Application of novel pH sensitive isoniazid-heptamethine carbocyanine dye conjugates against prostate cancer cells

Article abstract of DOI:10.1691/ph.2020.0521

Recent studies have shown that monoamine oxidase A (MAOA) is significantly expressed in malignant prostate cancer (PCa) and plays an important role in tumorigenesis indicating its potential to serve as a target for PCa treatment. Here, we choose the small molecule isoniazid as the MAOA inhibition functionality and incorporated it in the tumor-targeting moiety of heptamethine carbocyanine dyes via a pH sensitive hydrazone bond to design and synthesize novel MAOA inhibitor isoniazid-heptamethine carbocyanine dye conjugates. Cytotoxicity assay in PC-3 cells shows that all conjugates possessed improved antitumor efficacy compared with isoniazid. The tested compounds also demonstrated a moderate MAOA inhibitory effect. In conclusion, these results indicate that these conjugates exert antitumor effects by delivering the MAOA-inhibiting moiety to PCa cells.

Full text of DOI:10.1691/ph.2020.0521

ORIGINAL ARTICLES
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, China
Application of novel pH sensitive isoniazid–heptamethine carbocyanine
dye conjugates against prostate cancer cells
1
1
1
1
1,
XIAO-GUANG YANG , YAN-FENG LI , YAN-JUN WANG , RUI-HENG KONG , DUN WANG *
Received May 2020, accepted June 12, 2020
*Corresponding author: Dun Wang, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, China
wangduncn@hotmail.com
Pharmazie 75: 412-416 (2020)
doi: 10.1691/ph.2020.0521
Recent studies have shown that monoamine oxidase A (MAOA) is significantly expressed in malignant prostate
cancer (PCa) and plays an important role in tumorigenesis indicating its potential to serve as a target for PCa
treatment. Here, we choose the small molecule isoniazid as the MAOA inhibition functionality and incorporated
it in the tumor-targeting moiety of heptamethine carbocyanine dyes via a pH sensitive hydrazone bond to design
and synthesize novel MAOA inhibitor isoniazid-heptamethine carbocyanine dye conjugates. Cytotoxicity assay
in PC-3 cells shows that all conjugates possessed improved antitumor efficacy compared with isoniazid. The
tested compounds also demonstrated a moderate MAOA inhibitory effect. In conclusion, these results indicate
that these conjugates exert antitumor effects by delivering the MAOA-inhibiting moiety to PCa cells.
generation of hydrogen peroxide (H2O2) belonging to the reactive
oxygen species (ROS), which may cause cell damage and promote
tumorigenesis and tumor development. Recently, a close correlation
between high MAOA expression and PCa progression and poor
prognosis of patients was revealed (Wu et al. 2014; True et al. 2006).
Conversely, specific inhibition of MAOA suppresses the proliferation
of PCa cells and the growth of tumor xenografts (Wu et al. 2014;
Flam et al. 2010; Zhao et al. 2009). The growth and metastasis of
PCa xenograft in which the MAOA gene has been knocked out was
even reduced and eliminated (Liao et al. 2018; Xu et al. 2015; Wu et
al. 2015). Further studies have shown MAOA to enhance the growth,
invasion and metastasis of PCa cells by stabilizing the transcription
factor HIF1α as a result of the elevation of ROS and inducing epitheli-
al-mesenchymal transition (EMT) (True et al. 2006; Pheel et al. 2008;
Wu et al. 2014). Above findings demonstrate the vital function of
MAOA in PCa pathogenesis and suggest that MAOA may serve as a
potential target for PCa treatment.
1. Introduction
Prostate cancer (PCa) has become the second leading cause of cancer
deaths among Western males due to the growing of the elderly popu-
lation. Current approaches to the treatment of early stage PCa include
endocrine treatment, radiotherapy and surgical resection. However,
these treatments are not suitable for the treatment of advanced malignant
PCa (Aggarwal et al. 2011; Heidegger et al. 2013; Kgatle et al. 2016;
Stavridi et al. 2010). Hence, there is an urgent need to better understand
the mechanism of PCa progression and explore new targets in PCa.
