Targeted photoresponsive carbazole–coumarin and drug conjugates for efficient combination therapy in leukemia cancer cells

Article abstract of DOI:10.1016/j.bioorg.2020.103904

Phototriggered drug delivery systems (PTDDSs) facilitate controlled delivery of drugs loaded on photoactive platform to the target region under light stimulation. The present study investigated the synthesis and efficacy of carbazole–coumarin (CC)-fused heterocycles as a PTDDS platform for the photocontrolled release of a chemotherapeutic agent, chlorambucil, in an in vitro model of human breast and leukemia cancer cells. CC-fused heterocycles were constructed using 4-hydroxycarbazole as the starting material, and further modification of these heterocycles yielded two CC derivatives. CC-7 with an additional ? COOH group and CC-8 with the triphenylphosphonium (TPP) group, a mitochondria-targeting ligand introduced in the carbazole ring, dissolved in polar solvents and exhibited emission bands at 360 and 450 nm, respectively. The results indicate that visible light of 405 nm triggers the photolysis of the CC–drug conjugate and efficiently delivers the drug in both in vitro cancer cell models. Cytotoxicity evaluation indicates the suppression of proliferation of both types of cells treated with CC-8 under synergy effect combining drug potency and photosensitization. Further, the lower IC₅₀ of CC-8 toward leukemia cells suggests the efficacy of the TPP ligand in increasing the bioavailability of CC–drug conjugates in leukemia treatment. Studies on mitochondria-targeting drug delivery systems are required for improving the performance of anticancer drugs.

Full text of DOI:10.1016/j.bioorg.2020.103904

BioorganicChemistry100(2020)103904
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Targeted photoresponsive carbazole–coumarin and drug conjugates for
efficient combination therapy in leukemia cancer cells
⁎
Bing-Yen Wanga,e,f,g,h,i, Yen-Cheng Linb, Yi-Ting Laic, Jia-Yu Oub, Wen-Wei Changc,
,
⁎
Chih-Chien Chub,d,
a Division of Thoracic Surgery, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan
b Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung, Taiwan
c Department of Biomedical Science, Chung Shan Medical University, Taichung, Taiwan
d Department of Medical Education, Chung Shan Medical University Hospital, Taichung, Taiwan
e School of Medicine, Chung Shan Medical University, Taichung, Taiwan
f School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
g Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan
h Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung, Taiwan
i Center for General Education, Ming Dao University, Changhua, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phototriggered drug delivery systems (PTDDSs) facilitate controlled delivery of drugs loaded on photoactive
platform to the target region under light stimulation. The present study investigated the synthesis and efficacy of
carbazole–coumarin (CC)-fused heterocycles as a PTDDS platform for the photocontrolled release of a che-
motherapeutic agent, chlorambucil, in an in vitro model of human breast and leukemia cancer cells. CC-fused
heterocycles were constructed using 4-hydroxycarbazole as the starting material, and further modification of
these heterocycles yielded two CC derivatives. CC-7 with an additional − COOH group and CC-8 with the
triphenylphosphonium (TPP) group, a mitochondria-targeting ligand introduced in the carbazole ring, dissolved
in polar solvents and exhibited emission bands at 360 and 450 nm, respectively. The results indicate that visible
light of 405 nm triggers the photolysis of the CC–drug conjugate and efficiently delivers the drug in both in vitro
cancer cell models. Cytotoxicity evaluation indicates the suppression of proliferation of both types of cells
treated with CC-8 under synergy effect combining drug potency and photosensitization. Further, the lower IC50
of CC-8 toward leukemia cells suggests the efficacy of the TPP ligand in increasing the bioavailability of CC–drug
conjugates in leukemia treatment. Studies on mitochondria-targeting drug delivery systems are required for
improving the performance of anticancer drugs.
Phototriggered drug delivery systems
Coumarin
Carbazole
Chlorambucil
Triphenylphosphonium ligands
Mitochondrial targeting
Photodynamic therapy
Synergy effect
1. Introduction
photocontrolled to achieve the chemotherapeutic outcome. Accord-
ingly, photolabile chromophores that are responsive to ultraviolet (UV),
Phototriggered drug delivery systems (PTDDSs) have attracted
much attention because light stimulation provides excellent spatial and
temporal control over the release of loaded drugs by photoactive car-
riers under an active pathway.[1–3] Moreover, light can be externally
applied and easily tuned to a desired wavelength and is suitable for
versatile therapeutic purposes.[4] Generally, a PTDDS has a tailor-made
chemical structure composed of a drug molecule caged within a pho-
tolabile building block and targeting ligands. After reaching the de-
signated position, the PTDDS accumulates topically in a dormant state
until light stimulation is activated. The release of drugs can thus be
visible, and near-infrared (NIR) light are crucial to the development of
an effective PTDDS.
