Anticoagulant activity of singlet oxygen released from a water soluble endoperoxide by thermal cycloreversion

Article abstract of DOI:10.1039/d1ra02569d

Singlet oxygen generated by photosensitization has limited potential in vivo due to light attenuation in tissues. However, controlled chemical generation of this reactive oxygen species is likely to open new therapeutic spaces to explore. The fact that its activity is limited by the rate of cycloreversion reaction and the diffusion distance of the excited state molecular oxygen species, is a clear advantage, considering the serious side effects of off-target anticoagulants. In this work, we present novel 1,4-naphthalene endoperoxides as potential anti-coagulant agents due to thermal release of singlet oxygen. This journal is

Full text of DOI:10.1039/d1ra02569d

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Anticoagulant activity of singlet oxygen released
from a water soluble endoperoxide by thermal
cycloreversion†
Cite this: RSC Adv., 2021, 11, 14513
ab
Meina Liu,ab Esma Ucar,c Ziang Liu,ab Lei Wang,
Li Yang,b Jiawei Xud
*
ab
*
and Engin U. Akkaya
Singlet oxygen generated by photosensitization has limited potential in vivo due to light attenuation in
tissues. However, controlled chemical generation of this reactive oxygen species is likely to open new
therapeutic spaces to explore. The fact that its activity is limited by the rate of cycloreversion reaction
and the diffusion distance of the excited state molecular oxygen species, is a clear advantage,
considering the serious side effects of off-target anticoagulants. In this work, we present novel 1,4-
naphthalene endoperoxides as potential anti-coagulant agents due to thermal release of singlet oxygen.
Received 1st April 2021
Accepted 9th April 2021
DOI: 10.1039/d1ra02569d
rsc.li/rsc-advances
allow much exibility in their utilization. However, in recent
years aromatic endoperoxides,10 with their tunable rates of
singlet oxygen release,11 attracted attention as they appear to be
Introduction
Among reactive oxygen species, singlet oxygen has a unique
place as it is the most reactive, and short-lived.1 Other than
being a useful chemical agent,2 it is known to be primarily
responsible for the photodynamic action3 and various critical
biological processes, including regular maintenance.4
Within the last decade, there has been accumulation of
evidence5 suggesting a role for singlet oxygen in hemostasis,
which is the system of generation and destruction of thrombi,
which involve opposing actions of coagulation and thrombol-
ysis. The cellular part of the hemostatic process involves the
thrombocytes and endothelial cells for coagulation and the
polymorphonuclear granulocytes (PMN) for thrombolysis. PMN
are known6 to generate singlet oxygen enzymatically by the
action of NADPH-oxidase and myeloperoxidase. The short life-
time of singlet oxygen, may help to put a spatio-temporal limit
to anticoagulant action.
more amenable for biological utilization.
Both photogenerated singlet oxygen12 and chemically
generated (hypochlorite/chloramine reaction) singlet oxygen13
was shown to have thrombolytic action in vitro. Unfortunately,
these two methods will have limited value in vivo, because of the
strong light attenuation in tissues, and impractical nature of
producing large concentrations of hypochlorite and chloramine
or hydrogen peroxide, on demand. Thus, we posit that the most
viable option is to generate singlet oxygen by the thermal
cycloreversion of aromatic endoperoxides.
Experimental
Materials and instrumentation
All commercial chemicals were used as supplied unless other-
wise indicated. Anhydrous solvents were obtained from
a Solvent Purication System. Column chromatography was
performed using silica gel (200–300 mesh). 1H and 13C NMR
spectra were recorded on Bruker Avance II 400 MHz or Bruker
Avance III 500 MHz. Signal splitting patterns were described as
singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m)
with coupling constants (J) in hertz (Hz). High resolution mass
spectra (HRMS) were recorded with an Agilent mass spectrom-
Singlet oxygen can be generated by the intermediacy of
photosensitizers under irradiation of ground state molecular
oxygen, or alternatively by chemical reactions of phosphine/
phosphite ozonides;7 also, molybdate,8 chloramine and HOCl
reactions with H2O2.9 The chemical methods mentioned above
all require strongly oxidant inorganic species which would not
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong eter. Reactions were monitored by thin-layer chromatography
Road, 116024, Dalian, China. E-mail: leiwang@dlut.edu.cn; eua@dlut.edu.cn
bDepartment of Pharmaceutical Science, School of Chemical Engineering, Dalian
using Merck TLC Silica gel 60 F254.
