Design, synthesis, and evaluation of monoamine oxidase A inhibitors–indocyanine dyes conjugates as targeted antitumor agents

Article abstract of DOI:10.3390/molecules24071400

Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH) and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized. The synthesized compounds G1–G13 were evaluated in vitro for their cytotoxicity against PC-3 cells using the MTT assay, and molecular docking studies were performed. Results showed that most tested compounds exhibited improved antitumor efficacy compared with INH. Moreover, conjugates G10 and G11 showed potent anticancer activity with IC₅₀ values (0.85 and 0.4 μM respectively) comparable to that of doxorubicin (DOX). This may be attributable to the preferential accumulation of these conjugates in tumor cells. G10, G11, and G12 also demonstrated moderate MAOA inhibitory activities. This result and the results of molecular docking studies were consistent with their cytotoxicity activities. Taken together, these data suggest that a combination of the MAOA inhibitor INH with tumor-targeting heptamethine cyanine dyes may prove to be a highly promising tool for the treatment of advanced prostate cancer.

Full text of DOI:10.3390/molecules24071400

molecules
Article
Design, Synthesis, and Evaluation of Monoamine
Oxidase A Inhibitors–Indocyanine Dyes Conjugates
as Targeted Antitumor Agents
Xiao-Guang Yang 1,†, Yan-Hua Mou 2,†, Yong-Jun Wang 3, Jian Wang 1, Yan-Yu Li 1,
Rui-Heng Kong 1, Meng Ding 1, Dun Wang 1,* and Chun Guo 1,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China; xiaog_yang@163.com (X.-G.Y.);
jianwang@syphu.edu.cn (J.W.); 18345384016@163.com (Y.-Y.L.); krh2735@163.com (R.-H.K.);
dingmeng74376@126.com (M.D.)
School of life sciences and biological pharmacy, Shenyang Pharmaceutical University, Shenyang 110016,
China; mouyanhua2018@163.com
2
3
Wuya College of innovation, Shenyang Pharmaceutical University, Shenyang 110016, China;
i_maple@163.com
*
Correspondence: wangduncn@163.com (D.W.); chunguo@syphu.edu.cn (C.G.); Tel.: +86-13940196276 (D.W.);
+86-024-43520226 (C.G.)
†
These authors contributed equally.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Rainer Riedl
Received: 18 March 2019; Accepted: 6 April 2019; Published: 10 April 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the
oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant
association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression
and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH)
and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized.
The synthesized compounds G1–G13 were evaluated in vitro for their cytotoxicity against PC-3 cells
using the MTT assay, and molecular docking studies were performed. Results showed that most tested
compounds exhibited improved antitumor efficacy compared with INH. Moreover, conjugates G10 and
G11 showed potent anticancer activity with IC50 values (0.85 and 0.4
of doxorubicin (DOX). This may be attributable to the preferential accumulation of these conjugates in
tumor cells. G10 G11, and G12 also demonstrated moderate MAOA inhibitory activities. This result and
µM respectively) comparable to that
,
the results of molecular docking studies were consistent with their cytotoxicity activities. Taken together,
these data suggest that a combination of the MAOA inhibitor INH with tumor-targeting heptamethine
cyanine dyes may prove to be a highly promising tool for the treatment of advanced prostate cancer.
Keywords: monoamine oxidase A; prostate cancer; heptamethine cyanine dyes; isoniazid;
molecular docking
1. Introduction
Global prostate cancer (PCa) incidence and mortality have substantially increased, compounded
by an increase in the proportion of the elderly population and in the frequency of diagnosis.
Although the majority of cases with metastatic PCa respond to the available therapeutic modalities,
hormone-refractory PCa and advanced metastatic PCa remain inevitable [1–4]. Therefore, novel and
highly effective treatments with high tumor-targeting specificity, fewer side effects, and improved
efficacy are desirable for hormone-refractory PCa and metastatic PCa.
Molecules 2019, 24, 1400; doi:10.3390/molecules24071400
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1400
2 of 13
Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the
oxidative deamination of monoamine neurotransmitters including serotonin (5-hydroxytryptamine),
catecholamines, and biogenic amines [5–8]. This process subsequently generates their catalytic
by-product hydrogen peroxide (H2O2), a predominant source of reactive oxygen species (ROS), which
may predispose cancer cells to DNA damage, leading to tumor initiation and progression. Thus,
although MAOA was initially recognized as a crucial neurotransmitter regulator, emerging evidence
revealed its significant roles in oncogenesis [
the overexpression of MAOA could promote the invasion and metastasis of PCa cells by mediating
epithelial-mesenchymal transition (EMT) [13 14]. Conversely, the inhibition of MAOA by the inhibitor
clorgyline attenuated the proliferation of PCa cells and inhibited tumor growth and metastasis in
mouse xenograft models [15 19]. Therefore, MAOA inhibitors can serve as a potentially promising
9–12]. Moreover, recent studies have also reported that
,
–
agent for the effective therapy of advanced human PCa.
Near-infrared (NIR) heptamethine carbocyanine dyes are a novel class of heterocyclic polymethine
cyanine analogues with cancer imaging ability due to their high extinction coefficients and large
Stokes shifts, exhibiting characteristic fluorescence emission in the range of 700–1000 nm [20–22].
Recent in vitro and in vivo studies revealed that hypoxia—one of the elements of the tumor
endogenous microenvironment—induced the preferential uptake of NIR cyanine dyes by human cancer
cells and tissues through the hypoxia inducible factor-1
(HIF-1 /OATPs) signaling axis [23 32]. Second, tumor cells usually possess higher negative
mitochondrial transmembrane potentials than normal cells. Therefore, some lipophilic cations
preferentially accumulate in the mitochondria of tumor cells [33 34]. The higher mitochondrial membrane
α/organic anion-transporting polypeptides
α
–
,
potential in tumor cells may play a critical role in the preferential accumulation of heptamethine
cyanine dyes in tumors owing to the lipophilic and electropositive characteristics of these dyes [35].
Wang et al. confirmed that NIR heptamethine cyanine dyes IR-780 and MHI-148 could be retained in the
mitochondria of tumor cells, increase ROS production, decrease mitochondrial membrane potential, and
induce tumor cell apoptosis [36]. Taken together, these findings suggest that heptamethine carbocyanine
dyes hold great promise for tumor targeting and to some extent possess antitumor efficacy.
Isoniazide (INH) is a small molecule inhibitor of MAOA and is now clinically used as the first-line
anti-tuberculosis medicine. Studies have shown that INH hydrazone and N-acetylated derivatives are
more efficient and less hepatotoxic than INH because of the blockage of the terminal amino group of
hydrazine [37–39]. Herein, we designed and synthesized a series of novel conjugates (Figure 1) through
the conjugation of structurally diverse heptamethine carbocyanine dyes with INH via hydrazine
bond and investigated their cytotoxicity against PC-3 cells. Further, molecular docking studies were
performed to elucidate the binding mode of these conjugates with MAOA. MAOA inhibition assay
was also carried out.
Figure 1. Structures of the designed compounds.

Molecules 2019, 24, 1400
3 of 13
2. Results and Discussion
2.1. Chemistry
The synthetic route for the type
I
compounds is outlined in Scheme 1. Intermediates
1
and
2
were both
synthesized by electrophilic substitution of 5-substitutional 3H-indole. The synthesis of key intermediate
was achieved according to procedures described in the literature [26]. Briefly, both intermediates
and condensed with via base-catalyzed (AcONa) aldol condensation reaction to give intermediate
The target compounds G1 G3 were generated through the coupling reaction of intermediate with INH
in the presence of dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP).
4
1
2
3
4.
–
4
Scheme 1. Synthesis of target compounds G1–G3. Reagents and conditions: (a) Y-(CH2)nCOOH,
toluene, 100 ◦C, 20–25 h; (b) Y-R4 or 1,4-butane sultone, MeCN, 60 ◦C, 30–40 h; (c) Intermediates
1
◦
and
2, AcONa, Ac2O, 60 C, 1 h; (d) INH, dicyclohexylcarbodiimide (DCC), dimethylaminopyridine
(DMAP), dichloromethane (DCM), 24 h, r.t.
As illustrated in Scheme 2, symmetric heptamethine cyanine dye intermediate
6 was synthesized
by one-step base-catalyzed (Et3N) aldol reaction of intermediates
heptamethine cyanine dye intermediate was afforded by the two-step aldol condensation reaction of
intermediates , and in Ac2O with pyridine as catalyst. Finally, the desired compounds G4 G12
were generated by the coupling of with INH in anhydrous DCM with DCC and DMAP acting
1 and 2. However, asymmetric
6
1,
2
5
–
6
as coupling agents. Meanwhile, the type III compound G13 was obtained in the sequence of steps
outlined in Scheme 3.
Scheme 2. Synt◦hesis of target compounds G4
–
G12. Reagents and conditions: (a) i. Intermediate
2,
◦
Et3N, Ac2O, 60 C, 30 min; ii. Intermediate 1, Py, 70 C, 1 h; (b) INH, DCC, DMAP, DCM, r.t., 24 h.

Molecules 2019, 24, 1400
4 of 13
Scheme 3. Synthesis of compound G13. Reagents and conditions: (a) Intermediate 5, Et3N, Ac2O,
◦
◦
60 C, 30 min; (b) 3-(5-Carboxypentyl)-1,1,2-trimethyl-1H-benzo[e]indol-3-ium bromide, Py, 70 C, 1 h;
(c) INH, DCC, DMAP, DCM, r.t., 24 h.
2.2. Cytotoxicity against PC-3
The antitumor activities of G1–G13 were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay using a human PC-3 cell line. The IC50 values of G1–G13
against PC-3 cells are summarized in Table 1.
Table 1. Anti-proliferative activities of compounds against PC-3 cells (IC50, µM). DOX: doxorubicin.
Compound
R1
R2
R3
R4
n
Y
IC50 (µM)
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
INH
DOX
OMe
Br
OMe
Br
OMe
Me
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
-
Me
Me
Et
Me
Me
Et
Me
Me
Me
Me
Me
Me
-
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
5
5
5
5
5
5
5
5
5
3
5
5
-
Br
Br
Br
Br
Br
Br
-
-
-
Br
Br
Br
-
18.41
75.00
13.29
46.66
5.06
37.07
28.59
101.80
90.53
0.85
0.40
1.55
10.54
>200
0.21
OMe
Me
OMe
OMe
Me
OMe
H
Me
Me
Br
−
(CH2)4SO3
(CH2)4SO3
(CH2)4SO3
−
−
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
MTT assay results revealed that nearly all tested compounds showed improved antitumor
activity when compared with their parent compound, the MAOA inhibitor INH. The moderate
cytotoxicity of these conjugates may be due to their preferential uptake by PC-3 cells mediated by
OATPs and subsequent accumulation in the mitochondria of PC-3 cells, disrupting mitochondrial
activities. The fact that INH exhibited negligible cytotoxicity may be attributed to its poor passive
diffusion into PC-3 cells. G10
,
G11 (IC50 values of 0.85 and 0.40
µM, respectively) possessed potent
cytotoxicity comparable to that of doxorubicin (DOX) (IC50 = 0.21
µM). G10 and G11 have very similar
chemical structures except for the different chain length of carboxyalkyl connected to the N atom in
indole moieties. Their structural similarity may explain their similar antitumor efficacy. These results
indicated that the conjugation with INH was conducive to the enhancement of anti-tumor activity
against PC-3 in vitro.

Molecules 2019, 24, 1400
5 of 13
2.3. MAOA Inhibitory Activity of G10, G11, and G12
Because the prostate cancer cell line LNCaP expressed a relatively high level of MAOA [17
,40],
we tested the inhibitory effect of compounds G10 G11, and G12 on MAOA using LNCaP cells (Table 2).
,
Our data showed that three synthesized novel compounds displayed an enhanced inhibitory action
on MAOA levels as compared with the parent compound INH, while clorgyline—another MAOA
inhibitor—exhibited the most potent inhibitory effect. The fact that heptamethine dye–INH conjugates
possessed higher MAOA inhibition efficacy than INH may be attributed to the modification of the
terminal hydrazine group in INH. This assay result is consistent with that of the cell viability assay,
suggesting that the tested compounds inhibited cell growth by suppressing MAOA activity.
Table 2. Monoamine oxidase A (MAOA) inhibitory activity of G10–G12 in LNCaP cells (IC50, nM).
Compound G10
G11
G12
Isoniazid
Clorgyline
IC50 (nM)
221.1
469.5
660.6
>1000
1.8
2.4. In Silico Molecular Docking
To clarify the underlying mechanism of action of these compounds at the molecular level, molecular
docking of 2BXR and G11 was performed. 2BXR is a human monoamine oxidase A (hMAOA).
De Colibus et al. revealed that the active binding site of hMAOA was formed primarily by a single
cavity consisting of residues 210–216 (all active site residues: Tyr69, Gln74, Val91, Val93, Leu97, Ile207,
Phe208, Ser209, Val210, Glu216, Cys323, Ile325, Ile335, Leu337, Met350, Phe352, Tyr407, Tyr444) [41].
Since G11 showed the best in vitro antitumor activity, it was selected as the ligand for this docking
analysis. The molecular docking analysis showed the heptamethine chain and indole moieties of G11
could be integrated closely with the hydrophobic active site cavity (Figure 2) with the low binding energy
of (
∆
G)
−
12.27 kcal/mol. The docked pose of G11 (in 2BXR) was enclosed by a partially hydrophobic
cavity with amino acid residues including Tyr69, Val93, Leu97, Ile180, Ile207, Phe208, Phe208, Val210,
Cys323, Ile325, Ile335, Leu337, Met350, Phe352, Tyr407, and Tyr444. A hydrogen bond interaction
between O44 and Ser209 was also observed.
Figure 2. Molecular docking model of compound G11 interacting with the active site of 2BXR. (a)
The docking site of G11 (blue) in 2BXR. (b) 3D ligand interactions diagram. (c) 2D ligand interactions
diagram. (d) The structure of G11.

Molecules 2019, 24, 1400
6 of 13
3. Materials and Methods
3.1. Materials
All reagents and solvents were obtained from commercial sources and were used as received
unless otherwise stated. Phenylhydrazine hydrochloride, 4-bromophenylhydrazine hydrochloride,
4-methylphenylhydrazine hydrochloride, 4-methoxyphenylhydrazine hydrochloride, 6-bromohexanoic
acid, ethyl 4-bromobutyrate, and 1-bromobutane were purchased from Meryer (Shanghai, China).
3-Methyl-2-butanone, 3-methyl-2-pentanone, 1,4-butane sultone, and isoniazid were purchased from
Aladdin (Shanghai, China). Clorgyline was obtained from Bide Pharmatech Ltd. (Shanghai, China).
All other reagents were synthesized in our laboratory. 1640 DMEM and 0.25% trypsin EDTA were obtained
from Gibco (Sigma-Aldrich, St. Louis, MO, USA). (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide). MTT reagent was acquired from Sigma-Aldrich (St. Louis, MO, USA). PC-3 and LNCaP cells
were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
3.2. Molecular Docking
The crystal structure of MAOA with a resolution of 3 Å was retrieved from the Protein Data
Bank (PDBID: 2BXR). AutoDock 4.2 suite, an automated docking tool that uses a Lamarckian genetic
algorithm (LGA), was used to perform the molecular docking simulations [42]. Briefly, all the bound
water molecules, ligands, and co-factors were eliminated from the protein, and the polar hydrogen
was added to the proteins. Gasteiger charges were computed in AutoDockTools 4.2 [43]. 2D structures
of G11 were constructed using ChemBioDraw ultra12.0 and were transformed to 3D. Subsequently,
3D structures of the aforementioned compounds were also deduced and then the structure was
energetically minimized by using an MMFF94 force field.
In the docking process the grid point maps were calculated using AutoGrid 4.2. The program
AutoGrid was used to generate the grid maps. Each grid was centered at the structure of the corresponding
receptor. The grid dimensions for the grid maps were fixed at a spacing of 0.375 Å with the grid box of
size 90
default parameters for docking run included number of final conformations, 100; population size, 150;
maximum number of energy evaluations, 2.5
106, and maximum number of generations, 27,000. Some
×
70
×
60 (number of points in x-, y-, and z- axes for both proteins). For each docking experiment,
×
low-binding-energy docked poses were investigated in detail for their binding interactions with the binding
cavity of the target protein, using Discovery Studio Visualizer (DSV). The docked compound complexes
were built using the low-free-energy binding positions.
3.3. Cytotoxicity Evaluation
The in vitro cytotoxicity activity of G1
PC-3 cells were incubated in 96-well plates at a density of 3
were exposed to various concentrations of tested compounds, INH and DOX, respectively for 96 h.
–
G13 were measured by the colorimetric MTT assay. Briefly,
×
103 cells/well for 24 h. Then, the cells
After incubation, 10
37 C. Next, the medium was replaced with 200
µ
L of MTT (5 mg/mL) solution was added and the plates were incubated for 4 h at
◦
µL of DMSO to dissolve the formed formazan crystals.
The absorbance was measured at 570 nm on a microplate reader (Synergy-HT, BioTek Instruments,
Winooski, VT, USA).
3.4. MAOA Inhibition Activity Assay
In a 10-mm dish, 6
After 24 h, cells were treated with various concentrations (10 pM, 1 nM, 100 nM, 10 µM) of the tested
compounds G10 G11 G12, isoniazid, and clorgyline for 48 h. The reaction products were extracted
×
105 LNCaP cells were plated in medium supplemented with 10% FBS.
,
,
and MAOA activities were measured as described in the introduction for the Cell MAOA Assay Kit
(Shanghai Chengong Biotechnology, CHN, Lot No: 1-201838-10). The MAOA activity was assessed
based on the substrate p-tyramine level after treatment with MAOB inhibitor pargyline.

Molecules 2019, 24, 1400
7 of 13
3.5. General Procedure for the Synthesis of Compounds G1–G3
(E)-2-Chloro-3-(hydroxymethylene)-cyclohex-1-enecarbaldehyde (
3) (500 mg, 2.9 mmol), 5-methoxyl-
1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bromide (1a) (2.23 g, 5.8 mmol) and sodium acetate
◦
(401 mg, 2.9 mmol) were suspended in acetic anhydride, the mixture was stirred at 60 C for 1 h. Next the
resulting solution was poured into water. Precipitation was allowed to proceed for 6 h and then mixture
was filtered to obtain intermediate 4a as a gold solid. DCC (104 mg, 0.506 mmol) and DMAP (6 mg,
0.051 mmol) were added to a solution of 4a (400 mg, 0.506 mmol) in DCM in an ice-salt bath. INH (58 mg,
0.422 mmol) was added dropwise, the mixture was stirred for 24 h, and was then filtered. Filtrate was
evaporated to dryness under reduced pressure and the residue was purified by silica gel column to yield
G1 (50 mg, 13%). G2 and G3 were prepared by the same procedure as G1 (Supplementary Materials).
2-(2-(3-(2-(5-Methoxyl-1-(5-carboxypentyl)-3,3-dimethyl-indol-2-ylidene)ethylidene)-2-chloro-1-cyclohexen-1-
yl)vinyl)-5-methoxyl-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-3,3-dimethyl-3H-indol-1-ium bromide (G1).
MS (ESI) m/z: 862.4 [M + H]+. 1H NMR (400 MHz, CDCl3)
δ 8.63–8.56 (br, 2H), 8.14–8.12 (m, 2H),
7.89–7.83 (br, 2H), 7.11 (d, 1H), 6.89 (s, 2H), 6.87 (s, 2H), 6.84 (s, 2H), 6.14 (d, 1H), 6.00 (d, 1H), 5.92 (d, 1H),
4.09–4.01 (br, 2H), 3.94–3.87 (br, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 2.58–2.54 (br, 2H), 2.50–2.45 (br, 2H),
2.43–2.34 (br, 2H), 2.23 (d, 4H), 1.83–1.78 (br, 2H), 1.77–1.70 (br, 6H), 1.65 (s, 6H), 1.63 (s, 6H), 1.56 (d, 4H).
13C NMR (151 MHz, CD3OD)
δ 180.10, 174.51, 172.99, 172.86, 166.31, 159.81, 159.74, 150.86, 149.56, 144.20,
144.16, 144.02, 143.94, 141.91, 141.89, 136.92, 136.90, 126.93, 126.90, 124.92, 124.73, 122.96, 118.49, 114.75,
112.89, 112.82, 109.80, 101.80, 101.67, 66.69, 56.26, 50.57, 50.54, 48.94, 45.15, 45.08, 38.22, 34.22, 28.15, 28.11,
27.89, 27.65, 27.15, 27.12, 27.06, 26.94, 25.88, 23.76, 21.97, 15.26.
2-(2-(3-(2-(5-Bromo-1-(5-carboxypentyl)-3,3-dimethyl-indol-2-ylidene)ethylidene)-2-chloro-1-cyclohexen-1-
yl)vinyl)-5-bromo-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-3,3-dimethyl-3H-indol-1-ium bromide (G2).
1
MS (ESI) m/z: 960 [M + H]+. H NMR (400 MHz, CD3OD)
δ 8.71 (d, 2H), 8.42 (d, 1H), 8.39 (d, 1H), 7.81
(dd, 2H), 7.71 (d, 2H), 7.69–7.65 (m, 1H), 7.58–7.57 (m, 1H), 7.56 (dd, 1H), 7.55 (d, 1H), 7.28 (d, 1H), 7.27
(d, 1H), 6.30 (d, 1H), 6.28 (d, 1H), 2.75–2.71 (br, 2H), 2.69 (t, 2H), 2.36 (t, 2H), 2.20 (t, 2H), 1.92–1.89
(m, 4H), 1.79 (dt, 2H), 1.73 (d, 12H), 1.71–1.65 (m, 4H), 1.58 (dt, 7.7 Hz, 2H), 1.50–1.44 (m, 4H). 13
C
NMR (151 MHz, DMSO) δ 175.40, 171.45, 163.96, 156.11, 150.48, 147.97, 143.87, 141.01, 140.49, 139.78,
132.07, 130.69, 130.45, 129.95, 129.63, 128.38, 124.89, 124.77, 124.56, 123.74, 121.51, 110.78, 108.44, 108.17,
108.15, 107.96, 99.69, 99.62, 93.24, 48.85, 45.44, 40.14, 33.45, 33.24, 28.71, 28.12, 27.72, 27.43, 27.22, 26.61,
26.51, 26.15, 26.00, 25.47, 25.22, 24.97, 21.27, 19.66.
2-(2-(3-(2-(5-Methoxyl-1-(5-carboxypentyl)-5-ethyl-3-methyl-indol-2-ylidene)ethylidene)-2-chloro-1-cyclohexen-1-
yl)vinyl)-5-methoxyl-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-5-ethyl-3-methyl-3H-indol-1-ium bromide (G3).
MS (ESI) m/z: 891.5 [M + H]+. 1H NMR (400 MHz, CDCl3)
δ 8.59 (s, 1H), 8.21 (d, 1H), 8.14 (d, 1H), 7.77–7.70
(br, 2H), 7.68–7.62 (br, 1H), 7.57–7.50 (br, 1H), 7.18–7.10 (br, 1H), 7.07 (d, 1H), 6.97 (d, 2H), 6.91–6.87 (m, 1H),
6.85 (d, 1H), 6.16 (d, 2H), 5.98 (d, 1H), 4.06–3.97 (br, 2H), 3.95–3.88 (br, 2H), 3.83 (d, 6H), 2.56–2.50 (br, 2H),
2.50–2.44 (br, 2H), 2.38–2.32 (br, 2H), 2.30–2.21 (br, 4H), 2.13–2.07 (m, 2H), 2.00–1.94 (br, 4H), 1.73–1.69
(m, 4H), 1.64 (d, 6H), 1.53–1.48 (br, 2H), 1.44–1.39 (br, 2H), 1.26 (d, 2H), 1.21 (d, 6H). 13C NMR (151 MHz,
CD3OD) δ 174.66, 171.35, 171.17, 166.41, 159.80, 159.75, 150.87, 143.53, 143.29, 142.20, 142.13, 141.71, 137.99,
137.96, 128.75, 127.04, 126.93, 125.23, 125.14, 122.93, 118.44, 114.84, 114.79, 112.68, 112.55, 112.19, 109.74,
102.33, 102.11, 56.26, 55.73, 55.70, 55.66, 55.64, 45.16, 45.05, 35.77, 35.75, 35.39, 34.18, 28.11, 28.04, 27.92, 27.36,
27.10, 25.84, 21.92, 21.47, 15.23, 15.01, 14.92, 9.03.
3.6. General Procedure for the Synthesis of Compounds G4–G6
1-Phenylamino-5-phenylimino-1,3-pentadiene hydrochloride (5) (500 mg, 1.76 mmol),
1-(5-carboxypentyl)-2,3,3,5-tetramethyl-3H-indol-1-ium bromide (1a) (1.30 g, 3.52 mmol)◦, and
triethylamine (1 mL) were dissolved in acetic anhydride, and the mixture was stirred at 60 C for
1 h. Afterwards, the resulting solution was added to water and allowed to stand for 1 h followed by
extraction with ethyl acetate. The organic layer was dried over Mg2SO4 and concentrated. The residue

Molecules 2019, 24, 1400
8 of 13
was purified by chromatography on silica gel to get 6a. Next, DCC (86 mg, 0.419 mmol) and DMAP
(5 mg, 0.04 mmol) were added to a solution of 6a (300 mg, 0.419 mmol) in DCM in an ice-salt bath.
After cooling to room temperature, INH (52 mg, 0.377 mmol) was added, and the mixture was stirred
for 24 h. After filtration, the filtrate was evaporated in vacuo and the residue was purified by silica gel
column to yield G4 (60 mg, 17%). G5 and G6 were prepared by the same procedure as G4.
2-(7-(1-(5-carboxypentyl)-1,3-dihydro-3,3,5-trimethyl-2H-indol-2-ylidene)-1,3,5-heptatrien-1-yl)-1-(6-(2-
isonicotinoylhydrazinyl)-6-oxohexyl)-3,3,5-trimethyl-3H-indol-1-ium bromide (G4). MS (ESI) m/z: 756.4
1
[M + H]+. H NMR (400 MHz, CD3OD)
δ
8.73 (d, 2H), 7.87 (dd, 2H), 7.83 (d, 2H), 7.57–7.49 (br, 1H),
7.28 (s, 2H), 7.20 (d, 1H), 7.18 (d, 1H), 7.14 (t, 2H), 6.51 (t, 1H), 6.43 (t, 1H), 6.25 (d, 1H), 6.19 (d, 1H),
4.05 (dt, 4H), 2.39 (d, 6H), 2.36 (t, 2H), 2.25–2.18 (br, 2H), 1.88–1.82 (m, 2H), 1.81–1.76 (m, 4H), 1.68
(s, 2H), 1.66 (s, 6H), 1.65 (s, 6H), 1.57 (dt, 2H), 1.51–1.43 (m, 2H). 13C NMR (151 MHz, MeOD)
δ 177.39,
174.89, 170.49, 166.76, 166.66, 159.54, 156.55, 151.24, 151.10, 142.14, 142.12, 141.94, 138.33, 130.83, 128.94,
126.46, 126.38, 125.30, 125.14, 123.16, 118.65, 114.71, 114.69, 112.48, 112.25, 112.23, 109.97, 104.82, 104.73,
66.87, 56.40, 55.53, 45.00, 44.97, 36.22, 35.46, 34.38, 28.38, 28.24, 27.79, 27.59, 27.24, 26.31, 26.06, 22.12,
15.44, 9.23.
2-(7-(5-methoxyl-1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-1,3,5-heptatrien-1-yl)-5-
methoxyl-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-3,3-dimethyl-3H-indol-1-ium bromide (G5). MS (ESI)
1
m/z: 788 [M + H]+. H NMR (400 MHz, CDCl3)
δ
8.71 (d, 2H), 8.63–8.57 (br, 2H), 7.89–7.80 (br, 2H), 7.72
(d, 1H), 7.66 (d, 2H), 7.52–7.39 (m, 1H), 7.15–7.10 (m, 1H), 7.03–7.10 (br, 1H), 6.98 (d, 1H), 6.90 (d, 1H),
6.86 (s, 2H), 6.53–6.42 (br, 1H), 6.25 (d, 1H), 5.93 (d, 1H), 3.96–3.86 (br, 2H), 3.82 (s, 3H), 3.82 (s, 3H), 3.48
(q, 2H), 2.31 (d, 2H), 1.79–1.61 (br, 6H), 1.58 (s, 3H), 1.57 (s, 3H), 1.56–1.53 (m, 2H), 1.52–1.48 (br, 2H),
1.44–1.39 (br, 2H), 1.21 (t, 2H). 13C NMR (151 MHz, MeOD)
δ 178.22, 174.91, 173.45, 172.13, 166.65, 159.65,
159.52, 156.50, 151.68, 151.21, 151.11, 144.33, 144.19, 141.94, 137.22, 130.81, 126.38, 123.15, 114.66, 114.65,
113.28, 112.65, 112.47, 110.96, 110.53, 109.98, 104.35, 104.32, 56.40, 50.42, 49.57, 44.96, 44.94, 36.93, 36.89,
34.38, 28.23, 28.08, 28.01, 27.58, 27.15, 26.56, 26.07, 24.62, 22.67.
2-(7-(5-methoxyl-1-(5-carboxypentyl)-1,3-dihydro-5-ethyl-3-methyl-2H-indol-2-ylidene)-1,3,5-heptatrien-1-
yl)-5-methoxyl-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-5-ethyl-3-methyl-3H-indol-1-ium bromide (G6).
1
MS (ESI) m/z: 816.6 [M + H]+. H NMR (400 MHz, CD3OD)
δ
8.73 (s, 2H), 7.84 (d, 2H), 7.80 (d, 2H),
7.51–7.43 (br, 1H), 7.19 (t, 2H), 7.03 (t, 2H), 6.97–6.95 (m, 1H), 6.95–6.94 (m, 1H), 6.49 (t, 1H), 6.42 (t, 1H),
6.27 (d, 1H), 6.21 (d, 1H), 4.05 (dd, 4H), 3.84 (s, 3H), 3.83 (s, 3H), 2.36 (t, 2H), 2.32 (dd, 2H), 2.26–2.13
(br, 4H), 1.85–1.81 (m, 2H), 1.81–1.77 (m, 4H), 1.70–1.67 (br, 2H), 1.66 (s, 3H), 1.65 (s, 3H), 1.61–1.56
(br, 2H), 1.52–1.44 (br, 2H). 13C NMR (151 MHz, MeOD)
δ 177.36, 174.89, 170.49, 166.76, 166.66, 159.54,
156.55, 151.24, 151.10, 151.05, 142.14, 142.12, 141.94, 138.33, 130.83, 128.94, 126.38, 125.30, 125.14, 123.16,
118.65, 114.71, 114.69, 112.48, 112.25, 112.23, 109.97, 104.82, 104.73, 66.87, 56.40, 55.53, 49.57, 45.00, 44.97,
36.22, 35.46, 34.38, 28.38, 28.24, 27.79, 27.59, 27.24, 26.31, 26.06, 22.12, 20.54, 15.44, 9.23.
3.7. General Procedure for the Synthesis of Compounds G7–G13
1-Phenylamino-5-phenylimino-1,3-pentadiene hydrochloride (5) (500 mg, 1.76 mmol) and
4-(5-methoxy-2,3,3-trimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate (2a) were added to acetic
◦
anhydride with continuous stirring at 50 C for 1 h. The mixture was poured into diethyl ether
after cooling to room temperature. Then, precipitate (A) was filtered and used in further reaction
without any purification.
1-(5-Carboxypentyl)-2,3,3,5-tetramethyl-3H-indol-1-ium bromide (1a) (352 mg, 0.956 mmol) was
added to a solution of (500 mg, 0.956 mmol) in 25 mL pyridine. The resulting solution was stirred
at 60 ◦C for 30 min. Diethyl ether was added in excess to the mixture to obtain the precipitate after
A
cooling to room temperature. The resulting mixture was acidified with hydrochloric acid to get 6d
Subsequently, DCC (92 mg, 0.445 mmol) and DMAP (5 mg, 0.04 mmol) were added to a solution of 6d
(300 mg, 0.445 mmol) in DCM in an ice-salt bath. After the mixture was cooled to room temperature,
INH (76 mg, 0.557 mmol) was added slowly. The mixture was stirred for 24 h and then filtered. Filtrate
.

Molecules 2019, 24, 1400
9 of 13
was evaporated to dryness in vacuo and the residue was purified by silica gel to yield G7 (80 mg, 23%).
G8–G13 were prepared by the same procedure as G7.
3H-Indolium,2-(7-(5-methoxy-1-(4-sulfonatobutyl)-3,3-dimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-
1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-1,3-dihydro-3,3,5-trimethyl-, inner salt (G7). MS (ESI) m/z: 816.3
[M + Na]+. 1H NMR (400 MHz, CDCl3)
δ 8.63 (s, 1H), 8.58–8.55 (br, 2H), 7.79–7.76 (br, 2H), 7.69 (s, 1H),
7.46–7.34 (m, 2H), 7.11–7.06 (br, 2H), 7.02 (d, 1H), 6.99 (s, 1H), 6.86 (d, 1H), 6.74 (s, 2H), 6.39–6.32 (br, 2H),
6.24–6.17 (br, 1H), 6.03–5.93 (br, 1H), 4.04–3.97 (br, 2H), 3.85–3.77 (br, 2H), 3.46 (s, 3H), 3.04–2.99 (m, 2H),
2.41–2.35 (br, 2H), 2.28 (s, 3H), 2.07–2.02 (br, 2H), 1.88–1.83 (br, 2H), 1.71–1.65 (m, 4H), 1.45 (s, 12H), 1.33
(d, 2H). 13C NMR (151 MHz, CD3OD)
δ 174.66, 173.09, 171.11, 166.60, 166.40, 159.70, 150.91, 150.75,
144.27, 142.15, 141.74, 141.54, 136.74, 135.32, 129.82, 126.32, 124.88, 124.70, 123.75, 122.94, 122.60, 118.47,
114.63, 112.88, 112.30, 110.87, 109.73, 103.44, 56.23, 55.55, 51.58, 50.49, 49.61, 44.92, 44.44, 43.57, 34.24,
27.95, 27.77, 27.71, 27.22, 27.00, 25.92, 23.31, 21.06.
3H-Indolium,2-(7-(5-methoxy-1-(4-sulfonatobutyl)-3,3-dimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-
5-methoxy-1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-, inner salt (G8). MS (ESI)
m/z: 832.3 [M + Na]+. 1H NMR (400 MHz, CDCl3)
δ 8.46–8.35 (br, 2H), 7.72–7.62 (br, 2H), 7.47 (d, 2H),
6.99–6.90 (br, 2H), 6.87–6.83 (br, 1H), 6.82–6.76 (br, 2H), 6.75–6.70 (br, 2H), 6.64–6.60 (br, 1H), 6.60–6.55
(br, 1H), 6.32–6.22 (br, 2H), 6.05–5.98 (br, 1H), 5.97–5.89 (br, 1H), 3.83–3.75 (br, 2H), 3.71 (s, 3H), 3.52
(s, 3H), 2.92–2.82 (br, 2H), 2.33–2.19 (br, 2H), 1.93–1.82 (br, 2H), 1.57–1.43 (m, 6H), 1.39 (s, 6H), 1.34 (s, 6H),
1.29–1.25 (m, 4H). 13C NMR (151 MHz, CD3OD)
δ 174.84, 172.15, 171.92, 166.84, 166.59, 159.52, 159.43,
151.12, 150.96, 144.19, 144.08, 141.91, 137.19, 137.16, 128.85, 126.48, 125.17, 124.99, 123.16, 122.84, 118.69,
114.67, 114.63, 112.55, 112.48, 109.95, 104.52, 104.32, 66.85, 56.41, 55.61, 51.83, 50.42, 50.34, 45.01, 44.82,
43.64, 34.41, 28.10, 28.03, 27.33, 27.15, 26.08, 23.51, 23.42.
3H-Indolium,2-(7-(5-methoxy-1-(4-sulfonatobutyl)-3,3-dimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-
1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-, inner salt (G9). MS (ESI) m/z: 780.3 [M
1
+ H]+. H NMR (400 MHz, CD3OD)
δ
8.68 (d, 2H), 7.95–7.89 (m, 1H), 7.86 (d, 2H), 7.79 (t, 1H), 7.70–7.64
(m, 1H), 7.57–7.46 (m, 1H), 7.39 (d, 1H), 7.36–7.31 (m, 2H), 7.26–7.23 (m, 1H), 7.17–7.14 (m, 1H), 6.99
(dd, 1H), 6.54–6.45 (m, 2H), 6.40 (d, 1H), 6.12 (d, 1H), 4.21–4.15 (m, 2H), 4.02–3.98 (m, 2H), 3.85 (s, 3H),
2.89 (t, 2H), 2.35 (t, 2H), 2.01–1.94 (m, 2H), 1.96–1.93 (m, 2H), 1.83–1.77 (m, 4H), 1.69 (s, 6H), 1.66 (s, 6H),
1.58–1.55 (br, 2H). 13C NMR (151 MHz, MeOD)
δ 174.90, 174.49, 170.13, 166.66, 160.34, 151.12, 150.96,
150.25, 144.81, 144.11, 141.86, 136.69, 129.54, 128.91, 126.61, 125.21, 124.98, 124.78, 123.16, 122.82, 118.64,
115.00, 113.64, 112.52, 110.86, 109.90, 106.59, 102.93, 66.87, 56.44, 54.82, 51.71, 51.10, 45.40, 44.34, 34.42,
28.25, 27.83, 27.78, 27.51, 27.22, 26.13, 23.48, 15.44.
2-(7-(5-Methoxy-1-butyl-3,3,5-trimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-1-(6-(2-isonicotinoylhydraz
inyl)-4-oxobutyl)-1,3-dihydro-3,3-dimethyl-3H-indolium bromide (G10). MS (ESI) m/z: 686.4 [M − Br]+. 1H
NMR (600 MHz, CDCl3)
δ 8.61 (d, 2H), 7.84 (d, 2H), 7.67–7.61 (m, 2H), 7.60–7.53 (m, 1H), 7.34–7.28
(br, 1H), 7.13 (s, 2H), 7.10 (d, 1H), 7.06 (s, 1H), 6.91 (d, 1H), 6.88 (s, 1H), 6.83 (d, 1H), 6.62–6.54 (br, 1H),
6.35 (dd, 2H), 5.89 (d, 1H), 4.19–4.14 (br, 2H), 3.91–3.84 (m, 2H), 3.82 (s, 3H), 3.14 (td, 2H), 2.70–2.64
(br, 2H), 2.34 (s, 3H), 2.10–2.04 (m, 2H), 1.75–1.69 (m, 2H), 1.60 (s, 6H), 1.57 (s, 6H), 0.97 (t, 3H). 13C NMR
(151 MHz, MeOD) δ 173.68, 171.84, 170.86, 165.09, 160.04, 156.87, 152.97, 151.03, 150.54, 149.88, 144.64,
144.46, 142.21, 141.67, 136.89, 135.35, 130.01, 126.79, 126.65, 123.94, 123.23, 122.95, 114.87, 113.15, 110.87,
109.91, 105.64, 103.38, 56.42, 50.86, 49.71, 47.36, 45.23, 43.87, 31.70, 30.80, 28.18, 27.84, 27.33, 23.97, 21.25,
21.12, 14.23.
2-(7-(5-Methoxy-1-butyl-3,3,5-trimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-1-(6-(2-isonicotinoylhydraz
inyl)-4-oxohexyl)-1,3-dihydro-3,3-dimethyl-3H-indolium bromide (G11). MS (ESI) m/z: 714.5 [M − Br]+. 1H
NMR (400 MHz, CDCl3)
δ 8.62 (s, 2H), 7.67 (s, 4H), 7.43–7.36 (br, 1H), 7.11 (d, 1H), 7.08 (s, 1H), 6.95
(t, 2H), 6.89 (d, 1H), 6.86 (dd, 1H), 6.55–6.48 (m, 1H), 6.40 (t, 1H), 6.06 (d, 1H), 5.98 (d, 1H), 3.90 (s, 4H),
3.83 (s, 3H), 2.34 (s, 3H), 1.63 (s, 6H), 1.60 (s, 6H), 1.54–1.50 (m, 2H), 1.43 (dq, 2H), 1.32 (d, 2H), 0.96 (t, 3H).
13C NMR (151 MHz, CD3OD)
δ 173.32, 172.08, 171.28, 164.43, 159.87, 156.96, 152.77, 151.27, 150.28, 149.73,

Molecules 2019, 24, 1400
10 of 13
145.36, 144.53, 142.33, 141.70, 136.93, 135.45, 130.00, 126.65, 126.43, 123.92, 123.21, 122.90, 114.80, 112.98,
111.00, 109.89, 105.29, 103.47, 66.86, 56.42, 50.74, 49.78, 45.12, 44.55, 35.21, 30.74, 28.15, 28.02, 27.89, 27.30,
26.52, 21.26, 21.10, 15.43, 14.24.
2-(7-(1-Butyl-3,3,5-trimethyl-2H-indolin-2-ylidene)-1,3,5-heptatrien-1-yl)-5-bromo-1-(6-(2-isonicotinoylhydraz
inyl)-4-oxohexyl)-1,3-dihydro-3,3-dimethyl-3H-indolium bromide (G12). MS (ESI) m/z: 778.3 [M
−
Br]+. 1H
NMR (600 MHz, CDCl3) 8.60 (s, 2H), 7.75 (t, 1H), 7.70–7.65 (br, 2H), 7.57 (t, 1H), 7.41 (t, 1H), 7.36 (d, 1H),
δ
7.29 (s, 1H), 7.07 (d, 1H), 6.92 (s, 1H), 6.90 (d, 1H), 6.85 (d, 1H), 6.46 (dd, 2H), 6.17 (d, 1H), 5.90 (d, 1H),
4.03–3.98 (br, 2H), 3.84 (s, 3H), 3.80–3.76 (br, 2H), 2.36–2.29 (br, 2H), 1.78–1.75 (m, 2H), 1.72–1.67 (br, 4H),
1.64 (s, 6H), 1.57 (s, 6H), 1.50–1.46 (br, 2H),1.45– 1.42 (m, 2H), 0.96 (t, 3H). 13C NMR (151 MHz, CD3OD)
δ
175.73, 174.00, 167.39, 165.87, 160.66, 156.27, 154.33, 150.69, 150.18, 148.38, 145.15, 143.60, 143.53, 142.60,
136.21, 132.08, 127.55, 126.77, 126.22, 122.96, 122.74, 116.16, 115.09, 114.04, 111.64, 109.59, 107.76, 102.00,
66.68, 56.29, 51.37, 47.13, 45.62, 34.43, 30.84, 28.05, 27.42, 27.39, 27.16, 27.11, 27.03, 26.05, 20.87, 13.99.
1H-Benz[e]indolium,2-(7-(3,3-dimethyl-1-(4-sulfonatobutyl)benz(e)indolin-2-ylidene)hepta-1,3,5-trien-1-yl)-
1-(6-(2-isonicotinoylhydrazinyl)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-, inner salt (G13). MS (ESI) m/z: 872.5
1
[M + H]+. H NMR (400 MHz, CDCl3)
δ 8.65 (d, 1H), 8.55 (s, 4H), 7.99–7.91 (m, 2H), 7.84–7.79 (br, 4H),
7.74 (s, 2H), 7.68 (d, 1H), 7.60–7.56 (br, 1H), 7.53–7.46 (m, 2H), 7.42–7.37 (br, 1H), 7.35 (d, 2H), 7.31–7.29
(m, 1H), 7.22–7.10 (br, 1H), 6.61–6.52 (br, 1H), 6.50–6.43 (br, 1H), 6.37–6.28 (br, 1H), 6.26–6.17 (br, 1H),
4.17–4.07 (br, 2H), 4.07–3.95 (br, 2H), 3.08–3.00 (br, 2H), 2.47–2.31 (br, 2H), 2.07 (s, 4H), 1.98–1.89
(br, 2H), 1.78 (s, 6H), 1.75 (s, 6H), 1.58–1.50 (m, 2H). 13C NMR (151 MHz, MeOD)
δ 174.85, 174.48,
174.24, 166.89, 166.59, 151.07, 151.00, 150.94, 144.53, 142.01, 141.11, 134.80, 134.64, 133.26, 133.23, 131.69,
131.05, 130.82, 129.44, 128.85, 128.66, 128.62, 127.17, 125.92, 125.84, 125.14, 124.94, 123.31, 123.27, 123.15,
122.82, 118.67, 112.50, 112.08, 112.05, 104.47, 66.88, 55.63, 54.82, 52.13, 51.82, 45.00, 43.65, 34.41, 28.36,
27.61, 27.12, 26.10, 23.79, 23.52, 15.43.
4. Conclusions
In this study, we developed a series of novel near-infrared heptamethine dye–INH conjugates.
Evaluation of IC50 values indicated that nearly all conjugates showed moderate antitumor efficacy.
Among them, conjugates G10 and G11 exhibited strong cytotoxicity comparable to doxorubicin in
inhibiting PC-3 cell growth while INH showed negligible in vitro antitumor activity. Furthermore,
MAOA inhibitory activities of G10, G11, and G12 and molecular docking analysis were consistent
with the cell viability results, suggesting that the enhanced antitumor activity of these conjugates may
be attributed to the preferential accumulation in cancer cells mediated by OATPs and the increased
MAOA inhibitory activity derived from INH N-acylation. All these above results suggested that
a combination of the MAOA inhibitor INH and tumor-specific targeting heptamethine cyanine dyes
may provide a promising tool for the treatment of advanced prostate cancer.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/7/1400/
s1.
Author Contributions: Conceptualization, D.W. and C.G.; methodology, X.-G.Y., Y.-H.M. and Y.-J.W.; software,
J.W. and M.D.; formal analysis, X.-G.Y. and R.-H.K.; investigation, X.-G.Y., Y.-H.M., Y.-Y.L.; data curation, X.-G.Y.;
writing—original draft preparation, X.-G.Y.; writing—review and editing, D.W. and X.-G.Y.; supervision, D.W.
and C.G.; project administration, D.W. and C.G.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Prof. Lihui Wang for his technical support in MAOA
Inhibition Activity Assay.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 1400
11 of 13
References
1.
Aggarwal, R.; Ryan, C.J. Castration-resistant prostate cancer: Targeted therapies and individualized
treatment. Oncologist 2011, 16, 264–275. [CrossRef]
2.
Heidegger, I.; Massoner, P.; Eder, I.E.; Pircher, A.; Pichler, R.; Aigner, F.; Bektic, J.; Horninger, W.; Klocker, H.
Novel therapeutic approaches for the treatment of castration-resistant prostate cancer. J. Steroid Biochem.
Mol. Biol. 2013, 138, 248–256. [CrossRef]
3.
4.
5.
6.
7.
8.
9.
Kgatle, M.M.; Kalla, A.A.; Islam, M.M.; Sathekge, M.; Moorad, R. Prostate cancer: Epigenetic alterations,
risk factors, and therapy. Prostate Cancer. 2016, 2016, 1–11. [CrossRef] [PubMed]
Stavridi, F.; Karapanagiotou, E.M.; Syrigos, K.N. Targeted therapeutic approaches for hormone-refractory
prostate cancer. Cancer Treat. Rev. 2010, 36, 122–130. [CrossRef] [PubMed]
Mou, Q.; Ma, Y.; Zhu, X.; Yan, D. Inhibition of excessive monoamine oxidase A/B activity protects against
stress-induced neuronal death in huntington disease. J. Control. Release 2016, 230, 34–44.
Shih, J.C.; Chen, K.; Ridd, M.J. Monoamine oxidase: From genes to behavior. Annu Rev. Neurosci. 1999, 22,
197–217. [CrossRef]
Wu, H.F.; Chen, K.J.; Shih, C. Site-directed mutagenesis of monoamine oxidase A and B: Role of cysteines.
Mol. Pharmacol. 1993, 43, 888–893. [PubMed]
Sandi, C.; Haller, J. Stress and the social brain: Behavioural effects and neurobiological mechanisms. Nat. Rev.
Neurosci. 2015, 16, 290–304. [CrossRef]
Sun, W.; Choi, J.; Cha, Y.; Koo, J. Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer.
Int. J. Mol. Sci. 2017, 18, 2775. [CrossRef]
10. Liu, F.; Hu, L.; Ma, Y.; Huang, B.; Xiu, Z.; Zhang, P.; Zhou, K.; Tang, X. Increased expression of monoamine
oxidase A is associated with epithelial to mesenchymal transition and clinicopathological features in
non-small cell lung cancer. Oncol. Lett. 2018, 15, 3245–3251. [CrossRef]
11. Li, P.C.; Siddiqi, I.N.; Mottok, A.; Loo, E.Y.; Wu, C.H.; Cozen, W.; Steidl, C.; Shih, J.C. Monoamine oxidase A
is highly expressed in classical Hodgkin lymphoma. J. Pathol. 2017, 243, 220–229. [CrossRef]
12. Schwartz, T.L. A neuroscientific update on monoamine oxidase and its inhibitor. CNS Spectrums 2013, 18,
25–33. [CrossRef]
13. True, L.; Coleman, I.; Hawley, S.; Huang, C.Y.; Gifford, D.; Coleman, R.; Beer, T.M.; Gelmann, E.; Datta, M.;
Mostaghel, E.; et al. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc.
Natl. Acad. Sci. USA 2006, 103, 10991–10996. [CrossRef]
14. Peehl, D.M.; Coram, M.; Khine, H.; Reese, S.; Nolley, R.; Zhao, H. The significance of monoamine oxidase-A
expression in high grade prostate cancer. J. Urol. 2008, 180, 2206–2211. [CrossRef]
15. Liao, C.P.; Lin, T.P.; Li, P.C.; Geary, L.A.; Chen, K.; Vaikari, V.P.; Wu, J.B.; Lin, C.H.; Gross, M.E.; Shih, J.C.
Loss of MAOA in epithelia inhibits adenocarcinoma development, cell proliferation and cancer stem cells in
prostate. Oncogene 2018, 37, 5175–5190. [CrossRef]
16. Zhao, H.; Flamand, V.; Peehl, D.M. Anti-oncogenic and pro-differentiation effects of clorgyline, a monoamine
oxidase A inhibitor, on high grade prostate cancer cells. BMC Med. Genom. 2009, 2, 55. [CrossRef]
17. Xu, S.; Adisetiyo, H.; Tamura, S.; Grande, F.; Garofalo, A.; Roy-Burman, P.; Neamati, N. Dual inhibition of
survivin and MAOA synergistically impairs growth of PTEN-negative prostate cancer. Br. J. Cancer 2015
113, 242–251. [CrossRef]
,
18. Wu, J.B.; Lin, T.P.; Gallagher, J.D.; Kushal, S.; Chung, L.W.; Zhau, H.E.; Olenyuk, B.Z.; Shih, J.C. Monoamine
oxidase A inhibitor-near-infrared dye conjugate reduces prostate tumor growth. J. Am. Chem. Soc. 2015, 137,
2366–2374. [CrossRef]
19. Flamand, V.; Zhao, H.; Peehl, D.M. Targeting monoamine oxidase A in advanced prostate cancer. J. Cancer
Res. Clin. 2010, 136, 1761–1771. [CrossRef]
20. Alan, S.; Waggoner, P.P. Cyanine Dye as Labeling Reagents for Detection of Biological and othEr Materials
by Luminescence Methods. U.S. Patent 5627027, 6 May 1997.
21. Chen, X.; Peng, X.; Cui, A.; Wang, B.; Wang, L.; Zhang, R. Photostabilities of novel heptamethine
3H-indolenine cyanine dyes with different N-substituents. J. Photochem. Photobiol. A 2006, 181, 79–85.
[CrossRef]

Molecules 2019, 24, 1400
12 of 13
22. Song, F.; Peng, X.; Lu, E.; Zhang, R.; Chen, X.; Song, B. Syntheses, spectral properties and photostabilities of
novel water-soluble near-infrared cyanine dyes. J. Photochem. Photobiol. A 2004, 168, 53–57. [CrossRef]
23. Khemthongcharoen, N.; Jolivot, R.; Rattanavarin, S.; Piyawattanametha, W. Advances in imaging probes
and optical microendoscopic imaging techniques for early in vivo cancer assessment. Adv. Drug Deliv. Rev.
2014, 74, 53–74. [CrossRef] [PubMed]
24. Shi, C.; Wu, J.B.; Chu, G.; Li, Q.; Wang, R.; Zhang, C.; Zhang, Y.; Kim, H.L.; Wang, J.; Zhau, H.E.; et al.
Heptamethine carbocyanine dye-mediated near-infrared imaging of canine and human cancers through the
HIF-1α/OATPs signaling axis. Oncotarget 2014, 5, 10114–10126. [CrossRef] [PubMed]
25. Luo, S.; Tan, X.; Qi, Q.; Guo, Q.; Ran, X.; Zhang, L.; Zhang, E.; Liang, Y.; Weng, L.; Zheng, H.; et al.
A multifunctional heptamethine near-infrared dye for cancer theranosis. Biomaterials 2013, 34, 2244–2251.
[CrossRef]
26. Henary, M.; Pannu, V.; Owens, E.A.; Aneja, R. Near infrared active heptacyanine dyes with unique
cancer-imaging and cytotoxic properties. Bioorg. Med. Chem. Lett. 2012, 22, 1242–1246. [CrossRef]
27. Yang, X.; Shi, C.; Tong, R.; Qian, W.; Zhau, H.E.; Wang, R.; Zhu, G.; Cheng, J.; Yang, V.W.; Cheng, T.; et al. Near
IR Heptamethine Cyanine Dye-Mediated Cancer Imaging. Clin Cancer Res. 2010, 16, 2833–2844. [CrossRef]
[PubMed]
28. Wu, J.B.; Shao, C.; Li, X.; Shi, C.; Li, Q.; Hu, P.; Chen, Y.T.; Dou, X.; Sahu, D.; Li, W.; et al. Near-infrared
fluorescence imaging of cancer mediated by tumor hypoxia and HIF1alpha/OATPs signaling axis.
Biomaterials 2014, 35, 8175–8185. [CrossRef]
29. Hong, G.; Antaris, A.L.; Dai, H. Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng. 2017
1, 0010. [CrossRef]
,
30. Tan, X.; Luo, S.; Wang, D.; Su, Y.; Cheng, T.; Shi, C. A NIR heptamethine dye with intrinsic cancer targeting,
imaging and photosensitizing properties. Biomaterials 2012, 33, 2230–2239. [CrossRef]
31. Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. A review of NIR dyes in cancer targeting and imaging. Biomaterials
2011, 32, 7127–7138. [CrossRef]
32. Sevick-Muraca, E.M. Translation of near-infrared fluorescence imaging technologies: Emerging clinical
applications. Annu. Rev. Med. 2012, 63, 217–231. [CrossRef]
33. Lv, Q.; Wang, D.; Yang, Z.; Yang, J.; Zhang, R.; Yang, X.; Wang, M.; Wang, Y. Repurposing antitubercular
agent isoniazid for treatment of prostate cancer. Biomater. Sci. 2018, 7, 296–306. [CrossRef]
34. Lv, Q.; Yang, X.; Wang, M.; Yang, J.; Qin, Z.; Kan, Q.; Zhang, H.; Wang, Y.; Wang, D.; He, Z.
Mitochondria-targeted prostate cancer therapy using a near-infrared fluorescence dye-monoamine oxidase
A inhibitor conjugate. J. Control. Release 2018, 279, 234–242. [CrossRef]
35. Wang, G.L.; Jiang, B.H.; Rue, E.A.; Semenza, G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS
heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 1995, 92, 5510–5514. [CrossRef]
36. Wang, Y.; Liu, T.; Zhang, E.; Luo, S.; Tan, X.; Shi, C. Preferential accumulation of the near infrared
heptamethine dye IR-780 in the mitochondria of drug-resistant lung cancer cells. Biomaterials 2014, 35,
4116–4124. [CrossRef]
37. Oliveira, P.F.M.; Guidetti, B.; Chamayou, A.; Andre-Barres, C.; Madacki, J.; Kordulakova, J.; Mori, G.;
Orena, B.S.; Chiarelli, L.R.; Pasca, M.R.; et al. Mechanochemical synthesis and biological evaluation of novel
isoniazid derivatives with potent antibumercular activity. Molecules 2017, 22, 1457. [CrossRef]
38. Pahontu, E.; Ilies, D.C.; Shova, S.; Oprean, C.; Paunescu, V.; Olaru, O.T.; Radulescu, F.S.; Gulea, A.; Rosu, T.;
Draganescu, D. Synthesis, characterization, antimicrobial and antiproliferative activity evaluation of Cu(II),
Co(II), Zn(II), Ni(II) and Pt(II) complexes with isoniazid-derived compound. Molecules 2017, 22, 650.
[CrossRef]
39. Vila-Vicosa, D.; Victor, B.I.; Ramos, J.; Machado, D.; Viveiros, M.; Switala, J.; Loewen, P.C.; Leitao, R.;
Martins, F.; Machuqueiro, M. Insights on the mechanism of action of INH-C10 as an antitubercular prodrug.
Mol. Pharm. 2017, 14, 4597–4605. [CrossRef]
40. Wu, J.B.; Shao, C.; Li, X.; Li, Q.; Hu, P.; Shi, C.; Li, Y.; Chen, Y.T.; Yin, F.; Liao, C.P.; et al. Monoamine oxidase
A mediates prostate tumorigenesis and cancer metastasis. J. Clin. Invest. 2014, 124, 2891–2908. [CrossRef]

Molecules 2019, 24, 1400
13 of 13
41. Colibus, L.D.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D.E.; Mattevi, A. Three-dimensional structure
of human monoamine oxidase A (MAOA): Relation to the structures of rat MAOA and human MAOB.
Proc. Natl. Acad. Sci. USA 2005, 102, 12684–12689. [CrossRef]
42. Morris, G.M.; Goodsell, D.S.; Halliday, R.S.; Huey, R.; Hart, W.E.; Belew, R.K.; Olson, A.J. Automated Docking
Using a Lamarckian Genetic Algorithm and an Empirical Binding Free Energy Function. J. Comput. Chem.
1998, 19, 1639–1662. [CrossRef]
43. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4
and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30,
2785–2791. [CrossRef]
Sample Availability: Samples of the compounds G1–G13 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).