Among the synthetic phototriggers with well-established photolytic
mechanisms, coumarin-based derivatives that undergo efficient photo-
lysis exhibit unique features such as efficient fluorescence visualization
and a tunable absorption wavelength.[5,6] Photolysis involving facile
CeO bond cleavage upon light irradiation produces the free drug and
solvent-trapped coumarin as the byproduct.[7] Moreover, coumarin
substituted at the 7-position with an electron-donating group is known
to exhibit red-shifted absorption and emission in the blue-green light
⁎ Corresponding authors at: Department of Medical Applied Chemistry, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung 40201, Taiwan
(C.-C. Chu). Department of Biomedical Science, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Rd., Taichung 40201, Taiwan (W.-W. Chang).
E-mail address: jrchu@csmu.edu.tw (C.-C. Chu).
https://doi.org/10.1016/j.bioorg.2020.103904
Received 1 April 2020; Received in revised form 27 April 2020; Accepted 29 April 2020
Availableonline05May2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

B.-Y. Wang, et al.
BioorganicChemistry100(2020)103904
region. For example, 7-aminocoumarin, which has remarkable fluor-
escence quantum yield, has been employed as an optical brightener,
fluorescent probe, and phototrigger.[8] Lin et al. reported the effective
photorelease of anticancer drugs regulated by either a one- or two-
photon excitation process, and combining the intrinsic fluorescence of
the coumarin for tracing a biodistribution makes these coumarin-based
materials a favored PTDDS candidate for biomedical applications.
[9–11]
Moreover, CC-7 exhibits considerably high solubility in polar solvents,
such as dimethyl sulfoxide, which is commonly used as a cosolvent with
H2O in biomedicine experiments.
As illustrated in Fig. 1c, to further increase the antiproliferative
activity of the CC–drug conjugates, we introduced a triphenylpho-
sphonium (TPP) group into the carbazole ring to yield CC-8 through a
facile amide-coupling reaction.[19–21] The TPP group, which has a
delocalized cationic property, has been proven to be an efficient mi-
tochondria-targeting ligand. TPP was reported to accumulate 5- to 10-
fold in cytoplasm from extracellular space under the plasma membrane
potential (Δψp = − 30 to − 60 mV) and then further accumulate 100-
to 500-fold in the mitochondrial matrix under the mitochondrial
membrane potential (Δψm = − 150 to − 170 mV).[22] Accordingly,
the cellular uptake efficiency for conjugates can be enhanced, and the
phototrigger-released species is capable of damaging mitochondrial
DNA (mtDNA), leading to mitochondria dysfunction and eventually cell
death.
In this paper, we introduce a carbazole–coumarin (CC) fused het-
erocycle as the PTDDS platform for performing the photocontrolled
release of anticancer drugs.[12] Because the amine moiety of the car-
bazole serves as an electron donor and the lactone moiety of the cou-
marin acts as an electron acceptor, the coumarin building block, which
has a “push–pull” nature, undergoes a photolytic reaction under more
extended wavelengths to visible light.[13–17] Zheng et al. reported that
CC derivatives possess one- and two-photon fluorescence probes for
imaging carbon monoxide in living tissues.[18] By exploiting the
photocontrolled release and other unique features of CC, we develop
robust synthetic routes for preparing CC–drug conjugates. Herein, we
use a historical nitrogen mustard, chlorambucil, as the drug linkage in
chemotherapeutic regimens. The secondary amine group on the car-
bazole ring also permits facile chemical modification of a site-specific
targeting ligand, resulting in the conjugate system having high effec-
tiveness in cancer treatment.
2.2. Visible-light-triggered photorelease of CC–drug conjugate
Because of their fused heterocyclic structure, CC-7 and CC-8 dis-
solved in methanol solution exhibits π-π* absorption and emission
bands centered at approximately 360 and 450 nm, respectively, which
is attributed to intrinsic optical property of the CC heterocycle (Fig. 2).
[12,18] We further examined fluorescence quantum yields (QYs) for the
dilute solutions of CC derivatives (1 × 10−5 M), and two fluorescence
QYs were comparable (approximately 0.19 for CC-7 and 0.21 for CC-8)
with the photoluminescence reference of 9,10-diphenylanthrance.[23]
According to the photo-SN1 cleavage mechanism, photochemical reac-
tion and fluorescence represent the two main deactivation pathways of
the first singlet excited state (S1).[7,24] Therefore, the photolabile CeO
bond that connects the CC moiety with chlorambucil can be simulta-
neously cleaved in a protic solvent (e.g., methanol or H2O) when the CC
derivatives photoluminesce. Moreover, this photolytic liberation is
usually combined with marked fluorescence enhancement and can thus
be monitored through fluorescence measurement. Although the CC
derivatives have an absorption maximum located in the UV region, the
weak absorption tail toward wavelengths longer than 400 nm enables
visible-light-triggered photocleavage, which may cause less damage to
biological specimens.
2. Results and discussion
2.1. Synthesis of CC–drug conjugates
Commercially available 4-hydroxycarbazole was selected as the
starting material for constructing the carbazole–coumarin fused het-
erocycles. As illustrated in Fig. 1a, the first step in the formation of CC-1
involved Lewis-acid-catalyzed Pechmann condensation by using ethyl
acetoacetate for the lactone synthesis; after methylation of the amine in
the carbazole ring to obtain CC-2, the allylic group in the coumarin ring
was further oxidized by SeO2, followed by subsequent reduction with
NaBH4 to yield CC-3 with a allyl alcohol, which enabled conjugation of
the anticancer drug chlorambucil through DCC-promoted esterification.
