β-Maleimide substituted meso-arylporphyrins: Synthesis, transformations, physico-chemical and antitumor properties

Article abstract of DOI:10.1016/j.dyepig.2019.107760

The maleimide moiety is widely used in drug design. To explore the properties of maleimide containing photosensitizers we obtained a series of new β-maleimide functionalized meso-arylporphyrins through acylation of β-amino group in porphyrins with maleic anhydride followed by condensation of maleic acid monoamides. The selective reactivity of porphyrin maleimides toward thiols was demonstrated using mercaptocarboranes and cysteine. New derivatives retained the ability of tetrapyrrolic macrocyclic compounds to absorb light in visible spectral region and to generate triplet states and reactive oxygen species upon photoactivation. Importantly, illumination of cells loaded with a cell permeable 2-{3-[(o-carboran-1′-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-tetraphenylporphyrin triggered rapid (within the initial minutes) generation of superoxide anion radical concomitantly with a decrease of mitochondrial membrane potential, and then the loss of the plasma membrane integrity and cell death. Thus, the maleimide-substituted porphyrins represent a new chemotype of polyfunctionalized compounds for an in-depth investigation as photosensitizers in cancer and beyond.

Full text of DOI:10.1016/j.dyepig.2019.107760

Journal Pre-proof
β-Maleimide substituted meso-arylporphyrins: Synthesis, transformations, physico-
chemical and antitumor properties
Valentina A. Ol'shevskaya, Viktoriya M. Alpatova, Alexandra S. Radchenko, Alla
A. Ramonova, Albina S. Petrova, Victor V. Tatarskiy, Andrei V. Zaitsev, Elena G.
Kononova, Nikolay S. Ikonnikov, Alexey A. Kostyukov, Anton E. Egorov, Mikhail M.
Moisenovich, Vladimir A. Kuzmin, Natalya A. Bragina, Alexander A. Shtil
PII:
S0143-7208(19)31290-2
DOI:
https://doi.org/10.1016/j.dyepig.2019.107760
DYPI 107760
Reference:
To appear in: Dyes and Pigments
Received Date: 4 June 2019
Revised Date: 22 July 2019
Accepted Date: 28 July 2019
Please cite this article as: Ol'shevskaya VA, Alpatova VM, Radchenko AS, Ramonova AA, Petrova
AS, Tatarskiy VV, Zaitsev AV, Kononova EG, Ikonnikov NS, Kostyukov AA, Egorov AE, Moisenovich
MM, Kuzmin VA, Bragina NA, Shtil AA, β-Maleimide substituted meso-arylporphyrins: Synthesis,
transformations, physico-chemical and antitumor properties, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.107760.
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β-Maleimide substituted meso-arylporphyrins: Synthesis, transformations,
physico-chemical and antitumor properties
a
a,b
c
Valentina A. Ol’shevskaya , Viktoriya M. Alpatova , Alexandra S. Radchenko , Alla A.
d
e
f,g,h
a
Ramonova , Albina S. Petrova , Victor V. Tatarskiy , Andrei V. Zaitsev , Elena G.
a
a
c
c
Kononova , Nikolay S. Ikonnikov , Alexey A. Kostyukov , Anton E. Egorov , Mikhail M.
d
c
b
f,g
Moisenovich , Vladimir A. Kuzmin , Natalya A. Bragina , Alexander A. Shtil
a
A.N.Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 119991
Moscow, Russian Federation
b
MIREA - Russian Technological University, M.V. Lomonosov Institute of Fine Chemical
Technologies, 119571 Moscow, Russian Federation
c
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow,
Russian Federation
d
Department of Biology, Moscow State University, 119991 Moscow, Russian Federation
e
RUDN University, 117198 Moscow, Russian Federation
f
Blokhin National Medical Research Center of Oncology, 115478 Moscow, Russian Federation
g
Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russian Federation
h
National University of Science and Technology "MISIS", 119991 Moscow, Russian Federation
1

Highlights
1. A panel of new β-maleimide-substituted meso-arylporphyrins was synthesized.
2. Reactivity of porphyrin maleimides towards S-nucleophiles was studied.
3. Flash photolysis showed the ability of new compounds to generate ROS.
4. Complexes with albumin were determined for maleimide and succinimide porphyrins.
5. Selected compounds induced rapid photonecrosis in colon carcinoma cells.
.
Abstract
The maleimide moiety is widely used in drug design. To explore the properties of
maleimide containing photosensitizers we obtained a series of new β-maleimide functionalized
meso-arylporphyrins through acylation of β-amino group in porphyrins with maleic anhydride
followed by condensation of maleic acid monoamides. The selective reactivity of porphyrin
maleimides toward thiols was demonstrated using mercaptocarboranes and cysteine. New
derivatives retained the ability of tetrapyrrolic macrocyclic compounds to absorb light in visible
spectral region and to generate triplet states and reactive oxygen species upon photoactivation
Importantly, illumination of cells loaded with a cell permeable 2-{3-[(o-carboran-1’-
yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-tetraphenylporphyrin triggered rapid (within the
initial minutes) generation of superoxide anion radical concomitantly with a decrease of
mitochondrial membrane potential and then the loss of the plasma membrane integrity and cell
death. Thus, the maleimide-substituted porphyrins represent a new chemotype of
polyfunctionalized compounds for an in-depth investigation as photosensitizers in cancer and
beyond.
Key words: porphyrins, maleimides, carboranes, fluorescence, singlet oxygen,
phototoxicity.
2

1
. Introduction
During the last few decades porphyrins and their analogues have attracted significant
interest due to their exploration in various fields such as dyes for photovoltaic solar cells [1,2],
chemical sensors [3,4], molecular electronic components [5], non-linear optic materials [6,7],
catalysts [8], nanomaterials [9,10], and as photosensitizers in photodynamic therapy (PDT) of
cancer and other diseases [11-14]. Progress in antitumor PDT is associated with advances in the
design of non-toxic efficacious photosensitizers capable of providing the maximum therapeutic
effect while sparing the surrounding non-malignant tissues.
Among the tetrapyrrolic macrocyclic systems, the tetraarylporphyrins are of great
importance as a source of new photosensitizers due to their availability and diversity of
structurally functionalized, highly potent derivatives. The aryl substituted porphyrins are the
synthetic analogues of natural porphyrins and chlorins. Derivatives of arylporphyrins and their
metal complexes are of practical use in the creation of a variety of anticancer drugs, including
the agents for PDT. Furthermore, porphyrins are basic compounds for the preparation of chlorins
and bacteriochlorins that are used for a deeper tumor photodamage due to a stronger absorption
in the visible and near-infrared region [15]. Therefore, the design of new porphyrins with
particular structural fragments is of considerable importance since it gives to the porphyrin
system new specific properties and extends the areas of application.
To improve PDT efficiency of photosensitizers a number of approaches have been
developed to conjugate the photosensitizer with another structural moiety using a covalent bond
[
16-20]. A considerable attention has been paid to the conjugates of porphyrins with proteins and
peptides [21]. The advantage of such conjugates is that they make it possible to overcome the
low water solubility of tetrapyrrolic compounds and reduce their aggregation. Selective
conjugation of porphyrins to peptides is based on thiol-targeting reactions. Modification of
porphyrins with functionalities capable of reacting with cysteine thiol groups is a useful strategy
for regioselective delivery of the photosensitizer to target cells [17].
Bioconjugation via the maleimide moiety has emerged as an excellent approach for a
variety of applications, in particular, therapeutic antibody-drug conjugates [22-25], structural
investigations of complex cellular compartments [26] and design of targeted delivery systems
[
27]. The maleimide-substituted porphyrin derivatives are of significant interest. The maleimide
units represent the substrates for thiol Michael addition reactions [28]. The presence of two cis-
oriented carbonyl groups in the maleimide structure makes its electron-deficient double bond
extremely reactive towards cysteine thiol groups of proteins at physiological pH, with the
formation of relatively stable succinimidyl thioether bonds. Commonly the Michael addition
process provides a high selectivity, high yields of products, good reaction rates, insensitivity to
3

oxygen or water, and a compatibility with various thiol structures. Moreover, the thiol group
displays a higher nucleophilicity compared to the hydroxyl and amino groups at physiological
pH, thereby reacting preferentially with the double bond of maleimide. A series of porphyrin-
maleimide derivatives have been used for the preparation of efficient cell penetrating peptide-
porphyrin conjugates which are suitable for use in targeted PDT. The regiospecific
bioconjugation of maleimide porphyrins to the peptide backbone can improve the selectivity and
broaden the spectra of therapeutic applications [17, 29].
In this study we describe a new panel of β-maleimide-substituted porphyrins. A variety of
synthetic approaches have been developed to synthesize β-substituted porphyrins [30-34]. The β-
substituents can shift the electronic absorption bands to the near infrared region; these systems
may find use in PDT [33] and elsewhere [35]. There is available information on the improved
antitumor properties of tetrapyrrole photosensitizers containing boron polyhedra in PDT as
compared with their non-boronated analogs [34,36,37]. Considering our long-term interest in
boronated porphyrins as antitumor agents we report the synthesis, characterization of β-
maleimide-substituted porphyrins and their reactivity towards S-nucleophiles including
mercaptocarboranes. Among the series of new derivatives, the boron containing 2-{3-[(o-
carboran-1’-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-tetraphenylporphyrin demonstrated
good properties in vitro and in cell based test systems, suggesting its potential as an antitumor
photosensitizer.
2
. Results and Discussion
2
.1. Synthesis
β-Maleimide-substituted porphyrins 1-5 were synthesized using meso-arylporphyrins 6-8
38,39] as starting compounds. For their preparation we exploited the availability of β-nitro-
[
substituted porphyrins which can be easily synthesized in a good yield by the regioselective
nitration of corresponding porphyrin copper complexes with copper(II) nitrate [40]. We tried to
obtain 2-nitro-derivatives of porphyrins 6-8 on the basis of metal-free derivatives, using an
excess of copper nitrate in CH Cl /acetic acid/acetic anhydride mixture, suggesting the in situ
2
2
formation of a copper complexes followed by simultaneous nitration under reflux. As a result for
all porphyrins the desired products were obtained but the yields of copper(II) 2-
nitroporphyrinates were dependent on the meso-substituent in the porphyrin macrocycle. In the
case of porphyrins 6, 8 the 2-nitro-substituted copper complexes were obtained in ~ 45-50%
yields while for pentafluorophenyl substituted porphyrin 7 the yield of desired product was
almost quantitative (97%). In this regard, copper(II) 2-nitroporphyrinates 9-11 were obtained in a
good yield (81-98%) by nitration of the corresponding porphyrin copper complexes with
copper(II) nitrate at room temperature in CH Cl /acetic acid/acetic anhydride mixture.
2
2
4

