Synthesis of boronated meso-arylporphyrins via copper-catalyzed 1,3-dipolar cycloaddition reaction and their binding ability towards albumin and low density lipoproteins

Article abstract of DOI:10.1016/j.jorganchem.2020.121248

A series of novel meso- triazoloporphyrin-carborane conjugates was prepared in good yields via the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of Zn(II) or Pd(II) 5,10,15,20-tetrakis[4-(2-propargyloxy)-2,3,5,6-tetrafluorophenyl]porphyri

Full text of DOI:10.1016/j.jorganchem.2020.121248

Journal Pre-proof
Synthesis of boronated meso-arylporphyrins via copper-catalyzed 1,3-dipolar
cycloaddition reaction and their binding ability towards albumin and low density
lipoproteins
Valentina A. Ol'shevskaya, Andrei V. Zaitsev, Anton V. Makarenkov, Elena G.
Kononova, Alina A. Markova, Alexey A. Kostyukov, Anton E. Egorov, Mikhail A.
Klimovich, Olga A. Koroleva, Vladimir A. Kuzmin
PII:
S0022-328X(20)30150-9
DOI:
https://doi.org/10.1016/j.jorganchem.2020.121248
JOM 121248
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 20 December 2019
Revised Date: 20 March 2020
Accepted Date: 22 March 2020
Please cite this article as: V.A. Ol'shevskaya, A.V. Zaitsev, A.V. Makarenkov, E.G. Kononova, A.A.
Markova, A.A. Kostyukov, A.E. Egorov, M.A. Klimovich, O.A. Koroleva, V.A. Kuzmin, Synthesis of
boronated meso-arylporphyrins via copper-catalyzed 1,3-dipolar cycloaddition reaction and their binding
ability towards albumin and low density lipoproteins, Journal of Organometallic Chemistry (2020), doi:
https://doi.org/10.1016/j.jorganchem.2020.121248.
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Synthesis of Boronated meso-Arylporphyrins via Copper-catalyzed 1,3-
Dipolar Cycloaddition Reaction and Their Binding Ability Towards
Albumin and Low Density Lipoproteins
Valentina A. Ol'shevskayaa,*, Andrei V. Zaitseva, Anton V. Makarenkova, Elena G. Kononovaa,
Alina A. Markovaa, Alexey A. Kostyukovb, Anton E. Egorovb, Mikhail A. Klimovichb, Olga A.
Korolevab, Vladimir A. Kuzminb
a A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
119991, Moscow, Russian Federation
b Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334, Moscow,
Russian Federation
Graphical abstract
A series of novel meso- triazoloporphyrin-carborane conjugates were prepared in good yields
starting from propargyl derivatives of tetrakis(pentafluorophenyl)porhyrin and tetrakis(4-
hydroxyphenyl)porphyrin and azidometyl carborane. Their photophysical properties,
complexation with LDL, triplet states, quantum yields and cytotoxicity were investigated.
Abstract
A series of novel meso- triazoloporphyrin-carborane conjugates was prepared in good yields via
the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of Zn(II) or Pd(II)
5,10,15,20-tetrakis[4-(2-propargyloxy)-2,3,5,6-tetrafluorophenyl]porphyrin or Zn(II) 5,10,15,20-
tetra(p-propargyloxyphenyl)porphyrin with 1-azidomethyl-o-carborane in CH2Cl2. Zinc
metalloporphyrins were demetallated under acidic conditions to afford the corresponding free-
base triazolo-carborane porphyrins in excellent yields. All new compounds were characterized
by MS, NMR and UV-vis spectroscopy and their photophysical properties were studied.
Key words
porphyrins, triazoles, carboranes, fluorescence, triplet state, phototoxicity.

Highlights
Novel triazole-carborane porphyrin conjugates were synthesized.
Synthesized compounds were structurally characterized by conventional spectroscopy.
Binding constants for albumin and LDL with synthesized compounds were obtained.
Singlet oxygen quantum yields of synthesized compounds were measured.
All compounds are nontoxic and show pronounced photoinduced cytotoxicity.
1. Introduction
Porphyrins and their derivatives represent an important class of macroheterocycles that
have important applications in a number of fields, including catalysis [1], polymer synthesis [2],
materials [3]. As photoactive compounds they are used as photosensitizers (Ps) in photodynamic
therapy (PDT) for treatment of oncological cancer diseases [4]. PDT is based on excitation of Ps
with light of specific wavelengths initiating of type I and type II photochemical reactions that
may result in the primary tumor destruction via the local generation of reactive oxygen species
(ROS) [5-7]. PDT is a complex modality for destruction of cancer cells, and results of the
treatment depend on multiple parameters including the chemical and photochemical properties of
the PS, light delivery system, and subcellular localization of Ps in organelles of the tissue.
Numerous porphyrin derivatives as bases for PDT application have been reported and
some of them are currently used in preclinical and clinical trials [8]. Nevertheless, intensive
investigations are carried out on the improvement of the structural characteristics of Ps in the
hope to get newer derivatives with the higher anticancer PDT activity. One of the approaches
includes the functionalization of porphyrins with well-established pharmacophore structural
fragments. Small nitrogen heterocycles are substituents of particular interest, providing sites for
metal coordination, hydrogen bonding, alkylation, and thus affecting on the electronic and
physico-chemical properties of the porphyrin macrocycle.
Among nitrogen heterocycles five-membered 1,2,3-triazoles are important scaffold due to
their extensive biological activities. Copper(I)-catalyzed 1,3-dipolar cycloaddition of azides and
alkynes leading to 1,4-substituted 1,2,3-triazoles plays an important role in the preparation of
biologically active compounds [9]. It employs stable and readily available chemical reagents
(azides and alkynes) which react rapidly at ambient temperature, tolerates a wide range of
functional groups, including unprotected alcohols, carboxylic acids, and amines, and shows little
sensitivity to steric factors [10-12]. This simple reaction is considered as an efficient approach
for linking two organic fragments by triazole bridge. But the role of triazoles is not limited to the
role of a convenient linker for organic groups. A great number of compounds containing 1,2,3-