However, the overall yield for the three steps was less than 10%, mainly
due to the low solubility of CC-2 in organic solvents during hetero-
geneous oxidation.
As shown in Fig. 2a and 2b, the UV–visible and fluorescence spectra
have a time-dependent profiles upon irradiation with a 405 nm laser
diode (LD). After light exposure, the absorption and emission profiles
are still centered at the same wavelengths for the nonirradiated sam-
ples, but the absorbance and fluorescence intensity changed; the
emission intensity dramatically increased as the absorbance slightly
decreased during light irradiation for 10 min. Notably, the fluorescence
of CC-8 was much more strongly enhanced than that of CC-7 upon light
excitation for the same concentration (1 × 10−4 M). Comparing two
compounds under light exposure, we assumed that CC-7 may undergo
self-aggregation in a polar solvent, leading to the severe self-quenching
of fluorescence; by contrast, the TPP moiety grafted onto CC-8 might
improve the conjugate’s dispersive ability in both polar and nonpolar
solvents, preventing molecular aggregation and self-quenching. Ac-
cording to one previous study, the fluorescence increment is combined
with the photolytic reaction of the coumarin-derived photocages, and
the marked fluorescence enhancement during photocleavege is bene-
ficial to the visualization of the photolytic process and release of drugs
inside living cells.[7]
Alternatively, as shown in Fig. 1b, we developed a synthetic route
based on
a modified condensation reaction using ethyl 4-chlor-
oacetoacetate for coumarin formation. The CC-4 bearing an electro-
philic allyl chloride was thus prepared through the catalysis of me-
thanesulfonic acid. To increase the solubility of CC-4, the amine in the
carbazole was reacted with excess tert-butyl bromoacetate in the pre-
sence of K2CO3 to yield CC-5 functionalized with an acid-labile tert-
butyl ester (OtBu) group. However, the hydrolyzation of the allyl
chloride of CC-5 in an acetone/water (1:1) mixture failed to obtain an
alcohol group. We assumed that the nucleophilic substitution in a protic
solvent tended to result in the formation of a carbocation in the allylic
position, thus leading to undesirable intramolecular side reactions on
the CC ring rather than an intermolecular nucleophilic attack by H2O.
Stronger nucleophile and aprotic media are crucial for performing the
substitution reaction. Accordingly, the chlorambucil drug was first
treated with K2CO3 to provide a carboxylate ion to act as a stronger
nucleophile, and then subsequent substitution with CC-5 was success-
fully performed in dimethylformamide (DMF) solution to yield the
OtBu-protected CC-6. Compared with the first pathway, this route with
an overall yield of approximately 40% involves fewer synthetic steps
and is an efficient and robust method for the construction of CC–drug
conjugates, especially drugs containing COOH groups. Notably, the
OtBu group can be quantitatively removed in an acidic environment,
and the final CC-7 bearing an additional COOH group derived from the
amine group in carbazole enables further chemical modification.
The photoinduced release of chlorambucil drugs was monitored by
reverse-phase high-performance liquid chromatography (HPLC). As il-
lustrated in Fig. 3a, CC-7 and chlorambucil were identified as the elu-
tion peaks at 3.5 and 3.9 min under 10% of H2O in methanol as a
mobile phase, respectively. The amount of CC-7 decreased and free
drug gradually accumulated in the solution as the duration of irradia-
tion with a 405 nm LD increased. Fig. 3b illustrates the photolytic
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B.-Y. Wang, et al.
BioorganicChemistry100(2020)103904
Fig. 1. Synthesis of carbazole–coumarin (CC) derivatives 1–8. Conditions: (i) ethyl acetoacetate, ZnCl2, and EtOH; (ii) CH3I, K2CO3, and DMF; (iii) a. SeO2, PhMe; b.
NaBH4, CH3OH/THF (20:1); (iv) ethyl 4-chloroacetoacetate and CH3SO3H; (v) tert-butyl bromoacetate, K2CO3, and DMF; (vi) chlorambucil, K2CO3, and DMF; (vii)
CF3COOH; (viii) (3-aminopropyl)triphenylphosphonium bromide, HATU, Hünig’s base, and DMF; (ix) CH3COOK, DMF.
process in which the CeO linkage is cleaved in polar protic solvent
under light excitation, and a chlorambucil molecule is then released
from a CC–drug conjugate; however, only the free drug molecules were
observed in the chromatogram. Moreover, photo-triggered drug re-
leased from CC-8 was also eluted at 5.1 min and accumulated under 5%
of H2O in methanol as a mobile phase, but neither CC-8 nor photo-
dissociated counterpart was observed in the chromatogram. Although
the fragments were incompletely recovered in HPLC analysis during
light stimulation, the results clearly indicate that visible-light-triggered
photocleavage efficiently releases the free drug molecules in the two
systems. Fig. 3c plots the correlation between laser irradiation time and
concentration of drug released, calculated from the peak areas relative
to a chlorambucil standard solution (100 μM). The free drug succes-
sively accumulated until a maximum value of approximately 50 μM for
CC-7 and 30 μM for CC-8 was reached after irradiation for 20 min.