Synthesis of compound 2. 1,4-Dimethylnaphthalene (1)
University of Technology, 2 Linggong Road, 116024, Dalian, China
(0.44 g, 2.8 mmol, 1 equiv.) was dissolved in dichloromethane
cDepartment of Chemistry, Bilkent University, 06800 Ankara, Turkey
(16.0 mL) and degassed with argon. Under an argon atmo-
sphere, N-bromosuccinimide (2 equiv.) and benzoyl peroxide
(0.07 equiv.) were added and the suspension was degassed to
give a yellow suspension. The reaction mixture was heated
dCollege of Pharmacy, Liaoning University of Traditional Medicine, 110847 Shenyang,
China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra02569d
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under reux argon atmosphere for 5 hours. The reaction The mixture was diluted with CH2Cl2, washed with H2O and the
mixture was cooled to room temperature and then washed with solvent was removed in vacuum. The residue was puried by
2.0 M HCl (2 ꢀ 15 mL), 2.0 M NaOH (2 ꢀ 20 mL), brine, and column chromatography with 10 : 1 (vol/vol) CH2Cl2/MeOH to
1
dried with MgSO4. The solvent was evaporated to yield an off- get the desired product (36 mg, 0.048 mmol) in 30% yield. H-
white powder as the crude product, which was puried by NMR (500 MHz, CDCl3) d 8.17–8.15 (m, 2H), 7.54–7.53 (m,
column chromatography with 1 : 1 (vol/vol) dichloromethane/ 2H), 7.44 (s, 2H), 5.00 (s, 4H), 3.68–3.64 (m, 44H), 3.55–3.53 (m,
petroleum ether to yield the desired product in 50% yield. 4H), 3.37 (s, 6H); 13C NMR (125 MHz, CDCl3) d 134.2, 132.1,
Used without further purication.
126.0, 125.8, 124.6, 72.0, 71.8, 70.69, 70.66, 70.62, 70.59, 70.58,
Synthesis of compound 3. Hexaethyleneglycol monomethyl 70.5, 69.5, 59.0; HRMS (ESI) m/z calcd for C38H64O14Na [M + Na]+
ether (2.0 equiv.) was dissolved in dry THF (5.0 mL) under argon 767.4188, found 767.4188.
atmosphere. Aer that, sodium hydride (2.0 equiv.) was added
Attempted synthesis of compound 7. Compound 6 (0.020 g,
and the mixture was stirred under reux for 1 h. Next compound 0.027 mmol) was dissolved in 1.0 mL D2O. The reaction mixture
2 (0.040 g, 0.17 mmol, 1.0 equiv.) dissolved in THF (1.0 mL) was was cooled to 0 ꢁC. Methylene blue was added into the solution
added. The reaction mixture was stirred for 12 h under reux. and mixture was stirred for 8 hours under oxygen atmosphere.
Then, the crude mixture was concentrated in vacuum. The During the reaction, 630 nm LED lamps (red light irradiation)
mixture was diluted with CH2Cl2, washed with H2O and the was used. Aer removal of the methylene blue by cation
solvent was removed in vacuum. The residue was puried by exchange resin, the crude product by analysed was shown to be
column chromatography with 10 : 1 (vol/vol) CH2Cl2/MeOH to an intractable mixture, made more difficult to handle by the
get the desired product (36.0 mg, 0.048 mmol) in 50% yield. 1H short halives of the endoperoxides (with 1,4 and 5,8-bridges,
NMR (400 MHz, CDCl3) d 8.21–8.19 (m, 1H), 8.06–8.03 (m, 1H), presumably).