A mitochondrial outer membrane enzyme, monoamine oxidase A
(MAOA), regulates the monoamine neurotransmitter level in the body
by oxidative deamination. The critical role of MAOA in maintaining
neurotransmitter balance for brain function has long been recognized
(Jolene et al. 2015; Shih et al. 1999; Wu et al. 1993; Sandi et al. 2015).
Nevertheless, MAOA is suggested to play an important role in tumor-
igenesis (Sun et al. 2017; Liu et al. 2017; Li et al. 2017; Schwartz
2013). The process of oxidative deamination is accompanied by the
Fig. 1: Structures of designed compounds.
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Fig. 2: Synthesis of designed compounds Z1-Z10. Reagents and conditions: (a) AcONa, C2H5OH, 60 °C; (b) NaH, THF, r.t.; isoniazid, CH3OH, r.t.; (c) intermediate 4, DMSO, r.t.
Given these results, we herein chose isoniazid, a small molecule
hydrazine inhibitor of MAOA, which is an anti-tuberculosis drug.
Considering the fact that MAOA is present in the central nervous
system and other peripheral tissues, we integrated near-infrared
(NIR) heptamethine carbocyanine dyes into the moiety of isoni-
azid for tumor targeting in order to achieve tumor-selectivity and
reduce side effects.
The selective uptake of NIR heptamethine carbocyanine dyes
into several types of cancer including PCa has been demon-
strated. They could preferentially accumulate and retain in cancer
cells but not in normal cells (Yang et al. 2010; Xiao et al. 2013).
The mechanism of preferential uptake of NIR carbocyanine dyes
into tumors are hypoxia inducible factor 1α/organic anion-trans-
porting polypeptides (HIF1α/OATPs) signaling axis and higher
negative mitochondrial transmembrane potentials in tumor cells
(Khemthongcharoen et al. 2013; Shi et al. 2014; Luo et al. 2013;
Henary et al. 2012; Wu et al. 2014; Hong et al. 2017; Tan et al.
2012; Luo et al. 2011; Sevick-Muraca et al. 2012; Lv et al. 2012;
Lv et al. 2018; Wang et al. 1995). In this study, we designed and
synthesized tumor-specific conjugates where tumor-targeting
NIR heptamethine carbocyanine dyes were covalently bound to
isoniazid via a hydrazone bond (Fig. 1). Their cytotoxicity against
PC-3 cells was investigated and MAOA inhibitory activity was
also evaluated.
2.2. Cytotoxicity evaluation
Cytotoxicity of the synthesized conjugates was evaluated by MTT
assay in human PC-3 cells. Doxorubicin (DOX) was used as posi-
tive control. IC50 values of compounds Z1-Z10 against PC-3 cells
are shown below in Table 1.
Table: Cytotoxicity assay of compounds Z1-Z10 against PC-3 cells
(IC50, μM)
Compd.
Z1
R1
H
R2
H
R3
R4
IC50 (μM)
-
(CH2)4SO3-
(CH2)4SO3Na
Z2
H
H
(CH2)5COOH (CH2)5COOH
24.57
-
Z3
CH3 CH3 (CH2)5COOH (CH2)5COOH
Z4
-
-
(CH2)4SO3-
OCH3 OCH3 (CH2)4SO3-
CH3 CH3
(CH2)4SO3-
(CH2)4SO3Na
(CH2)4SO3Na
(CH2)4SO3Na
12.54
43.71
38.04
11.35
21.42
16.02
10.34
>200
0.48
Z5
Z6
Z7
Br
-
Br
H
(CH2)5COOH (CH2)5COOH
(CH2)4SO3-
(CH2)4SO3Na
(CH2)5COOH (CH2)5COOH
Z8
Z9
H
-
Br
CH3
-
Z10
Isoniazid
DOX
(CH2)4SO3-
(CH2)4SO3Na
-
-
-
-
-
-
-
2. Investigations and results
2.1. Synthesis
The cytotoxicity assay showed that isoniazid indicated no obvious
cytotoxicity with IC >200 μM. By comparison, all compounds
exhibited preferable50antitumor efficacy with IC at the range
of 10-45 μM. Evaluation of IC values revealed 5t0hat conjugates
possessed a 5-20 times higher5p0 otency in inhibiting PC-3 cells
growth than their parent compound, isoniazid.
The synthetic route for compounds Z1-Z10 is illustrated in Fig.
2. Substituted indoles 1 and 2 which could be the same or not and
were both prepared by quaternization reaction described in our
previous study (Yang et al. 2019) reacted with 2-chloro-3-(hy-
droxymethylene)cyclohex-1-enecarbaldehyde (3) in the presence
of sodium acetate to afford intermediate 4. p-Hydroxybenzenecar-
baldehyde (5) was converted into its sodium salt by treatment with
sodium hydride, the corresponding salt condensed with isoniazid
and intermediate 6 was thus formed. The production of compounds
Z1-Z10 were carried out between intermediates 4 and 6 via substi-
tution reaction.
2.3. MAOA inhibitory activity
MAOA inhibitory effects of compounds Z4, Z7, Z9 and Z10 were
investigated in a LNCaP cell line which possesses a detectably high
expression level of MAOA (Xu et al. 2015; Wu et al. 2014). (Table 2).
Pharmazie 75 (2020)
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ORIGINAL ARTICLES
Table 2: MAOA inhibitory activity of compounds in LNCaP cells (IC50,
nM).
4.3. MAOA inhibition activity assay
About 6×105 LNCaP cells were plated in a 10-mm dish with 10% FBS medium for 24
h. After that, 10 pM, 1 nM, 100 nM, 10 μM cells of the tested compounds, isoniazid
or clorgyline were added and kept for 48 h. Finally, MAOA activities were exam-
ined by Cell MAOA Assay Kit (Shanghai Chen gong Biotechnology, CHN, Lot No:
1-201838-10) according to the instruction. The MAOA activity was determined based
on the substrate p-tyramine level after treatment with MAOB inhibitor pargyline.
Compd.
IC50 (nM)
Z4
Z7
Z9
Z10
Isoniazid
>1000
840.7
421.7
854.2
512.3
4.4. Synthesis
Experimental data indicated that with isoniazid for comparison, four
conjugates showed a significant increase in MAOA inhibition efficacy.
The result seems consistent with that of the cytotoxicity assay.
4.4.1. 2-(2-((2-chloro-3-(2-(3,3-dimethyl-1,5-disubstituted-2H-indol-2-
ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-3,3-dimethyl-1,5-disubstitut-
ed-3H-indolium (4)
To a round-bottomed flask equipped with a magnetic stirrer were added 3,3-dimethyl-
1,5-disubstituted-3H-indole (1, 1.0 equiv), 3,3-dimethyl-1,5-disubstituted-3H-indole
(2, 1.0 equiv), 2-chloro-3-(hydroxymethylene)cyclohex-1-enecarbaldehyde (3, 1.0
equiv) and ethanol. The above solution was added sodium acetate (1.0 equiv) at one
time. The reaction was kept at 60 °C and stirred for 5 h and then poured into ice-water.
The mixture was still stirred for 6 h. Finally, after filtered, the filter cake was washed
with ether and gold solid intermediate 4 was obtained after drying in vacuum.
3. Discussion
As MAOA showed its potential as a therapeutic target of advanced
PCa, finding a suitable MAOA inhibitor becomes more and more
crucial.IsoniazidisoneoftheearliesthydrazineMAOAinhibitorsand
the first line antituberculotic. Based on the principle of conventional
drug in new use, we regard isoniazid as a potential therapeutic drug
for PCa treatment. However, isoniazid showed serious side effects
due to the lack of selective tumor uptake. Hence, we combined isoni-
azid with heptamethine carbocyanine by hydrazone bond, which can
transport isoniazid effectively to tumor tissue. Studies have shown
that isoniazid hydrazone and N-acetylated derivatives are more effi-
cient and less hepatotoxic than isoniazid because of the blockage of
the terminal amino group of hydrazine (Oliveira et al. 2017; Pahontu
et al. 2017; Vila-Vicosa et al. 2017). Apparently, these conjugates
showed some antitumor activity according to the results of MTT
assay. We reasoned moderately increased cytotoxicity of these
conjugates may be attributed to including of heptamethine carbocy-
anine functionality in their structures which result in its selectivity
toward PC-3 cells mediated by OATPs and finally accumulation in
the mitochondria of PC-3 cells, disrupting the normal function of
mitochondrial. The fact that isoniazid exhibited negligible cytotox-
icity may be due its poor passive diffusion into PC-3 cells. Above
results indicated that incorporating heptamethine cyanine dyes
for tumor targeting with the moiety of isoniazid was conducive to
augmentation of in vitro anti-tumor activity against PC-3. Besides,
some conjugates also exhibited obvious MAOA inhibitory effect.
Thus, the marked antitumor activity of heptamethine carbocy-
anine-isoniazid conjugates may be attributed to preferential accu-
mulation of conjugates mediated by OATPs and increased MAOA
inhibitory effect derived from N-acylation of terminal hydrazine
group of isoniazid. In conclusions, through combination of MAOA
inhibitor isoniazid and tumor-targeting heptamethine carbocyanine
dyes, we obtained novel MAOA inhibitor-heptamethine carbocy-
anine dyes conjugates. Compared with isoniazid these conjugates
demonstrated improved in vitro antitumor efficacy as well as better
MAOA inhibitory activity suggesting their antitumor ability may
derive from tumor targeting and MAOA inhibition. Strategy in this
article may provide a new platform for future generation of human
PCa therapeutics.
4.4.2. Sodium 4-(2-isonicotinoylhydrazinyl)phenoxide (6)
Dry THF (30 mL) and p-hydroxybenzaldehyde (5, 1.0 g, 8.19 mmol, 1.0 equiv) was
added a 100 ml round-bottomed flask equipped with a magnetic stirrer. NaH (197 mg,
8.19 mol, 1.0 equiv) was added to the solution and the suspension thus formed was
stirred at room temperature for 2 h. After filtration of the mixture, a brown solid was
obtained which was then added to anhydrous methanol (30 ml) followed by addition
of isoniazid (1.24 g, 9.01 mmol, 1.1 equiv). Condensation reaction was carried out at
room temperature for 5 h. The reaction was filtered to yield 6 (2.0g, 93.3%).
4.4.3. General procedure for the synthesis of compounds Z1-Z10
Intermediate 4 (1.0 equiv) together with intermediate 6 (1.2 equiv) were added to
DMSO. The resultant solution was stirred at room temperature overnight and then
poured into dry ether. The mixture was allowed to stand overnight and filtered. The
crude material was purified by silica gel column (eluted with methylene dichloride/
methanol 5:1) to yield the product.
Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3-dimethyl-1-(4-
sulfonylbutyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-3,3-dime-
1
thylindol-1-yl)butane-1-sulfonate (Z1). MS(ESI) m/z: 932.4 [M-Na+2H]+; H NMR
(600 MHz, DMSO-d6) δ 8.76 (d, J=5.7 Hz, 2H), 8.46 (s, 1H), 7.85-7.79 (m, 5H),7.77
(s, 1H), 7.49 (d, J=7.5 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 7.35 (t, J=7.6 Hz, 2H), 7.28
(d, J=8.3 Hz, 1H), 7.18 (t, J=7.4 Hz, 2H), 6.26 (d, J=14.2 Hz, 2H), 4.14 (s, 4H), 2.75
(s, 4H), 1.96 (s, 3H), 1.76 (d, J=6.7 Hz, 5H), 1.72 (dd, J=13.3, 6.5 Hz, 5H), 1.60 (t,
J=37.7 Hz, 2H), 1.27 (s, 12H); 13C NMR (101 MHz, DMSO-d6) δ 171.51, 161.82,
161.50, 160.90, 150.29, 149.55, 148.52, 142.01, 140.94, 140.52, 140.48, 129.66,
128.49, 128.31, 124.78, 122.34, 121.51, 121.31, 115.02, 111.34, 100.50, 50.67, 48.53,
43.59, 27.19, 26.02, 23.71, 22.48, 20.72.
2-(2-((2-(4-(2-Isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3-dimethyl-1-(5-carboxy-
pentyl)-2H-indol-2
-ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-
indolium bromide (Z2). MS(ESI) m/z: 888.54 [M-Br]+; 1H NMR (600 MHz, CD3OD)
δ 8.75 (d, J = 6.0 Hz, 2H), 8.34 (d, J = 30.6 Hz, 1H), 7.98 (s, 1H), 7.95 (d, J = 8.9 Hz,
2H), 7.91 (d, J = 6.1 Hz, 2H), 7.71 (d, J = 8.6 Hz, 1H), 7.41-7.35 (m, 3H), 7.31-7.23
(m, 4H), 7.21 (t, J = 7.4 Hz, 2H), 6.86 (d, J = 8.7 Hz, 1H), 6.19 (d, J = 14.2 Hz, 1H),
4.11 (t, J = 7.5 Hz, 4H), 2.78 (t, J = 6.0 Hz, 3H), 2.63 (t, J = 6.0 Hz, 1H), 2.19 (q, J
= 7.5 Hz, 4H), 2.11-2.07 (m, 1H), 1.83 (dq, J = 22.7, 7.6 Hz, 4H), 1.73 (s, 3H), 1.68
(dt, J = 14.9, 7.4 Hz, 4H), 1.48 (dq, J = 22.4, 7.6 Hz, 4H), 1.37 (s, 8H), 1.35-1.28
(m, 4H); 13C NMR (101 MHz, CD3OD) δ 182.41, 173.67, 164.60, 164.49, 163.16,
151.10, 150.90, 143.61, 142.96, 142.53, 142.48, 131.48, 129.93, 129.78, 126.21,
123.35, 123.20, 122.92, 116.45, 112.12, 101.23, 50.30, 49.85, 49.71, 49.64, 49.50,
49.43, 49.28, 49.21, 49.07, 49.00, 48.79, 48.57, 48.36, 45.11, 38.84, 28.17, 27.89,
27.28, 25.22, 22.40, 18.37.
4. Experimental
4.1. Materials
2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3,5-trimethyl-1-(5-carboxy-
pentyl)-2H-indol-2-ylidene]ethylidene]-1-cyclohexen-1-yl]vinyl]-3,3,5-trimethyl-
1-(5-carboxypentyl)-3H-indolium bromide (Z3). MS(ESI) m/z: 916.50 [M-Br]+; 1H
NMR (600 MHz, CD3OD) δ 8.73 (s, 2H), 8.32 (s, 1H), 7.89 (d, J=13.3 Hz, 5H),
7.34-7.04 (m, 7H), 6.12 (d, J=14.0 Hz, 2H), 4.05 (s, 4H), 2.73 (s, 4H), 2.34 (s, 6H),
2.15 (s, 4H), 2.10-1.98 (m, 3H), 1.77 (s, 4H), 1.64 (s, 5H), 1.42 (s, 5H), 1.32 (s,
10H), 1.22-1.09 (m, 2H); 13C NMR (101 MHz, CD3OD) δ 173.25, 164.51, 163.99,
163.12, 151.10, 150.96, 142.64, 142.37, 141.42, 136.59, 131.46, 130.21, 129.85,
124.00, 123.21, 122.44, 116.41, 111.80, 100.96, 66.87, 50.52, 50.18, 49.64, 49.43,
49.21, 49.00, 48.79, 48.57, 48.36, 45.11, 38.63, 28.33, 28.17, 27.85, 27.19, 25.21,
22.41, 21.34, 21.28, 15.44.
All reagents and solvents were commercially available. Isoniazid and 1,4-butane
sultone were purchased from Aladdin (Shanghai, China). 6-Bromohexanoic acid
was purchased from Meryer (Shanghai, China). Thiazolyl blue tetrazolium bromide
(MTT) and other MTT assay reagents were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Clorgyline was purchased from Bide Pharmatech Ltd. (Shanghai, China).
PC-3 and LNCaP cells were obtained from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China). All chemicals and reagents employed were of analytical
or HPLC grade.