Overall, the spectroscopy and HPLC analyses confirm that visible light
of 405 nm wavelength is sufficient to drive the photochemical reaction.
is similar to the cytotoxic profile of pristine chlorambucil toward the
two cell lines (Fig. 4b). The half-maximum inhibitory concentration
(IC50) of CC-7 was much higher than 80 μM for the Jurkat T-cells and
approximately 66 μM for the BT-474 cells. This result suggests that CC-
7 is capable of causing cell death under high dosages, but drug re-
sistance to the nitrogen mustard derivatives was discovered in the two
cell lines, particularly the leukemia cells.
Fig. 4c presents the cytotoxicity profile of CC-8, a drug conjugate
with a TPP ligand that assists in drug accumulation inside the two cell
lines. The proliferation of both types of cell treated with CC-8 was ef-
fectively suppressed as the dosage was increased. Notably, for the
Jurkat T-cells, the IC50 dramatically decreased to approximately 20 μM,
suggesting that the TPP ligand plays a critical role in increasing the
bioavailability of CC–drug conjugates toward leukemia cells. Moreover,
this result implied that the resistance of the mustard drug was improved
by introducing a mitochondria-targeting ligand. CC-8 was assumed to
substantially accumulate in the mitochondria, and the mtDNA lacking a
self-repair mechanism could be permanently damaged by the drug
conjugates, eventually leading to cell death.
2.3. In vitro cytotoxicity evaluation toward human breast and leukemia
cancer cells
To further elucidate the improving drug potency under light assis-
tance, the two types of cell were treated with chlorambucil, CC-7, and
CC-8 at a lower dosage of 10 μM for 30 min, and all cell viability values
were discovered to be higher than 70% in a dark condition. After being
rinsed with buffer to remove extracellular drug molecules, the cell
suspension was exposed to light emitted by a 405 nm LD for 20 min and
then cultured under dark conditions for 48 h. According to the HPLC
analysis, the irradiation time allowed for maximum phototriggered
release of the anticancer drugs. As illustrated in Fig. 4d, approximately
90% of the BT-474 and Jurkat T-cells not treated with the drug survived
The nitrogen mustard, chlorambucil, is a common DNA-alkylating
agent used for chemotherapy and induces nonspecific cell apoptosis
through the accumulation of persistent DNA damage. Two human
cancer cell lines were selected as an in vitro platform for testing the
bioactivity of the CC conjugates by using a colorimetric MTT assay.
Fig. 4a shows that CC-7, a chlorambucil drug conjugate without TPP
ligands, exhibits concentration-dependent cytotoxicity to breast cancer
BT-474 cells and leukemia Jurkat T-cells under dark conditions, which
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BioorganicChemistry100(2020)103904
[26] The CC moiety may behave like a coumarin photocage, and
therefore, quenching the triplet excited state using ground-state mole-
cular oxygen (3O2) to yield cytotoxic singlet oxygen (1O2) is a possible
mechanism of inducing cell death. We also assumed that the enhanced
potency of the CC–drug conjugates toward leukemia cells was a sy-
nergic effect combining phototriggered anticancer drug release and
photosensitization.[27–29] The simultaneously released chlorambucil
and 1O2 contribute equally to nuclear DNA damage and eventually lead
to apoptotic cell death. Moreover, to assess the role of 1O2, a fluor-
esceinyl Cypridina luciferin analogue (FCLA) was employed as a “turn-
on” chemiluminescence probe.[30] The FCLA was readily oxidized by
1O2 and thus markedly enhanced the fluorescence at 524 nm. Fig. 5a
shows that the fluorescence of a methanol solution of CC-7 doped with
FCLA probe was gradually enhanced upon laser irradiation. The emis-
sion intensity of FCLA increased approximately 1.6-fold when the ir-
radiation duration was increased from 0 to 10 min, but further irra-
diation did not cause any further difference in intensity. This result is
consistent with the UV–vis, fluorescence, and HPLC analysis results for
the photoexcited CC–drug conjugates, confirming that photocleavage
and maximum 1O2 production occur on the same time scale. Further-
more, we synthesized a drug-free CC-9 as the model photosensitizer to
confirm the photosensitization effect of CC alone with Jurkat T-cells
(Fig. 1d). In a dark condition, CC-9 (10 μM) was nontoxic for Jurkat T-
cells but exhibited certain cytotoxicity with 60% of viable cells under
405-nm light irradiation. Compared with pristine chlorambucil and CC-
9, the drug-loaded CC-7 shows higher potency with only 50% of viable
cells at the same dosage. The result clearly indicates that the novel
CC–drug conjugate possesses synergistic therapeutic effect toward
cancer cells.