Demetallation of 2-nitroporphyrinates 9-11 was readily proceeded according to [41] in
H SO /CF COOH (1:1) system affording the free base 2-nitroporphyrins 17-19 in 83-90% yield.
2
4
3
The derivatives 17-19 are considered the key compounds for the synthesis of 2-
aminoporphyrins. The most efficient protocols to transform NO group into the amino group are
2
based on reduction with SnCl -HCl or NaBH -Pd/C [42, 43]. All 2-nitro-substituted porphyrins
2
4
9
, 12, 17-19 were smoothly reduced with NaBH and Pd/C [43] in CH Cl - MeOH to afford the
4 2 2
expected aminoporphyrins 20-24 in 71-79% yields as starting compounds for maleimide cycle
formation.
Ar
NO2
Ar
NH2
NH
N
N
NH
N
N
iii
Ar
Ar
Ar
Ar
HN
Ar
Ar
NO2
HN
1
1
7 M = 2H, Ar = C6H5
8 M = 2H, Ar = C6F5
19 M = 2H, C6H4-p-CF3
Ar
0 M = 2H, Ar = C H
6 5
1 M = 2H, Ar = C6F5
N
N
N
N
N
N
N
N
Ar
i
Ar
M
Ar
Ar
M
Ar
2
2
Ar
NH2
22 M = 2H, C6H4-p-CF3
Ar
Ar
N
N
N
6
M = 2H, Ar = C6H5
M = 2H, Ar = C6F5
M = 2H, Ar = C H -p-CF
9 M = Cu, Ar = C H
6 5
10 M = Cu, Ar = C6F5
11 M = Cu, Ar = C6H4-p-CF3
12 M = Zn, Ar = C6F5
Ar
M
Ar
7
8
1
1
1
1
N
6
4
3
3 M = Cu, Ar = C H5
6
4 M = Cu, Ar = C6F5
5 M = Cu, Ar = C6H4-p-CF3
23 M = Cu, Ar = C H
6
5
Ar
M =Zn, Ar = C6F
5
24
6 M = Zn, Ar = C H5
6
Scheme 1. Synthesis of 2-aminotetraarylporphyrins (i) Cu(NO ) *3H O, CH Cl , AcOH,
3
2
2
2
2
Ac O; (ii) CF COOH/H SO (1:1), r.t.; (iii) NaBH , Pd/C in CH Cl – MeOH, Ar, r.t.
2
3
2
4
4
2
2
The synthetic strategy to obtain new β-pyrrole maleimide derivatives involved acylation
of β-amino groups with maleic anhydride (Scheme 2). These reactions produced porphyrin
conjugates 25-29 containing the substituted maleic acid monoamides. Acylation of porphyrins
2
0, 21, 23 with maleic anhydride was carried out in AcOH for 50h at room temperature; the
corresponding monoamides were obtained in 20-82 % yields. These results demonstrated that the
product yields are dependent on the meso-substituents in the porphyrin macrocyle; the lowest
yield was obtained for 21 that contains pentafluorophenyl substituents. To increase the yield of
maleic acid monoamide for 21, we used the reaction of its zinc complex 24 with maleic
anhydride in MeCN under reflux. In this case the yield of the target product was 69% after
column chromatography on SiO . The same reaction conditions (MeCN, reflux) were used for
2
acylation of porphyrin 22 with maleic anhydride; porphyrin 27 was obtained in a good yield. The
use of MeCN instead of acetic acid for acylation is more convenient since the reaction time is
significantly reduced. However, MeCN as a solvent is not suitable for acylation of 20, 22, 23
since these porphyrins are insoluble in MeCN.
5

The subsequent thermal cyclization of porphyrin maleamic acids 25-29 was performed at
1
10 °C in Ac O in the presence of NaOAc producing the corresponding 2-maleimide-substituted
2
porphyrins 1-5 in 61-77% yields.
Scheme 2. Synthesis of 2-maleimide-substituted porphyrins: (i) for 20, 21, 23: maleic
anhydride, HOAc, r.t.; for 22, 24: maleic anhydride, MeCN, reflux; (ii) NaOAc, Ac O, 110 °C.
2
Substituted maleimides are a unique class of compounds used in chemical and biological
studies due to the activated double bond suitable for chemical transformations of different types.
Substituted maleimides can behave as dipolarophiles in the reaction of 1,3-dipolar cycloaddition
or as dienophiles in the Diels–Alder reaction, as well as acceptors in the Michael reaction with
O-, N- and S-nucleophiles. Since we were interested in the reactivity of prepared maleimides
with S-nucleophiles, we studied the Michael addition reaction of 1-mercapto-o-carborane 30 [44]
and 9-mercapto-m-carborane 31 [45] with porphyrins 1, 3 and 5.
6

Scheme 3. Synthesis of carborane-substituted porphyrins 32-36.
Functionalization of the maleimide double bond in 1, 3, 5 with carboranes 30, 31 was
performed in boiling CHCl in the presence of NaOAc affording the corresponding carborane
3
succinimidyl thioethers 32-35 in a good yield. Free base porphyrin 36 was obtained by the
removal of Zn from coordination sphere of porphyrin 35 under the action of trifluoroacetic acid
(Scheme 3). Since the maleimide is by far the most suitable functional group to be coupled to a
cysteine residue because the coupling reaction is highly specific and efficient [28], we
demonstrated the ability of maleimides 3, 4 to form conjugates with cysteine 37. Reactions were
carried out in a THF-H O system at room temperature in argon to produce 38, 39 in a
2
quantitative yield (Scheme 4). Of note, it is necessary to remove oxygen from solvents otherwise
the disulfide bond in the formed cystine would prevent the reaction with maleimides.
Scheme 4. Synthesis of cysteine-substituted porphyrins 38, 39.
7

Compounds 38, 39 contain functional amino and ester groups suitable for further
functionalization including their use in peptide synthesis. The structures of all newly prepared
1
11
11
1
19
compounds were identified by IR and H, B, B{ H}, F NMR spectroscopies and mass
spectrometry. (See Experimental and Supporting Information Figure S1-13 for details).
2
2
.2. Physico-chemical properties
.2.1. UV–visible absorption spectra
The absorption spectra of the new compounds in DMSO solutions are shown in Figure
A. Spectra were normalized [0; 1] to their respective QX10 maxima (QY00 for 5). Spectra for 1-3,
2, 34 are characteristic of metal free porphyrins [46, 47] showing the intense Soret bands
1
3
around λ=420 nm and four Q-bands of lower intensities at longer wavelengths. For 5 containing
2
+
Zn , the spectra showed three Q-bands and the Soret band was slightly red-shifted (4 nm) (Table
). The major Q-bands for all the compounds were located at λ=516 nm with exception of 5
λ=554 nm) and 2 (λ=508 nm). Introduction of the pentafluorophenyl group (porphyrin 2) led to
1
(
2
+
a 7-10 nm blue shift of all the bands. Zn derivative 5 displayed a 10 nm red shift and less
number of Q-bands with significant red-shift relative to the metal free 2.
2
.2.2. Fluorescence spectra
The emission spectra of porphyrins 1-3, 5, 32, 34 were normalized to the respective
Q*X00 (1, 32, 3, 34) or Q*X01 (5, 2), the maxima are shown in Figure 1B. All the spectra
demonstrated Q*X00 and Q*X01 emission bands typical of porphyrins [46, 47] (see Table 1 for the
band positions). Trifluoromethyl-substituted porphyrins 3 and 34 showed a 8 nm red shift of
Q*X00 bands relative to the 2-maleimide-substituted porphyrin 1. No correlation was found
between the substituent in porphyrins 2, 5, 32, 34 and the red shift of Q*X00 bands. Similar to the
absorption spectra, all the bands in the fluorescence spectra of the pentafluorophenyl substituted
porphyrin 2 are blue-shifted by 13 nm relative to maleimideporphyrin 1, and the band intensities
2
+
were inversed. The pentafluorophenyl-substituted porphyrin 5 (Zn complex) displayed
fluorescence bands which values are significantly blue-shifted (λ=598 nm and λ=650 nm)
compared to other zinc containing porphyrins (~ λ=650 nm and λ=715 nm) [48,49].
8

(A)
(B)
Figure 1. Normalized absorption (A) and fluorescence (B) spectra of the porphyrin
derivatives and zoomed Q-bands in the 450-700 nm region. See Experimental for details.
Table 1. UV–visible absorption and fluorescence data in DMSO solution.
Porphyrin
λmax, nm
Q bands
Soret band
420
Emission
653, 718
653, 718
598, 650
640, 706
650, 715
650, 715
1
516, 551, 594, 647
516, 550, 593, 648
517, 554, 593, -
508, 543, 583, 635
516, 549, 590, 645
516, 548, 592, 646
3
2
421
5
424
2
3
414
420
3
4
420
The most intensive peaks are marked in bold. Standard deviation ± 1 nm.
9

2
.2.3. Fluorescence lifetimes and quantum yields, singlet oxygen quantum yields
Fluorescence lifetimes and quantum yields in DMSO (measured relative to rhodamine
G) for all tested porphyrin derivatives are summarized in Table 2 and Figure 2. Values of
6
2
+
quantum yields of fluorescence varied from 0.04 for 34 to 0.01 for Zn -containing 5. The decay
curves fit to single-exponential functions with χ2< 1.6 except for 5 that required a two-
2
+
exponential fit. The short decay time for 5 indicates that Zn conferred a shorter lifetime of S
1
state [50]. The pentafluorophenyl-substituted 2 had the longest lifetime τ = 10.52 ns and an
f
average quantum yield of fluorescence (~0.01). Boronated derivatives had average values τ ~8-9
f
2
+
ns and the highest φ (0.03-0.04). Values of φ and τ for Zn derivative 5 were the lowest in the
f
f
f
series corroborating the published data [50].
Fluorescence, being dependent largely on the inner π-electron system of the macrocycle,
is unlikely to be affected by periphery substituents that do not distort the macrocycle’s planarity.
In general, the quantum yields of fluorescence should be ~ 0.1 in the free tetraphenylporphyrin
(TPP) and ~ 0.04 for a zinc-TPP in an organic solvent [50-52]. In fact, the obtained values for
investigated porphyrins are lower than for TPP. This difference can be explained by influence of
the maleimide group on the central macrocyclic π system. However, the ratio of values φ and τ
f
f
2
+
of Zn containing 5 (φ = 0.01, τ = 1.37 ns) to its free base analog 2 (φ = 0.02, τ = 10.52 ns) are
f
f
f
f
in good agreement with the reference data.
Table 2. Fluorescence quantum yields and lifetimes, and quantum yield of singlet oxygen
generation in DMSO.
Compound
a, c
1
b
φf (DMSO)
τf (DMSO), ns
8.20 ± 0.01
8.39 ±0.02
1.37 ±0.01
10.52 ±0.02
8.73 ±0.05
9.08 ±0.08
φ O
2 (DMSO)
1
0.03
0.69
0.81
0.77
0.92
0.70
0.91
32
0.03
5
2
3
0.01
0.02
0.03
34
0.04
a,b
c
Reproducibility ±10%. The optical density was matched for tested compounds and
reference at the excitation wavelengths (see Experimental)
.
1
0