triazole moieties have been reported to show a broad spectrum of biological activities, including
fungicidal, antimicrobial, anticonvulsant, analgesic, and antitumor behavior [13-16]. Triazole
compounds containing three nitrogen atoms in the five-membered aromatic ring are able to bind
with a variety of enzymes and receptors in biological system via diverse non-covalent
interactions, and thus modulating versatile biological activities [17] Moreover, this heterocycle is
stable towards metabolic degradation and can improve the stability of molecules. So, coupling
porphyrins to triazoles may lead to the multi-chromophore conjugates with improved biological
activity possessing the potential for medical applications. With regard to porphyrins, it is
extremely important to have available and simple synthetic methods for functionalization of
macrocycle backbone in order to obtain derivatives with desirable characteristics. Via click
chemistry, porphyrins have been connected with various structural units such as fullerene [18],
ferrocene [19], carbohydrate [20-21] and also with triazole units and peripheral alkyl chains [22-
23] in a single molecular framework for the study of their photophysical and therapeutic
properties.
Polyhedral carboranes due to their unique chemical and structural properties[24] has
attracted much attention as promising systems for various practical applications, including the
biomedical ones [25].They also prospective for drug therapy as the hydrophobic pharmacophores
targeting of biomolecules with suitable hydrophobic binding sites [26-27]. As a class of efficient
photosensitizers, porphyrin conjugates with boron polyhedra have been under extensive study for
PDT [28], since it was evidenced that carboranyl-substituted porphyrins demonstrated a higher
PDT efficiency than comparable non-boronated analogues [29]. Furthermore, conjugation of
carborane polyhedra to porphyrins yields dual efficacy antitumor photo/radiosensitizers allowing
to carry out simultaneously both neutron capture therapy (BNCT) and PDT [30-31]. For the
preparation of triazole-substituted boronated porphyrins meso-tetraarylporphyrins were used due
to their straightforward syntheses and the availability of a wide variety of aryl-functionalized
derivatives [32]. Most aryl derivatives do not change the electronic properties of the porphyrin
macrocycles chromophore in any major way [33], but this does not apply to meso-tetrakis-
(pentafluorophenyl)porphyrin which contain 20 strongly electron-withdrawing fluorine atoms
having a significant inductive effect on porphyrin core. So the chemical and physical properties
of this porphyrin and its functional derivatives may be altered when compared to non-fluorinated
phenyl-substituted macrocycles [34-39]. The presence of pentafluorophenyl groups in this
macrocycle enables its further simple modification at the peripheral parts by aromatic
nucleophilic substitution [40-41]. In living organisms porphyrins, as prosthetic groups of
enzymes, play an important role for electron transport in oxidation– reduction processes of cells
and oxygen transport [42]. So, extensive studies have been carried out with respect to their

interaction with some cell compartments for estimation their therapeutic value. By considering
the anticancer significance of porphyrins and biological dependence of the peripheral
substituents of porphyrins on their properties it was contemplated to synthesize new porphyrin
structures combining porphyrin macrocycle (fluorinated and parent phenyl-substituted),
carborane and 1,2,3-triazole moiety within one molecule and to study binding ability and
transport of prepared conjugates with albumin and low density lipoproteins (LDL).
2.Results and discussion
2.1. Synthesis
In this work for the preparation of porphyrins functionalized with carborane polyhedra
and
1,2,3-triazole
heterocycle
commercially
[43]
available
5,10,15,20-
tetrakis(pentafluorophenyl)porphyrin
(1)
and 5,10,15,20-tetrakis(4-
hydroxyphenyl)porphyrin (2) [44] were used as starting compounds. Porphyrin 1 containing four
pentafluorophenyl substituents easily enters into the nucleophilic para-regioselective SNAr
aromatic substitution reactions with various nucleophiles [45-49] and this reaction widely
applied in pharmaceutical and chemical research, providing a broadly useful platform for the
modification of aromatic ring scaffolds. In our study the functionalization of porphyrin 1 was
achieved through two simple reaction steps. It was found that zinc (3) [50] or palladium (4) [51]
complexes of porphyrin 1 readily reacted with excess of propargyl alcohol to afford porphyrins
5, 6 via the substitution of fluorine atoms in the para position of pentafluorophenyl units with
four propargyloxy groups. The reaction was performed in boiling THF in the presence of NaOH
(powder). The crude products were purified by column chromatography to afford the porphyrins
5 and 6 with a yield of 94% and 92%, respectively. Porphyrins 5, 6 are valuable substrates to use
in Huisgen copper catalyzed 1,3-dipolar cycloaddition reaction [52-53] which give an
opportunity to easily connect various types of molecular units. We have found that reaction of 1-
azidomethyl-o-carborane (7) [54] with alkynyl-substituted porphyrins 5 and 6 under “click-
chemistry” conditions proceeded smoothly and chemoselectively by heating in CH2Cl2 affording
boronated porphyrin-triazole conjugates 8, 9 in 72-79 % yields. Free base conjugate 10 was
obtained in 95% yield by the removal of zinc from compound 8 under the action of
trifluoroacetic acid in CHCl3 – MeOH mixture (Scheme 1.).