2.4. Mitochondrial stress test in cancer cells treated with CC–drug
conjugates
To confirm the mitochondria targeted by CC-8 with a TPP ligand,
we employed a Seahorse extracellular flux analyzer to monitor mi-
tochondrial stress through the change in oxygen consumption and pH in
the extracellular microenvironment. Cancer cells are characterized by
aerobic glycolysis, which is known as the Warburg effect, to generate
more ATP and reactive oxygen species than are produced in normal
cells by the Krebs cycle.[31] This characteristic also indicates the mi-
tochondria dysfunction that can result from the occurrence of mi-
tochondrial DNA mutations; therefore, suppression of mitochrondrial
DNA replication also results in delayed tumor growth.[32] The acu-
mination of defective mitochondrial respiration was observed in benign
tumors but increased mitochondrial respiration was discovered to sus-
tain the proliferation of malignant tumors.[33] Some reports have also
suggested that targeting mitochondrial respiration or mitochondrial
DNA replication through DNA-alkylating agents (e.g., chlorambucil)
may serve as potential cancer therapy strategies.[34,35] As shown in
Fig. 6a, the real-time monitoring for oxygen consumption (OCR) in
A549 lung cancer cells treated with CC-8 for 24 h clearly demonstrates
decreased basal and maximal respiration (i, iii) and ATP production (ii).
Compared with that of the nontreated cells, the OCR of the treated cells
Fig. 2. (a) UV–visible and (b) fluorescence spectra of CC-7 and CC-8 (1 × 10−4
M) upon 405 nm laser irradiation (0–10 min). The absorption λmax for CC-7 and
CC-8 is 360 nm, and the fluorescence quantum yields for CC-7 and CC-8 are
0.19 and 0.21, respectively, with respect to the 9,10-diphenylanthrance stan-
dard.
the laser exposure in the control experiment, indicating that incident
light of 405 nm wavelength is harmless to both cell lines. After both cell
lines were treated with chlorambucil, we discovered that more than
80% of the cells survived laser exposure, because the mustard drug is
insensitive to light of wavelength 405 nm; however, after treatment
with the CC–drug conjugates, the breast cancer and leukemia cells ex-
hibited different cytotoxic responses upon laser irradiation. The BT-474
cells remained insensitive to the incident light, but the cell proliferation
of the phototreated Jurkat T-cells was greatly suppressed. The percen-
tage of viable cells decreased to approximately 50% and 40% after
treatment with CC-7 and CC-8, respectively, implying that the IC50 was
lower than 10 μM. Unlike in the nonphototreated cells (Fig. 4a and 4c),
the CC moiety serving as a photoresponsive carrier system enhanced the
drug’s potency at a lower dosage under the assistance of visible light
illumination, and the drug conjugates with a TPP ligand exhibited op-
timal antiproliferative activity toward the leukemia cell line.[25] No-
tably, we found that CC-8 was nontoxic for the primary T-cells, and
more than 90% of the cells was survived in both dark and light-ex-
posure conditions. The result suggests that CC-8 can only “target” the
leukemia cancer cells and exhibit enhanced potency under light assis-
tance but cause no damage to the normal T-cells.
was lower; 121.2
0.8 versus 142.6
2.8 versus 98.6
7.2 pmol/min during basal
9.8 pmol/min during ATP
respiration and 79.3
production (Fig. 6b). These data imply that CC-8 can target mi-
tochondria and induce a basal level of mitochondrial damage without
light exposure. Promisingly, future examinations of mitochondrial DNA
replication and cell proliferation after light exposure could demonstrate
the full potential of the CC–drug conjugates in cancer therapy.
Because coumarin-based photocages have been proven using tran-
sient spectroscopy to exhibit efficient triplet–triplet absorption in the
UV-to-visible light region, we assumed that the photoinduced anti-
proliferative activity is caused by the combination of the mustard drug’s
potency and the photosensitization effect of the CC fused heterocycle.
3. Conclusion
In this study, we synthesized a photoresponsive drug carrier based
on a carbazole–coumarin heterocycle. This platform permits visible-
light-triggered drug release and. Through a tailor-made synthetic
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BioorganicChemistry100(2020)103904
Fig. 3. (a) HPLC chromatograms of the CC-drug conjugates under 405 nm laser irradiation (0–60 min); mobile phase for CC-7: MeOH/H2O = 90:10, for CC-8:
MeOH/H2O = 95:5. (b) A visible-light-triggered photolytic and drug release process. (c) Accumulated concentration of chlorambucil released from CC-7 and CC-8
upon laser irradiation.
protocol, the chemotherapy drug (chlorambucil) and site-specific tar-
geting ligand (TPP) can be readily conjugated with the coumarin and
carbazole moieties, respectively. Compared with the pristine drug, the
hybrid drug system resulted in considerably lower IC50 toward leu-
kemia Jurkat T-cells under 405 nm laser irradiation. This was pre-
sumably due to a synergic effect of phototriggered anticancer drug re-
lease and photosensitization of the carbazole–coumarin moiety. A
mechanistic investigation of mitochondrial targeting and method of
further improving drug performance is now in progress.
purification. 1H (400 MHz) and 13C (75 MHz) NMR spectra were re-
corded on a Varian Mercury Plus 400 MHz spectrometer at room
temperature using CDCl3, DMSO‑d6, methanol‑d4, or D2O as the sol-
vents. Spectral processing (Fourier transform, peak assignment, and
integration) was performed using MestReNova 6.2.1 software. Matrix-
assisted laser desorption ionization/time-of-flight (MALDI–TOF) MS
was performed on a Bruker AutoFlex III TOF/TOF system in positive ion
mode using either 2,5-dihydroxybenzoic acid or α-cyano-4-hydro-
xycinnamic acid as the desorption matrix. UV–Vis absorption spectra
was performed on
a Thermo Genesys 10S UV–Vis spectrometer.
Fluorescence emission spectra was recorded on a Hitachi F-2500
spectrometer. HPLC was performed on a JASCO instrument equipped
with a UV–970 UV–Vis detector covering the wavelengths from 200 to
900 nm. The HPLC analysis was conducted at 25 °C using Dr. Maisch
ReproSil-100 C18 column (250 × 4.6 mm, reverse phase with 5 um
porous spherical silica). A solvent mixture of methanol (90%) and water
(10%) was used as a mobile phase and the flow rate was maintained at
4. Experimental section
4.1. Materials and instruments
The chemical reagents and organic solvents for materials synthesis
were obtained as high-purity reagent-grade from commercial suppliers
including Sigma-Aldrich and TCI chemicals and used without further
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BioorganicChemistry100(2020)103904
Fig. 4. Cytotoxicity under dark conditions: (a) CC-7, (b) chlorambucil, and (c) CC-8 toward breast cancer BT-474 (♢) and leukemia Jurkat T (○) cells; (d) photo-
cytotoxicity under 405 nm laser diode (LD) irradiation—(i) control, (ii) LD only, (iii) chlorambucil + LD, (iv) CC-7 + LD, (v) CC-8 + LD toward cancer BT-474 (◆)
and leukemia Jurkat T (●) cells. The drug dosage was 10 μM, at which the cell viability was higher than 70% in dark condition.
Fig. 5. (a) Fluorescence spectra of FCLA added to CC-7 as the photosensitizer upon 405 nm light irradiation (0–20 min). (b) Time courses for the increment of
fluorescence intensity under light exposure.
1.0 cm3/min. Photolysis of CC-conjugates were carried out by using
and the precipitate was collected and purified by flash column chro-
matography using solvent gradient elution (ethyl acetate:hexane = 6:4
to 2:8, Rf = 0.3) to obtain CC-1 as white solid (20%).1H NMR
(400 MHz, DMSO) δ = 11.91 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.74 (d,
J = 8.6 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.47 – 7.51 (m, 2H), 7.32 (t,
J = 7.5 Hz, 1H), 6.28 (s, 1H). 13C NMR (100 MHz, DMSO): δ = 160.84,
155.57, 150.43, 143.11, 140.19, 126.74, 123.17, 122.85, 121.13,
120.80, 112.23, 111.63, 110.50, 110.01, 108.78, 19.59.
405-nm laser diodes under a power density of 1.9 W/cm2.
4.2. Synthetic procedures for CC-3
The CC-3 was synthesized following a modified procedure based on
Zheng’s report.[18] An ethanol solution of 4-hydroxycarbazole
(366.4 mg, 2.0 mmol) and ZnCl2 (381.7 mg, 2.8 mmol) was added by
ethyl acetoacetate (840 μL, 6.7 mmol), and the mixture was stirred at
110 °C under N2 for 24 h. The mixture was then poured into ice water,
A DMF solution of CC-1 (327.0 mg, 1.3 mmol), K2CO3 (1.267 g,
9.1 mmol) was added dropwise by methyl iodide (1.63 ml, 20 mmol).
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B.-Y. Wang, et al.
BioorganicChemistry100(2020)103904
Fig. 6. Mitochondrial stress assay determined using a Seahorse extracellular flux analyzer. (a) Real-time trace monitoring for oxygen consumption rate (OCR) during
the (i) basal respiration, (ii) ATP production, and (iii) maximal respiration phases. Oligomycin and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone were
injected at 18 and 44 min, respectively, to induce the phase transition. (b) Quantitative analysis of OCR of basal respiration and ATP production in nontreated and
CC-8-treated cancer cells.
The mixture was stirred at 50 °C under N2 for 24 h. The solvent and
volatile were removed by vacuum, and the residue was extracted with
CH2Cl2 and water. The organic layer was collected and evaporated to
dryness to obtain CC-2 quantitatively. An anhydrous toluene solution of
SeO2 (55.5 mg, 0.5 mmol) and CC-2 (65.3 mg, 0.25 mmol) was stirred
at 110 °C under N2 for 48 h. After the reaction was cooled to room
temperature, the precipitate was filtered off, and the filtrate was eva-
porated to dryness. The residue redissolved in anhydrous methanol/
THF solutions (20:1) was added dropwise into a methanol solution of
NaBH4 (47.3 mg, 1.25 mmol), and stirred at 25 °C overnight. After
neutralized with 1 M HCl, the mixture was extracted with ethyl acetate
and brine. The organic layer was collected and evaporated to dryness,
and the crude was purified by flash column chromatography using
solvent gradient elution (CH2Cl2:CH3OH = 99:1 to 97:3, Rf = 0.1) to
obtain CC-3 as white solid (20%).1H NMR (400 MHz, DMSO) δ = 8.33
(d, J = 6.9 Hz, 1H), 7.70 – 7.75 (m, 2H), 7.64 – 7.53 (m, 2H), 7.42 –
7.34 (m, 1H), 6.40 (s, 1H), 5.69 (bs, 1H), 4.87 (d, J = 5.6 Hz, 2H), 3.95
(s, 3H). 13C NMR (100 MHz, DMSO): δ = 161.21, 158.72, 150.19,
143.46, 141.02, 126.83, 122.86, 121.98, 121.07, 120.66, 110.57,
109.43, 109.29, 107.04, 106.76, 60.18, 30.15.
1.36 (s, 9H). 13C NMR (100 MHz, CDCl3): δ = 166.71, 160.81, 151.11,
150.77, 143.09, 140.22, 126.60, 123.86, 121.68, 121.32, 121.04,
111.89, 110.98, 109.36, 108.59, 105.61, 83.18, 45.65, 27.93, 27.55.
A DMF solution of chlorambucil (84.1 mg, 0.276 mmol) and K2CO3
(41.7 mg, 0.3 mmol) was mixed at room temperature for 30 min. The
CC-5 (100 mg, 0.25 mmol) dissolved in DMF was added dropwise into
the mixture, followed by stirring for another 30 min. The solvent and
volatile were removed by vacuum, and the residue was purified by flash
column chromatography (CH2Cl2:CH3OH = 99:1, Rf = 0.6) to obtain
CC-6 as yellow solid (72%). 1H NMR (400 MHz, CDCl3) δ = 8.56 (d,
J = 7.9 Hz, 1H), 7.54 – 7.43 (m, 2H), 7.38 – 7.27 (m, 2H), 7.20 (d,
J = 8.7 Hz, 3H), 7.01 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H),
6.37 (s, 1H), 5.32 (s, 2H), 4.89 (s, 2H), 3.70 – 3.45 (m, 8H), 2.54 (t,
J = 7.5 Hz, 2H), 2.43 (t, J = 7.5 Hz, 2H), 2.07 – 1.84 (m, 2H), 1.35 (s,
9H). 13C NMR (100 MHz, CDCl3): δ = 172.69, 166.68, 160.95, 150.32,
144.30, 142.84, 140.12, 129.98, 129.66, 126.55, 123.80, 121.21,
120.96, 120.42, 112.02, 110.79, 109.04, 108.53, 105.60, 83.08, 61.28,
53.47, 45.56, 40.46, 33.84, 33.28, 29.64, 27.89, 26.51.
The CC-6 (120 mg, 0.198 mmol) was mixed with 5 ml of tri-
fluoroacetic acid and stirred for overnight. The volatile was removed by
vacuum, and the crude was recrystallized in CH3OH to obtain CC-7 as
yellow solid (80%). 1H NMR (400 MHz, DMSO)
δ = 8.34 (d,
4.3. Synthetic procedures for CC-7
J = 7.8 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H),
7.64 (d, J = 8.8 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.39 (t, J = 7.5 Hz,
1H), 7.03 (d, J = 8.3 Hz, 2H), 6.65 (d, J = 8.3 Hz, 2H), 6.35 (s, 1H),
5.48 (s, 2H), 5.37 (s, 2H), 3.68 (s, 8H), 1.97 – 1.69 (m, 2H). 13C NMR
(100 MHz, DMSO) δ = 173.04, 170.50, 160.60, 152.41, 150.33,
145.14, 143.76, 141.01, 130.02, 127.05, 122.93, 122.51, 121.54,
120.81, 112.51, 110.77, 109.89, 109.33, 108.49, 107.57, 62.17, 52.84,
45.02, 41.79, 33.93, 33.42, 27.22. MALDI-TOF-MS: Cacld. For
(M + H)+ C32H31Cl2N2O6: 609.16 Da; Found: 609.33 Da.
4-hydroxycarbazole (200 mg, 1.09 mmol), ethyl 4-chlor-
oacetoacetate (0.33 ml, 2.45 mmol), and methanesulfonic acid (2.5 ml.
38.1 mmol) were mixed and vigorously stirred for 2 h. The mixture was
then poured into ice water to remove excess acid. The precipitate was
collected and purified by flash column chromatography (CH2Cl2,
Rf = 0.4) to obtain CC-4 as dark-yellow solid (54%). 1H NMR
(400 MHz, DMSO) δ = 11.99 (s, 1H), 8.30 (d, J = 7.7 Hz, 1H), 7.82 (d,
J = 8.6 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.51 (m, 2H), 7.33 (t,
J = 7.7 Hz, 1H), 6.57 (s, 1H), 5.10 (s, 2H). 13C NMR (100 MHz, DMSO):
δ = 160.11, 152.22, 150.25, 142.53, 139.52, 126.25, 122.30, 122.21,
120.35, 111.69, 110.59, 109.52, 108.39, 54.96, 41.99, 40.15, 39.94,
39.73, 39.52, 39.31, 39.10, 38.89.