Figure 2. Time course of fluorescence decay of porphyrins in DMSO.
.2.4. Triplet states of new porphyrin derivatives
2
The flash photolysis was used to investigate the ability of the new compounds to generate
ROS upon photoactivation. Excitation by the visible light (400-510 nm or 480-560 nm) in the
oxygen free organic solvents (ethanol, propanol-1, DMSO) induced a short-lived triplet state of
porphyrins. Differential absorption spectra of the triplet states (see Figure 3 for 32 as an
example) showed the absorption bands at 440–510 nm and 540-750 nm and bleaching of S- and
Q- bands. Spectra were recorded after a single pulsed photoexcitation (480-560 nm, 80 J). The
kinetics decay of the triplet states followed a first-order law A = A ×exp(-t/τ ), where А is the
∆
∆
0
т
∆
triplet-triplet absorption at a given time, А is triplet-triplet absorption immediately after light
∆
0
illumination, t is time. The triplet lifetime is τ = 1/k , where k is the constant of the triplet state
т
т
т
kinetics decay. Lifetimes of the triplet states for 1-3, 5, 32 and 34 varied insignificantly within a
low millisecond range.
Figure 3. Transient triplet-triplet absorption spectrum of 32 (2 µM) in propanol-1 (200 µs
after flash) and the kinetic trace of triplet at 440 nm.
1
1

Quenching of the triplet states for 1-3, 5, 32 and 34 by oxygen leads to the formation of
1
the singlet oxygen (see Figure S14 for O phosphorescence spectra; rose bengal (RB) is a
2
reference compound). Measurements of quantum yields of the singlet oxygen were carried out at
high concentrations of oxygen in solutions; all the porphyrin triplets transferred energy to the
singlet oxygen. Quantum yields in the solutions are presented in Table 2. All the new compounds
showed a significant quantum yield of the singlet oxygen (0.69-0.92). Thus, the quantum yields
of singlet oxygen and the triplet state of porphyrin derivatives were almost coincident, making
these compounds perspective as photosensitizers for PDT.
2
.2.5. Stable complexes with HSA
We were interested whether 1-3, 5, 32, 34 can form complexes with HSA, a major drug
carrier in the body. Figure S15 shows the absorption spectra depending on HSA concentrations
(32 as an example). The equilibrium dissociation constants (calculated by formula 4, see
Experimental) are shown in Table 3. Compounds 1 and 2 showed the biggest affinity to HSA.
2
+
Introduction of the carborane or CF substituents, as well as Zn , somewhat weakened the
3
ability of drug-albumin complex formation, most probably due to a steric hindrance [53].
Table 3. Dissociation constants (K ) of porphyrin-HSA complexes.
d
6
Compound
K × 10 , M
d
1
2
3
5
0.83± 0.25
1.10± 0.34
2.09± 0.73
4.14± 1.76
3.39± 0.96
1.84± 0.52
3
3
2
4
Altogether, testing in several cell free systems demonstrated that our new maleimide-
substituted porphyrins largely retained the characteristics suitable for PDT. All tested derivatives
fluoresced in the visible spectral region that makes them convenient for detection in the cells.
Most importantly, peripheral moieties or metal ion in the coordination sphere of the tetrapyrrolic
macrocycle did not seriously alter the ability to generate ROS upon light activation and form
stable complexes with albumin. Therefore we set out to investigate these compounds in cell
based assays and evaluate their relevance as candidates for antitumor PDT.
1
2

2
2
.3. Biological properties
.3.1. Dark and light toxicity
We tested dark cytotoxicity of 1-3, 5, 32 and 34 by treating HCT116 human colon
carcinoma cell line with each of these compounds (up to 50 µM) for 72 h followed by an MTT-
test. No discernible cell growth inhibition or death was registered (data not shown). Next, we
monitored intracellular accumulation of 1-3, 5, 32 and 34. The HCT116 cells were loaded with 5
µM of each compound for 24 h, washed with cold saline and immediately analyzed by flow
cytometry. Figure S16 shows the histograms of cell associated fluorescence after excitation at
4
05 nm and emission at 710 nm and 780 nm. The most pronounced accumulation was detected
for 32.
Next, we evaluated light activated cell death after loading the HCT116 cells with 1-3, 5,
0
3
2 and 34 (0-50 µM each, 24 h at 37 C, 5% CO ) and illumination with a 420 nm laser. Cell
2
viability was assessed by an MTT test 2 h post illumination. Data in Table 4 showed that 32 was
the most potent photosensitizer: IC was in a low micromolar range of concentrations. Also, we
5
0
2
illuminated cells with a 650 nm laser (Figure 1A; 5 J/cm ) after the same loading. Only 32
2
triggered a pronounced death whereas 1-3, 5 and 34 were inert. At 30 J/cm compound 5 was
slightly active (not shown). Together, based on the appropriate physico-chemical characteristics
in cell free systems, the lack of dark cytotoxicity, good cell permeability and a potent
phototoxicity, we selected 32 for further experiments as a candidate antitumor photosensitizer.
Table 4. Phototoxicity of porphyrins 1-3, 5, 32, 34.
Compound
IC , µM
50
1
2
3
5
13.2
32.6
>50
7.8
3
3
2
4
1.1
>50
The HCT116 cells were loaded with 5 µM of each compound for 24 h, washed with
2
saline, illuminated with a 420 nm laser (1.5 J/cm ) and incubated in a fresh medium
0
supplemented with the MTT reagent for 2 h at 37 C, 5% CO . IC values are mean of 3
2
50
independent measurements with a <10% error. See Experimental for details.
1
3

2
.3.2. Intracellular distribution
0
Cells were exposed to 5 µM 32 for 24 h at 37 C, 5% CO to achieve maximum
2
accumulation (see Figure S16). Figure 4 demonstrates that 32 can be visualized in the cytoplasm
of HCT116 cells. No co-localization with lysosomes, mitochondria, endosomes or nuclei was
detectable using the respective fluorescent probes. Similar patterns of intracellular localization
were observed for 1-3 and 34 (Figure S17) indicating that new maleimideporphyrins and
succinimideporphyrins are distributed across the organelle-free cytoplasm.
Figure 4. Cytoplasmic localization of compound 32 in HCT116 cells.
0
Cells were exposed to 1 µM 32 for 1 h at 37 C, 5% CO , washed and visualized by a confocal
2
laser scanning microscopy. A, D, G: autofluorescence of 32, B: LysoTracker Green DND-26, E:
transferrin-FITC, H: MitoTracker Green FM; C: merge of A and B; F: merge of D and E; I:
merge of G and H. N: nuclei. Bar, 10 µm.
2
.3.3. Lethal cell photodamage upon photoactivation of 32.
To evaluate the perspective of 32 as an antitumor photosensitizer we investigated the
events that lead to death of cells loaded with this compound. We were interested whether ROS
generation by 32 detectable in a cell free system (Table 2), translates into intracellular free
oxygen burst upon illumination of cells loaded with this compound. In so doing we used the
-
MitoSOX Red probe for intracellular visualization of О and the MitoTrackerCMXROS Red
2
1
4

dye sensitive to mitochondrial transmembrane electric potential. As expected, in intact HCT116
cells MitoSOX Red fluorescence was localized largely to mitochondria (Figure 5A-C). However,
illumination of cells loaded with 32 led to an instant (by 30 sec) increase of MitoSOX Red
fluorescence whereas MitoTrackerCMXROS Red fluorescence decreased (Figure 5D,E). Merged
images (Figure 5F) showed that, in illuminated cells, MitoSOX Red fluorescence was no longer
attributable to viable (that is, maintaining their membrane potential) mitochondria. To
simultaneously monitor mitochondrial damage and cell death, the DNA intercalating agent
propidium iodide (PI) was added to the cell culture prior to illumination. PI staining is a hallmark
of the loss of plasma membrane integrity [34,54]. The mitochondrial membrane potential
dropped dramatically within the initial 1-2 min post illumination (Figure 6A-D). This effect was
paralleled by increased cell permeability for PI. As shown in Figure 6E, F, PI alone did not
affect the morphology of non-illuminated cells that remained PI-negative over the time of
experiment. In contrast, as soon as 5 min post illumination PI entered individual cells; by 10 min
the dye was clearly detectable in the nuclei and perinuclear areas (Figure 6G,H). Thus, ROS
generation coincident with the drop of the mitochondrial membrane potential preceded the influx
of PI into illuminated cells. Together with data in Figure 4, these results indicated that, although
3
2 lacks a specifically mitochondrial accumulation, the intracellular oxidative burst upon light
activation can damage mitochondria and probably other membrane organelles. Most importantly,
the plasma membrane permeability for PI indicates a non-repairable cell photodamage.
-.
Figure 5. Detection of О in HCT116 cells.
2
0
Cells were loaded with 5 µM 32 for 24 h at 37 C, 5% CO prior to laser confocal
2
microscopy. А-C: no light; D-F: 30 sec after photoactivation. Shown are intracellular distribution
-
of О sensor MitoSOX Red (green; excitation 488/emission 510-600 nm); nuclear dye Hoechst
2
3
3342 (blue; excitation 405/emission 415-480 nm); mitochondrial probe MitoTracker Red
CMXROS (red; excitation 543/emission 560-620 nm). Bar, 10 µm.
1
5