Scheme 1. Synthesis of carboranyl triazole conjugates 8-10 based on porphyrin 1.
To study the influence of porphyrin chromophore structure on the properties of boronated
triazole conjugates we have synthesized similar derivatives based on non-fluorinated porphyrin 2
as starting compound. Reaction between porphyrin 2 and excess of propargyl bromide in DMF in
the presence of K2CO3 (Williamson coupling reaction) led to the formation of 5,10,15,20-
tetra(propargyloxyphenyl)porphyrin (11) [55] in 85% yield. To avoid undesirable metalation of
porphyrin ring with copper ions during click reaction zinc complex (12) was synthesized in 92%
yield by reaction of porphyrin 11 with Zn(OAc)2 in CH2Cl2-MeOH mixture at ambient
temperature. Next, the terminal triple bonds of propargylated porphyrin 12 were introduced into
reaction with azide 7 in the presence of copper(II) acetate/sodium ascorbate catalyst under argon
atmosphere at boiling in a mixture of solvent CH2Cl2/H2O leading to porphyrin 13 containing
1,2,3-triazole linkers between porphyrin macrocycle and carborane polyhedra (Scheme 2.).

OH
O
NH
N
N
NH
N
N
Br
K2CO3 / DMF
Zn(OAc)2
HO
OH
O
O
HN
HN
2
11
OH
O
N
N
N
O
Zn
O
O
N
N
N
7
N
N
N
N
N
N
1) (CH3
)3O BF4
O
O
Zn
O
O
Sodium Ascorbate/ Cu(OAc)2
2) CH3OH
N
N
CH2Cl2 / H2
O
N
N
N
N
N
N
12
13
O
N
N
N
N
H3C
N
N
O
4 BF4
NH
N
N
O
O
HN
N
N
N
N
N
N
CH3
H3
C
14
O
N
N
H3C
N
Scheme 2. Synthesis of carboranyl triazole conjugates 13, 14 based on porphyrin 2.
Alkylation of porphyrin 13 with the excess of trimethyloxonium tetrafluoroborate [56] in CH2Cl2
resulted in non-metallated tetracationic triazolium salt 14 in quantitative yield. This reaction is
accompanied by the zinc removal from the coordination sphere of porphyrin. We suggest that the
zinc removal occurs after the final treatment of the reaction mass with MeOH, which, interacting
with the remaining trimethyloxonium tetrafluoroborate, gives HBF4 acid, which removes zinc
from the coordination sphere of porphyrin [57]. The structures of all newly prepared compounds
1
11
11
19
were identified by IR and H, B, B {1H}, F NMR spectroscopies and mass spectrometry.
The IR spectra of boronated compounds 8, 9, 10, 13 and 14 contains absorption band at 2580-
2588 cm-1 assigned to the BH-stretching vibration in the closo-carborane polyhedron. The
formation of propargyl-substituted porphyrins 5, 6, 11 and 12 was confirmed by the presence of
an absorption band in the region of 2119-2130 cm-1 due to the stretching vibration of the

1
terminal C≡C triple bond. H NMR spectra of porphyrins 5, 6, 8 – 14 exhibit singlet signals of
the β-pyrrole protons at δ = 8.86-9.25 ppm and broadened singlets of the porphyrin NH protons
at δ = –2.77- –2.86 ppm for porphyrins 10, 11 and 14. The presence of characteristic singlet
1
signals in H NMR corresponding to triazolyl =CH protons in the region of 8.26 – 9.34 ppm
confirmed the formation of triazole ring. The formation of porphyrin triazoles was also
1
confirmed by the presence in H NMR spectra for porphyrins 8 – 10, 13 of two types of
methylene proton groups in the region of 5.72-5.81 ppm and 5.37 – 5.58 ppm, respectively. The
carborane CH protons for 8 – 10, 13 were observed at δ = 4.92 - 5.28 ppm. 19F NMR spectra are
also in good agreement with the structures of porphyrins 8 – 10 demonstrating two sets of
fluorine nuclei signals of tetrafluorophenylfragments corresponding to multiplet ones at δ -137.3
– -139.5 ppm (eight o-fluorine atoms) and at δ -152.6 – -156.1 ppm (eight m-fluorine atoms),
respectively. The 11B {1H} NMR spectra for porphyrins 8 – 10, 13 were sufficiently well-
resolved to allow integration and contained set of signals between δB - 1.0 – (-19.0) ppm
characteristic for the closo-structure of carborane polyhedra.
2.2. Physico-chemical properties
2.2.1. Absorption spectra
The absorption spectra of compounds 8, 9, 10, 13, 14 have an intense absorption in the
region of 400-425 nm (S-band) and absorption in the red region of 500-660 nm (Q-band) (Fig.
1), which allowed us to consider these compounds as potential photosensitizers for PDT.
Fig. 1. (A) Absorbance spectra of 8, 9, 10 compounds in acetonitrile; (B) Absorbance
spectra of 13, 14 compounds in acetonitrile.

2.2.2. Complexation with HSA
HSA has a complex molecular structure and the structure of binding sites is well known
[58]. During complexation, porphyrin primarily binds to FA1 site located in subdomain IB of
human serum albumin, and the rigidity of the structure of porphyrin increases and fluorescence
increases as a result [59-60]. Formation of complexes between 8, 13 and HSA strongly changed
the spectral characteristics. Binding constant determined from the absorption spectra (Fig. 2A)
was Kb_abs = 3.5×105 M-1 for 8 and Kb_abs = 2.2×105 M-1 for 13. Binding constant calculated from
the fluorescence spectra (Fig. 2B) estimated to be Kb_fl = 3.3×105 M-1 for 8. Similar values of
these constants indicated that the replacement of hydrogen atoms with fluorine atoms in the
structure of boronated mesoarylporphyrins caused no significant changes in binding constants of
complexes. In the complexes of the porphyrin with albumin, a rigid molecular structure is
formed, and relaxation of the excited singlet state of the porphyrin to the ground state becomes
difficult. For this reason, the lifetime of fluorescence of the porphyrin in the complex with HSA
is increased. The measured fluorescence lifetime for 8 in propanol-1 is 1.6 ns. Meanwhile in the
complex with albumin the fluorescence lifetime is 3.2 ns which is consistent with the assumed
strong interaction between the porphyrin and the amino acids in the binding site of albumin.
Fig. 2. (A) Absorbance spectra of 8 in phosphate buffer, pH = 7.0 with HSA (3.2 × 10-6 – 5 × 10-
5 М). Inset: Experimental values of θ as a function of the protein concentration (425 nm).
(B) Fluorescence spectra of 8 (phosphate buffer, pH = 7.0, λex 424 nm) with HSA (8 × 10-7 – 5 ×
10-5 М). Inset: Experimental values of θ as a function of the protein concentration (λem = 652
nm).
2.2.3. Complexation with LDL
Generally lipoproteins are soluble complexes of proteins (apolipoproteins) and lipids that
provided transport of lipids to their sites in living systems [61]. The ability of the synthesized
compounds to form complexes with low density lipoproteins (LDL) is an important property for