4.4. Synthetic procedure for CC-8
(3-aminopropyl)triphenylphosphonium bromide was synthesized
following the reported procedure.[36] An anhydrous DMF solution of
CC-7 (79 mg, 0.129 mmol), HATU (49.3 mg, 0.129 mmol), and Hünig’s
base (0.23 ml, 1.29 mmol) was added by the phosphonium salt (50 mg,
0.155 mmol), and the mixture was stirred for 3 days under N2. After
complete consumption of the CC-7, the solvent and volatile was re-
moved by vacuum, and the crude was purified by flash column chro-
matography (CH2Cl2:CH3OH = 20:1, Rf = 0.5). The final product was
recrystallized in CH3OH to obtain CC-9 as yellow solid (50%). 1H NMR
(400 MHz, CDCl3) δ = 8.43 (d, J = 8.1 Hz, 1H), 7.83 – 7.14 (m, 20H),
7.09 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H), 6.29 (s, 1H), 5.21 (s,
A DMF solution of CC-4 (100 mg, 0.35 mmol), K2CO3 (342 mg,
2.47 mmol) was added dropwise by tert-butyl bromoacetate (2.6 ml,
17.7 mmol). The mixture was stirred at 60 °C under N2 for 24 h. The
solvent and volatile were removed by vacuum, and the residue was
extracted with CH2Cl2 and water. The organic layer was collected and
evaporated to dryness. The crude was purified by flash column chro-
matography (ethyl acetate:hexane = 1:4, Rf = 0.2) to obtain CC-5 as
yellow solid (70%). 1H NMR (400 MHz, CDCl3)
δ = 8.55 (d,
J = 7.9 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H),
7.37 – 7.27 (m, 2H), 7.24 (d, J = 8.7 Hz, 1H), 6.39 (s, 1H), 4.90 (s, 2H),
7

B.-Y. Wang, et al.
BioorganicChemistry100(2020)103904
2H), 4.86 (s, 2H), 3.70 (t, J = 6.2 Hz, 4H), 3.62 (t, J = 6.2 Hz, 4H),
3.41 (m, 2H), 3.04 (m, 2H), 2.61 (t, J = 7.4 Hz, 2H), 2.48 (t,
J = 7.4 Hz, 2H), 2.07 – 1.92 (m, 2H), 1.90 – 1.75 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ = 172.89, 168.37, 161.23, 150.76, 150.24,
144.47, 143.35, 140.37, 135.44, 135.41, 133.27, 133.17, 130.74,
130.61, 130.23, 129.87, 126.69, 123.68, 121.15, 121.01, 120.66,
118.05, 117.19, 112.20, 110.55, 109.20, 108.94, 108.13, 106.41,
77.48, 77.16, 76.84, 61.44, 53.66, 45.85, 40.70, 39.37, 34.03, 33.44,
26.72, 22.06, 20.29, 19.75. MALDI-TOF-MS: Cacld. For M+
C53H51Cl2N3O5P: 910.29 Da; Found: 910.02 Da.
respectively. All the inhibitors were purchased from Ailgent
Technologies, Inc., Santa Clara, CA, USA), inserts with activated probes
and inhibitors were inserted into cell-seeded wells and sent into
Seahorse Analyzer for measurement. The raw data were outputted and
analyzed by the excel template provided by the manufacturer of
Seahorse Analyzer.
Acknowledgments
The authors would like to thank Changhua Christian Hospital and
Ministry of Science and Technology (MOST) of Taiwan, for financially
supporting this research under grant numbers: 105-CCH-ICO-128,
MOST-105-2119-M-040-001 and 106-2113-M-040-001.
4.5. Synthetic procedure for CC-9
The CC-9 was synthesized following the same procedure for pre-
paring CC-7. A DMF solution of potassium acetate (0.232 g, 2.36 mmol)
and CC-5 (47.1 mg, 0.12 mmol) was stirred at room temperature for
overnight. The solvent and volatile were removed by vacuum, and the
residue was extracted with CH2Cl2 and brine. The organic layer was
collected and evaporated to obtain tert-butyl ester-protected CC-9 as
yellow oil. The product was then treated with 3 ml of trifluoroacetic
acid and stirred for overnight. The volatile was removed by vacuum,
and the crude was recrystallized in CH3OH to obtain CC-9 as orange
solid (60%). 1H NMR (400 MHz, DMSO) δ = 13.19 (bs, 1H), 8.34 (d,
J = 7.7 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H),
7.64 (d, J = 8.8 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.39 (t, J = 7.5 Hz,
1H), 6.38 (s, 1H), 5.47 (s, 2H), 5.37 (s, 2H), 2.21 (s, 3H). 13C NMR
(100 MHz, DMSO) δ = 170.07, 169.87, 159.98, 151.77, 143.15,
140.40, 126.42, 122.29, 121.91, 120.91, 120.18, 110.16, 108.70,
106.95, 61.58, 48.63, 44.39, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10,
38.89, 20.63.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103904.
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