Figure 6. Rapid photodamage of HCT116 cells loaded with porphyrin 32.
A-D: cells were loaded with MitoTracker Green FM (1 µМ) for 30 min in the absence (A) or
presence of 5 µM 32 (B-D) followed by washing with saline. A,B: no light; C, D: 1 min or 2 min,
respectively, after illumination with a 405 nm laser. E-H: cells were given 10 µg/ml PI for 10
min prior to imaging. E, no 32, F-H: cells loaded with 5 µМ 32 and then either left in the dark
(F) or illuminated with a 405 nm laser and photographed 5 min (G) or 10 min (H) post
illumination. In each image: cells were counterstained with DAPI (blue). Bar, 10 µm.
Overall, aiming at conjugation of a biologically active moiety to the known
photoactivatable scaffold, we synthesized and characterized a new series of β-functionalized
maleimide-substituted meso-arylporphyrins. The maleimide units were highly specific for thiols
through the Michael addition. New compounds can be used for porphyrin-driven, site-selective
modifications of cysteine residues in proteins. Recently these residues have been shown to be
important for covalent but reversible binding to and potent inhibition of the enzyme critical for
cell division [54; see also 28 for recent review].
Along with the maleimide moiety, our new porphyrin derivatives carried diverse meso-
2
+
2+
aryl substituents including fluorine groups, metal centers (Zn , Cu ) in the coordination sphere
of the tetrapyrrolic macrocycle, as well as succinimide units in β-position of macrosycle
functionalized with the boron polyhedron (carborane cage) or cysteine. The resulting compounds
represented bulky structures decorated with individual chemical groups for a variety of
biological functions. Multifunctionality of new compounds re-iterates the suitability of the
tetrapyrrolic macrocycle as a versatile core in medicinal chemistry.
Importantly, in aqueous and/or organic solutions the substituents did not alter a major
biomedical property of porphyrins, that is, the ability to generate ROS upon light activation.
Furthermore, the selected compounds formed stable complexes with the carrier protein albumin
in a physiological buffer. Nevertheless, the behavior of individual new derivatives in cell culture
1
6

varied. Although all porphyrins tested in cell-based assays were non-toxic in the dark within as
long as 72 h, their differential intracellular accumulation and phototoxicity made not each
compound therapeutically perspective. Compound 2-{3-[(o-carboran-1’-yl)thio]pyrrolidine-2,5-
dione-1-yl}-5,10,15,20-tetraphenylporphyrin (32) demonstrated reasonably good photochemical
properties in vitro. Furthermore, the carborane moiety did not seriously alter the 32:HSA
binding, corroborating our experimental and in silico findings with other monocarboranyl
substituted tetrapyrrolic photosensitizers [53]. Together with a high intracellular uptake, 32 can
be considered a lead in the series.
Most importantly, 32 was the most potent in sensitizing HCT116 colon carcinoma cells to
light. A burst of ROS and a drop of mitochondrial membrane potential occurred within the initial
minutes post cell illumination. These events preceded rapid (by 10 min) loss of the plasma
membrane integrity followed by a dramatically decreased reduction of the MTT reagent by
mitochondrial dehydrogenases. This mode of cell death is reminiscent of the previously reported
fast photonecrosis triggered by illumination of cells loaded with the monocarboranyl- or
polyfluorinated tetracarboranylchlorins [14,34]. Still, 32 differed from these photosensitizers in
the patterns of intracellular distribution. Boronation of chlorin e6 amide conferred its localization
in the membrane organelles and the ability to traverse across the artificial membranes [34].
However, the carborane containing 32 accumulated in the organelle-free cytoplasm rather than in
the organelles. Thus, the carborane cage alone does not necessarily render the compound affine
to biomembranes. Nevertheless, an extensive ROS generation by photoactivated cytoplasmic 32
was sufficient to quickly trigger irreversible damage of the vital compartments such as
mitochondria and the plasma membrane.
Conclusion
This study analyzed the synthesis and properties of novel porphyrins functionalized with
the maleimide substituent at the β-position of the macrocycle. The maleimide units readily
interacted with mercaptocarboranes and cysteine via Michael addition reactions; no catalyst was
needed for these transformations. The new chemotype is particularly pertinent to the
development of anticancer agents with manifold activities, the photosensitizers and beyond. In
particular, the maleimide moiety can be useful in the design of protein kinase inhibitors via
selective targeting of the cysteine residues. Individual new compounds demonstrated a
remarkable ability to produce singlet oxygen (quantum yields > 70%). The peripheral
substituents provided no steric hindrance for the formation of stable complexes with serum
albumin, a major biological carrier. The cell permeable 2-{3-[(o-carboran-1’-yl)thio]pyrrolidine-
2
,5-dione-1-yl}-5,10,15,20-tetraphenylporphyrin was virtually non-toxic in the dark whereas
rapid generation of superoxide anion radical and a necrotic death were observed after
1
7

illumination of tumor cells loaded with this compound. Overall, the new chemotype is
perspective for an in-depth investigation as a source of multifunctional anticancer drug
candidates.
3
. Experimental
3
3
.1. Synthesis
.1.1. General information
Reagents were from Sigma-Aldrich unless specified otherwise. All reactions were
performed in an atmosphere of dry argon. The starting free base porphyrins, 5,10,15,20-
tetraphenylporphyrin (6) [38], 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (7) [39] and
5
,10,15,20-tetrakis[4-(trifluormethyl)phenyl]porphyrin (8) [38] were prepared according to
published procedures. All solvents were dried as recommended in standard protocols. Chemical
1
shifts (δ) were referenced to the residual solvent peak (CDCl and (CD ) CO, H: 7.26 ppm and
3
3 2
1
11
19
2
.05, respectively) for H, external BF ·OEt for B and external CFCl for F. IR spectra were
3 2 3
recorded on a Bruker FTIR spectrometer Tensor 37 in KBr tablets. Merck silica gel L 0.040–
.080 mesh was used for column chromatography. The identities of new compounds were
0
verified by TLC on Sorbfil and Kieselgel 60 F254 (Merck) plates. The UV-Vis spectra were
measured on a spectrophotometer Carl Zeis Specord M 40 in CH Cl . MALDI mass spectra for
2
2
carborane-substituted porphyrins were recorded on a Brucer autoflex speed time-of-flight (TOF)
mass obtained mass spectrometer (Bruker Daltonics Inc., Germany) equipped with a solid-state
ultraviolet (UV) laser of 355 nm (1 kHz repetition rate, 1000 shots for each spectrum) and
operated in positive reflectron mode. MALDI mass spectra were recorded by using stainless-
steel targets (MTP 384 ground steel; Bruker Daltonics Inc., Germany) containing 384 cells for
the deposition of the analyte mixed with matrix; the most intense peaks were given for each
compound. The APCI mass-spectra were registered on the Finnigan LCQ Advantage tandem
dynamic mass-spectrometer (USA), equipped by octapole ion trap mass analyzer with the
Surveyor MS pump and the nitrogen generator Schmidlin-Lab (Germany). Nitrogen 70/10
served as a sheath and auxiliary gas. The flow rate of acetonitrile was 350 µl/min. The
о
о
temperature of the vaporizer was 400 С. The temperature of the heated capillary was 150 С, the
electric potential between the needle and the counter electrode 6.0 kV. Samples (100 µM in
acetonitrile solution) were introduced into the ion source through the Reodyne injector with 5 µl
loop. Acetonitrile (Merck) was used for gradient analysis. For data collection the X Calibur
version 1.3 was used.
3
.1.2. General procedures for preparation of copper(II) complexes of porphyrins.
A solution of Cu(OAc) ·H O (4.6 mmol) in methanol (10 mL) was added to a solution of
2
2
corresponding porphyrin (1.15 mmol) in methylene chloride (50 mL). The resulting mixture was
1
8

stirred for 1.5 h at room temperature with TLC monitoring (CHCl –hexane 1:2). Then the
3
reaction mixture was poured into water and extracted with methylene chloride. The organic layer
was dried over Na SO , and the solvent was removed under reduced pressure. The residue was
2
4
used in the reactions without purification.
,10,15,20-(tetraphenylporphyrinato)copper(II) (13) [56] (757 mg, yield 97%). UV-Vis
5
-3
+
(
CH Cl ) λ , (εх10 ) nm: 414 (611), 539 (29). APCI-MS Found: [M] 676.16; ‘C H CuN ’
2
2
max
44 28
4
+
requires [M] 676.26.
5
,10,15,20-{tetrakis[4-(trifluoromethyl)phenyl]porphyrinato}copper (II) (15) [57] (1080
-3
mg, yield 99 %). UV-Vis (CH Cl ) λ , (εх10 ) nm: 416 (60.2), 544 (2.40). APCI-MS Found:
2
2
max
+
+
[
M] 948.11; ‘C H CuF N ’ requires [M] 948.26.
48 24 12 4
3
.1.3. General procedures for nitration of porphyrin copper(II) and zinc(II) complexes.
To a solution of corresponding porphyrin copper complex (1.13 mmol) in methylene
chloride (90 mL) Cu(NO ) ·3 H O (2.30 mmol) in the mixture of acetic acid (5 mL) and acetic
3
2
2
anhydride (2 mL) was added, and reaction mixture was stirred for 3 h at room temperature, with
TLC monitoring (CHCl –hexane 1:2). After completion the reaction the solution was washed
3
with water (200 mL), then with Na CO solution, the organic phase was separated and dried over
2
3
Na SO . After removal of the solvent under reduced pressure, the residue was purified by
2
4
column chromatography on silica gel using a CH Cl –hexane system (3:7).
2
2
2
-Nitro-5,10,15,20-(tetraphenylporphyrinato)copper(II) (9) [58] (798 mg, yield 98%).
-3
-1
UV-vis (CH Cl ): λ , nm (εх10 ) 423 (94), 548 (6.9), 592 (4.5). IR (KBr) ν , сm : 3435
2
2
max
max
+
(
NН), 2922 (СН), 1524 (NO ), 1342 (NO ). MALDI-MS Found: [M] 721.15; ‘C H CuN O ’
2 2 44 27 5 2
+
requires [M] 721.26.
2
-Nitro-5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato)copper(II) (10) was obtained
-3
under refluxe for 4h, [41] (1186 mg, yield 97%). UV-vis (CH Cl ): λ , nm (εх10 ) 416 (225),
2
2
max
-1
5
43(18), 588 (16.2). IR (KBr) νmax, сm : 3435 (NН), 2956 (СН), 1520 (NO ), 1493 (CF), 1293
2
+
+
(
CF), 1339 (NO ). APCI-MS Found: [M] 1081.74; ‘C H CuF N O ’ requires [M] 1081.07.
2 44 7 20 5 2
{
2-Nitro-5,10,15,20-tetrakis[4-(trifluoromethyl)phenyl]porphyrinato}copper (II) (11)
-3
[
57] (909 mg, yield 81%). UV-vis (CH Cl ): λ , nm (εх10 ) 418 (112), 547 (11.8), 590 (8.67).
2
2
max
-1
+
MS IR (KBr) νmax, сm : 3389 (NН), 1527 (NO ), 1326 (CF). APCI-MS Found: [M] 993.89;
2
+
‘
C H CuF N O ’ requires [M] 993.26.
4
8
23
12
5
2
[
2-Nitro-5,10,15,20- tetrakis(pentafluorophenyl)porphyrinato]zinc(II) (12) [59].
To a solution of 5,10,15,20-tetraphenylporphyrinporphyrin zinc complex (16) [60] 500
mg (0.48 mmmol) in methylene chloride (50 mL) Cu(NO ) ·3 H O (200 mg, 0.84 mmol) in the
3
2
2
mixture of acetic acid (5 mL) and acetic anhydride (2 mL) was added, and reaction mixture was
stirred for 1,5 h under reflux, with TLC monitoring (CHCl –hexane 1:1). After the reaction was
3
1
9

complete the solution was washed with water (100 mL), then with Na CO solution, the organic
2
3
phase was separated and dried over Na SO . After removal of the solvent under reduced
2
4
pressure, the residue was purified by column chromatography on silica gel using a methylene
-3
chloride-hexane system (4:6). Yield 500 mg (96%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 426
2
2
max
-1
(
243), 558 (19.4), 603 (14.0). IR (KBr) νmax, сm : 3392 (NH), 1521 (NO ), 1501 (CF), 1337
2
1
(
NO ), 1286 (CF). H NMR, δ (CDCl , 400 MHz), ppm: 8.94 (m, 6H, β-H), 9.26 (s, 1H, β-H).
2
H
3
1
9
F NMR, δ (CDCl , 376 MHz), ppm: –136.5 (d, 2F, J = 16.5 Hz), –136.8 (d, 4F, J = 22.0 Hz),
F
3
–
137.5 (d, 2F, J = 16.5 Hz), –150.5 (t, 1F, J = 22.0 Hz), –150.7 (t, 1F, J = 22.00 Hz), –151.3 (t,
F, J = 19.3 Hz), –160.9 (t, 2F, J = 16.5 Hz), –161.3 (t, 4F, J = 19.3 Hz), –161.7 (t, 2F, J = 19.3
2
+
+
Hz). APCI-MS Found: [M] 1082.98; ‘C H F N O Zn’ requires [M] 1082.91.
44
7
20
5
2
3
.1.4. General procedure of demetallation of copper complexes (9-11).
To the corresponding copper (II) complex of 2-nitroporphyrin (0.5 mmol) concentrated
H SO (5 mL) and CF COOH (5 mL) were added and the reaction was stirred for 3 h at room
2
4
3
temperature. After the demetallation (TLC monitoring, CHCl -hexane 1:2), the mixture was
3
poured into water, extracted with CH Cl (50 mL), washed with a saturated solution of NaHCO
3
2
2
and water. The organic layer was dried over anhydrous Na SO . After removal of the solvent
2
4
under reduced pressure, the residue was purified by column chromatography on silica gel using
CH Cl -hexane system (1:1) as an eluent.
2
2
2
-Nitro-5,10,15,20-tetraphenylporphyrin (17) (287 mg, yield 87%). UV-Vis (CH Cl )
2
2
-3
-1
λmax, (εх10 ) nm: 425 (200), 527 (14.5), 568 (4.1), 604 (4.1), 651 (8.3). IR (KBr) ν , сm :
max
1
3
327 (NH), 1523 (NO ), 1347 (NO ). H NMR, δ (CDCl , 400 MHz), ppm: –2.50 (br s, 2H,
2
2
H
3
NH), 7.82 (m, 12 H, Ph), 8.28 (m, 6 H, Ph), 8.36 (dd, 2H, J = 7.9, 1.2 Hz, Ph), 8.80 (d, 1H, J =
4
.6 Hz, β-H), 8.82 (d, 1H, J = 4.6 Hz, β-H), 8.99 (d, 2H, J = 4.9 Hz, β-H), 9.04 (d, 1H, J = 5.2
+
Hz, β-H), 9.12 (d, 1H, J = 5.2 Hz, β-H), 9.16 (s, 1H, β-H). MALDI-MS Found: [M] 659.23;
+
‘
C H N O ’ requires [M] 659.73.
4
4
29
5
2
2
-Nitro-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (18) [41] (459 mg, yield 90%).
-3
UV-Vis (CH Cl ) λ , (εх10 ) nm: 420 (100), 516 (7.71), 557 (3.10), 591 (3.86), 656 (2.29). IR
2
2
max
-1
1
(
KBr) νmax, сm : 3334 (NH), 1522 (NO ), 1360 (NO ), 1499 (CF), 1201 (CF). H NMR, δ
2 2 H
(
CDCl , 400 MHz), ppm: –2.79 (s, 2H, NH), 8.83 (dd, 2H, J = 7.2, 4.8 Hz, β-H), 9.06 (m, 4H, J
3
1
9
=
5.7 Hz, β-H), 9.19 (s, 1H, β-H). F NMR, δ (CDCl , 376 MHz), ppm: –136.1 (dd, 2F, J =
F 3
21.6, 6.5 Hz), –136.4 (dd, 4F, J = 22.7, 6.5 Hz), –137.2 (dd, 2F, J = 21.6 Hz, J = 6.5 Hz), –
149.4 (m, 1F, J = 20.6 Hz), –149.6 (m, 1F, J = 20.6 Hz), –150.3 (m, 2F), –160.3 (m, 2F, J =
20.6, 6.5 Hz), –160.7 (m, 4F, J = 19.5, 4.3 Hz), -161.1 (td, 2F, J = 20.6, 5.4 Hz). APCI-MS
+
+
Found: [M] 1019.96; ‘C H F N O ’ requires [M] 1019.54.
4
4
9
20
5
2
2
0

2
-Nitro-5,10,15,20-tetrakis[4-(trifluoromethyl)phenyl]porphyrin (19) [57] (387 mg, yield
-3
8
3%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 428 (183), 525 (14.1), 564 (3.49), 602 (4.03), 660
2 2 max
-1
1
(
6.75). IR (KBr) νmax, сm : 3336 (NH), 1530 (NO ), 1324 (CF). H NMR, δ (CDCl , 400
2 H 3
MHz), ppm: –2.64 (s, 2H, NH), 7.99 (d, 2H, J = 7.9 Hz, Ph), 8.09 (d, 6H, J = 7.6 Hz, Ph ), 8.36
(d, 8 H, J = 7.3 Hz, Ph), 8.72 (dd, 2H, J = 7.9, 4.8 Hz, β-H), 8.92 (dd, 2H, J = 8.9, 5.1 Hz, β-H),
1
9
8
.98 (dd, 2H, J = 11.4, 5.1 Hz, β-H), 9.04 (s, 1H, β-H). F NMR, δ (CDCl , 376 MHz), ppm: –
F 3
+
6
2.2 (s, 3F), –62.1 (d, 9F, J = 16.5 Hz). APCI-MS Found: [M] 932.22; ‘C H F N O ’
4
8
25 12
5
2
+
requires [M] 931.73.
.1.5. General procedure for preparation of 2-amino-5,10,15,20-tetraarylporphyrins (20-
3
2
4).
To a mixture of the corresponding 2-nitro-substituted porphyrin (0.3 mmol) and 10%
palladium on activated charcoal (30 mg) in dichloromethane – methanol (2:1) system (30 ml)
sodium borohydride (1.5 mmol) was added under argon atmosphere in the dark. The reaction
mixture was stirred at room temperature for 30 min. After completion of the reaction (TLC
control) the reaction mass was filtered, and the organic phase was washed with water and dried
over Na SO . The solvent was removed under reduced pressure. The residue was purified by
2
4
column chromatography using CH Cl -hexane system (2:3) as an eluent to afford the appropriate
2
2
2
-amino-5,10,15,20-tetraarylporphyrin.
-Amino-5,10,15,20-tetraphenylporphyrin (20) [61] (149 mg, yield 79 %). UV-Vis
2
-3
(
CH Cl ) λ , (εх10 ) nm: 424 (76.3), 523 (8.8), 560 (3.6), 596 (3.9), 650 (4.4). IR (KBr) νmax,
2 2 max
-1
1
сm : 3460 (NH). H NMR, δ (CDCl , 400 MHz), ppm: –2.73 (br s, 2H, NH), 4.47 (br s, 2H,
H
3
NH ), 7.75 (d, 10H, J = 5.4 Hz, Ph), 7.81 (d, 2H, J = 7.9 Hz, Ph), 8.14 (m, 4H, Ph), 8.22 (d, 4H,
2
J = 6.4 Hz, Ph,), 8.53 (d, 1H, J = 3.8 Hz, β-H), 8.59 (br s, 1H, β-H), 8.71 (br s, 1H, β-H), 8.75
+
(
d, 1H, J = 3.8 Hz, β-H), 8.80 (br s, 3H, β-H). MALDI-MS Found: [M] 630.65; ‘C H N ’
4
4
31
5
+
requires [M] 629.75.
2
-Amino-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (21) [32] (211 mg, yield
-3
7
1%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 420 (100), 516 (7.71), 557 (3.1), 591 (3.86), 656
2 2 max
-1
1
(
2.29). IR (KBr) νmax, сm : 3314 (NH), 1498 (CF), 1150 (CF). H NMR, δ (CDCl , 400 MHz),
H 3
ppm: –2.60 (br s, 2H, NH), 4.70 (br s, 2H, NH ), 7.87 (br s, 1H, β-H), 8.66 (d, 1H, J = 4.8 Hz, β-
2
H), 8.79 (d, 1H, J = 4.5 Hz, β-H), 8.85 (dd, 3H, J = 10.0, 4.5 Hz, β-H), 8.89 (d, 1H, J = 4.2 Hz,
1
9
β-H). F NMR, δ (CDCl , 376 MHz), ppm: –135.5 (d, 2F, J = 17.1 Hz), –136.6 (d, 6F, J = 21.3
F
3
Hz), –149.8 (d, 1F, J = 21.3 Hz), –151.5 (d, 2F, J = 21.3 Hz), –152.0 (d, 1F, J = 21.3 Hz), –
+
1
9
59.6 (dd, 2F, J = 36.3, 17.1 Hz), –161.6 (d, 6F, J = 21.3 Hz). APCI-MS Found: [M+H]
+
91.19; ‘C H F N ’ requires [M+H] 990.56.
44
12 20
5
2
1