4.2. Cytotoxicity evaluation
Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3-dimethyl-1-(4-
sulfonylbutyl)-2H-4,5-benz[e]indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-
3,3-dimethyl-4,5-benz[e]indol-1-yl)butane-1-sulfonate (Z4). MS (ESI) m/z: 1054.39
[M+H]+; 1H NMR (600 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.36 (s, 1H), 8.30 (s, 1H),
8.29 (s, 1H), 8.10 (s, 1H), 8.08 (d, J=5.3 Hz, 2H), 8.06 (s, 1H), 7.82 (d, J=8.8 Hz, 2H),
7.65 (t, J=7.5 Hz, 2H), 7.52 (t, J=7.4 Hz, 2H), 6.43 (s, 1H), 6.40 (s, 1H), 4.41-4.30
(m, 4H), 2.77 (s, 4H), 2.56-2.51 (m, 4H), 1.95 (s, 11H), 1.92-1.84 (m, 6H), 1.79 (dd,
J=14.6, 7.3 Hz, 4H); 13C NMR (151 MHz, CD3OD) δ 175.36, 174.81, 151.07, 150.43,
144.51, 142.24, 141.96, 141.01, 140.93, 135.19, 134.93, 133.47, 133.30, 131.89,
Cytotoxicity evaluation of compounds Z1-Z10 were determined in PC-3 cells by
MTT assay. In brief, PC-3 cells were incubated in 96-well plates at a density of 3000
cells per well for one day. Next, these cells were treated with various concentrations
of tested compounds, isoniazid or DOX and incubated for four days. Then, the plates
were added 10 μl of MTT (5 mg/ml) solution and incubated for 4 h at 37 °C. Finally,
the medium was replaced with 200 μL DMSO in order to make the formed formazan
crystals dissolved. The absorbance was tested at 570 nm on a microplate reader
(Synergy-HT, Bio Tek Instruments, Winooski, VT, USA).
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ORIGINAL ARTICLES
131.10, 129.33, 129.22, 128.78, 128.16, 126.18, 123.39, 123.18, 123.13, 112.27,
102.00, 52.37, 52.01, 51.79, 49.85, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36,
45.25, 27.92, 27.57, 27.35, 23.59, 22.12.
Acknowledgments: This study was financially supported by the Innovative Talent
Support Program of Higher Education in Liaoning province (No. 46).
Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3-dimethyl-5-
methoxy-1-(4-sulfonylbutyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)
vinyl)-3,3-dimethylindol-1-yl) butane-1-sulfonate (Z5). MS(ESI) m/z: 992.40
[M-Na+2H]+; 1H NMR (600 MHz, DMSO-d6) δ 8.72 (d, J=6.0 Hz, 2H), 8.42 (s, 1H),
7.79 (dd, J=4.6, 1.5 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.68 (s, 1H), 7.66 (s, 1H), 7.28
(s, 1H), 7.27 (s, 1H), 7.23 (d, J=8.3 Hz, 2H), 7.13 (d, J=2.4 Hz, 2H), 6.87 (d, J=2.4
Hz, 1H), 6.86 (d, J=2.4 Hz, 1H), 6.14 (s, 1H), 6.11 (s, 1H), 4.10-4.03 (m, 4H), 3.70 (s,
6H), 2.68 (s, 4H), 1.94-1.88 (m, 2H), 1.71 (dt, J=14.4, 7.2 Hz, 5H), 1.65 (dd, J=14.7,
7.6 Hz, 5H), 1.61 (s, 1H), 1.23 (s, 10H), 1.22-1.17 (m, 4H); 13C NMR (101 MHz,
CD3OD) δ 173.14, 172.67, 159.95, 159.72, 151.11, 144.37, 144.20, 141.75, 137.11,
137.06, 131.45, 127.41, 123.19, 122.27, 116.43, 114.95, 114.78, 113.08, 112.82,
110.01, 109.94, 102.03, 100.93, 65.22, 56.46, 56.40, 51.81, 50.75, 50.41, 45.17,
45.02, 28.34, 28.19, 27.35, 23.59.
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Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3,5-trimethyl-1-(4-
sulfonylbutyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)vinyl)-3,3,5-trime-
1
thylindol-1-yl)butane-1-sulfonate (Z6). MS(ESI) m/z: 982.4 [M+H]+; H NMR (600
MHz, DMSO-d6) δ 8.72 (d, J=5.7 Hz, 2H), 8.41 (s, 1H), 7.80-7.76 (m, 3H), 7.75 (s,
1H), 7.71 (s, 1H), 7.69 (s, 1H), 7.26 (s, 2H), 7.23 (dd, J=7.9, 4.1 Hz, 4H), 7.12 (s, 1H),
7.11 (s, 1H), 6.17 (s, 1H), 6.15 (s, 1H), 4.10-4.03 (m, 4H), 2.69 (s, 4H), 2.26 (s, 6H),
1.90 (d, J=4.8 Hz, 2H), 1.74-1.67 (m, 5H), 1.64 (dd, J=14.1, 7.2 Hz, 5H), 1.60 (s, 1H),
1.21 (s, 10H), 1.20-1.15 (m, 4H); 13C NMR (101 MHz, DMSO-d6) δ 171.10, 161.48,
161.45, 160.89, 150.25, 148.55, 141.09, 140.38, 140.02, 139.89, 134.48, 129.63,
128.88, 128.38, 122.97, 121.64, 120.88, 115.04, 111.07, 100.26, 50.69, 48.45, 43.61,
27.51, 27.20, 26.07, 23.72, 22.45, 20.86, 20.75.