the delivery of photosensitizers to cancer cells. Effective delivery of the photosensitizer can also
occur through the interaction of fluorophenyl boronated meso-arylporphyrins with LDL. The
binding constants for 8, 9, and 10 with LDL calculated on the basis of fluorescence spectra were
Kb_flu = 4.0×108, 4.7×108 and 7.4×108 M-1, respectively. The binding constants for LDL are
significantly larger than the constants for complexes with albumin. However, the mechanisms of
interaction of fluorophenyl boronated meso-arylporphyrins with albumin differs significantly
than the one with LDL. For albumin, interaction within its drug binding site is typical for small
molecular weight compounds whereas complexation with LDL occurs when the porphyrin
passes from the aqueous to the organic phases inside the vesicles formed by LDL molecules.
This non-specific binding of the porphyrin to the LDL lipid phase is characterized by very high
constant. According to [62], the vesicles consist of approximately 800 units of phospholipids.
Taking into account the amounts of the protein that interact with the drug, the true binding
constants for LDL (Kb(LDL) are ~3 orders of magnitude smaller (Kb(LDL)=K(b_flu) 800) and
/
approximately correspond to the binding constants calculated for HSA.
2.2.4. Triplet states
Using flash photolysis, we obtained the spectral-kinetic characteristics of the triplet state
of 9 in propanol. Photoexcitation of the oxygen free 9 solution was carried out in the S-band.
Differential absorption spectra of the triplet of 9 (Fig. 3) correspond to the bleaching of S/Q
bands and the formation of two absorption bands at 440-500 and 530-800 nm. The triplet
lifetime was 0.6 ms. The quenching of 9 triplets by oxygen occurs with a constant typical for
triplet quenching kq = 2×109 M-1 s-1 as a result of energy transfer to oxygen and generation of
singlet oxygen.

Fig. 3. Differential triplet-triplet absorption spectra of 9 ( ̴
10-7 M) in propanol-1 (200 μs after
flash). Inset: kinetic trace of the triplet at 710 nm, kT =1.6×103 s-1.
2.2.5. Quantum yields
The phosphorescence spectra of singlet oxygen (Fig. 4) in acetonitrile (λmax ~ 1270 nm) were
obtained upon irradiation (xenon lamp, photoexcitation at 511 nm for 9, 10, 14, 549 nm for 8,
13) of air-saturated solutions.
The quantum yields of singlet oxygen for 8, 9, 10, 13, 14 in acetonitrile were determined
by comparison with the standard tetraphenylporphyrin (TPP) (Fig 4.) [63-64].
Fig. 4. Phosphorescence spectra of singlet oxygen (1O2) in acetonitrile.
Singlet oxygen forms as a result of quenching of the triplet by the ground state oxygen and
singlet oxygen quantum yield correlates with quantum yield of triplet state. According to heavy
atom effect introduction of Zn (8, 13) and Pd (9) into the structure of these compounds enhances
intersystem crossing and results in the increase of quantum yield of triplet state. At the same time
singlet oxygen quantum yield increases (8, 9, 13).
TPP
0.60
Porphyrin
Ф (1O2)
8
9
10
13
14
0.75
0.80
0.66
0.72
0.67
Table 1. Quantum yields of singlet oxygen Ф (1O2) for porphyrins in air-saturated solutions of
acetonitrile.

Quantum yield of triplet states for all new compounds remains sufficient (~0.6-0.8), so
that singlet oxygen is formed as a result of quenching of the triplet by the ground state of
oxygen.
Thus, fluorophenyl boronated meso-arylporphyrins 8, 9, 13 have high quantum yield of singlet
oxygen and this peculiarity could make these compounds potential new photosensitizers.
2.3. Dark and light toxicity
All studied photosensitizers showed low dark cytotoxicity. 50% cell death in the dark did
not reach a concentration in the range studied (0.1–50 µM) (Fig. 5.). The control compound in
the dark experiment was doxorubicin at the same concentrations as the photosensitizers.
Doxorubicin exhibits cytotoxicity upon HCT116 cells and is a suitable positive control in the
dark cytotoxicity experiment at low IC50 values of the experimental compounds. Photoinduced
cytotoxicity of the studied porphyrins was characterized; 50% cell death in the light experiment
occurred at submicromolar concentrations (Fig. 6.) (IC50 values are shown in Table 2.). The
highest photo-induced cytotoxicity showed compound 9. The compound 8 showed the average
value in the synthesized series. IC50 values one order higher were shown by the compounds 10,
13, 14. Non-boronated porphyrins, 5,10,15,20-tetraphenylporphyrin (TPP) and 5,10,15,20-
tetrakis(pentafluorophenyl)porphyrin (F-TPP) did not exhibit dark and photo-induced
cytotoxicity. The introduction of carborane-containing substituents on the periphery of porphyrin
macrocycle increases the photosensitivity of HCT116 cells, while the rather low dark
cytotoxicity of the synthesized carboranes remains. This data are consistent with our previous
results [65]. All studied compounds show slight dark cytotoxicity with pronounced photoinduced
cytotoxicity, which characterizes them as potential photosensitizers for antitumor PDT.