7.59–7.54 (m, 2H), 7.40 (d, J ¼ 8.0 Hz, 1H), 7.30–7.28 (m, 1H),
5.01 (s, 2H), 3.69–3.65 (m, 24H), 3.40 (s, 3H), 2.71 (s, 3H); 13C
Results and discussion
NMR (100 MHz, CDCl3) d 126.5, 125.9, 125.8, 125.6, 124.8, 124.6,
72.0, 71.9, 70.70, 70.65, 70.62, 70.61, 70.59, 70.58, 70.53, 69.3,
59.0, 19.5; HRMS (ESI) m/z calcd for C25H38O7Na [M + Na]+
473.2510, found 473.2513.
Here in this work, we targeted the synthesis of PEG-
functionalized naphthalene endoperoxides of the structures
shown in Fig. 1.
Synthesis of compound 4. Compound 3 (0.02 g, 0.04 mmol)
was dissolved in D2O (1.0 mL). The reaction mixture was cooled
to 0 ꢁC. Methylene blue was added into the solution and mixture
was stirred for 8 hours under oxygen atmosphere. During the
reaction, 630 nm lamp (red light irradiation) was used. Aer
removal of the methylene blue by mixing with 0.5 g cation
Bromomethyl and 1,4-(bis-bromomethyl)naphthalenes were
reacted with hexaethyleneglycol monomethyl ether in the
presence of NaH in THF. The products were puried by
1
exchange resin (Chelex 100, Na+ form) and ltration. H NMR
(400 MHz, D2O) d 7.43–7.29 (m, 4H), 6.89 (d, J ¼ 8.0 Hz, 1H), 6.80
(d, J ¼ 8.0 Hz, 1H), 4.43–4.32 (m, 2H), 3.65–3.55 (m, 24H), 3.28
(s, 3H), 1.84 (s, 3H).
Synthesis of compound 5. 1,4-Dimethylnaphthalene (1)
(0.44 g, 2.8 mmol, 1 equiv.) was dissolved in dichloromethane
(16 mL) and degassed with argon. Under an argon atmosphere,
N-bromosuccinimide (3 equiv.) and benzoyl peroxide (0.1
equiv.) were added and the suspension was degassed to give
a yellow suspension. The reaction mixture was heated under
reux argon atmosphere for 9 hours. The reaction mixture was
cooled to room temperature and then washed with 2.0 M HCl (2
ꢀ 15 mL), 2 M NaOH (2 ꢀ 20 mL), brine, and dried with MgSO4.
The solvent was evaporated to yield an off-white powder as the
crude product, which was puried by column chromatography
with 1 : 1 (vol/vol) dichloromethane/petroleum ether to yield
1
the desired product in 80% yield. H-NMR (400 MHz, CDCl3)
d 8.22–8.21 (m, 2H), 7.68–7.66 (m, 2H), 7.49 (s, 2H), 4.94 (s, 4H).
Synthesis of compound 6. Hexaethylene glycol monomethyl
ether (4 equiv.) was dissolved in dry THF (5.0 mL) under argon
atmosphere. Aer that, sodium hydride (4.0 equiv.) was added
and the mixture was stirred under reux for 1 h. Next compound
5 (0.0526 g, 0.17 mmol, 1.0 equiv.) dissolved in THF (1.0 mL)
was added. The reaction mixture was stirred for 12 h under
Fig. 1 Synthesis of the PEG-functionalized, water soluble naphthalene
reux. Then, the crude mixture was concentrated in vacuum. endoperoxides.
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chromatography. The reaction with photogenerated singlet pathways, intrinsic pathway and the common pathway of
oxygen was carried out in D2O, using methylene blue as coagulation. Partial thromboplastin time (PTT) measures
photosensitizer and a red LED array for light source at 4 ꢁC.
a number of coagulation factors such as brinogen,
Endoperoxides were puried by column chromatography. prothrombin, proaccelerin, anti-hemophilic factor, Stuart–
Doubly-PEGylated naphthalene 7, did not react very well, and Prower factor, plasma thromboplastin antecedent, and Hage-
produce an intractable mixture of the desired endoperoxide 7 man factor. The prothrombin time (PT) is a measure of the
(15–20%), unreacted material 6 and 5,8-endoperoxide. Thus, we clotting via the extrinsic pathway. TT and FIB measures
focussed on the mono-PEGylated compound 4 with high reac- specically the rate of brin clot formation, which maybe more
tion yield (>98%). The half-life of the endoperoxide was deter- sensitive changes produced by singlet oxygen.