5-Bromo-2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(5-bromo-3,3-di-
methyl-1-(5-carboxypentyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)
vinyl)-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolium bromide (Z7). MS(ESI) m/z:
1044.29 [M-Br]+;1H NMR (600 MHz, CD3OD) δ 8.73 (d, J=5.1 Hz, 2H), 8.32 (s, 1H),
7.94 (s, 1H), 7.92-7.89 (m, 3H), 7.88 (d, J=5.1 Hz, 2H), 7.55 (s, 2H), 7.49 (s, 1H),
7.48 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 7.18 (s, 1H), 6.18 (s, 1H), 6.16 (s,
1H), 4.06 (t, J=7.2 Hz, 4H), 2.75 (t, J=5.6 Hz, 4H), 2.14 (t, J=7.3 Hz, 4H), 2.07-2.02
(m, 2H), 1.76 (dt, J=14.6, 7.3 Hz, 4H), 1.68-1.59 (m,5H), 1.45-1.38 (m, 5H), 1.34 (s,
11H), 1.32 (s, 1H); 13C NMR (101 MHz, CD3OD) δ 182.37, 173.22, 164.89, 163.12,
151.10, 150.81, 144.64, 143.11, 142.90, 142.56, 132.77, 131.51, 130.04, 126.81,
123.81, 123.20,119.04, 116.45, 113.73, 101.66, 101.39, 66.89, 50.37, 45.28, 38.80,
28.04, 27.83, 27.23, 25.21, 22.34, 15.43.
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Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3-dimethyl-1-(4-
sulfonylbutyl)-2H-indol-2-ylidene) ethylidene]-1-cyclohexen-1-yl)vinyl)-3,3-dimeth-
yl-4,5-benz[e]indol-1-yl)butane-1-sulfonate (Z8). MS(ESI) m/z: 1004.37 [M+H]+; 1H
NMR (400 MHz, CD3OD) δ 8.76 (s, 2H), 8.45-8.33 (m, 1H), 8.14 (d, J=8.9 Hz, 2H),
7.99 (q, J=10.4 Hz, 5H), 7.92 (s, 2H), 7.72-7.55 (m, 2H), 7.49 (d, J=7.5 Hz, 1H), 7.39
(d, J=5.3 Hz, 2H), 7.32 (d, J=8.5 Hz, 3H), 7.21 (dd, J=15.8, 8.2 Hz, 1H), 6.41-6.25 (m,
1H), 6.22 (d, J=14.0 Hz, 1H), 4.31 (s, 2H), 4.15 (s, 2H), 3.01-2.87 (m, 4H), 2.84 (s,
4H), 2.18-1.87 (m, 10H), 1.74-1.60 (m, 6H), 1.44-1.30 (m, 6H); 13C NMR (101 MHz,
CD3OD) δ 175.70, 172.78, 164.47, 164.21, 163.17, 151.10, 143.65, 143.49, 143.00,
142.87, 142.42, 142.27, 142.13, 140.85, 135.34, 133.48, 133.34, 131.86, 131.53,
131.08, 129.88, 129.75, 129.20, 128.77, 126.22, 126.04, 125.86, 123.33, 123.21,
123.14, 116.52, 112.28, 112.13, 111.84, 101.58, 101.37, 100.97, 100.76, 66.87, 52.28,
52.08, 51.84, 50.26, 50.04, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 45.24,
44.75, 28.26, 28.20, 27.81, 27.54, 27.22, 27.17, 25.22, 23.58, 22.42.