Fig. 5. Dark cytotoxity
Fig. 6. Photoinduced cytotoxity
Compound
IC50, μM
Light experiment
3.9±1.2
1.2±0.6
12.9±5.3
11.1±4.2
10.9±3.1
>50
Dark experiment
>50
>50
>50
>50
>50
8
9
10
13
14
>50
TPP
>50
0.6±0.1
>50
0.6±0.1
F-TPP
doxorubicin

Table 2. IC50 values of photosensitizers (MTT-assay)
3. Experimental
3.1. Synthesis and characterization
3.1.1 General Information
All reactions were performed in an atmosphere of dry argon. All solvents were dried
according to the standard protocols. Dichloromethane was purified by distillation over calcium
hydride. Tetrahydrofuran (THF) was stored over sodium benzophenone and distilled before use.
11
19
1H and B and F NMR spectra were recorded on a Bruker Avance-400 spectrometer
operating at 400.13 MHz for 1H NMR, 128.28 MHz for 11B NMR and 376.5 MHz for 19F NMR.
1
Chemical shifts (δ) were referenced to the residual solvent peak (CD3)2CO, H: 2.05 ppm;
1
1
1
11
CDCl3, H: 7.26 ppm; THF-D8 H: 3.58 and 1.73 ppm) for H, external BF3·OEt2 for B and
19
external CFCl3 for F. IR spectra were recorded on a Brucker FTIR spectrometer Tensor 37 in
KBr tablets. Merck silica gel L 0.040–0.08 mesh was used for column chromatography. The
identities of new compounds were verified on Sorbfil plates. The UV-vis spectra were measured
on a spectrophotometer Jasco UV 7800 series in CH3CN, CHCl3 and THF. The mass spectra
were obtained using VISION 2000 (MALDI) mass spectrometer, the most intense peaks were
given for each compound.
The identities of new compounds were verified on TLC 60 F254 plates (Merck). Merck
silica gel L 0.04-0.08 mesh was used for column chromatography (elution with chloroform-
hexane 2 : 1, chloroform, and chloroform—methanol 10 : 1).
3.1.2 Synthesis of compounds 5-6 and 8-14
3.1.2.1. 5,10,15,20-Tetrakis[4-(2-propargyloxy)-2,3,5,6-tetrafluorophenyl]porphyrinato zinc
(II) (5). To a solution of porphyrin 3 (100 mg, 0.096 mmol) in THF (20 mL) propargyl alcohol
(54 mg, 0.96 mmol) (~ 30 μL) and powdered NaOH (38 mg, 0.96 mmol) were added and the
mixture was boiled for 4 h under argon atmosphere. Then the reaction mixture was poured into
water (50 mL), extracted with CH2Cl2 (2ꢀ×ꢀ20 mL), the extracts were washed with water (3ꢀ×ꢀ20
mL), dried over Na2SO4, filtered and solvent was removed in vacuo. The residue was purified by
column chromatography on silica gel, using CHCl3— hexane mixture (2 : 1) as an eluent.

Yield 107 mg (94%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3): 416 (281.8), 545 (24.0). IR (KBr)
1
νmax, cm-1: 2925 (porphyrin СН), 2124 (C≡C). Н NMR (400.13 MHz, (CD3)2CO), δ: 9.18 (s,
8H, β-pyrrole), 5.30 (d, J = 2.5 Hz, 8H, CH2), 3.34 (d, J = 2.5 Hz, 4H, C≡CH). 19F NMR (376.5
MHz, (CD3)2CO), δ: –139,1 (m, 8F), –156,5 (m, 8F). MS (MALDI): m/z [М+] calculated for:
C56H20F16N4O4Zn 1180.052, found: 1180.036.
3.1.2.2. 5,10,15,20-Tetrakis[4-(2-propargyloxy)-2,3,5,6-tetrafluorophenyl] porphyrinato
palladium (II) (6). To a solution of porphyrin 4 (100 mg, 0.093 mmol) in THF (20 mL)
propargyl alcohol (52 mg, 0.93 mmol) (~ 29 μL) and powdered NaOH (37 mg, 0.93 mmol) were
added and the mixture was boiled for 4 h under argon atmosphere. Then the reaction mixture was
poured into water (50 mL), extracted with CH2Cl2 (2ꢀ×ꢀ20 mL), the extracts were washed with
water (3ꢀ×ꢀ20 mL), dried over Na2SO4, filtered and solvent was removed in vacuo. The residue
was purified by column chromatography on silica gel, using CHCl3 – hexane mixture (2 : 1) as
an eluent. Yield 105 mg (92.5%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3): 410 (312.6), 520 (36.9),
1
552 (26.1). IR (KBr) νmax, cm-1: 2920 (porphyrin СН), 2130 (C≡C). Н NMR (400.13 MHz,
(CD3)2CO), δ: 8.96 (s, 8H, β-pyrrole), 5.23 (d, J = 2.5 Hz, 8H, CH2), 2.96 (d, J = 2.5 Hz, 4H,
19
C≡CH). F NMR (376.5 MHz, (CD3)2CO), δ: –139.0 (m, 8F), –156.4 (m, 8F). MS (MALDI):
m/z [М+] calcd. for C56H20F16N4O4Pd 1222.026, found 1222.004.
3.1.2.3. 5,10,15,20-Tetrakis{4-[(1-o-carboranylmethyl-1,2,3-triazol-4-yl)methyloxy]-2,3,5,6-
tetrafluorophenyl}porphyrinato zinc (II) (8). To a solution of porphyrin 5 (100 mg, 0.085
mmol) and carborane 7 (102 mg, 0.51 mmol) in CH2Cl2 (15 mL), a solution of Cu(OAc)2 (2 mg,
0.0102 mmol) in water (0.3 mL) was added, and then a solution of sodium ascorbate (4 mg,
0.0168 mmol) in water (0.3 mL) was added with stirring. The reaction mixture was boiled for 48
h, cooled to room temperature, washed with water (3ꢀ×ꢀ20 mL), dried over Na2SO4, filtered and
solvent was removed in vacuo. The dry residue was washed with toluene (50 mL), dried in
vacuo. Yield 127 mg (75.3%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3): 419 (427.0), 550 (28.5). IR
(KBr) νmax, cm-1: 2925 (porphyrin СН), 2586 (ВН). 1Н NMR (400.13 MHz, (CD3)2CO), δ: 9.17
(s, 8H, β-pyrrole), 8.58 (s, 4Н, triazole СН), 5.72 (s, 8Н, СН2), 5.54 (s, 8Н, СН2), 4.92 (br.s, 4Н,
11
carborane СН). B NMR (128.28 MHz, (CD3)2CO), δ: 2.2 (d, J=147 Hz, 4B), – 4.8 (d, J=132
Hz, 4B), –9.6 (d, J=150 Hz, 8B), –10.2 (d, J=129 Hz, 8B), –12.2 (d, J=142 Hz, 16B). 19F NMR
(376.5 MHz, (CD3)2CO), δ: –138.3 (m, 8F), –156.1 (m, 8F). MS (MALDI): m/z [М+] calcd. for
C68H72B40F16N16O4Zn 1977.893 found 1977.855.