ꢁ
mined by NMR in D2O, and found to be 53 minutes at 37 C
The results were supportive of our expectations. Within the
(Fig. 2). The NMR spectrum in the aliphatic region was crowded concentration range studied (0–10 mM) APTT (Fig. 3a) and PT
with PEG and methyl peaks, however aromatic region of the (Fig. 3b) did not show signicant changes, but TT (Fig. 3c) and
spectra can be used to study the rate of progression from the FIB time (Fig. 3d), and the brinogen concentration changed in
endoperoxide back to naphthalene core. The disappearance of accordance with anti-coagulant activity (Fig. 3e).
the olenic protons at 6.8–7.0 ppm was coupled to the emer-
gence of the aromatic set at 8.0–8.2 ppm. This is in accordance hemostatic equilibrium can be, and most likely is multi-faceted,
with the expected changes on the ejection of singlet oxygen. there is already literature data suggesting that brinogen is
While it is possible that the effect of singlet oxygen on
In vitro tests of anti-coagulant activity of the endoperoxide 4 oxidized by singlet oxygen at the methionine sites, blocking the
ꢁ
at 37 C were carried out using rat blood plasma. As a control, formation of polymeric brils and thus clotting. In addition,
compound 3 was used to isolate the role of singlet oxygen oxidized brinogen is converted by thrombin into a plasmin-
precisely. Rat blood were collected into plastic tubes with ogen activator. Plasminogen when hydrolytically activated is
sodium citrate (citrate/blood: 1/9, v/v) for plasma anti- transformed into plasmin, which is the major factor for the
coagulation. Aer centrifugation at 3000 rpm for 10 minutes, breakdown to brin polymers.
300 mL of plasma was mixed with 30 mL of endoperoxide with
a nal concentration of 0–10 mM and incubated for 30 min at
37 ꢁC. Then, prothrombin time (PT), activated partial throm-
boplastin time (APTT), thrombin time (TT) and brinogen (FIB)
were analyzed by a blood coagulation analyzer (Sysmex CA-500)
with commercial kits following the manufacturer's instructions.
The experiments were conducted in multiplicates (n ¼ 3), and
the results are presented as mean ꢂ standard deviation (SD).
Activated Partial Thromboplastin Time (APTT) measures the
overall speed at which blood clots by means of two distinct
Fig. 3 (a–e) Coagulation data and oxidative inactivation of fibrinogen.
Treated plasma was incubated with 0–10 mmol Lꢃ1 (final concentra-
Fig. 2 Gradual thermal conversion of the endoperoxide 4 (top spec- tion) compound 3 (control, black squares) or compound 4 (red dia-
trum) to its naphthalene precursor 3 at 37 ꢁC in D2O. Spectra from top monds) for 30 minutes (37 ꢁC). Various coagulation parameters were
to bottom: 0, 10 minutes, 30 minutes, 1 h, 2 h, 3 h, respectively.
acquired by blood coagulation analyzer.
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H. S. Tang, Y. Huang, Y. W. Lin, Z. Cao and W. M. He,
Green Chem., 2020, 22, 1720–1725.
Conclusions
3 A. P. Castano, T. N. Demidova and M. R. Hamblin,
Photodiagn. Photodyn. Ther., 2004, 1, 279–293.
4 I. E. Kochevar, Sci. STKE, 2004, pe7.
5 (a) T. W. Stief, U. Feek, A. Ramaswamy, V. Kretschmer,
H. Renz and J. Hareed, Thromb. Res., 2001, 104, 361–370;
(b) T. W. Stief, Clin. Appl. Thromb./Hemostasis, 2007, 26,
225–239.
