of two novel types of LSD1i [31-33]. The most widely studied and
potent LSD1i are cyclopropylamine containing compounds, derived from
tranylcypromine (2-PCPA) [34, 35], and two of these compounds ORY-1001 (1) and
GSK2879552(2), are in clinical development for the treatment of acute myeloid
leukemia [35-37].
HDACs are enzymes responsible for removing the acetyl group from acetylated
lysine residues located on histone as well as nonhistone proteins [38, 39]. Such
posttranslational modifications are crucial for the regulation of gene expression and
cellular processes, including apoptosis, cell cycle, growth and differentiation [40].
The HDAC family consists of 18 proteins, which can be grouped into four different
classes based on their sequence homology: class I (HDACs 1-3 and 8), class II
(HDACs 4, 5, 6, 7, 9, 10), and class IV (HDAC 11) are all zinc-dependent
deacetylases, and class III called Sirtuins (Sirtuins1-7) are the NAD+-dependent
HDACs [41]. Abnormal expression of HDACs has been implicated in the initiation
and progression of diverse cancers [42, 43]. Histone deacetylase inhibition has proven
to be a valuable epigenetic strategy for cancer treatment [44]. Five HDACs inhibitors
(HDACi) (SAHA [45], romidepsine [46], belinostat [47], panobinostat [48] and
chidamide [49]) have been approved by FDA (Fig. 2), and several other HDACi are
currently in different phases of clinical development [50, 51]. HDACi have also been
combined with other cancer therapeutics including those targeting DNMT[52], Bcl-2
[53], EGFR [54]or conventional chemotherapeutic agents, such as paclitaxel,
gemcitabine, fluorouracil and platinum compounds to treat cancers[55], and showed
more effective anticancer activities, many different combination strategies are at
various stages of clinical trials against various cancers[56, 57].
Recent studies have revealed that there are intimate functional link between the
LSD1 and HDACs. LSD1 and HDACs are found in the same cellular complexes, for
example, HDAC1, HDAC2 and LSD1 are the integral subunits of CoREST and
NuRD corepressor complexes [7, 58]. HDAC1 deacetylates LSD1 at K374 in the
substrate binding lobe, which affects the histone 3 binding and gene expression
activity of LSD1 [59]. Overexpression of HDAC5 stabilizes LSD1 protein and
decreases the level of H3K4 methylation, whereas loss of HDAC5 diminishes LSD1
protein stability and demethylation activity [60]. HDACi increases H3K4 methylation
via transcriptional repression of histone demethylases [61, 62], while inhibition of

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LSD1 activity leads to enhanced histone acetylation and sensitizes cancer cells to
HDACi-induced apoptosis [63]. HDACi and LSD1i have been tested as combination
anticancer agents and showed synergistic effect in human breast cancer [64, 65],
AML [66] and glioblastoma multiforme [63, 67].
Both LSD1 and HDACs are overexpressed in many human cancers, resulting in
aberrant silencing of tumor suppressor genes, inhibition of their activity can prevent
cancer growth, migration and invasion. More importantly, simultaneous inhibition of
LSD1 and HDACs exhibits synergistic anticancer activity. Therefore, there is a great
interest in developing inhibitors targeting both LSD1 and HDACs for epigenetic
therapy of cancers. Herein, a series of tranylcypromine derivatives with
pharmacophore characteristics of HDACi were synthesized and evaluated as
LSD1/HDACs dual inhibitors.
2. Results and discussion
2.1. Chemistry
Compounds 1-7 and 8-11 were synthesized using the routes described in Schemes
1-2. A commercially available trans-2-phenylcyclopropylamine hydrochloride was
reacted with (Boc)2O in dry dichloromethane to give compound 1. Compound 1 was
treated
with
methyl
4-(bromomethyl)benzoate
or
methyl
3-(4-
bromomethyl)cinnamate in dry DMF, in the presence of NaH, to give the 2 and 8,
which were then hydrolyzed with Lithium hydroxide to afford compounds 3 and 9,
respectively. Condensation of 3 or 9 with the corresponding amino-compounds in dry
DMF at room temperature in the presence of HBTU provided compounds 4 and 10,
respectively. The target compounds 5a-b and 11a-b were finally obtained by de-
protection of 4a-b and 10a-b with trifluoroacetic acid in dichloromethane. For the
synthesis of hydroxamate compound 7, compound 4c was treated with hydroxylamine
in the presence of KOH to produce 6, which was de-protected with TFA to generate
compound 7.
2.2. In vitro LSD1, MAO-A and MAO-B inhibition
All the synthesized target compounds and SAHA were tested for their inhibitory
activity against LSD1 in vitro. The data are summarized in Table 1. 2-PCPA was
chosen as positive control. As shown in Table 1, all of the compounds with the
exception of exhibited moderate to potent inhibitory activities against LSD1 with IC50
values ranging from 1.20 to 7.16 µM, more potent than 2-PCPA, while SAHA
showed no inhibition against LSD1. Among them, compounds 7 (IC50 = 1.20 µM)
showed the most potent anti-LSD1 activities, which are 25 times higher than that of 2-
PCPA. As LSD1 belongs to the FAD-dependent monoamine oxidases family
including MAO-A and MAO-B, we also analyzed the inhibitory effects of synthesized
compounds against MAO-A and MAO-B to assess their selectivity, clorgyline and R-
(-)-deprenyl were chosen as positive control for MAO-A and MAO-B, respectively.
All the compounds displayed weak MAO-A inhibitory activities, while had no
significant effects on MAO-B activities. The results showed that these compounds
exhibited better selectivity for LSD1 over MAO A and MAO B, compared to 2-PCPA.
2.3. In vitro HDAC inhibition
To explore the characteristics of target compounds on HDAC inhibition, the
inhibitory effects against selected recombinant HDACs-HDAC1, HDAC2 and