3.1.2.4. 5,10,15,20-Tetrakis {4-[(1-o-carboranylmethyl-1,2,3-triazol-4-yl)methyloxy]-2,3,5,6-
tetrafluorophenyl} porphyrinato palladium (II) (9). To a solution of porphyrin 6 (100 mg,
0.085 mmol) and carborane 7 (98 mg, 0.49 mmol) in CH2Cl2 (15 mL), a solution of Cu(OAc)2 (2
mg, 0.0102 mmol) in water (0.3 mL) was added, and than a solution of sodium ascorbate (4 mg,
0.0168 mmol) in water (0.3 mL) was added with stirring. The reaction mixture was boiled for 48
h, cooled to room temperature, washed with water (3ꢀ×ꢀ20 mL), dried over Na2SO4, filtered and
solvent was removed in vacuo. The dry residue was washed with toluene (50 mL), dried in
vacuo. Yield 132 mg (79.2%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3): 407 (280.4), 478 (11.8),
1
518 (32.3), 550 (21.7), 595 (2.2). IR (KBr) νmax, cm-1: 2920 (porphyrin СН), 2584 (ВН). Н
NMR (400.13 MHz, (CD3)2CO), δ: 9.25 (s, 8H, β-pyrrole), 8.62 (s, 4Н, triazole СН), 5.80 (s,
8Н, СН2), 5.58 (s, 8Н, СН2), 4.98 (br.s, 4Н, carborane СН). 11B NMR (128.28 MHz, (CD3)2CO),
δ: –2.2 (d, J=150 Hz, 4B), –4.8 (d, J=131 Hz, 4B), –8.9 (d, J=150 Hz, 8B,), –10.1 (d, J=130 Hz,
19
8B,), –12.1 (d, J=140 Hz, 16B). F NMR (376.5 MHz, (CD3)2CO), δ: –139.5 (m, 8F), –154,1
(m, 8F). MS (MALDI): m/z [М+] calcd. for C68H72B40F16N16O4Pd 2019.868 found 2019.845.
3.1.2.5. 5,10,15,20-Tetrakis{4-[(1-o-carboranylmethyl-1,2,3-triazol-4-yl)methoxy]-2,3,5,6-
tetrafluorophenyl} porphyrin (10). To a solution of porphyrin 8 (50 mg, 0.025 mmol) in a
mixture of CHCl3 (5mL) and MeOH (5mL), TFA (1 mL) was added and the mixture was stirred
for 1 h at room temperature. Then the reaction mixture was poured into water (10 mL), washed
with water to a neutral medium, evaporated, passed through a layer of silica gel, using CHCl3–
MeOH (10 : 1) mixture as an eluent. Yield 46 mg (95%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3):
412 (261.3), 506 (27.5), 584 (15.9), 657 (3.2). IR (KBr) νmax, cm-1: 2924 (porphyrin СН), 2580
1
(ВН). Н NMR (400.13 MHz, (CD3)2CO), δ: 9.24 (s, 8H, β-pyrrole), 8.62 (s, 4Н, triazole СН),
5.81 (s, 8Н, СН2), 5.57 (s, 8Н, СН2), 4.95 (br.s, 4Н, carborane СН), –2,86 (s, 2H, porphyrin
NH). 11B NMR (128.28 MHz, (CD3)2CO), δ: –2.2 (d, J=148 Hz, 4B), –4.8 (d, J=130 Hz, 4B), –
19
9.5 (d, J=150 Hz, 8B), –11.6 (d, J=132 Hz, 8B), –12.4 (d, J=138 Hz, 16B). F NMR (376.5
MHz, (CD3)2CO), δ: –137.3 (m, 8F), –152.6 (m, 8F). MS (MALDI): m/z [М+] calcd. for
C68H74B40F16N16O4 1915.980 found 1915.964.
3.1.2.6. 5,10,15,20-Tetra(p-propargyloxyphenyl) porphyrin (11) was prepared according to
[55] with several changes.
To a solution of porphyrin 2 (100 mg, 0.144 mmol) in anhydrous DMF (5 mL) propargyl
bromide (137 mg, 1.15 mmol) and K2CO3 (159 mg, 1.15 mmol) were added and the mixture was
0
stirred at 20 C for 24 h under argon atmosphere. Then the reaction mixture was poured into
CHCl3 (20 mL), washed with water (5ꢀ×ꢀ50 mL), dried over MgSO4, and solvent was removed in