6 (a) M. J. Steinbeck, A. U. Khan and M. J. Karnovsky, J. Biol.
Chem., 1992, 267, 13425–13433; (b) C. C. Winterbourn and
A. J. Kettle, Antioxid. Redox Signaling, 2013, 18, 642–660.
7 P. D. Bartlett, G. D. Mendenhall and D. L. Durham, J. Org.
Chem., 1980, 45, 4271–4274.
In summary, we present a water soluble endoperoxide with
a potential to inhibit clotting and enhance thrombolysis. Our
methodology is novel because of the fact that we use endoper-
oxides, which are more likely to be non-toxic at cell and organ
level compared to hypochlorites. Optimized derivatives of these
compounds are likely to nd utility in a number of diseases that
feature blood clots, such as pulmonary embolism, myocardial
infarction, and stroke. The most common use would be for
ischemic stroke, by either systemic administration or through
the application of an arterial catheter directly to the site of the
peripheral arterial thrombi. Our work in further assessment of
the therapeutic potential of endoperoxide delivered singlet
oxygen is in progress.
8 K. Boehme and H. D. Brauer, Inorg. Chem., 1992, 31, 3468–
3471.
9 (a) A. Greer, Acc. Chem. Res., 2006, 39, 797–804; (b)
D. F. Evans and M. W. Upton, J. Chem. Soc., Dalton Trans.,
1985, 1141–1145.
Conflicts of interest
There are no conicts to declare.
10 (a) S. Kolemen, T. Ozdemir, D. Lee, G. M. Kim, T. Karatas,
J. Yoon and E. U. Akkaya, Angew. Chem., Int. Ed., 2016, 55,
3606–3610; (b) M. Klapper and T. Linker, Chem.–Eur. J.,
2015, 21, 8569–8577.
Acknowledgements
The authors acknowledge support from LiaoNing Revitalization
Talents Program (XLYC1902001, XLYC1907021) and the
Fundamental Research Funds for the Central Universities
(DUT19RC(3)009).
11 (a) W. Fudickar and T. Linker, Angew. Chem., Int. Ed., 2018,
57, 12971–12975; (b) E. Ucar, D. Xi, O. Seven, C. Kaya,
X. J. Peng, W. Sun and E. U. Akkaya, Chem. Commun.,
2019, 55, 13808–13811; (c) S. Ayan, G. Gunaydin,
N. Yesilgul-Mehmetcik, M. E. Gedik, O. Seven and
E. U. Akkaya, Chem. Commun., 2020, 56, 14793–14796.
12 (a) M. O. Senge and M. W. Radomski, Photodiagn. Photodyn.
Ther., 2013, 10, 1–16; (b) S. Houthoofd, M. Vuylsteke,
S. Mordon and I. Forneau, Photodiagn. Photodyn. Ther.,
2020, 29, 101568.
Notes and references
1 (a) P. Ogilby, Photochem. Photobiol. Sci., 2010, 9, 1543–1560;
(b) M. Bregnhøj, M. Westberg, F. Jensen and P. R. Ogilby,
Phys. Chem. Chem. Phys., 2016, 18, 22946–22961.
2 (a) W. B. He, L. Q. Gao, X. J. Chen, Z. L. Wu, Y. Huang, Z. Cao,
X. H. Xu and W. M. He, Chin. Chem. Lett., 2020, 31, 1895–
1898; (b) K. J. Liu, J. H. Deng, J. Yang, S. F. Gong,
Y. W. Lin, J. Y. He, Z. Cao and W. M. He, Green Chem.,
2020, 22, 433–438; (c) L. Y. Xie, Y. S. Bai, X. Q. Xu, X. Peng,
13 (a) T. W. Stief, V. Kretschmer, B. Kosche, M. O. Doss and
H. Renz, Ann. Hematol., 2001, 80, 189–194; (b) T. W. Stief,
J. Kurz, M. O. Doss and J. Fareed, Thromb. Res., 2000, 97,
473–480.
14516 | RSC Adv., 2021, 11, 14513–14516
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