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HDAC5, which are typically found in association with LSD1, were determined by a
fluorescence-based assay. SAHA and TMP269 were used as positive controls. The
data are showed as IC50 values in Table 2 below. All target compounds showed good
to excellent inhibitory activities against HDAC1 and HDAC2, while had no effect on
HDAC5 except for compound 7 and 11a, which were found to be moderately active
with IC50 values in micromolar range. Compounds 7 and 11a displayed the most
potent HDAC1/2 inhibitory activity with IC50 values 12, 15, 25, and 23 nM,
respectively, similar to that of SAHA.
2.4. In vitro anti-proliferative activity
All synthesized target compounds together with the reference compound SAHA
were explored for their anti-proliferative activity in five different cancer cell lines,
human gastric cancer cells (MGC-803), human breast cancer cell (MCF-7), human
colorectal cancer cell (SW-620), human prostate cancer cell (PC-3) and human lung
cancer cell (A-549). The results are summarized in Table 3. All synthesized
compounds manifested evident antiproliferative activities against five cancer cell lines.
Compounds 7 and 11a displayed potent broad-spectrum growth inhibitory activities
against all the tested cell lines with IC50 ranging from 0. 81 µM to 5.48 µM and 1.41
µM to 6.24 µM, respectively. Compound 7 showed higher antiproliferative activities
than SAHA against all tested five human cancer cell lines with the exception of PC-3.
Compound 7 proved to be 10-fold more potent than SAHA in the case of MGC-803.
2.5. Western blot analysis
To determine whether target compounds are cell-active LSD1/HDACs dual
inhibitors, the effects of selected compound 7 on the methylation levels of LSD1
substrates H3K4 and H3K9 and acetylation levels of H3, the biomarkers of HDACs
inhibition, were analyzed. After treatment of MGC-803 cells for 24 h with compound
7 at different concentrations (0, 1.0 and 2.0 µM), the amounts of H3K4me2 and
H3K9me2 were dose dependently elevated, while the levels of H3K4me3 was not
affected. These results validated that compound 7 is cell-active LSD1 inhibitor (Fig.
3A). As expected, exposure to compound 7 also induced a marked acetylation of
histone H3 in a dose-dependent manner, suggesting that compound 7 is an effective
HDAC inhibitor. It is noteworthy that while the in vitro HDACs inhibitory activity of
compound 7 was comparable to that of SAHA, compound 7-induced acetylated
histone H3 was more evident (Fig. 3B). These results revealed that compound 7 can
inhibit LSD1 and HDACs in cells, which were consistent with its strong inhibitory
activities against LSD1, HDACs and cancer cells.
2.6. Apoptotic assay
Flow cytometry assay was performed to evaluate the ability of compound 7 to
induce apoptosis in MGC-803 cells. Treatment in MGC-803 cells with compound 7 at
different concentrations (0, 2.5, 5.0, 7.5 µM) for 24 h, resulted in 4.82%, 26.77%,
37.20% and 42.60% cells apoptosis, respectively (Fig. 4A), whereas after treatment
with compound 7 (0, 2.5, 5.0, 7.5 µM) for 48 h, the percentage of apoptotic cells were
7.47%, 72.40%, 85.9% and 91.0%, respectively (Fig. 4B). The results showed that
compound 7 markedly increased the cellular apoptosis in a concentration and time-
dependent manner, which might result from concerted inhibition of LSD1 and
HDACs activity.

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2.7. Analysis of mitochondrial membrane potential
Loss of mitochondrial membrane potential (MMP) plays an important role in the
apoptotic process and is lethal to the cells. The remarkable apoptosis induced by
compound 7 led us to investigate whether this compound had an effect on the MMP.
After MGC-803 cells were treated with different concentrations of compound 7 (0,
2.5, 5.0, 7.5 µM) for 48 h, the polarization of the MMP decreased remarkably as
compared with the control group in a dose-dependent manner (Fig.5). The results
showed that compound 7-induced cellular apoptosis might be mediated via the
mitochondrial pathway.
2.8. Docking study
In order to investigate the binding model of compound 7 and LSD1, we carried
out molecular docking study adopting the software MOE 2015.10. A co-structure
figure of the N-substituted tranylcypromine derivate (GSK2699537) with LSD1
which covalently binds to FAD-C4a (Fig. 6A) has been reported in previous study
[68], but the X-ray structure is not released in the Protein Data Bank. In this study,
compound 7 is a N-substituted tranylcypromine derivative as well as GSK2699537, so
we speculate that they have the similar binding mode. Here, the protein LSD1 (PDB
ID: 2V1D) containing a free cofactor FAD, H3K4 mimetic peptide as substrate and
CoREST as corepressor was selected as the docking receptor. The covalent docking
was not supported in the present version of MOE 2015.10, therefore, we used the non-
covalent docking method to mimic covalent docking process and the position
occupied by substrate was selected as the docking site on the basis of hypothesis of
covalently inhibitory mechanism [69] in this study.
In the present study, compound 7 can be successfully docked into the substrate-
binding site. Among the top 20 score’s conformers, there are two main type
conformations. The first type is the one adopting a similar pose to the adduct formed
by GSK2699537 with FAD-C4a which occupies more than half of the docking
conformations. And all the other docking conformations appears the same type called
as the second type which is the one appearing the opposite pose to GSK2699537.
Based on the hypothesis of covalently inhibitory mechanism, the first type
conformation is considered in the study. Here, the docking pose with the highest score
is shown in Fig. 6B. The α-C atom of cyclopropylamine moiety situates at a distance
of 3.50 Å to FAD-C4a and the N atom of cyclopropylamine moiety is located 5.0Å
from the Flavin, according to the covalent inhibitor hypothesis, which tends to form
the covalent bond between FAD-C4a and cyclopropane. Meanwhile, hydroxamic acid
carbonyl forms a hydrogen bond with Gln358, amine of amide forms a polar contact
with Asp556, and amine of tranylcypromine forms salt-bridge with Ala809. Alkyl
chain is buried into a hydrophobic pocket surrounded by Phe538, Leu693, Leu536,
Phe382, Trp531, Leu677 and Trp695. In addition, the phenyl group connecting with
amide is located in the hydrophobic regions which formed by Phe538, Ala539,
Tyr761and Ala809, while the cyclopropylamine moiety extensively forms
hydrophobic interactions with flavin ring, His564, Val333, Thr335, Thr310, Phe538
and Trp695. All these interactions indicate that compound 7 could well dock into the
substrate-binding pocket of LSD1.
Compound 7 was also docked into the active site of HDAC2 to explain its potent
inhibitory activity (23nM). Due to compound 7 is quite similar to Vorinostat (SAHA)
which was published by Lauffer et al [70], 4LXZ was selected as the docking receptor.
The docking conformation with the highest score indicates that compound 7 fits the