2
-Amino-5,10,15,20-tetrakis[4-(trifluoromethyl)phenyl]porphyrin (22) (198 mg, yield
-3
7
3%, purple solid). UV-Vis (CH Cl ) λ , (εх10 ) nm: 424 (87.2), 523 (11.4), 558 (5.11), 595
2 2 max
-1
1
(
5.36), 651 (5.55). IR (KBr) νmax, сm : 3403 (NH), 3313 (NH ), 1325 (CF). H NMR, δ
2 H
(
CDCl , 400 MHz), ppm: –2.70 (br s, 2H, NH), 4.43 (br s, 2H, NH ), 7.72 (s, 1H, β-H), 8.03 (dd,
3
2
6
H, J = 7.9, 3.8 Hz, Ph), 8.10 (d, 2H, J = 7.9 Hz, Ph), 8.31 (dd, 8 H, J = 11.8, 8.0 Hz, Ph), 8.46
(d , 1H, J = 4.8 Hz, β-H), 8.69 (dd, 2H, J = 12.3, 4.7 Hz, β-H), 8.77 (s, 1H, β-H), 8.78 (dd, 2H, J
19
=
10.5, 4.8 Hz, β-H). F NMR, δ (CDCl , 376 MHz), ppm: –62.2 (s, 3F), –62.0 (s, 9F). APCI-
F 3
+
+
MS Found: [M+H] 902.91; ‘C H F N ’ requires [M+H] 902.74.
48
28 12
5
[
2-Amino-5,10,15,20-tetraphenylporphyrinato]copper (II) (23) [62] (160 mg, yield 77%).
-3
-1
UV-Vis (CH Cl ) λ , (εх10 ) nm: 423 (147), 548 (12.4), 596 (7.6). IR (KBr) ν , сm : 3371
2
2
max
max
+
+
(
NH). APCI-MS Found: [M] 691.08; ‘C H CuN ’ requires [M] 691.28.
44 29 5
[
2-Amino-5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato]zinc (II) (24) [59] (237
-3
mg, yield 75%, green solid). UV-Vis (CH Cl ) λ , (εх10 ) nm: 409 (109), 556 (10.3), 605
2
2
max
-1
1
(
8.85). IR (KBr) νmax, сm : 3392 (NH), 1492 (CF), 1338 (CF). H NMR, δ (CDCl , 400 MHz),
H 3
ppm: 4.57 (br s, 2H, NH ), 7.88 (s, 1H, β-H), 8.75 (d, 1H, J = 4.5 Hz, β-H), 8.93 (m, 5H, β-H).
2
1
9
F NMR, δ (CDCl , 376 MHz), ppm: –136.0 (dd, 2F, J = 24.7, 5.5 Hz), –136.9 (d, 6F, J = 19.2
F
3
Hz), –150.6 (t, 1F, J = 19.2 Hz), –152.3 (q, 2F, J = 24.0 Hz), -152.7 (t, 1F, J = 22.0 Hz), –160.0
+
(
d, 2F, J = 35.7 Hz), –161.9 (m, 6F, J = 44.0, 13.7 Hz). APCI-MS Found: [M] 1052.35;
+
‘
C H F N Zn’ requires [M] 1052.92.
4
4
9
20
5
3
.1.6. Preparation of maleinoylamino-substituted porphyrins (25, 26, 28).
To 2-aminosubstituted porphyrin (0.2 mmol) in glacial acetic acid (15 mL) maleic
anhydride (59 mg, 0.6 mmol) was added, and the reaction was stirred under argon for 50 h at
room temperature. The solution was evaporated, the resulting solid was dissolved in
dichloromethane (20 mL), washed with water and dried over Na SO . After removal of the
2
4
solvent in vacuo, the residue was purified by column chromatography on silica gel using
CH Cl -MeOH system (9:1) as an eluent.
2
2
2
-(Maleinoylamino)-5,10,15,20-tetraphenylporphyrin (25) (120 mg, yield 82%). UV-Vis
-3
(
CH Cl ) λ , (εх10 ) nm: 424 (166), 520 (20), 555 (5.8), 593 (6.6), 651 (4.1). IR (KBr) νmax,
2 2 max
-1
1
сm : 3328 (NH), 2853.26 (CH), 1720 (C=O), 1610 (amide I), 1596 (C=C), 1539 (amide II). H
NMR, δ (CDCl , 400 MHz), ppm: –2.80 (br s, 2H, NH), 5.42 (d, 1H, J = 12.4 Hz, CH=CH),
H
3
6
.34 (d, 1H, J = 12.4 Hz, CH=CH), 7.79 (s, 9H, Ph), 7.89 (t, 2H, J = 7.0 Hz, Ph), 7.96 (d, 1H, J
=
6.7 Hz, Ph), 8.21 (br s, 8H, Ph), 8.68 (d, 1H, J = 4.5 Hz, β-H), 8.80 (br s, 3H, β-H), 8.91 (d,
+
3
H, J= 9.9 Hz, β-H), 9.34 (br s, 1H, NHCO). APCI-MS Found: [M+H] 728.51; ‘C H N O ’
4
8
34
5
3
+
requires [M+H] 728.81.
2
2

2
-(Maleinoylamino)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (26) (43 mg, yield
-3
2
0%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 424 (166), 520 (20), 555 (5.8), 593 (6.6), 651 (4.1).
2 2 max
-1
1
IR (KBr) νmax, сm : 3341 (NH), 1723 (C=O), 1612 (amide I), 1591 (C=C), 1532 (amide II). H
NMR, δ (CDCl , 400 MHz), ppm: –2.72 (br s, 2H, NH), 4.87 (d, 1H, J = 6.0 Hz, CH=CH),
H
3
5
.70 (d, 1H, J = 2.9 Hz, CH=CH), 8.63 (d, 2H, J = 4.1 Hz, β-H), 8.82 (s, 3H, β-H), 9.00 (d, 2H,
1
9
J = 4.8 Hz, β-H), 9.12 (br s, 1H, NHCO). F NMR, δ (CDCl , 376 MHz), ppm: –136.1 (d, 1F,
F
3
J = 16.5 Hz), –136.5 (d, 6F, J = 16.5 Hz), –137.4 (d, 1F, J = 16.5 Hz), –148.6 (d, 1F, J = 19.2
Hz), –150.6 (t, 3F, J = 22.0 Hz), –159.1 (d, 1F, J = 16.5 Hz), –160.9 (d, 6F, J = 22.0 Hz), –
+
+
1
62.0 (d, 1F, J = 8.3 Hz). APCI-MS Found: [M] 1088.22; ‘C H F N O ’ requires [M]
48 13 20 5 3
1
087.62.
2-(Maleinoylamino)-5,10,15,20-tetraphenylporphyrinato]copper (II) (28) (123 mg, yield
[
-3
-1
7
8%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 424 (97), 546 (10.8), 580 (4.7). IR (KBr) ν , сm :
2
2
max
max
+
3
381 (NH), 1722 (C=O), 1610 (amide I), 1539 (amide II). APCI-MS Found: [M] 789.18;
+
‘
C H CuN O ’ requires [M] 789.34.
4
8
31
5
3
3
.1.8. Preparation of maleinoylamino-substituted porphyrins (27, 29).
To a solution of the corresponding 2-amino-substituted porphyrin (0.2 mmol) in MeCN
15mL) maleic anhydride (59 mg, 0.6 mmol) was added, and reaction was refluxed for 11 h
(
under argon. The mixture was poured into water (80 mL), extracted with dichloromethane and
dried over Na SO . After removal of the solvent in vacuo, the residue was purified by column
2
4
chromatography on silica gel using CH Cl -MeOH system (95:5) as an eluent.
2
2
2
-(Maleinoylamino)-5,10,15,20-tetrakis[4-(trifluoromethyl)phenyl]porphyrin (27) (152
-3
mg, yield 76%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 428 (93.8), 526 (10.5), 565 (2.90), 601
2
2
max
-1
(
4.81), 656 (3.10). IR (KBr) νmax, сm : 3397 (NН), 1726 (С=О), 1616 (amide I), 1531 (amide
1
II), 1325 (СF). H NMR, δ ((CD ) CO, 400 MHz), ppm: –2.51 (br s, 2H, NH), 4.85 (dd, 1H, J
H
3 2
=
2.9, 9.7 Hz, CH=CH), 5.78 (dd, 1H, J = 4.6, 2.0 Hz, CH=CH), 8.03 (m, 6H, Ph), 8.18 (d, 2H,
1
9
J = 6.5 Hz, Ph), 8.32 (m, 6H, Ph), 8.49 (d, 2H, J = 8.4 Hz, Ph), 8.93 (m, 7H, β-H). F NMR, δ
F
+
(
(CD ) CO, 376 MHz), ppm: –62.9 (s, 3F), –62.6 (t, 9F, J = 4.5 Hz). APCI-MS Found: [M]
3 2
+
1
000.40; ‘C H F N O ’ requires [M] 999.80.
52 29 12 5 3
[
2-(Maleinoylamino)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato]zinc (II) (29)
-
3
(
(
(
159 mg, yield 69%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 419 (129), 550 (7.58), 628 (6.13). IR
2 2 max
-1
1
KBr) νmax, сm : 3340 (NН), 1729 (С=О), 1632 (С=C), 1496 (CF), 1254 (CF). H NMR, δ
H
CDCl , 400 MHz), ppm: 5.32 (dd, 1H, J = 3.7, 1.4 Hz, CH=CH), 5.82 (dd, 1H, J = 7.5, 4.6 Hz,
3
CH=CH), 8.76 (d, 1H, J = 4.5 Hz, β-H), 8.81 (s, 1H, β-H), 8.88 (d, 2H, J = 4.5 Hz, β-H), 8.94 (s,
1
9
3
H, β-H), 9.18 (br s, 1H, NHCO). F NMR, δ (CDCl , 376 MHz), ppm: –133.1 (d, 1F, J = 19.2
F 3
Hz), –134.8 (d, 2F, J = 19.2 Hz), –136.2 (br s, 2F), –136.8 (br s, 4F), –151.9 (dd, 4F, J = 52.2,
2
3