vacuo. The residue was purified by column chromatography on silica gel, using CHCl3 as an
eluent.
Yield 101 mg (85%). UV-vis (CHCl3): λmax, nm (ε ꢀ×ꢀ10-3): 422 (311), 519 (12.1), 556 (8.0), 592
(4.7), 651 (5.3). IR (KBr) νmax, cm-1: 2922 (porphyrin СН), 2125 (C≡C). 1Н NMR (400.13 MHz,
CDCl3), δ: 8.86 (s, 8H, β-pyrrole), 8.13 (d, J=8.4 Hz, 8H, Ph), 7.35 (d, J=8.5 Hz, 8H, Ph), 4.98
(d, J=2.3 Hz, 8H, СН2), 2.69 (t, J=2.3 Hz, 4H, C≡СН), –2.77 (br.s, 2H, porphyrin NH). MS
(MALDI): m/z [М+] calcd. for C56H38N4O4 830,289 found 830,258.
3.1.2.7. 5,10,15,20-Tetra (p-propargyloxyphenyl) porphyrinato zinc (II) (12). To a solution
of of porphyrin 11 (50 mg, 0.06 mmol) in a mixture of CH2Cl2 (20 mL) and methanol (2 mL)
Zn(OAc)2 (43 mg, 0.24 mmol) was added and the mixture was stirred at 20 0C for 3 hours. Then
the reaction mixture was washed with water (4ꢀ×ꢀ10 mL), dried over MgSO4, and solvent was
removed in vacuo.
Yield 49 mg (92%). UV-vis (CHCl3): λmax, nm (ε ꢀ×ꢀ10-3): 425 (183), 553 (7.3), 595 (3.3). IR
(KBr) νmax, cm-1: 2923 (porphyrin СН), 2119 (C≡C). 1Н NMR (400.13 MHz, CDCl3), δ: 8.86 (s,
8H, β-pyrrole), 8.15 (d, J=6.5 Hz, 8H, Ph), 7.37 (d, J=6.5 Hz, 8H, Ph), 4.99 (d, J=2.3 Hz, 8H,
CH2), 2.70 (t, J=2.36 Hz, 4H). MS (MALDI): m/z [М+] calcd. for C56H36N4O4Zn 892,203 found
892,176.
3.1.2.8. 5,10,15,20-Tetrakis {4-[(1-o-carboranylmethyl-1,2,3-triazol-4-yl)methyloxy] phenyl}
porphyrinato zinc (II) (13).
To a solution of porphyrin 12 (50 mg, 0.056 mmol) and carborane 7 (67 mg, 0.336 mmol) in
CH2Cl2 (5 mL), a solution of Cu(OAc)2 (1 mg, 0.0056 mmol) in water (0.3 mL) was added, and
than a solution of sodium ascorbate (2 mg, 0.0084 mmol) in water (0.3 mL) was added with
stirring. The reaction mixture was boiled for 48 h, cooled to room temperature, washed with
water (3ꢀ×ꢀ15 mL), dried over MgSO4, filtered and solvent was removed in vacuo. The dry
residue was washed with diethyl ether (40 ml), dried in vacuo.
Yield 68 mg (75%). UV-vis (THF): λmax, nm (ε ꢀ×ꢀ10-3): 427 (30.2), 460 (32.1), 558 (7.1), 598
(3.9). IR (KBr) νmax, cm-1: 2927 (porphyrin СН), 2588 (BH). 1Н NMR (400.13 MHz, THF-D8),
δ: 8.90 (s, 8H, β-pyrrole), 8.26 (s, 4Н, triazole СН), 8.14 (d, J=6.2 Hz, 8H, Ph), 7.45 (d, J=6.2
Hz, 8H, Ph), 5.52 (s, 8Н, СН2), 5.37 (s, 8Н, СН2), 5.28 (br.s, 4Н, carborane СН). 11В NMR
(128.28 MHz, THF-D8), δ: –1.8 (d, J=147 Hz, 4B), –4.7 (d, J=130 Hz, 4B), –9.4 (d, J=151 Hz,
8B), –11.6 (d, J=128 Hz, 8B), –12.5 (d, J=140 Hz, 16B). MS (MALDI): m/z [М+] calcd. for
C68H88B40N16O4Zn 1689,048 found 1689,012.

3.1.2.9. 5,10,15,20-Tetrakis {4-[(1-o-carboran-1-yl)methyl-1,2,3-triazol-3-y-4-yl) methyloxy]
phenyl} porphyrin terakistetrafluoroborate (14). To a solution of compound 13 (50 mg,
0.031 mmol) in CH2Cl2 (20 mL) trimethyloxonium tetrafluoroborate (39 mg, 0.27 mmol) was
added with stirring and the reaction mixture was stirred at room temperature for 1 hour (TLC
control). After that, methanol (5 mL) was added and the reaction mixture was stirred at room
temperature for another 1 hour. The solvents were removed in vacuo, crude product was washed
with diethyl ether (20 mL) and CH2Cl2 (10 ml), dried in vacuo.
Yield: 62 mg (99.5%). UV-vis (CH3CN): λmax, nm (ε ꢀ×ꢀ10-3): 418 (300), 517 (12.5), 551 (8.4),
1
592 (4.7), 648 (4.7). IR (KBr) νmax, cm-1: 2921 (porphyrin СН), 2580 (BH). Н NMR (400.13
MHz, THF-D8), δ: 9.34 (s, 4H, triazole CH), 8.86 (m, 8H, β-pyrrole), 8.17 (m, 8H, Ph), 7.56 (m,
8H, Ph), 5.98 (s, 8H, N-CH2-C), 5.88 (s, 8H, C-CH2-O), 5.14 (br.s, 4H, carborane CH), 4.78 (s,
11
12H, N+-CH3), –2.77 (s, 2H, NH). В NMR (128.28 MHz, THF-D8), δ: –1.0 (s, 4B, BF4), –2.3
(d, J=146 Hz, 4B), –4.2 (d, J=161 Hz, 4B), –10.0 (d, J=148 Hz, 8B), –12.2 (d, J=164 Hz, 20B),
–19.0 (d, J=187 Hz, 4B). MS (MALDI): m/z [М4+/4] calcd. for C72H102B44F16N16O4 509.059
found 509.015.
3.2. Spectroscopic measurements
Electronic absorption spectra were recorded for porphyrin solutions in acetonitrile on a
UV-3101РС spectrophotometer (Shimadzu, Japan) in the range of 370–800 nm in a quartz
cuvette with the optical path length 1 cm at room temperature. Solutions in DMSO (5 μM) were
analyzed on a UV-3101РС spectrophotometer (Shimadzu, Japan) in the range of 360–700 nm in
a quartz cuvette with the optical path length 1 cm at room temperature. Steady-state singlet
oxygen phosphorescence measurements were carried out on a FluoTime 300 fluorimeter
equipped with a NIR PMT Module H10330-45 (Hamamatsu, Japan). A Xe-lamp was used as an
excitation source. Fluorescence lifetimes were measured by the time correlated single photon
counting using a FluoTime 300 fluorimeter (PicoQuant). Fluorescence spectra were obtained
using a FluoTime 300 fluorimeter (PicoQuant GmbH, Germany). Excitation laser pulse (405 nm)
frequency was set at 40 MHz bin width 60 ps. Fluorescence decays were fitted using a FluoFit
software (PicoQuant). The triplet-triplet absorption spectra and the kinetics decay of the triplet
states were measured using a conventional flash photolysis setup (optical path length 20 cm,
excitation by a Xe-lamp through multi-band blue-green optical absorption filters at 400–510 nm,
80 J, 20 μs [66]). Signals were recorded on a PMT-38 photomultiplier (MELZ, USSR) at 400–
800 nm. The solutions were degassed before use. The binding constants of porphyrins with HSA