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active site appropriately (Fig. 7A, B). The catalytic Zn2+ ion situated at the bottom of
the active site could form coordinate bonds with Asp181, His183, and Asp269 as well
as the carbonyl oxygen of the hydroxamate of compound 7. Meanwhile, the carbonyl
oxygen and amine of the hydroxamic acid moiety have a hydrogen-bond interaction
with Tyr308, His146, respectively. The alkyl linker group is located in the narrow
lipophilic tube formed by Gly154, Phe155, His183, Phe210 and Leu276, and the
amine nitrogen forms a polar contact with Asp104. The cap group can be well
accommodated in the hydrophobic groove on the rim of active site of HDAC2
(Fig.7B). Besides, the relative long cap group is stabilized by the salt-bridge through
amine with Glu103.
2.9 Theoretical evaluation of ADMET properties
Computer predictions of absorption, distribution, metabolism, elimination and
toxicity (ADMET) properties of the target compounds was performed utilizing the
molinspiration property calculator and preADMET. The results are presented in Table
4. All the tested compounds followed Lipinski’s rule of 5, indicating that these
compounds would not be expected to cause problems with oral bioavailability.
Additionally, all the evaluated compounds showed TPSA range 61.4-90.2 Ų (<140 Ų),
suggesting good permeability and transport of the compounds in the cellular plasma
membrane. The studied compounds are found to be non-carcinogenicity and non-
inhibitors of CYP-3A4 and CYP-2C19, which are important enzymes involved in drug
metabolism.
3. Conclusions
In conclusion, a series of tranylcypromine derivatives with pharmacophore
characteristics of HDACi have been designed, synthesized and evaluated as dual
inhibitors against LSD1 and HDACs. Compound 7 displayed the most potent
inhibitory activity against HDAC1 and HDAC2 with IC50 of 15 nM and 23 nM,
respectively. It also showed potent inhibition against LSD1 with IC50 of 1.20 µM.
Western blotting showed that compound 7 dose-dependently increased the amount of
H3ac, H3K4me2 and H3K9me2 in MGC-803, as expected with LSD1/HDACs dual
inhibitors. In vitro cell growth inhibition assays indicated that compound 7 possessed
potent antiproliferative activity with IC50 values of 0.81~5.48 µM against five cancer
cell lines, and is more potent than SAHA. Compound 7 could induce apoptosis of
MGC-803 cells accompanied with decrease of the MMP. Molecular docking of the
compound 7 into the active binding sites of LSD1 and HDAC2 was performed and the
result suggested that compound 7 could bind well with these two sites. These data
support further studies for the rational design of more efficient LSD1/HDACs dual
inhibitors for cancer treatment.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
Reagents and solvents were purchased from commercial sources, when necessary,
were purified and dried by standard methods. Melting points were determined on an
X-5 micromelting apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance III HD 400 MHz and 100 MHz spectrometer at room
temperature, using TMS as an internal standard. Chemical shifts were reported in ppm
(δ). Spin multiplicities were described as s (singlet), d (doublet), dd (double doublet),
t (triplet), br (broad signal), or m (multiplet). Coupling constants were reported in

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hertz (Hz). High resolution mass spectrometry (HRMS) was recorded on a Bruker
MicrOTOF-Q III Micro mass spectrometer by electrospray ionization (ESI).
4.1.2. General procedure for synthesis of compounds 2 and 8.
To a stirred solution of compound 1 (1.0 equiv) in dry DMF was added NaH (3.0
equiv) and methyl 4-(bromomethyl)benzoate (1.1 equiv) or methyl 3-(4-
bromomethyl)cinnamate (1.1 equiv) at 0oC under N2. The above mixture was stirred
for 0.5 h at room temperature. The mixture was diluted with EtOAc and the organic
phase was washed with water and brine, dried with anhydrous Na2SO4, filtered,
concentrated under reduced pressure, and purified by flash column chromatography
on silica gel, to afford the pure product 2 and 8.
4.1.2.1. methyl 4-(((tert-butoxycarbonyl)(2-phenylcyclopropyl)amino)methyl)
benzoate (2)
1
Colorless oil, Yield: 67.3%. H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.4 Hz, 2H),
7.30 (d, J = 8.0 Hz, 2H), 7.25-7.20 (m, 2H), 7.17-7.13 (m, 1H), 7.03 (d, J = 7.6 Hz,
2H), 4.64 (d, J = 16.0 Hz, 1H), 4.44 (d, J = 16.0 Hz, 1H), 3.91 (s, 3H), 2.71-2.68 (m,
1H), 2.16-2.11 (m, 1H), 1.42 (s, 9H), 1.31-1.25 (m, 1H), 1.18-1.14 (m, 1H). HRMS
(ESI) calcd for C23H27NNaO4 [M + Na]+: 404.1832, Found: 404.1833.
4.1.2.2. (E)-methyl 3-(4-(((tert-butoxycarbonyl)(2-phenylcyclopropyl)amino)methyl)
phenyl) acrylate (8)
Colorless oil, Yield: 71.1%. 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 16.0 Hz, 1H),
7.47 (d, J = 8.0 Hz, 2H), 7.27-7.21 (m, 4H), 7.17-7.13 (m, 1H), 7.04 (d, J = 7.2 Hz,
2H), 6.42 (d, J = 16.0 Hz, 1H), 4.61 (d, J = 14.8 Hz, 1H), 4.39 (d, J = 14.8 Hz, 1H),
3.82 (s, 3H), 2.69-2.67 (m, 1H), 2.16-2.12 (m, 1H), 1.43 (s, 9H), 1.31-1.22 (m, 1H),
1.20-1.16 (m, 1H). HRMS (ESI) calcd for C25H29NNaO4 [M + Na]+: 430.1989, Found:
430.1990.
4.1.3. General procedure for synthesis of compounds 3 and 9.
Lithium hydroxide (5.0 equiv) was added to a solution of compound 3 or 9 in THF
-H2O (1:1) and stirred at room temperature for 8-11 h. The mixture pH was adjusted
to 2-3 by the addition of 1.0 M hydrochloric acid, and the resultant precipitate was
collected by filtration and dried to give compounds 3 and 9, which was used in the
next reaction without further purification.
4.1.3.1. 4-(((tert-butoxycarbonyl)(2-phenylcyclopropyl)amino)methyl)benzoic acid
(3)
White solid, Yield: 86.7%, Mp:135-136oC. 1H NMR (400 MHz, DMSO-d6) δ 12.84
(br, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.25-7.21 (m, 2H), 7.14
(t, J = 7.2 Hz, 1H),7.08 (d, J = 7.2 Hz, 2H), 4.60 (d, J = 16.0 Hz, 1H), 4.41 (d, J =
16.0 Hz, 1H), 2.68-2.61 (m, 1H), 2.19-2.14 (m, 1H), 1.34-1.27 (m, 10H), 1.19-1.13
(m, 1H). HRMS (ESI) calcd for C22H25NNaO4 [M + Na]+: 390.1676, Found:
390.1677.
4.1.3.2. (E)-3-(4-(((tert-butoxycarbonyl)(2-phenylcyclopropyl)amino)methyl)phenyl)
acrylic acid (9)
White solid, Yield: 84.9%, Mp: 146-147oC. 1H NMR (400 MHz, DMSO) δ 12.40 (br,
1H), 7.66 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 16.0 Hz, 1H), 7.27-7.20 (m, 4H), 7.14 (t, J

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= 7.2 Hz, 1H), 7.06 (d, J = 7.2 Hz, 2H), 6.51 (d, J = 16.0 Hz, 1H), 4.56 (d, J = 16.0
Hz, 1H), 4.37 (d, J = 16.0 Hz, 1H), 2.63-2.59 (m, 1H), 2.17-2.12 (m, 1H), 1.35-1.23
(m, 10H), 1.18-1.13 (m, 1H). HRMS (ESI) calcd for C24H27NNaO4 [M + Na]+:
416.1832, Found: 416.1831.
4.1.4. General procedure for synthesis of compounds 4 and 10.
Triethylamine (3.0 equiv) was added to a solution of compounds 3 (1.0 equiv) or 9
(1.0 equiv) and HBTU (1.5 equiv) in dry DMF at 0oC under nitrogen. After the
mixture was stirred for 0.5 h, appropriate amino-compounds (1.5 equiv) was added
and stirred for additional 12 h at room temperature. The mixture was diluted with
EtOAc and the organic phase was washed with water and brine, dried over anhydrous
Na2SO4, concentrated under reduced pressure, and purified by flash column
chromatography on silica gel, to afford the pure product 4 and 10.
4.1.4.1. tert-butyl 4-(hydroxycarbamoyl)benzyl(2-phenylcyclopropyl)carbamate (4a)
Yellowish solid, Yield: 54.6%, Mp:185-186oC.1H NMR (400 MHz, DMSO-d6) δ
11.18 (s, 1H), 9.03 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.25-
7.21 (m, 2H), 7.14 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 7.2 Hz, 2H), 4.57 (d, J = 16.4 Hz,
1H), 4.38 (d, J = 16.4 Hz, 1H), 2.65-2.61 (m, 1H), 2.18-2.12 (m, 1H), 1.34-1.23 (m,
10H), 1.18-1.11 (m, 1H). HRMS (ESI) calcd for C22H26N2NaO4 [M+Na]+: 405.1785,
Found: 405.1787.
4.1.4.2. tert-butyl4-((2-((tert-butoxycarbonyl)amino)phenyl)carbamoyl)benzyl(2-
phenylcyclopropyl)carbamate (4b)
White solid, Yield: 69.1%, Mp:68-69oC. 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s,
1H), 8.72 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.55-7.51 (m, 2H), 7.37 (d, J = 8.0 Hz,
2H), 7.26-7.08 (m, 7H), 4.62 (d, J = 16.0 Hz, 1H), 4.44 (s, J = 16.0 Hz, 1H), 2.66-
2.60 (m, 1H), 2.20-2.14 (m, 1H), 1.44 (s, 9H), 1.36-1.23 (m, 10H), 1.23-1.15 (m, 1H).
HRMS (ESI) calcd for C33H39N3NaO5 [M+Na]+: 580.2782, Found: 580.2783.
4.1.4.3. methyl 7-(4-(((tert-butoxycarbonyl)(2-phenylcyclopropyl)amino)methyl)
benzamido)heptanoate (4c)
Colorless oil, Yield 61.2%. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (t, J = 5.6 Hz, 1H),
7.79 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.25-7.21 (m, 2H), 7.14 (t, J = 7.6
Hz, 1H), 7.07 (d, J = 7.6 Hz, 2H), 4.57 (d, J = 16.0 Hz, 1H), 4.39 (d, J = 16.0 Hz, 1H),
3.57 (s, 3H), 3.23 (q, J = 6.4 Hz, 2H), 2.64-2.59 (m, 1H), 2.29 (t, J = 7.6 Hz, 2H),
2.18-2.12 (m, 1H), 1.54-1.48 (m, 4H), 1.34 (s, 9H), 1.31-1.24 (m, 5H), 1.18-1.13 (m,
1H). HRMS (ESI) calcd for C30H40N2NaO5 [M+Na]+: 531.2829, Found: 531.2834.
4.1.4.4.(E)-tert-butyl4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)benzyl(2-phenylcyclo-
propyl)carbamate (10a)
White solid, Yield: 52.3%, Mp:137-138 oC.1H NMR (400 MHz, DMSO-d6) δ 10.76 (s,
1H), 9.04 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 16.0 Hz, 1H), 7.26-7.21 (m,
4H), 7.14 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H), 6.44 (d, J = 16.0 Hz, 1H), 4.55
(d, J = 16.0 Hz, 1H), 4.36 (d, J = 16.0 Hz, 1H), 2.63-2.59 (m, 1H), 2.17-2.12 (m, 1H),
1.35-1.27 (s, 10H), 1.19-1.13 (m, 1H). HRMS (ESI) calcd for C24H28N2NaO4 [M +
Na]+: 431.1941, Found: 431.1946.
4.1.4.5.
(E)-tert-butyl
4-(3-((2-((tert-butoxycarbonyl)amino)phenyl)amino)-3-

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oxoprop-1-en-1-yl)benzyl(2-phenylcyclopropyl)carbamate (10b)
White solid, Yield: 70.7%, Mp: 83-84oC.1H NMR (400 MHz, CDCl3) δ 8.26 (br, 1H),
7.75 (d, J = 15.6 Hz, 1H), 7.63 (br, 1H), 7.49-7.40 (m, 3H), 7.27-7.16 (m, 7H), 7.06
(d, J = 7.8 Hz, 2H), 6.91 (br, 1H), 6.53 (d, J = 15.6 Hz, 1H), 4.64 (d, J = 16.0 Hz, 1H),
4.43 (d, J = 16.0 Hz, 1H), 2.72-2.67 (m, 1H), 2.17-2.13 (m, 1H), 1.53 (s, 9H), 1.43 (s,
9H), 1.33-1.26 (m, 1H), 1.24-1.16 (m, 1H). HRMS (ESI) calcd for C35H41N3NaO5 [M
+ Na]+: 606.7064, Found: 606.7062.
4.1.5 . Procedure for synthesis of tert-butyl 4-((7-(hydroxyamino)-7-oxoheptyl)
carbamoyl)benzyl(2-phenylcyclopropyl)carbamate (6)
KOH (2.8g, 50.9mmol) and NH2OH.HCl (2.34g, 34.3mmol) were dissolved,
respectively, in 7 mL and 12 mL MeOH to get solution A and solution B. Next
solution A was added dropwise to solution B. After filtering the precipitate (KCl), a
mix solution of NH2OK and NH2OH was obtained. Compound 4c (0.254 g, 0.5 mmol)
was dissolved in 5mL NH2OK solution and stirred overnight. After the reaction was
complete, it was evaporated under vacuum. The residue was acidified with 1 N HCl to
a pH 3-4 and then extracted with EtOAc. The organic layer was washed with brine
and dried over Na2SO4 overnight. The crude material was purified via flash
chromatography to afford the compound 6 (104 mg, yield 41.1%). White solid, Mp:
1
61-62oC. H NMR (400 MHz, DMSO) δ 10.35 (s, 1H), 8.67 (s, 1H), 8.40 (t, J = 5.6
Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.23-7.21 (m, 2H), 7.14 (t,
J = 7.2 Hz, 1H), 7.08 (d, J = 7.2 Hz, 2H), 4.57 (d, J = 16.0 Hz, 1H), 4.40 (d, J = 16.0
Hz, 1H), 3.23 (q, J = 6.4 Hz, 2H), 2.64-2.60 (m, 1H), 2.16-2.12 (m, 1H), 1.94 (t, J =
7.2 Hz, 2H), 1.51-1.48 (m, 4H), 1.35 (s, 9H), 1.30-1.23 (m, 5H), 1.18-1.13 (m, 1H).
HRMS (ESI) calcd for C29H40N3O5 [M + H]+: 510.2962, Found: 510.2965.
4.1.6. General procedure for synthesis of compounds 5, 7 and 11.
CF3COOH (20 equiv) was added to a solution of compounds 4a-b, 6 or 10 (1.0 equiv)
in CH2Cl2 at 0oC. The reaction mixture was warmed to room temperature and stirred
at the same temperature. Upon completion, the mixture was concentrated under
vacuum; the residue was dissolved in CH2Cl2, washed with saturated NaHCO3 and
brine, dried over anhydrous Na2SO4, concentrated under vacuum, and purified by
flash column chromatography on silica gel, to afford the pure product.
4.1.6.1. N-hydroxy-4-(((2-phenylcyclopropyl)amino)methyl)benzamide (5a)
1
Yellowish solid, Yield
:
74.1%, Mp:103-104oC. H NMR (400 MHz, DMSO-d6) δ
11.26 (br, 1H), 9.03 (br, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.22-
7.18 (m, 2H), 7.10 (t, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 2H), 3.83 (s, 2H), 2.27-
2.19 (m, 1H), 1.87 (s, 1H), 1.07-1.02 (m, 1H), 0.97-0.92 (m, 1H). HRMS (ESI) calcd
for C17H19N2O2 [M + H]+: 283.1441, Found: 283.1443.
4.1.6.2. N-(2-aminophenyl)-4-(((2-phenylcyclopropyl)amino)methyl)benzamide (5b)
White solid, Yield: 77.4%, Mp:115-116oC. 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s,
1H), 7.92 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.23-7.15 (m, 3H), 7.10 (t, J =
7.2 Hz, 1H), 7.00-6.95 (m, 3H), 6.79 (dd, J = 8.0, 1.2 Hz, 1H), 6.60 (t, J = 7.2 Hz,
1H), 4.90 (s, 2H), 3.85 (s, 2H), 3.06 (br, 1H), 2.23-2.19 (m, 1H), 1.88-1.83 (m, 1H),
13
1.06-1.01 (m, 1H), 0.97-0.92 (m, 1H). C NMR (101 MHz, DMSO-d6) δ 165.64,
145.10, 143.62, 142.96, 133.25, 128.56, 128.18, 128.08, 127.15, 126.90, 125.97,