3
3.0 Hz), –160.5 (br s, 1F), –161.5 (br s, 2F), –161.8 (br s, 4F), –161.8 (br s, 1F). APCI-MS
+
+
Found: [M] 1150.12; ‘C H F N O Zn’ requires [M] 1150.98.
4
8
11 20
5
3
3
.1.9. General procedure for preparation of maleimide substituted 5,10,15,20-
tetraarylporphyrins (1-5).
To a solution of maleinoylaminoporphyrin (0.15 mmol) in acetic anhydride (8 mL)
о
NaOAc (10 mg, 0.12 mmol) was added and reaction was heated at 110 С for 5 h under argon.
Then the reaction was treated with water (50 mL), extracted with dichloromethane (2x 25 mL).
The combined organic layers were dried over Na SO . After evaporating of the solvent the
2
4
residue was purified by column chromatography on silica gel using CH Cl as an eluent.
2
2
2
-(3’-Maleimido)-5,10,15,20-tetraphenylporphyrin (1) (82 mg, yield 77%). UV-Vis
-3
(
CH Cl ) λ , (εх10 ) nm: 420 (559), 518 (22.4), 552 (8.20), 592 (7.08), 648 (8.20). IR (KBr)
2 2 max
-1
1
νmax, сm : 3323 (NH), 1720 (C=O). H NMR, δ (CDCl , 400 MHz), ppm: –2.70 (br s, 2H, NH),
H
3
6
.46 (d, 2H, J = 1.9 Hz, CH=CH), 7.59 (m, 2H, Ph), 7.78 (t, 10H, J = 7.1 Hz, Ph), 8.04 (br s,
H, Ph), 8.25 (m, 6H, Ph), 8.61 (br s, 1H, β-H ), 8.81 (m, 4H, β-H), 8.93 (d, 2H, J = 3.2 Hz, β-
2
+
+
H). APCI-MS Found: [M+H] 710.84; ‘C H N O ’ requires [M+H] 710.79.
48
32
5
2
2
-(3’-Maleimido)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (2) (114 mg, yield
-3
7
1%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 415 (169), 510 (12.3), 543 (3.15), 587 (4.72), 641
2 2 max
-
1
1
(
(
(
1.21). IR (KBr) νmax, сm : 3331 (NH), 1727 (C=O), 1499 (CF), 1353 (CF). H NMR, δ
H
CDCl , 400 MHz), ppm: –2.86 (br s, 2H, NH), 6.98 (s, 2H, CH=CH), 8.83 (s, 2H , β-H), 8.87
3
19
dd, 2H, J = 7.0 Hz, J = 4.8 Hz, β-H), 8.94 (d, 1H, J = 5.0 Hz, β-H), 8.99 (s, 2H, β-H). F
NMR, δ (CDCl , 376 MHz), ppm: –133.6 (dd, 2F, J = 22.0, 5.5 Hz), –135.7 (dd, 2F, J = 23.5,
F
3
6
.5 Hz), –136.4 (dd, 4F, J = 13.8, 8.3 Hz), –150.8 (m, 3F, J = 19.3 Hz), –151.3 (t, 1F, J = 19.3
+
Hz), –161.0 (m, 6F), –161.4 (t, 2F, J = 19.3 Hz). APCI-MS Found: [M+H] 1070.61;
+
‘
C H F N O ’ requires [M+H] 1070.60.
4
8
11 20
5
2
2
-(3’-Maleimido)-5,10,15,20-tetrakis[4-(trifluoromethyl)phenyl]porphyrin (3) (90 mg,
-3
yield 61 %). UV-Vis (CH Cl ) λ , (εх10 ) nm: 418 (256), 517 (18.7), 548 (5.30), 590 (5.85),
2
2
max
-1
1
6
48 (4.54). IR (KBr) νmax, сm : 3323 (NH), 1723 (C=O), 1324 (CF). H NMR, δ (CDCl , 400
H 3
MHz), ppm: –2.79 (s, 2H, NH), 6.55 (s, 2H, CH=CH), 7.91 (d, 2H, J = 7.9 Hz, Ph), 8.06 (m, 6H,
Ph), 8.18 (d, 2H, J = 7.8 Hz, Ph), 8.36 (m, 6H, Ph), 8.62 (d, 1H, J = 4.8 Hz, β-H), 8.72 (s, 1H, β-
1
9
H), 8.75 (m, 2H, β-H), 8.81 (d, 1H, J = 4.8 Hz, β-H), 8.90 (s, 2H, β-H). F NMR, δ (CDCl ,
F
3
+
3
76 MHz), ppm: –62.7 (s, 3F), –62.1 (s, 3F), –62.0 (s, 6F). APCI-MS Found: [M+H] 982.82;
+
‘
C H F N O ’ requires [M+H] 982.78.
5
2
28 12
5
2
[
2-(3’-Maleimido)-5,10,15,20-tetraphenylporphyrinato]copper (II) (4) (102 mg, yield
-3
8
8%, dark red solid). UV-Vis (CH Cl ) λ , (εх10 ) nm: 416 (214), 542 (10.3), 577 (2.3). IR
2 2 max
2
4

-1
+
(
KBr) νmax, сm : 3440 (NH), 1719 (C=O). MALDI-MS Found: [M] 771.21; ‘C H CuN O ’
48 29 5 2
+
requires [M] 771.32.
[
2-(3’-Maleimido)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato]zinc (II) (5) (129
-3
mg, yield 76%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 421 (523), 553 (22.0), 590 (6.32). IR (KBr)
2
2
max
-1
1
νmax, сm : 3440 (NH), 1723 (C=O), 1499 (CF), 1285 (CF). H NMR, δ (CDCl , 400 MHz),
H
3
ppm: 6.91 (s, 2H, CH=CH), 8.77 (d, 1H, J = 4.8 Hz, β-H), 8.90 (m, 2H, β-H), 8.97 (m, 4H, β-H).
1
9
F NMR, δ (CDCl , 376 MHz), ppm: –135.9 (dd, 2F, J = 24.3, 5.5 Hz), –139.2 (dd, 1F, J =
F
3
2
3.0, 5.5 Hz), –139.6 (dd, 4F, J = 16.5, 8.2 Hz), –155.6 (t, 1F, J = 19.2 Hz), –156.1 (q, 3F, J =
9.2 Hz), –164.9 (dd, 6F, J = 46.7, 22.0 Hz), –165.2 (dd, 2F, J = 42.7, 6.2 Hz). APCI-MS
1
+
+
Found: [M] 1133.16; ‘C H F N O Zn’ requires [M] 1132.97.
4
8
9
20
5
2
3
.1.10. Preparation of carborane succinimidyl thioethers of 5,10,15,20-
tetraarylporphyrins (32-35).
To a solution of maleimidoporphyrin (0.06 mmol) in CHCl (10 mL) mercaptocarborane
3
(12 mg, 0.07 mmol) and NaOAc (5 mg, 0.06 mmol) were added and reaction mixture was heated
at reflux under argon for 8 h. After completion of the reaction (TLC control) the solvent was
removed under reduced pressure. The residue was purified by column chromatography on silica
gel using CH Cl as an eluent.
2
2
2
-{3-[(o-Carboran-1’-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-
-3
tetraphenylporphyrin (32) (44 mg, yield 83%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 421 (288),
2
2
max
-1
5
19 (14.8), 553 (4.93), 592 (4.62), 648 (5.06). IR (KBr) νmax, сm : 3438 (NH), 3054 (carborane
1
CH), 2581 (BH), 1724 (C=O). H NMR, δ (CDCl , 400 MHz), ppm: –2.72 (br s, 2H, NH), 2.55
H
3
(dd, 1H, J = 18.8, 6.4 Hz, CH), 2.99 (dd, 1H, J = 18.8, 10.2 Hz, CH), 3.69 (dd, 1H, J = 10.2, 6.4
Hz, CH), 4.10 (br s, 1H, carborane CH), 7.80 (m, 12H, Ph), 8.24 (m, 8H, Ph), 8.65 (d, 1H, J =
4
.8 Hz, β-H), 8.73 (s, 1H, β-H), 8.81 (m, 2H, β-H), 8.88 (d, 1H, J = 5.1 Hz, β-H), 8.95 (dd, 2H, J
1
1
=
6.4, 4.8 Hz, β-H). B NMR, δ , (CDCl , 128 MHz), ppm: –0.8 (d, 1B, J= 151 Hz), –4.4 (d,
B 3
+
1
8
B, J = 144 Hz), –9.2 (d, 4B, J = 142 Hz), –12.3 (d, 4B, J = 168 Hz). MALDI-MS Found: [M]
+
86.99; ‘C H B N O S’ requires [M] 886.08.
50
43 10
5
2
2
-{3-[(m-Carboran-9’-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-
-3
tetraphenylporphyrin (33) (47 mg, yield 88%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 420 (290),
2
2
max
-1
5
17 (14.5), 556 (4.83), 591 (4.63), 649 (5.02). IR (KBr) νmax, сm : 3331 (NH), 3054 (carborane
1
CH), 2603 (BH), 1723 (C=O). H NMR, δ (CDCl , 400 MHz), ppm: –2.72 (br s, 2H, NH), 2.75
H
3
(
dd, 1H, J = 18.8, 5.1 Hz, CH), 2.91 (dd, 2H, J = 18.9, 9.4 Hz, CH ), 3.00 (br s, 2H, carborane
2
CH), 7.77 (dd, 12H, J = 12.7, 6.8 Hz, Ph), 8.17 (dd, 2H, J = 18.8, 6.4 Hz, Ph), 8.25 (dd , 6H, J =
8.6, 6.8 Hz, Ph), 8.63 (d, 1H, J = 4.5 Hz, β-H), 8.76 (s, 1H, β-H), 8.83 (dd, 3H, J= 17.5, 4.5
1
1
1
Hz, β-H), 8.87 (d, 1H, J = 5.1 Hz, β-H), 8.94 (s, 2H, β-H). B NMR, δ , (CDCl , 128 MHz),
B
3
2
5

9
ppm: –0.7 (br s, 1B, B ), –6.3 (d, 2B, J = 135 Hz), –9.7 (d, 1B, J = 147 Hz), –13.7 (d, 5B, J =
+
1
59 Hz), –17.3 (d, 1B, J = 180 Hz). MALDI-MS Found: [M] 886.41; ‘C H B N O S’
5
0
43 10
5
2
+
requires [M] 886.08.
-{3-[(m-Carboran-9’-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-tetrakis[4-
trifluoromethyl)phenyl]porphyrin (34) (66 mg, yield 95%). UV-Vis (CH Cl ) λ , (εх10 ) nm:
2
-3
(
2
2
max
-1
4
19 (323), 517 (21.8), 551 (6.17), 592 (7.08), 648 (5.58). IR (KBr) νmax, сm : 3329 (NH), 3060
1
(
carborane CH), 2606 (BH), 1725 (C=O), 1324 (CF). H NMR, δ (CDCl , 400 MHz), ppm: –
H
3
2
.74 (br s, 2H, NH), 2.85 (d, 2H, J= 6.5 Hz, CH ), 2.98 (s, 1H, CH), 3.08 (br s, 2H, carborane
2
CH), 8.03 (d, 2H, J= 8.2 Hz, Ph), 8.09 (t, 6H, J = 7.9 Hz, Ph), 8.35 (m, 8H, Ph), 8.65 (t, 1H, J =
.5 Hz, β-H), 8.71 (s, 1H, β-H), 8.77 (m, 2H, β-H), 8.83 (d, 1H, J = 5.1 Hz, β-H), 8.92 (s, 2H, β-
2
1
1
9
H). B NMR, δ , (CDCl , 128 MHz), ppm: –0.2 (s, 1B, B ), –7.1 (d, 2B, J= 163 Hz), –10.8 (d,
B
3
1
B, J = 147 Hz), –14.0 (d, 2B, J = 177 Hz), –15.4 (d, 2B, J= 173 Hz), –19.0 (d, 1B, J = 182
1
9
Hz), –23.0 (d, 1B, J = 182 Hz). F NMR, δ (CDCl , 376 MHz), ppm: –62.2 (s, 3F), –62.1 (d,
F
3
+
+
9
F, J= 4.1 Hz). MALDI-MS Found: [M] 1158.19; ‘C H B F N O S’ requires [M] 1158.08.
54 39 10 12 5 2
[
2-{3-[(m-Carboran-9’-yl)thio]pyrrolidine-2,5-dione-1-yl}-5,10,15,20-
tetrakis(pentafluorophenyl)porphyrinato]zinc (II) (35) (61 mg, yield 78%). UV-Vis (CH Cl )
2
2
-3
-1
λmax, (εх10 ) nm: 422 (336), 553 (17.0), 590 (4.56). IR (KBr) ν , сm : 3435 (NH), 3067
max
1
(
carborane CH), 2607 (BH), 1729 (C=O), 1494 (CF). H NMR, δ (CDCl , 400 MHz), ppm:
H
3
2
.83 (dd, 2H, J = 13.7, 6.7 Hz CH ), 3.63 (dd, 1H, J = 14.3, 6.3 Hz, CH), 3.08 (br s, 2H,
2
carborane CH), 9.10 (t, 1H, J = 4.9 Hz, β-H), 9.24 (d, 1H, J = 4.7 Hz, β-H), 9.30 (d, 5H, J = 4.9
1
1
9
Hz, β-H). B NMR, δ , (CDCl , 128 MHz), ppm: –0.5 (br s, 1B, B ), –6.4 (d, 2B, J = 161 Hz), –
B
3
1
9
9
.8 (d, 1B, J = 149 Hz), –13.7 (m, 5B, J = 156 Hz), –17.1 (d, 1B, J = 179 Hz). F NMR, δ
F
(
CDCl , 376 MHz), ppm: –134.8 (d, 1F, J = 19.2 Hz), –135.7 (d, 1F, J = 19.2 Hz), –136.0 (d,
3
2
F, J = 22.0 Hz), –136.8 (t, 3F, J = 24.7 Hz), –136.9 (d, 1F, J= 19.2 Hz), –148.9 (t, 1F, J = 22.0
Hz), –151.9 (quin, 3F, J = 19.2 Hz), –157.6 (t, 1F, J = 19.2 Hz), –161.7 (d, 6F, J = 19.2 Hz), –
+
1
63.4 (t, 1F, J = 16.2 Hz). MALDI-MS Found: [M] 1309.03; ‘C H B F N O SZn’ requires
50
21 10 20
5
2
+
[
M] 1309.26.
.1.11. Demetallation of porphyrin 35.
To a solution of porphyrin 35 (40 mg 0.03 mmol) in CH Cl (8.0 mL) CF COOH (1.0
3
2
2
3
mL) was added and the reaction mixture was stirred for 2 min at room temperature. After
demetallation (TLC monitoring, CHCl -hexane 2:1) the mixture was treated with water (30 mL),
3
extracted with CH Cl (10 mL), and the organic solution was dried over anhydrous Na SO .
2
2
2
4
After removal of the solvent under reduced pressure, the residue was washed with hexane to give
-3
porphyrin 36 (34 mg, yield 91%) as a dark violet solid. UV-Vis (CH Cl ) λ , (εх10 ) nm: 417
2
2
max
-1
(
313), 511 (15.7), 549 (3.81), 586 (4.63), 647 (2.02). IR (KBr) νmax, сm : 3331 (NH), 3065
2
6

1
(
carborane CH), 2609 (BH), 1715 (C=O), 1499 (CF). H NMR, δ (CDCl , 400 MHz), ppm: –
H 3
2
.89 (brs, 2H, NH), 2.63 (dd, 2H, J= 18.8, 5.6 Hz, CH ), 3.68 (dd, 1H , J = 18.8, 9.6 Hz, CH),
2
3
.87 (br s, 2H, carborane CH), 9.19 (s, 1H, β-H), 9.32 (br s, 3H, β-H), 9.46 (d, 1H, J = 4.3 Hz, β-
1
1
9
H), 9.50 (br s, 2H, β-H). B NMR, δ , (CDCl , 128 MHz), ppm: –2.3 (br s, 1B, B ), –5.8 (d,
B
3
2
–
1
2
2
B, J = 165 Hz), –8.9 (d, 1B, J = 154 Hz), –12.5 (d, 2B, J = 168 Hz), –13.8 (d, 2B, J = 168 Hz),
1
9
17.5 (d, 1B, J = 182 Hz), –20.6 (d, 1B, J = 182 Hz). F NMR, δ (CDCl , 376 MHz), ppm: –
F
3
36.9 (dd, 1F, J = 22.9, 6.5 Hz), –137.5 (dd, 1F, J = 22.9, 6.5 Hz), –139.2 (ddd, 2F, J = 50.5,
2.9, 6.2 Hz), –139.9 (dd, 4F, J = 24.8, 6.2 Hz), –151.5 (t, 1F, J = 20.6 Hz), –154.9 (t, 1F, J =
0.6 Hz), –155.3 (ddd, 2F, J = 20.5, 19.6, 5.1 Hz), –159.7 (td, 1F, J = 22.7, J= 6.2 Hz), –164.1
(m, 2F), –164.3 (dd, 2F, J = 16.5, 8.3 Hz), –164.5 (dd, 2F, J = 20.6, 8.3 Hz), –165.5 (ddd, 1F, J
+
+
=
22.9, 20.6, 8.2 Hz). MALDI-MS Found: [M] 1245.23; ‘C H B F N O S’ requires [M]
50 23 10 20 5 2
1
245.89.
.1.12. Reaction of cysteine with porphyrins 3, 4. General procedure.
To a solution (0.03 mmol) of maleimideporphyrins 3 or 4 in 5 ml THF-H O (3:2) system
3
2
L-cysteine methyl ester hydrochloride (6.0 mg, 0.04 mmol) was added and reaction was stirred at
room temperature for 8 h under argon. After completion of reaction (TLC control), the reaction
mixture was treated with water (20 mL) and Et N (0.2 mL), extracted with CHCl (2x10 mL),
3
3
dried over anhydrous Na SO and concentrated in vacuo to give the desired compounds.
2
4
2
-[3-(S-Cysteinyl)pyrrolidine-2,5-dione-1-yl]-5,10,15,20-tetrakis[4-(trifluoromethyl)
-3
phenyl]porphyrin (38) (31 mg, yield 93%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 419 (269), 518
2
2
max
-1
(
14.0), 549 (4.47), 592(5.03), 647(4.47) IR (KBr) νmax, сm : 3331(NH), 1724 (C=O), 1324 (CF).
1
H NMR, δ (CDCl , 400 MHz), ppm: –2.81 (brs, 2H, NH), 2.68 (dd, 2H, J = 19.1, 10.4 Hz,
H
3
NH ), 3.16 (d, 2H, J = 13.3 Hz, CH ), 3.27 (d, 2H , J = 13.1 Hz, CH ), 3.77 (t, 3H, J = 9.0 Hz,
2
2
2
CH ), 3.97 (t, 1H, J = 6.9 Hz, CH), 4.37 (t, 1H, J = 6.9 Hz, CH), 8.07 (t, 8H, J = 6.4 Hz, Ph),
3
8
4
6
.30 (m, 8H, Ph), 8.63 (d, 1H, J = 3.1 Hz, β-H), 8.70 (d, 1H, J = 3.1 Hz, β-H), 8.74 (d, 2H, J =
1
9
.4 Hz, β-H), 8.81 (br s, 1H, β-H), 8.89 (br s, 2H, β-H). F NMR, δ (CDCl , 376 MHz), ppm: –
F
3
2.3 (d, 2F, J = 11.2 Hz), –62.1 (br s, 9F), –61.90 (d, 1F, J = 14.0 Hz). APSI-MS Found:
+
+
[
M+H] 1118.1; ‘C H F N O S’ requires [M+H] 1117.97.
56 37 12 6 4
{
2-[3-(S-Cysteinyl)pyrrolidine-2,5-dione-1-yl]-5,10,15,20-tetraphenylporphyrinato}
-3
copper (II) (39) (26 mg, yield 96%). UV-Vis (CH Cl ) λ , (εх10 ) nm: 417 (208), 541 (11.0),
2
2
max
-1
+
5
77 (2.88). IR (KBr) νmax, сm : 3448 (NH), 1721 (C=O). APSI-MS Found: [M] 906.14;
+
‘
C H CuN O S’ requires [M] 906.51.
5
2
38
6
4
3
.2. UV-visible and fluorescence spectroscopy
Electronic absorption spectra were recorded for new compounds (5 µM solutions in
DMSO) on a UV-3101РС spectrophotometer (Shimadzu, Japan) in the range of 300–800
2
7

nm in a quartz cuvette with the optical path length 1 cm at room temperature. Molar
extinction coefficients were determined on a Vibra AF-R220E, Shinko Denshi, Japan). Results
5
-1
-1
of repetitive measurements varied within 7% (ε440 = 2×10 M cm ). A Beer-Lambert function
was observed for all tested compounds at 1-10 µM. Fluorescence spectra were obtained by
analyzing the diluted solutions (absorbance values < 0.1 at the excitation wavelength) using a
FluoTime 300 fluorimeter (PicoQuant GmbH, Germany) with sampling interval 1 nm for
fluorescence and 2 nm for phosphorescence measurements. A Solea supercontinuum laser
(2.5 MHz, bandwidth 5 nm, 1×1 cm quartz cuvette) was used as an excitation source.
3
.3. Measurement of fluorescence quantum yield
Fluorescence quantum yields of 1-3, 5, 32, 34 in ethanol were measured with rhodamine
6
G as a standard (Q=0.94 in ethanol) and calculated using the equation (1) [63,64]:
2
2
Q = Q ×(I×OD ×n /I ×OD×n ) (1),
r
r
r
r
where Q is the quantum yield, I is the integrated intensity, OD is the optical density, and
n is the refractive index. R refers to rhodamine 6G. The optical density (<0.05 for all samples to
avoid the inner filter effect) was matched for tested compounds and reference Measurements
were performed in a quartz cell cuvette (optical path length 1 cm) at room temperature
.4. Measurements of steady-state singlet oxygen phosphorescence
.
.
3
Steady-state singlet oxygen phosphorescence measurements were carried out on a
FluoTime 300 fluorimeter equipped with NIR PMT Module H10330-45 (Hamamatsu, Japan)
coupled to a single photon counter TimeHarp TCSPC (PicoQuant Gmbh, Germany). Absorbance
of new porphyrins and RB in ethanol (at 530 nm for RB (EtOH); 523 nm for 1, 32, 3; 542 nm for
5
; 515 nm for 2 and 34) were matched with the reference value Φꢀ= 0.79 for RB in methanol
1
[
65, 66]. O ( ꢀg) phosphorescence was detected at 90º relative to the excitation beam. Corrected
2
emission spectra were recorded with the integration time 5 s between 1230 nm and 1330 nm.
Total phosphorescence intensities were calculated by integrating the emission band centered at
1
278 nm. Experiments were performed at room temperature.
Singlet oxygen quantum yields (Φꢀs) were determined using the method described in [67]
and the equation (2):
Φ∆s = (I ×I ×τ / I ×I ×τ )× Φ∆r (2),
r
∆s
r
s
∆r
s
where I and I represent the absorbed incident light, and I and I are singlet oxygen
s
r
ꢀs
∆r
integrated emission intensities at 1230-1330 nm for tested compound and the reference,
respectively. τ and τ are the singlet oxygen phosphorescence lifetimes in the reference and the
r
s
sample solutions; Φ∆r is the quantum yield of singlet oxygen of the reference compound. The
analysis did not require the calculation of the absorbed incident light Iabs because the optical
2
8