were determined using absorption and fluorescence spectroscopy. The binding constant Kb was
calculated from the curves according to Eq. (1) [67]:
⁄
[
]
ꢀ = (ꢁꢂ[ꢃꢄꢅ]) (1 + ꢁꢂ ꢃꢄꢅ )
(1)
where = ( F0)/(F∞ − F0) is the fraction of the dye bound to the protein; F0, F∞ and F
θ
F
−
are the fluorescence intensity or absorbance at [HSA] = 0 and at complete and
intermediate binding of the dye by the protein, respectively.
3.3. Cell culture and cytotoxicity
The human HCT116 colon adenocarcinoma cell line was from American Type Culture
Collection; ATCC, Manassas, VA. The HCT116 cell line was propagated in Dulbecco
modified Eagle’s medium (ThermoFisher Scientific, Waltham, MA). Medium was
supplemented with 5% fetal bovine serum (GE Healthcare Life Sciences; Chicago, IL), 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified CO2-
controlled atmosphere. Cells in logarithmic phase of growth were used in the experiments. New
compounds were dissolved in 10% aqueous DMSO (Paneco, Russia) as 10 mM stock solutions
followed by serial dilutions in culture medium immediately before experiments. All assays
were performed in 96-well microtiter plates (Nunc, USA). To each well 5×104 HCT116 cells
were plated and incubated overnight. Then for dark experiment experimental compounds and
рositive control doxorubicin (Teva, Netherlands) were added (final concentrations 0.1 µM-50
µM; DMSO < 0.5%), and cells were incubated for 48 h at 37 °C/20.9% O2/5%CO2. Cell
viability was assessed in an MTT test [68]. The IC50 values were defined as the concentrations
of the compound that inhibited MTT conversion by 50%. Light induced cell photodamage was
evaluated after the addition of compounds (final concentrations 0.1-50 µM) to НСТ116 cells in
96-well plates. Cells were incubated for 24 h at 37 °C/20.9% O2/5%CO2, washed with saline,
then 190 µl of fresh culture medium was added, and cells were illuminated with a white light
33 J/cm2. Cells were further incubated for 24 h at 37 °C/95% O2/5%CO2 followed by MTT
test. Cell viability (mean+S.D. of at least 3 independent experiments, each concentration in
duplicate) was expressed as a percentage of the optical density after MTT conversion in cells
treated with the respective compounds relative to untreated cells (100%).
4. Conclusion
Synthesis of novel caborane-substituted porphyrins was achieved using “click” chemistry
approach. Fuctionalization of Zn(II) or Pd(II) tetrakis(pentafluorophenyl)porhyrin and Zn(II)

tetrakis(4-hydroxyphenyl)porphyrin with propargyl alcohol yielded in the corresponding
propargyl derivatives which were successfully utilized as starting materials for the construction
of a novel series of porphyrin carborane-triazole conjugates in reaction with 1-azidomethyl-o-
carborane. The developed reaction conditions of the copper catalyzed 1,3-dipolar cycloaddtion
make it possible to obtain the desired carboranyl-triazole conjugates of porphyrins in moderate
to high yields (75-99%). Observed compounds showed great affinity to biomacromolecules like
albumin and LDL, a major biological carrier. Porphyrin complexes with Zn and Pd
demonstrated a good ability to produce singlet oxygen (quantum yields > 70%) and exhibited
significant photoinduced cytotoxicity. Synthesized porphyrin conjugates may be useful as
potential candidates for biological evaluations in PDT.
Acknowledgements
This work was performed with the financial support from Ministry of Science and Higher
Education of the Russian Federation using the equipment of Center for molecular composition
studies of INEOS RAS and Center “New Materials and Technologies” of IBCP RAS.
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Highlights
Novel triazole-carborane porphyrin conjugates were synthesized.
Synthesized compounds were structurally characterized by conventional spectroscopy.
Binding constants for albumin and LDL with synthesized compounds were obtained.
Singlet oxygen quantum yields of synthesized compounds were measured.
All compounds are nontoxic and show pronounced photoinduced cytotoxicity.

Declaration of interests
☑ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: