Visible light-controlled carbon monoxide delivery combined with the inhibitory activity of histone deacetylases from a manganese complex for an enhanced antitumor therapy

Article abstract of DOI:10.1016/j.jinorgbio.2021.111354

Multifunctional drugs with synergistic effects have been widely developed to enhance the treatment efficiency of various diseases, such as malignant tumors. Herein, a novel bifunctional manganese(I)-based prodrug [MnBr(CO)₃(APIPB)] (APIPB = N-(2-aminophen-yl)-4-(1H-imidazo[4,5-f] [1, 10] phenanthrolin-2-yl)benzamide) with inhibitory histone deacetylase (HDAC) activity and light-controlled carbon monoxide (CO) delivery was successfully designed and synthesized. [MnBr(CO)₃(APIPB)] readily released CO under visible light irradiation (λ > 400 nm) through which the amount of released CO could be controlled by manipulating light power density and illumination time. In the absence of light irradiation, the cytotoxic effect of [MnBr(CO)₃(APIPB)] on cancer cells was greater than that of the commercially available HDAC inhibitor MS-275. Consequently, with a combination of CO delivery and HDAC inhibitory activity, [MnBr(CO)₃(APIPB)] showed a remarkably enhanced antitumor effect on HeLa cells (IC₅₀ = 3.2 μM) under visible light irradiation. Therefore, this approach shows potential for the development of medicinal metal complexes for combined antitumor therapies.

Full text of DOI:10.1016/j.jinorgbio.2021.111354

Journal of Inorganic Biochemistry 216 (2021) 111354
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Visible light-controlled carbon monoxide delivery combined with the
inhibitory activity of histone deacetylases from a manganese complex for
an enhanced antitumor therapy
a
a
a
a
b
a,*
Hai-Lin Zhang , Ya-Ting Yu , Yi Wang , Qi Tang , Shi-Ping Yang , Jin-Gang Liu
a
Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, PR China
Key Lab of Resource Chemistry of MOE & Shanghai Key Lab of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, PR China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Multifunctional drugs with synergistic effects have been widely developed to enhance the treatment efficiency of
various diseases, such as malignant tumors. Herein, a novel bifunctional manganese(I)-based prodrug [MnBr
Manganese carbonyl complexes
Carbon monoxide
(
CO)
inhibitory histone deacetylase (HDAC) activity and light-controlled carbon monoxide (CO) delivery was suc-
cessfully designed and synthesized. [MnBr(CO)
(APIPB)] readily released CO under visible light irradiation (λ >
00 nm) through which the amount of released CO could be controlled by manipulating light power density and
illumination time. In the absence of light irradiation, the cytotoxic effect of [MnBr(CO) (APIPB)] on cancer cells
was greater than that of the commercially available HDAC inhibitor MS-275. Consequently, with a combination
of CO delivery and HDAC inhibitory activity, [MnBr(CO) (APIPB)] showed a remarkably enhanced antitumor
effect on HeLa cells (IC50 = 3.2 M) under visible light irradiation. Therefore, this approach shows potential for
the development of medicinal metal complexes for combined antitumor therapies.
3
(APIPB)] (APIPB = N-(2-aminophen-yl)-4-(1H-imidazo[4,5-f] [1, 10] phenanthrolin-2-yl)benzamide) with
Light-controlled release
Histone deacetylase inhibitor
Combined therapy
3
4
3
3
μ
1
. Introduction
(vorinostat, SAHA) [10], and short-chain fatty acids [11], have been
designed and synthesized for cancer treatment. Zn2 -dependent HDACIs
generally have a common pharmacophore, i.e., a zinc-binding group
(ZBG), a hydrophobic capping group, and a linker to connect the ZBG
and the capping group, among which the ZBG readily coordinates with
+
Histone deacetylases (HDACs) promote the removal of the acetyl
group from the lysine residue of the histone core and play significant
roles in the dynamic balancing process of lysine deacetylation and
acetylation [1,2]. However, an excessive HDAC activity causes the
aberrant expression of various genes, leading to chromatin condensation
and transcriptional repression associated with cancer cell proliferation,
metabolic disorders, and apoptosis regulation [3,4]. Correspondingly,
HDAC inhibitors (HDACIs) are potential antitumor agents that block
abnormal HDAC deacetylation to restore gene transcription, thereby
inhibiting cancer cell proliferation, angiogenesis, invasion, differentia-
tion, and metastasis; promoting cell cycle arrest and apoptosis; and
simultaneously enhancing host immune responses [5–7]. Therefore,
various kinds of HDACIs, including benzamides (entinostat, MS-275)
the active site Zn2 to inhibit HDAC activity (Fig. 1A) [12]. This struc-
ture provides a basis for exploring new HDACIs as anticancer agents.
HDACIs are mainly used to treat malignant tumors in the hemato-
logical system [13,14]. They have a relatively weaker effect on solid
tumors than HDACIs on hematological tumors. Therefore, the design
and preparation of novel HDACIs with combined chemo/photodynamic
therapy are effective strategies to enhance their anticancer activity.
+
Marmion and co-workers [15] developed a Pt complex of cis-
II
[Pt (NH
3
)
2
(malSAHAHꢀ 2)] that contains a SAHA inhibitor, and the
complex exhibited DNA binding and HDAC inhibitory activity; thus, the
treatment efficacy against tumors is enhanced. Mao and co-workers [16]
[
8], cyclic tetrapeptide (romidepsin, FK-228) [9], hydroxamic acids
Abbreviations: HDAC, Histone deacetylase; HDACI, HDAC inhibitor; GSH, Glutathione; ZBG, Zinc-binding group; Hb, Hemoglobin; photoCORM, Photo-activated
CO-releasing molecule; APIPB, N-(2-aminophen-yl)-4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzamide; PDO, 10-Phenanthroline-5,6-dione; HNCP, 2-(4-car-
boxyphenyl)imidazo(4,5-f)(1,10) phenanthroline; HOBT, 1-hydroxybenzotriazole; DCC, N,N-dicyclohexylcarbodiimide; PIPB, 4-(1H-imidazo[4,5-f][1,10]phenan-
throlin-2-yl)-N-phenylbenzamide; HbCO, Carboxyhemoglobin; PBS, Phosphate buffered saline; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
*
Corresponding author.
E-mail address: liujingang@ecust.edu.cn (J.-G. Liu).
https://doi.org/10.1016/j.jinorgbio.2021.111354
Received 18 August 2020; Received in revised form 24 December 2020; Accepted 3 January 2021
Available online 8 January 2021
0
162-0134/© 2021 Elsevier Inc. All rights reserved.

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
explored a series of cyclometalated Ir(III) complexes composed of a
hydroxamic acid inhibitor, which exhibits a synergistic inhibitory effect
on HDAC activity and photodynamic therapy by producing reactive
oxygen species under UV or visible light irradiation. In addition, HDACIs
have been incorporated with therapeutic gas-releasing molecules for
synergistic therapy. Zhang et al. reported a series of nitric oxide (NO)-
releasing molecules whose NO donor is connected to a HDACI via
various linkers. The released NO combined with the inhibitory effect of
HDACI can induce apoptosis and G1 phase arrest in HEL cells [17].
However, the uncontrollable delivery of NO molecules greatly limits the
practical application of gas therapy.
2. Materials and methods
2.1. Materials
All the reagents were used as purchased commercially without
further purification unless otherwise noted. 10-Phenanthroline-5,6-
dione (PDO) [42] and 2-(4-carboxyphenyl)imidazo(4,5-f)(1,10) phe-
nanthroline (HNCP) [43] were synthesized in accordance with previ-
ously reported methods.
Human cervical carcinoma cells (HeLa cells) were obtained from the
Shanghai Institute for Biological Sciences, the Chinese Academy of
Sciences (CAS, China).
As an emerging therapeutic gas molecule, carbon monoxide (CO) has
been applied to treat various diseases, including cancer [18,19].
Endogenous CO, a signaling molecule, plays significant roles in the
physiological activities of nerve transmission, cardiovascular regulation,
inflammation, apoptosis, and immune responses [20,21]. Notably, an
excessive amount of CO detracts the oxygen-binding capacity of hemo-
globin (Hb), resulting in serious cytotoxicity [22–24]. Therefore, a high
CO concentration is critical to show a desirable therapeutic efficacy
against cancer cells. However, the inherent toxicity of CO significantly
limits its direct application in cancer therapy. Consequently, stimulus-
responsive CO donor molecules for the controlled on-demand delivery
of CO have been developed; in this process, the release of CO is triggered
2
.2. Physical measurement
UV–vis absorption spectra, Fourier transform infrared (FT-IR)
1
spectra, ESI–MS spectra, fluorescence spectra, and H NMR spectra were
acquired in accordance with our previously described methods [44,45].
2
.3. Synthesis of APIPB
2 4 3
A cooled mixture of conc. H SO -HNO (60 mL, 2:1, v/v) was added
with 1,10-phenanthroline (4 g, 22 mmol) and KBr (4 g, 33 mmol) and
by different stimuli, such as pH [25,26], Glutathione (GSH) [27], H
2 2
O
◦
stirred at 130 C for 3 h. A yellow solution was cautiously neutralized
[
28,29], enzymes [30], magnetic heating [31,32], and light [33–35].
ꢀ
1
with NaOH solution (10 mol∙L ) until its pH was neutral. The resulting
mixture was extracted with CH Cl and dried with anhydrous Na SO
Various photo-activated CO-releasing molecules (photoCORMs) have
been widely explored because of their non-invasiveness, light maneu-
verability, and precise controllability of CO delivery [36–40], among
which the Mn(I)-based photoCORMs have been mostly studied due to
the feasibility to release CO under visible light with relative low-power
density. This can alleviate light-induced damage to healthy tissues and
cells [38]. In addition, as one of the essential trace elements for human
body, the Mn element can be effectively regulated by metabolism [41].
However, photoCORMs incorporated with HDACI for combined thera-
pies are lacking.
2
2
2
4
.
Afterward, solvents were removed, and PDO was obtained as a yellow
solid. Then, PDO (0.420 g, 2 mmol), 4-carboxybenzaldehyde (0.360 g,
2
.4 mmol), and CH
3
COONH
4
(3.2 g, 41.5 mmol) were mixed in 50 mL of
◦
CH
3
COOH and stirred at 130 C for 4 h. After being cooled to room
temperature, the solution was adjusted to pH 5.0 by adding NH
3
⋅H
25 wt%) solution while stirring, and HNCP was dried in vacuum. HNCP
0.34 g, 0.79 mmol), 1,2-diaminobenzene (0.325 g, 3 mmol), 1-hydrox-
(HOBT; 0.135 g, mmol), N,N-
2
O
(
(
ybenzotriazole
1
dicyclohexylcarbodiimide (DCC; 0.248 g, 1.2 mmol), and triethyl-
Herein, we prepared a novel manganese complex [MnBr(CO)
3
(A-
◦
amine (167
μ
L) were dissolved in 30 mL of DMF at 4 C for 2 h while
PIPB)] (APIPB N-(2-aminophen-yl)-4-(1H-imidazo[4,5-f] [1,10]
=
stirring. The resulting mixture was stirred at room temperature for 24 h
under a N atmosphere. The faint yellow solid was filtered, washed with
CH Cl and Et OH, and dried in vacuum. The reaction yielded 0.370 g
yield: 71.3%) of APIPB with the following properties: H NMR (400
MHz, DMSO‑d
): δ 13.95 (s, 1H), 9.84 (s, 1H), 9.07 (s, 2H), 8.99–8.97 (d,
phenanthrolin-2-yl)benzamide) as a bifunctional photoCORM for HDAC
activity inhibition and visible light-controlled CO delivery (Fig. 1B).
2
2
2
2
[
3
MnBr(CO) (APIPB)] contains a mimic of the classic benzamide HDAC
1
(
inhibitor MS-275, whose phenanthroline derivative serves as the
capping group of HDACI, benzamide functions as the ZBG, and benzene
participates as a linker to connect the ZBG and the capping group
6
2
7
1
4
H), 8.44–8.42 (d, 2H), 8.25–8.23 (d, 2H), 7.92–7.84 (ddd, 2H),
.21–7.19 (d, 2H), 7.02–6.98 (t, 1H), 6.82–6.80 (d, 1H), 6.64–6.61 (t,
(
Fig. 1A). The results showed that the HDAC inhibition efficiency of
MnBr(CO) (APIPB)] is higher than that of MS-275 in the dark, and CO
+
H), 5.05 (br, 1H) ppm. ESI–MS: m/z, [M + H] : calcd. 431.2, found
[
3
31.2.
was instantly released upon visible light irradiation. Consequently, the
anticancer efficacy against cancerous HeLa cells was remarkably
enhanced.
PIPB (PIPB
=
4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N-
phenylbenzamide) as a control ligand, which lacks the amino group on
the benzamide motif, was synthesized with a procedure similar to APIPB
1
synthesis, but o-phenylenediamine was replaced with aniline. H NMR
3 3
Fig. 1. (A) Design strategy of [MnBr(CO) (APIPB)]. (B) Schematic of [MnBr(CO) (APIPB)].
2

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
(
400 MHz, DMSO‑d
6
): δ 13.96 (s, 1H), 10.41(s, 1H), 9.06(s, 2H),
2.6. MTT assay
2.6.1. Cytotoxicity test in the dark
8
.98–8.96 (d, 2H), 8.45–8.43 (d, 2H), 8.22–8.20 (d, 2H), 7.91–7.84
ddd, 2H), 7.83–7.81 (d, 2H), 7.41–7.37 (t, 2H), 7.15–7.12 (t, 1H) ppm.
(
+
4
ESI–MS: m/z [M + H] : calcd. 416.2, found 416.2.
HeLa cells were plated in a 96-well plate at a density of 5 × 10 cells
◦
per well and incubated at 37 C in 5% CO
2
for 24 h. Then, they were
2
.4. Synthesis of [MnBr(CO)
3
(APIPB)]
incubated with different concentrations (0, 2, 5, 10, and 25
μ
M) of
[
MnBr(CO) (APIPB)], [MnBr(CO) (PIPB)], APIPB, and MS-275 for 16 h.
3
3
In this procedure, 0.302 g of 1.1 mmol Mn(CO)
5
Br and 0.430 g of
And the solution in each well was then aspirated and rinsed with PBS.
The background value was then measured with a microplate reader. An
mmol APIPB were added to 20 mL of CH Cl and heated at reflux for 5 h
2
2
under a N
2
atmosphere. The solution was cooled to room temperature
MTT solution (100
cubation for 3–4 h, the MTT residue in each well was aspirated, and 150
L of DMSO was added to lyse the formazan crystals. Cell viability was
μ
L, 0.5 mg/mL) was added to each well. After in-
◦
and stored at 4 C overnight. The obtained yellow solid was filtered,
washed with cold CH
yielded 0.590 g (yield: 90.6%) of [MnBr(CO)
MHz, DMSO‑d
2
Cl
2
, and collected by freeze-drying. The reaction
μ
1
3
(APIPB)]. H NMR (400
): δ 14.38 (s, 1H), 9.86 (s, 1H), 9.55–9.54 (d, 2H),
.19–9.17 (d, 2H), 8.44–8.42 (d, 2H), 8.27–8.26 (d, 2H), 8.17–8.13 (m,
H), 7.23–7.21 (d, 1H), 7.03–6.99 (t, 1H), 6.83–6.81 (d, 1H), 6.65–6.62
detected on the basis of the absorbance at 490 nm by using a multi-
functional microplate reader.