ACCEPTED MANUSCRIPT
125.58, 123.89, 116.74, 116.61, 52.73, 42.06, 25.00, 17.25. HRMS (ESI) calcd for
C23H23N3NaO [M + Na]+: 380.1733, Found: 380.1734.
4.1.6.3. N-(7-(hydroxyamino)-7-oxoheptyl)-4-(((phenylcyclopropyl)amino)methyl)
Benzamide (7)
White solid, Yield: 68.6%, Mp:140-141oC. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s,
1H), 8.66 (s, 1H), 8.37 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0
Hz, 2H), 7.21-7.17 (m, 2H), 7.11-7.07 (m, 1H), 6.96 (d, J = 6.8 Hz, 2H), 3.80 (s, 2H),
3.22 (q, J = 6.8 Hz, 2H), 2.96 (s, 1H), 2.21-2.17 (m, 1H), 1.94 (t, J = 7.2 Hz, 2H),
1.85-1.80 (m, 1H), 1.52-1.46 (m, 4H), 1.29-1.25 (m, 4H), 1.03-0.98 (m, 1H), 0.95-
0.90 (m, 1H). 13C NMR (101 MHz, DMSO) δ 169.55, 166.38, 144.56, 142.94, 133.38,
128.52, 128.13, 127.41, 125.94, 125.55, 52.73, 42.08, 32.71, 29.53, 28.83, 26.71,
25.57, 24.99, 17.23. RMS (ESI) calcd for C24H31N3NaO3[M + Na]+: 432.2258, Found:
432.2262.
4.1.6.4. (E)-N-hydroxy-3-(4-(((2-phenylcyclopropyl)amino)methyl)phenyl)acrylamide
(11a)
1
White solid, Yield: 57.7%, Mp:125-126oC. H NMR (400 MHz, DMSO-d6) δ 10.90
(br, 1H), 9.07 (br, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 16.0 Hz, 1H), 7.34 (d, J
= 7.8 Hz, 2H), 7.21-7.17 (m, 2H), 7.09 (t, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 2H),
6.50 (d, J = 16.0 Hz, 1H), 3.77 (s, 2H), 2.96 (br, 1H), 2.21-2.17 (m, 1H), 1.84-1.79 (m,
1H), 1.03-0.98 (m, 1H), 0.95-0.90 (m, 1H). 13C NMR (101 MHz, DMSO) δ 163.25,
143.04, 142.97, 138.49, 133.60, 128.95, 128.53, 127.70, 125.93, 125.55, 119.01,
52.80, 42.12, 25.02, 17.21. HRMS (ESI) calcd for C19H20N2NaO2 [M + Na]+:
331.1417, Found: 331.1419.
4.1.6.5. (E)-N-(2-aminophenyl)-3-(4-(((2-phenylcyclopropyl)amino)methyl)phenyl)
acryl Amide (11b)
White solid, Yield: 69.2%, Mp:142-143oC. 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s,
1H), 7.55-7.51 (m, 3H), 7.38-7.33 (m, 3H), 7.22-7.18 (m, 2H), 7.09 (t, J = 7.2 Hz,
1H), 6.98-6.85 (m, 4H), 6.75 (dd, J = 8.4, 1.6 Hz, 1H), 6.58 (td, J = 7.6, 1.2 Hz, 1H),
4.95 (s, 2H), 3.79 (s, 2H), 3.00 (br, 1H), 2.23-2.19 (m, 1H), 1.85-1.80 (m, 1H), 1.04-
0.99 (m, 1H), 0.96-0.91 (m, 1H). 13C NMR (101 MHz, DMSO) δ 164.03, 143.29,
142.95, 142.05, 139.97, 133.62, 129.02, 128.53, 127.89, 126.20, 125.95, 125.56,
125.14, 124.00, 122.07, 116.73, 116.46, 52.83, 42.15, 25.05, 17.22. HRMS (ESI)
calcd for C25H26N3O [M + H]+: 384.2070, Found: 384.2072.
4.2. LSD1, MAO-A and MAO-B inhibition assays
Inhibitory effects of the candidate compounds against LSD1, MAO-A and MAO-B
were evaluated following our previous method [31]. In brief, the candidate
compounds were incubated with the recombinant LSD1 and H3K4me2. After that, the
fluorescence was measured at excitation wavelength 530 nm and emission wavelength
590 nm with the addition of Amplex Red and horseradish peroxidase (HRP) in order
to evaluate the inhibition rate of the candidate compound. MAO inhibitory activities
were determined using a commercialized MAO-Glo assay kit from Promega,
according to the manufacturer’s protocol.
4.3. HDAC inhibition assay

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In vitro HDAC1 (BPS, Cat. No.50051), HDAC2 (BPS, Cat. No.50002) and HDAC5
(BPS, Cat. No.50005) assays were carried out by Shanghai Chempartner Co., Ltd in
Shanghai, China, using SAHA (Sigma, Cat. No. SML0061) as the reference
compound for HDAC1 and HDAC2, and TMP269 (MCE, Cat. No. HY-18360) as the
reference compound for HDAC5. Those compounds were tested over 10 serial
concentrations, 100 µM starting with 3-fold dilution. The general procedures were as
the following: 1x assay buffer (modified Tris Buffer) was prepared, and candidate
compounds were transferred to assay plate by Echo550 in 100% DMSO. Substrate
solution was made by preparing enzyme solution in 1x assay buffer and adding
trypsin and Ac-peptide substrate in 1x assay buffer. 15µL of enzyme solution or 1x
assay buffer was transferred to assay plate or for low control and incubated at room
temperature for 15 min. Substrate solution (10 µL) was then added to each well to
start reaction. The plates were incubated at room temperature for 60 min to allow the
fluorescence signal to develop. The fluorescence generated was monitored with
excitation at 355 nm and emission at 460 nm on an Synergy MX.
4.4. Anti-proliferative activity assays
Exponentially growing cells were seeded into 96-well plates at a concentration of
5×103 cells per well. After 24 h incubation at 37oC, the culture medium was removed
and replaced with fresh medium containing appropriate concentrations of each test
compound. The cells were incubated for another 72 h at 37°C in 5% CO2. Afterward,
20µL of MTT solution (5 mg/mL) was added to all wells and incubated for 4 h at
37oC. Then, the supernatant was discarded and 150 mL of DMSO was added to
dissolve the formazan product; the absorbance was measured using a microplate
reader at a wavelength of 490 nm. Each concentration was analyzed in triplicate and
the experiment was repeated three times. The average 50% inhibitory concentration
(IC50) was determined from the dose-response curves according to the inhibition ratio
for each concentration.
4.5. Flow cytometric analysis of cellular apoptosis
MGC-803 cells (5.0×104) were plated in 6-well plates and treated with increasing
doses (0, 2.5, 5.0 and 7.5 µM) of compound 7 at 37oC for 24 h or 48h. Cells were then
harvested and the Annexin-V-FITC/PI apoptosis kit (Biovision) was used according
to the manufacturer’s protocol to detect apoptotic cells. Ten thousand events were
collected for each sample and analyzed by Accuri C6 flow cytometer.
4.6. Detection of mitochondrial membrane potential
Mitochondria membrane potential was determined by the fluorescent dye JC-1. After
treatment with different concentrations of compound 7 under standard culture
conditions for 48 h, the cells were stained with 10 mg/mL JC-1 and incubated under
standard conditions for 30 min. The fluorescence intensity of cells was determined by
flow cytometry.
4.7. Western Blot
106 MGC-803 cells/well were incubated with compound 7 (0, 1.0, 2.0 µM) or SAHA
(2.0 µM) for 2 days. Histone proteins were extracted using EpiQuik total histone
extraction kit (Epigentek) according to the manufacturer’s protocol. Equivalent