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2-(2-((2-(4-(2-Isonicotinoylhydrazinyl)phenoxy)-3-(2-(5-bromo-3,3-dimethyl-1-(5-
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indolium bromide (Z9). MS(ESI) m/z: 966.37 [M-Br]+; 1H NMR (600 MHz, CD3OD)
δ 8.01 (d, J=14.5 Hz, 2H), 7.90 (d, J=8.7 Hz, 1H), 7.87 (d, J=4.8 Hz, 2H), 7.80 (d,
J=13.9 Hz, 1H), 7.47 (d, J=1.8 Hz, 1H), 7.43 (dd, J=8.4, 1.8 Hz, 1H), 7.38 (dd, J=14.6,
7.4 Hz, 2H), 7.34 (t, J=7.5 Hz, 1H), 7.23 (t, J=6.3 Hz, 1H), 7.20 (d, J=8.7Hz, 2H),
7.08 (d, J=8.5 Hz, 1H), 6.28 (d, J=14.5 Hz, 1H), 6.03 (d, J=13.9 Hz, 1H), 4.15 (t,
J=7.4 Hz, 2H), 3.97 (t, J=7.3 Hz, 2H), 2.78-2.70 (m, 4H), 2.14 (td, J=7.4, 3.1 Hz,
4H), 2.07-2.00 (m, 2H), 1.76 (ddt, J=29.6, 14.2, 7.2 Hz, 4H), 1.63 (dq, J=14.8, 7.4
Hz, 4H), 1.42 (dt, J=16.6, 8.0 Hz, 4H), 1.36-1.29 (m, 12H); 13C NMR (101 MHz,
CD3OD) δ 176.23, 171.44, 171.03, 151.09, 151.01, 147.26, 145.41, 144.28, 143.60,
143.54, 143.35, 143.28, 143.25, 143.16, 143.10, 142.88, 142.58, 142.43, 142.31,
132.65, 131.51, 130.07, 128.82, 128.10, 127.46, 126.77, 123.64, 123.49, 123.35,
123.24, 118.17, 116.41, 113.17, 113.12, 104.40, 100.89, 51.25, 50.61, 50.02, 45.77,
44.96, 38.60, 38.56, 28.49, 28.35, 28.18, 28.01, 27.93, 27.86, 27.81, 27.37, 27.30,
27.18, 27.13, 22.07.
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Sodium, 2-(2-((2-(4-(2-isonicotinoylhydrazinyl)phenoxy)-3-(2-(3,3,5-trimethyl-
1-(4-sulfonylbutyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl]vinyl]-3,3-
dimethyl-4,5-benz[e]indol-1-yl)butane-1-sulfonate (Z10). MS (ESI) m/z: 1040.37
[M+Na]+; 1H NMR (400 MHz, CD3OD) δ 8.74 (d, J = 5.7 Hz, 2H), 8.41 (s, 1H),
8.14-8.00 (m, 2H), 8.02-7.86 (m, 6H), 7.65-7.49 (m, 2H), 7.49-7.36 (m, 1H), 7.27
(d, J = 8.6 Hz, 2H), 7.20 (t, J = 8.5Hz, 3H), 6.22 (dd, J = 14.3, 4.8 Hz, 2H), 4.22 (d,
J = 6.7 Hz, 2H), 4.13 (d, J = 6.4 Hz, 2H), 2.90 (d, J = 5.6 Hz, 4H), 2.79 (d, J = 5.7
Hz, 4H), 2.37 (s, 3H), 2.14-1.85 (m, 10H), 1.65 (s, 6H), 1.35 (s, 6H); 3C NMR (101
MHz, CD3OD) δ 174.42, 173.73, 164.48, 164.03, 163.15, 151.09, 151.02, 142.92,
142.75, 142.62, 142.48, 142.36, 141.59, 141.37, 141.30, 141.06, 136.83, 136.55,
134.71, 133.24, 131.73, 131.51, 131.05,130.28, 129.84, 129.30, 128.63, 125.89,
124.01, 123.19, 122.99, 122.87, 122.70, 116.51, 112.07, 112.03, 101.60,100.46,
52.07, 51.91, 51.82, 51.79, 50.33, 50.17, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57,
48.36, 45.04, 44.94, 28.16, 27.85, 27.43, 27.30, 27.24, 25.25, 25.21, 23.58, 22.43,
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Conflicts of interest: None declared.
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