6
9
2
2.6.2. Cytotoxicity test under visible light irradiation
ꢀ
+
(
t, 1H), 4.99 (s, 2H) ppm. ESI–MS: m/z [M-3CO-Br ] : calcd. 485.1,
The process of cell plating in this procedure was the same as the steps
described in Section 2.6.1. After the cells were incubated with different
found 485.1. Anal. Calcd. for C29 BrMn (%): C, 53.64; H, 2.79;
18 6 4
H N O
N, 12.94. Found: C, 53.71; H, 2.78; N, 12.97.
In the synthesis of the control complex [MnBr(CO)
concentrations (0, 2, 5, 10, and 25
(CO)
μ
3
M) of [MnBr(CO) (APIPB)], [MnBr
3
(PIPB)], PIPB was
3
(PIPB)], APIPB, and MS-275 for 12 h, visible light was applied (λ
2
used instead of APIPB, and the remaining experimental steps were the
> 400 nm, 200 mW/cm , 10 min), and the cells were incubated for 4 h.
The same procedures as above were performed to obtain the final
absorbance at 490 nm by using a microplate reader.
1
same as above. H NMR (400 MHz, DMSO‑d
6
): δ 14.39 (s, 1H), 10.42 (s,
H), 9.55–9.54 (d, 2H), 9.18–9.17 (d, 2H), 8.45–8.44 (d, 2H), 8.25–8.23
d, 2H), 8.15 (brs, 2H), 7.83–7.82 (d, 2H), 7.41–7.38 (t, 2H), 7.15–7.12
1
(
(
ꢀ
+
t, 1H) ppm. ESI–MS: m/z [M-3CO-Br ] : calcd. 470.1, found 470.1.
BrMn (%): C, 54.91; H, 2.70; N, 11.04.
Found: C, 54.99; H, 2.69; N, 11.07.
3. Results and discussion
17 5 4
Anal. Calcd. for C29H N O
3
3.1. Synthesis and characterization of [MnBr(CO) (APIPB)]
2
2
.5. Light-triggered CO release
Manganese carbonyl complexes were synthesized in accordance with
the procedure illustrated in Scheme 1. Firstly, HNCP was obtained with
previously reported methods [42,43]. Then, it reacted with o-phenyl-
enediamine in the presence of HOBT and DCC, yielding the phenan-
.5.1. Measurement of CO release via the hemoglobin method
CO release was detected by spectrophotometrically measuring the
conversion of Hb to carboxyhemoglobin (HbCO) [46]. In brief, 4.2
μ
M
throline derivative APIPB. The final complex [MnBr(CO)
3
(APIPB)] was
Hb was dissolved completely in 10 mM phosphate buffered saline (PBS;
synthesized through the coordination of the APIPB ligand with Mn
1
pH = 7.4) and reduced by adding 1.6 mg of 9.2 mmol sodium dithionite
(CO)
(CO)
5
Br in a dichloromethane solution. The H NMR spectra of [MnBr
(APIPB)] revealed that the protons of phenanthroline in APIPB
(
2 3
SDT) in a N atmosphere. Next, [MnBr(CO) (APIPB)] was suspended in
3
D. I. water, deoxygenated with N
2
, and added to the above Hb solution.
experienced a downfield chemical shift when APIPB coordinated with
Mn(CO) Br, whereas the chemical shift of other protons in APIPB was
Then, the 4 mL solution was sealed immediately in a quartz cuvette, and
5
the mixture was irradiated with different intensities of visible light (λ >
almost unchanged (Figs. S1 ). This result indicated that APIPB success-
fully coordinated with the manganese center. Besides, the FT-IR spectra
4
00 nm). CO release was monitored by detecting the spectral changes
ꢀ 1
(
350–600 nm) in HbCO in the PBS solution at different intervals with a
of [MnBr(CO)
that could be assigned to the
These bands shifted to a lower wavenumber relative to the precursor
3
(APIPB)] showed strong peaks at 2030 and 1931 cm
UV–vis spectrophotometer. The UV adsorption spectral changes of the
solution at 420 nm were taken to calculate the concentration of the
released CO in accordance with Beer–Lambert’s law expressed in Eq. (1):
ν(CO) of the carbonyl group (Fig. 2A).
ꢀ
1
compound MnBr(CO)
spectra of [MnBr(CO)
5
(2054 and 1995 cm ). Furthermore, the UV–vis
3
(APIPB)] exhibited prominent absorption peaks
X% = EE
x
ꢀ E
0
’
× 100%
0
(1)
at 280 and 331 nm that extended to the visible region (Fig. 2B), where
the peaks in the UV region corresponded to the characteristic absorption
100
ꢀ E
3
peaks of APIPB. The visible absorption of [MnBr(CO) (APIPB)] indi-
where X% is the cumulative release percentage of CO, E100 is the
cated that the complex likely favored the visible light-triggered CO
release.
absorbance value when Hb is converted completely to HbCO, E
0
is the
′
initial absorbance value, and Ex and E
0
are the absorbance values after
light exposure and without light exposure, respectively.
3
.2. Visible light-controlled CO release
2
.5.2. Measurement of CO release by using a fluorescent probe
In addition to the Hb method, a fluorescent CO probe (FL-CO-1 +
PbCl ) was employed to measure the CO release. FL-CO-1 was synthe-
CO readily reacts with Hb to form HbCO, which shows characteristic
absorption changes with the Soret band (410–425 nm) and the Q-bands
540–570 nm) in the visible region; the Soret band is very sensitive to
the degree of Hb carboxylation [48]. Therefore, visible light-triggered
CO release from [MnBr(CO) (APIPB)] was measured with the Hb
method in accordance with Beer–Lambert’s law (Scheme S1). When the
solution of [MnBr(CO)
(APIPB)] was irradiated with visible light (λ >
2
(
sized in accordance with a previously reported method [47]. The
product of FL-CO-1 with CO generated a green fluorescence (excitation:
4
90 nm, emission: 516 nm). The probe system (5
μ
M probe +5
μ
M
3
PdCl ) was mixed with the [MnBr(CO) (APIPB)] solution and sealed in a
2
3
quartz cuvette. Then, the mixture was irradiated with visible light, and
the fluorescence spectra were obtained using a fluorescence spectro-
photometer in real time. The released CO was detected by determining
the fluorescence intensities at 516 nm.
3
4
00 nm), the UV–vis spectra of the reduced Hb experienced distinct
spectral changes; that is, the Soret band shifted from 430 to 420 nm, and
the Q-bands were developed at 540 and 570 nm, showing the charac-
teristic absorption peaks of HbCO (Fig. 3A).
The amount of CO released was investigated under the irradiation of
visible light with different power densities and complex concentrations.
3

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
Scheme 1. Synthesis route of manganese carbonyl complexes.
3 5
Fig. 2. FT-IR (A) and UV–vis spectra (B) of [MnBr(CO) (APIPB)], APIPB, and Mn(CO) Br.
The CO release rate reduced as exposure time under visible light irra-
diation at a certain power density was prolonged. Conversely, this rate
also significant for prolonging the drug efficacy and reducing the risk of
CO poisoning.
increased as the light power density was enhanced. The visible light
3
The visible light-triggered release of CO from [MnBr(CO) (APIPB)]
2
irradiation (λ > 400 nm, 400 mW/cm ) of the [MnBr(CO)
3
(APIPB)]
was further verified with a CO fluorescence probe (FL-CO-1 + PbCl
2
)
solution resulted in an immediate CO release that produced about 90%
of CO from the complex within 2 min (Fig. 3B). The amount of released
CO was positively proportional to the applied light intensity (Fig. 3B)
and the complex concentration (Fig. 3C). Under a dark condition, CO
[47] and FT-IR spectra. The fluorescence intensity of the probe in the
PBS solution increased significantly as the exposure time of [MnBr
(CO)
3
(APIPB)] under visible light irradiation was prolonged (Fig. 4A).
This result suggested that CO was released from the complex. The
2
+
0
was not released, suggesting the good stability of [MnBr(CO)
3
(APIPB)]
observed optical changes were attributed to the reduction of Pd to Pd
in the PBS solution in the absence of light irradiation. When visible light
was turned on, the CO was released immediately; once visible light was
turned off, the release of CO instantly stopped (Fig. 3D). As a result, the
repetitive on–off switching of visible light could readily control the
release of CO from the complex. Therefore, the release of CO from [MnBr
by CO, which mediated the Tsuji-Trost reaction of FL-CO-1 to produce
fluorescein (or 2,7-dichlorofluorescein) with strong yellow-green fluo-
rescence (Scheme S2). Because this process involves two steps, the
response time to detect CO is somewhat extended. Furthermore, the FT-
3
IR spectra of [MnBr(CO) (APIPB)] revealed that the characteristic ab-
ꢀ 1
(
CO)
3
(APIPB)] was completely controlled by light, and the CO concen-
sorption peaks of CO at 2030 and 1931 cm gradually diminished upon
visible light irradiation (Fig. 4B), indicating the escape of CO from
tration could be well controlled by manipulating the exposure time of
applied light, light intensity, and complex concentration. To note that
3
[MnBr(CO) (APIPB)].
3
the complex [MnBr(CO) (APIPB)] showed good stability after it was
stored in PBS solution under dark condition for 7 days (Fig. S5A), and
3.3. Assessment of in vitro cytotoxicity
still maintained excellent CO release performance under visible light (λ
2
>
400 nm, 200 mW/cm ) irradiation (Fig. S5B). This kind of CO release
The cytotoxic effects of [MnBr(CO) (APIPB)] were investigated
3
property could be useful for clinical practices requiring the precise
control of the on-demand dosage of CO for therapy. This property was
using HeLa cells as a model cell line. MS-257 is a benzamide derivative
and commercially available HDAC inhibitor used to treat hematological
4

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
Fig. 3. Changes in the UV–Vis spectra of HbCO, indicating the release of CO from [MnBr(CO)
3
(APIPB)] in a PBS solution under visible light irradiation (A). Release
3
of CO under visible light irradiation with different light intensities (B) and [MnBr(CO) (APIPB)] concentrations (C). Visible light controllability of [MnBr
(
3
CO) (APIPB)] for CO release by switching the visible light on/off (D).
Fig. 4. (A) Fluorescence spectral changes in the CO probe system (FL-CO-1 + PdCl
2 3
) with the addition of [MnBr(CO) (APIPB)] in the PBS solution under visible light
irradiation. (B) FT-IR spectral changes in [MnBr(CO) (APIPB)] solid sample under visible light irradiation.
3
malignancies and solid tumors. In this study, different concentrations of
the complex and MS-257 were incubated with HeLa cells at concentra-
3
results clearly suggested that [MnBr(CO) (APIPB)] elicited an excellent
HDAC inhibitory effect on cancerous cells even without releasing CO.
MTT cytotoxicity analysis was then performed using [MnBr
tions varying from 0
evaluate the enzyme inhibitory effect of [MnBr(CO)
Fig. 5A, the cytotoxic effect of the APIPB ligand was similar to that of
MS-275, and the viability of the [MnBr(CO) (APIPB)] (25 M)-treated
μM to 25
μ
M in the absence of light irradiation to
3
(APIPB)]. In
(CO)
mination. [MnBr(CO)
benzamide structure was taken as the control sample of [MnBr
(CO) (APIPB)]. This amino group functioned as a crucial reaction site of
ZBGs and determined the inhibitory activity of benzamide in HDACIs.
After the [MnBr(CO) (APIPB)] (25 M)-treated HeLa cells were irradi-
ated with visible light (λ > 400 nm, 200 mW/cm ) for 10 min, numerous
3
(APIPB)]-treated HeLa cells in the presence of visible light illu-
3
(PIPB)] that lacked the ortho amino group on the
3
μ
cells (53%) was lower than that of the MS-275-treated ones (68%) after
incubation under a dark condition. To note that significantly higher
3
viability of the [MnBr(CO)
3
(APIPB)]-treated normal LO2 cells was
3
μ
2
observed under the similar experimental conditions (Fig. S7B). These
5

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
Fig. 5. (A) MTT cytotoxicity assays of HeLa cells treated with Mn(CO)
5
3
Br, APIPB, [MnBr(CO) (APIPB)], and MS-275 at different concentrations under the dark
condition. (B) MTT cytotoxicity assays of HeLa cells treated with [MnBr(CO)
light irradiation conditions.
3
(PIPB)] and [MnBr(CO) (APIPB)] at different concentrations under the dark and visible
3
HeLa cells were killed, and their viability was less than 9% (Fig. 5B).
Conversely, the viability of the [MnBr(CO) (PIPB)]-treated cells (35%)
was relatively higher under the same experimental conditions. The
cytotoxicity difference between [MnBr(CO) (APIPB)] and [MnBr
CO) (PIPB)] was likely attributed to the absence of the ortho amino
group in the PIPB ligand, which rendered [MnBr(CO) (PIPB)] with a
neglected inhibitory effect on HDACs. IC50 of [MnBr(CO) (APIPB)] to-
ward HeLa cells under light irradiation was approximately 3.2 M,
which is less than the half of IC50 of [MnBr(CO) (PIPB)]. Consequently,
the anticancer effect of [MnBr(CO) (APIPB)] was improved by
Declaration of Competing Interest
The authors have no competing interests to declare.
Acknowledgement
3
3
(
3
3
3
This study was financially supported by the NSF of China (No.
21571062), the Program for Professor of Special Appointment (Eastern
Scholar) at Shanghai Institutions of Higher Learning.
μ
3
3
combining the pharmaceutical efficacy of CO and the enzyme inhibitory
activity of HDACIs. Therefore, the synthesized complex could be a
promising anticancer agent.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2021.111354.
4
. Conclusion
References
In this study, a novel bifunctional manganese carbonyl complex
(
[MnBr(CO)
3
(APIPB)]) that contains an HDAC inhibitor mimic was
[1] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo,
G. Kroemer, Oncogene 31 (2012) 1869–1883.
designed and successfully synthesized. This complex exhibited a high
HDAC inhibition efficiency and visible light-triggered CO release, whose
amount was readily controlled by tuning the applied light power den-
sity, illumination time, and complex concentration. The release of CO
[
2] C.H. Leung, H.J. Zhong, D.S.H. Chan, D.L. Ma, Coord. Chem. Rev. 257 (2013)
1
764–1776.
[3] M. Paris, M. Porcelloni, M. Binaschi, D. Fattori, J. Med. Chem. 51 (2008)
1
505–1529.
[
[
4] P. Bertrand, Eur. J. Med. Chem. 45 (2010) 2095–2116.
from [MnBr(CO)
action with hemoglobin, CO fluorescence probe (FL-CO-1 + PbCl
FT-IR spectroscopy. CO was instantly liberated and more than 90% of
CO was released from [MnBr(CO) (APIPB)] within minutes under
visible light irradiation (λ > 400 nm, 400 mW/cm ). The cytotoxic and
anticancer effects of [MnBr(CO) (APIPB)] against cancerous HeLa cells
3
(APIPB)] was spectroscopically confirmed via its re-
5] S.A.M. Thiagalingam, K.H. Cheng, H.J. Lee, N. Mineva, Ann. N. Y. Acad. Sci. 983
(2003) 84–100.
2
), and
[
[
[
6] P.A. Marks, V.M. Richon, R.A. Rifkind, J. Natl, Cancer Inst. 92 (2000) 1210–1216.
7] C.B. Yoo, P.A. Jones, Nat. Rev. Drug Discov. 5 (2006) 37–50.
8] T. Suzuki, T. Ando, K. Tsuchiya, N. Fukazawa, A. Saito, Y. Mariko, T. Yamashita,
O. Nakanishi, J. Med. Chem. 42 (1999) 3001–3003.
3
2
[
9] R. Furumai, A. Matsuyama, N. Kobashi, K.H. Lee, M. Nishiyama, H. Nakajima,
A. Tanaka, Y. Komatsu, N. Nishino, M. Yoshida, S. Horinouchi, Cancer Res. 62
3
in the dark were greater than those of MS-257. Under visible light
irradiation, cell mortality was remarkably enhanced with IC50 of as low
(
2002) 4916–4921.
[10] S. Grant, C. Easley, P. Kirkpatrick, Nat. Rev. Drug Discov. 6 (2007) 21–22.
[
11] N. Khan, M. Jeffers, S. Kumar, C. Hackett, F. Boldog, N. Khramtsov, X. Qian,
E. Mills, S.C. Berghs, N. Carey, P.W. Finn, L.S. Collins, A. Tumber, J.W. Ritchie, P.
B. Jensen, H.S. Lichenstein, M. Sehested, Biochem. J. 409 (2008) 581–589.
as 3.2
CO and the inhibitory effect of HDAC. Consequently, with a combination
of the CO release and the HDAC inhibitory activity, [MnBr(CO) (A-
PIPB)] showed an impressively enhanced antitumor effect on HeLa cells
under visible light irradiation. Thus, [MnBr(CO) (APIPB)] served as a
μM, which was attributed to the synergistic effect of the released
3
[12] C. Seidel, M. Schnekenburger, C. Zwergel, F. Gaascht, A. Mai, M. Dicato, G. Kirsch,
S. Valente, M. Diederich, Bioorg. Med. Chem. Lett. 24 (2014) 3797–3801.
[
[
[
13] R. Benedetti, M. Conte, L. Altucci, Antioxid. Redox Signal. 23 (2015) 99–126.
14] L. Verna, J. Whysner, G.M. Williams, Clin. Pharmacol. Ther. 71 (1996) 83–105.
15] D. Griffith, M.P. Morgan, C.J. Marmion, Chem. Commun. 44 (2009) 6735–6737.
3
novel example of an application that combined gas therapy with enzyme
inhibition for an efficacious tumor treatment.
[16] R.R. Ye, C.P. Tan, L. He, M.H. Chen, L.N. Ji, Z.W. Mao, Chem. Commun. 50 (2014)
1
0945–10948.
[
[
17] W.W. Duan, J. Li, E.S. Inks, J.C. Chou, Y.P. Jia, X.J. Chu, X.Y. Li, W.F. Xu, Y.
J. Zhang, J. Med. Chem. 58 (2015) 4325–4338.
Author statement
18] H.L. Yan, J.F. Du, S. Zhu, G.J. Nie, H. Zhang, Z.J. Gu, Y.L. Zhao, Small 15 (2019)
1
904382.
This manuscript or its contents in other forms has not been published
previously and/or is not under consideration for publication elsewhere
at the time of submission. And all the authors approved to publish this
work in Journal of Inorganic Biochemistry.
[
[
19] Y. Zhou, W.Q. Yu, J. Cao, H.L. Gao, Biomaterials 255 (2020) 120193.
20] R. Motterlini, L.E. Otterbein, Nat. Rev. Drug Discov. 9 (2010) 728–743.
[21] X.X. Yao, P. Yang, Z.K. Jin, Q. Jiang, R.R. Guo, R.H. Xie, Q.J. He, W.L. Yang,
Biomaterials 197 (2019) 268–283.
[
22] B. Wegiel, D. Gallo, E. Csizmadia, C. Harris, J. Belcher, G.M. Vercellotti,
N. Penacho, P. Seth, V. Sukhatme, A. Ahmed, P.P. Pandolfi, L. Helczynski,
A. Bjartell, J.L. Persson, L.E. Otterbein, Cancer Res. 73 (2013) 7009–7021.
6

H.-L. Zhang et al.
Journal of Inorganic Biochemistry 216 (2021) 111354
[
[
23] R. Foresti, M.G. Bani-Hani, R. Motterlini, Intensive Care Med. 34 (2008) 649–658.
24] G. D o¨ rdelmann, H. Pfeiffer, A. Birkner, U. Schatzschneider, Inorg. Chem. 50 (2011)
[35] S.H.C. Askes, G.U. Reddy, R. Wyrwa, S. Bonnet, A. Schiller, J. Am. Chem. Soc. 139
(2017) 15292–15295.
4
362–4367.
[36] S. Pordel, J.K. White, Inorg. Chim. Acta 500 (2020) 119206.
[37] P.C. Ford, Coord. Chem. Rev. 376 (2018) 548–564.
[
[
[
[
25] F. Zobi, A. Degonda, M.C. Schaub, A.Y. Bogdanova, Inorg. Chem. 49 (2010)
7
313–7322.
[38] M.N. Pinto, P.K. Mascharak, J. Photochem. Photobiol. C 42 (2020) 100341.
[39] M. Faizan, N. Muhammad, K.U.K. Niazi, Y.X. Hu, Y.Y. Wang, Y. Wu, H.M. Sun, R.
X. Liu, W.S. Dong, W.Q. Zhang, Z.W. Gao, Materials 12 (2019) 1643.
26] X. Ji, Z. Pan, C. Li, T. Kang, L.K.C. De La Cruz, L. Yang, Z. Yuan, B. Ke, B. Wang,
J. Med. Chem. 62 (2019) 3163–3168.
ˇ
27] C.J. Gao, X.H. Liang, Z.X. Guo, B.P. Jiang, X.M. Liu, X.C. Shen, ACS Omega 3
[40] T. Slanina, P. Sebej, Photochem. Photobiol. Sci. 17 (2018) 692–710.
(
2018) 2683–2689.
[41] Z. Liu, S.J. Zhang, H. Liu, M.L. Zhao, H.L. Yao, L.L. Zhang, W.J. Peng, Y. Chen,
Biomaterials 155 (2018) 54–63.
28] S. Fayad-Kobeissi, J. Ratovonantenaina, H. Dabir ´e , J.L. Wilson, A.M. Rodriguez,
A. Berdeaux, J. Dubois-Rand ´e , B.E. Mann, R. Motterlini, R. Foresti, Biochem.
Pharmacol. 102 (2015) 64–77.
[42] P.Y. Ma, F.H. Liang, D. Wang, Q.Q. Yang, Z.Q. Yang, D.J. Gao, Y. Yu, D.Q. Song, X.
H. Wang, Dyes Pigments 122 (2015) 224–230.
[
[
[
[
[
[
29] Z. Jin, Y. Wen, L. Xiong, T. Yang, P. Zhao, L. Tan, T. Wang, Z. Qian, B.L. Su, Q.
J. He, Chem. Commun. 53 (2017) 5557–5560.
[43] Y.Q. Wei, Y.F. Yu, K.C. Wu, Cryst. Growth Des. 7 (2007) 2262–2264.
[44] Y.H. Li, M. Guo, S.W. Shi, Q.L. Zhang, S.P. Yang, J.G. Liu, J. Mater. Chem. B 5
(2017) 7831–7838.
30] N.S. Sitnikov, Y. Li, D. Zhang, B. Yard, H. Schmalz, Angew. Chem. Int. Ed. 54
(
2015) 12314–12318.
[45] S.W. Shi, Y.H. Li, Q.L. Zhang, S.P. Yang, J.G. Liu, J. Mater. Chem. B 7 (2019)
1867–1874.
31] P.C. Kunz, H. Meyer, J. Barthel, S. Sollazzo, A.M. Schmidt, C. Janiak, Chem.
Commun. 49 (2013) 4896–4898.
[46] Q.J. He, D.O. Kiesewetter, Y. Qu, X. Fu, J. Fan, P. Huang, Y.J. Liu, G.Z. Zhu, Y. Liu,
Z.Y. Qian, X.Y. Chen, Adv. Mater. 27 (2015) 6741–6746.
32] H. Meyer, F. Winkler, P. Kunz, A.M. Schmidt, A. Hamacher, M.U. Kassack,
C. Janiak, Inorg. Chem. 54 (2015) 11236–11246.
[47] S.M. Feng, D.D. Liu, W.Y. Feng, G.Q. Feng, Anal. Chem. 89 (2017) 3754–3760.
[48] D. Wu, X.H. Duan, Q.Q. Guan, J. Liu, X. Yang, F. Zhang, P. Huang, J. Shen, X.
T. Shuai, Z. Cao, Adv. Funct. Mater. 29 (2019) 1900095.
33] E. Palao, T. Slanina, L. Muchov ´a , T. Solomek, L. Vítek, P. Klan, J. Am. Chem. Soc.
ˇ
´
1
38 (2016) 126–133.
34] T. Soboleva, H.J. Esquer, A.D. Benninghoff, L.M. Berreau, J. Am. Chem. Soc. 139
2017) 9435–9438.
(
7