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amounts of cell lysates were denatured, separated by SDS-PAGE and transferred onto
nitrocellulose membranes. After blocking with PBS containing 5% nonfat milk, the
membranes were incubated overnight at 4°C with specific primary antibodies,
followed by incubation with appropriate secondary antibodies. The immunoblots were
visualized by enhanced chemiluminescence detection kit from Thermo Fisher.
4.8. Molecular Docking
All molecular modeling studies were performed with MOE (The Molecular
Operating Environment) Version 2015.10. The crystal structure for LSD1 (PDB code:
4LXZ) and HDAC2 (PDB code: 4LXZ) were obtained from the RCSB protein data
bank. The docking procedure contained the preparation of protein and ligand and the
operation of docking. The preparation of protein structure was performed using the
Quickprep module, which contained the deletion of waters, the addition of hydrogen
atoms, the protonation and the repair of missing residues. The geometry optimization
of ligand structure mainly was executed by energy minimization and conformation
search. Next, compound 7 was docked into the LSD1 or HDAC2. Default triangle
matcher method was used for placement of ligand and the final conformation was
scored by GBVI/WSA dG. All these above treatments were formed in Amber 10:
EHT forcefield.
Acknowledgments
We are grateful for the financial support from the National Natural Science
Foundation of China Project (No. 81402793 for Ying-Chao Duan, No. 81502952 for
Yong-Cheng Ma and No. 21403200 for Li-Na Ding).
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Fig. 1. Reported representative LSD1 inhibitors.
Fig. 2. FDA approved HDAC inhibitors.
Fig. 3. Effect of histone methylation (A) and histone acetylation (B) in MGC-803 cells after 24 h
treatment with compound 7 at 1.0 and 2.0 µM or SAHA at 2.0 µM using western blot analysis.
Fig. 4. Quantitative analysis of apoptotic cells using PI/AnnexinV-FITC double staining and flow-
cytometry calculation. (A) MGC-803 cells were treated with compound 7 (0, 2.5, 5.0, 7.5 µM) for
24 h; (B) MGC-803 cells were treated with compound 7 (0, 2.5, 5.0, 7.5 µM) for 48 h.
Fig. 5. Changes of mitochondrial membrane potential after MGC-803 cells were treated with
different concentrations of compound 7 (0, 2.5, 5.0 and 7.5µM) for 48 h.
Fig. 6. Binding models of tranylcypromine derivates with LSD1. (A) The co-crystal structure of the
complex with GSK2699537 (yellow), FAD (green), and LSD1/CoREST. The residues are shown in
cyan lines, H-bonds are shown in black dash line. (B) Predicted binding model of compound 7 with
LSD1. Compound 7 and FAD are shown in cyan and green sticks, respectively; the residues are shown
in brick lines, H-bonds are shown in green dash line, and the distance of FAD and compound 7 is
shown in magenta.
Fig. 7. Predicted binding mode of compound 7 with HDAC2 (PDB: 4LXZ). (A) Interactions between
compound 1 and HDAC2 residues. Compound 7 is shown as cyan sticks and residues are shown as
brick lines. Zn2+ is shown as purple sphere. Hydrogen bonds are shown as green lines. (B) The
surface on the rim of the binding site of compound 7 with HDAC2. Green area represents hydrophobic
regions and red area represents exposed region.
Table 1 In vitro Inhibition of LSD1, MAO-A and MAO-B
Table 2 In vitro inhibitory activities of target compounds against HDAC isozymes
Table 3 In vitro antiproliferative activity of target compounds in five cancer cell lines
Table 4 Calculated physicochemical and ADME parameters the tested compounds
Scheme 1. Synthesis of compounds 1-7. Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2, 0oC-rt,
4h; (b) NaH, methyl 4-(bromomethyl)benzoate, dry DMF, 0oC-rt, 0.5 h; (c) LiOH, THF-H2O (1:1), rt,
overnight; (d) HBTU, Et3N, 0oC-rt, 2-6 h; (e) NH2OH, KOH, dry CH3OH, 0oC-rt, 3 h; (f) CF3COOH,
dry CH2Cl2, 0oC-rt, 3-8 h.
Scheme 2. Synthesis of compounds 8-11. Reagents and conditions: (a) NaH, methyl 3-(4-
Bromomethyl)cinnamate, dry DMF, 0oC-rt, 0.5 h; (b) LiOH, THF-H2O (1:1), rt, overnight; (c) HBTU,
Et3N, 0oC-rt, 2-4 h; (d) CF3COOH, dry CH2Cl2, 0oC-rt, 3-8 h.

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Table 1 In Vitro Inhibition of LSD1, MAO-A and MAO-B
IC50 (µM)a
Compounds
LSD1
MAO-A
MAO-B
4.09±0.36
7.16±0.54
1.20±0.09
2.21±0.33
3.85±0.73
> 100
45.84±3.17
50.07±3.06
21.11±2.03
39.41±1.75
36.89±2.27
> 100
> 100
> 100
87.09±2.96
> 100
5a
5b
7
11a
11b
> 100
> 100
SAHA
2-PCPA
Clorgyline
R(-)-deprenyl
29.31±2.35
4.72±1.04
2.95±0.81
ND
NDb
0.0036c
ND
ND
0.094c
a Data are represented as IC50 values (mean ± SD). All experiments were independently carried out
at least three times.
b ND: no detection
c Values are the mean of two experiments.

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Table 2 In vitro inhibitory activities of target compounds against HDAC isozymes
IC50 (nM)a
Compounds
HDAC1
HDAC2
HDAC5
1436
218
15
12
314
16
3103
413
23
25
491
28
NIb
NI
5a
5b
7
11a
11b
16840
2967
NI
SAHA
TMP269
NDc
990
ND
ND
a Values are the mean of two experiments.
b NI: no inhibition
c
ND: no detection

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Table 3 In vitro antiproliferative activity of target compounds in five cancer cell lines
IC50 (µM)a
SW-620
Compounds
MGC-803
MCF-7
PC-3
A-549
12.04±1.19 17.27±2.06 23.27±2.14 19.76±1.03 6.75±0.35
5a
5b
21.47±2.88 19.48±1.39
>32
21.69±2.31 5.36±0.46
0.81±0.07
2.46±0.32
4.28±1.10
6.24±1.21
2.35±0.13
4.03±0.88
5.48±0.54
5.62±0.67
1.34±0.12
1.41±0.18
7
11a
11b
SAHA
13.56±1.30 22.09±2.36 26.86±3.08 11.60±1.91 3.17±0.19
8.75±0.93 4.69±1.36 3.87±0.74 3.46±0.85 2.39±0.43
a IC50 values are expressed as mean ± SD from at least three independent experiments.

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Table 4 Calculated physicochemical and ADMET parameters of the target compounds
Lipinski’s Parameters
Comp
LogPa MWb nONc nOHNHd nviolationse TPSAf CYP-3A4g CYP-2C19h Carcino-rati
0
0
0
0
0
negative
negative
negative
negative
negative
2.1
3.5
3.2
2.7
4.2
282.3
357.5
409.5
308.4
383.5
4
4
6
4
4
3
4
4
3
4
61.4
67.2
90.5
61.4
67.2
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
5a
5b
7
11a
11b
a Calculated lipophilicity.
b Molecular weight.
c Number of hydrogen bond acceptor.
d Number of hydrogen bond donor.
e Number of violation from Lipinski's rule of five.
f Total polar surface area.
g In vitro Cytochrome P450 3A4 inhibition.
h In vitro Cytochrome P450 2C19 inhibition.
i 2 years carcinogenicity bioassay in rat.

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