Synthesis and biological evaluation of new water-soluble photoactive chlorin conjugate for targeted delivery

Article abstract of DOI:10.1016/j.ejmech.2017.12.062

A new water-soluble conjugate, consisting of a chlorin-based photosensitizing part, and a 4-arylaminoquinazoline moiety with high potential affinity to an epidermal growth factor receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), suitable for photodynamic therapy (PDT), was synthesized starting from methylpheophorbide-a in seven steps. An increased accumulation of this compound in A431 cells with high level of EGFR expression, in comparison with CHO and HeLa cells with low EGFR expression was observed. The prepared conjugate exhibits dark and photoinduced cytotoxicity at micromolar concentrations with IC_50dark/IC_50light ratio of 11–18. In tumor-bearing mice, the conjugate preferentially accumulates in the tumor tissue.

Full text of DOI:10.1016/j.ejmech.2017.12.062

Accepted Manuscript
Synthesis and biological evaluation of new water-soluble photoactive chlorin
conjugate for targeted delivery
Vasilii F. Otvagin, Alexander V. Nyuchev, Natalia S. Kuzmina, Ivan D. Grishin, Andrei
E. Gavryushin, Yuliya V. Romanenko, Oscar I. Koifman, Dmitrii V. Belykh, Nina N.
Peskova, Natalia Yu Shilyagina, Irina V. Balalaeva, Alexey Yu. Fedorov
PII:
S0223-5234(17)31094-2
10.1016/j.ejmech.2017.12.062
EJMECH 10040
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2017
Revised Date: 15 December 2017
Accepted Date: 16 December 2017
Please cite this article as: V.F. Otvagin, A.V. Nyuchev, N.S. Kuzmina, I.D. Grishin, A.E. Gavryushin, Y.V.
Romanenko, O.I. Koifman, D.V. Belykh, N.N. Peskova, N.Y. Shilyagina, I.V. Balalaeva, A.Y. Fedorov,
Synthesis and biological evaluation of new water-soluble photoactive chlorin conjugate for targeted
delivery, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.062.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of New Water-Soluble Photoactive
Chlorin Conjugate for Targeted Delivery
a
a
a
a
Vasilii F. Otvagin , Alexander V. Nyuchev , Natalia S. Kuzmina , Ivan D. Grishin , Andrei E.
b
c
c
d
Gavryushin , Yuliya V. Romanenko , Oscar I. Koifman , Dmitrii V. Belykh , Nina N.
a
a
a,e
a
Peskova , Natalia Yu. Shilyagina , Irina V. Balalaeva *, Alexey Yu.Fedorov *
a
Lobachevsky State University of Nizhny Novgorod, Gagarina av. 23, Nizhny Novgorod
6
03950, Russian Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
b
Ludwig-Maximilians-Universität, Butenandtstrasse 5-13, 81377, Munich, Germany
c
Research Institute of Macroheterocycles, Ivanovo State University of Chemical Technology,
1
53000 Ivanovo, Russian Federation
d
Institute of Chemistry, Komi Scientific Center, Urals Branch of the Russian Academy of
Sciences, 167982 Syktyvkar, Russian Federation
e
Sechenov First Moscow State Medical University, Trubetskaya str. 8-2, Moscow 119991,
Russian Federation
*
Corresponding authors
Prof. Alexey Yu. Fedorov
a
Lobachevsky State University of Nizhny Novgorod, Gagarina av. 23, Nizhny Novgorod 603950,
Russian Federation, Fax: +7 831-462-30-85, E-mail: afnn@rambler.ru
Dr. Irina V. Balalaeva
a
Lobachevsky State University of Nizhny Novgorod, Gagarina av. 23, Nizhny Novgorod 603950,
Russian Federation, Tel/Fax +7 831-462-32-13, E-mail: irin-b@mail.ru

ACCEPTED MANUSCRIPT
Keywords: chlorins, conjugate, PDT, targeted delivery, water-solubility, medicinal
chemistry.
Abstract
A new water-soluble conjugate, consisting of a chlorin-based photosensitizing part, and a 4-
arylaminoquinazoline moiety with high potential affinity to an epidermal growth factor
receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), suitable for
photodynamic therapy (PDT), was synthesized starting from methylpheophorbide-a in seven
steps. An increased accumulation of this compound in A431 cells with high level of EGFR
expression, in comparison with CHO and HeLa cells with low EGFR expression was
observed. The prepared conjugate exhibits dark and photoinduced cytotoxicity at micromolar
concentrations with IC50dark/IC50light ratio of 11-18. In tumor-bearing mice, the conjugate
preferentially accumulates in the tumor tissue.
1
. Introduction
Photodynamic therapy (PDT) is a clinically approved low-invasive procedure for the
treatment of oncological and dermatological diseases as well as an efficient method for
therapy of fungal, viral and bacterial infections [1-5]. The therapeutic effect of PDT in the
targeted tissue is based on photochemical conversion of oxygen by a preactivated by light
photosensitizer (PS), leading to the generation of singlet oxygen or active radical particles,
known as reactive oxygen species (ROS) [6,7]. Such species, in turn, are able to induce
cellular damage in the areas proximal to the PS localization. PDT method is well-established
in the treatment of superficial tumors, such as skin, head and neck cancer, as well as tumors of
hollow viscera, in particular, oral tumors, esophagus, stomach, colon, lung, breast, cervix,
prostate, and bladder cancer [1a, 1b]. Among the obstacles for wider application of PDT is
the low selectivity of the PS accumulation in tumor tissues. The accumulation of a

ACCEPTED MANUSCRIPT
photosensitizer in skin and mucous membranes leads to undesirable side effects such as
phototoxicity. A way to regulate the biodistribution of a photosensitizer and thus improve its
overall efficiency upon systemic administration is the application of carrier-linked drug
delivery [8]. The conjugation of a biologically active agent with macromolecules such as
antibodies, polysaccharides, lectins, serum proteins, peptides, growth factors and synthetic
polymers, as well as its incorporation into micelles or liposomes can dramatically change its
biodistribution and enhance its therapeutic potential [9].
The idea behind this work is the development of potentially therapeutically useful conjugates
which would (a) act as both PDT-photosensitizers and cytotoxic agents; (b) selectively
accumulate in tumor cells; (c) allow the fluorescence imaging of their tissue distribution; (d)
possess acceptable physical properties such as water solubility (Figure 1). Theranostic agents
for the simultaneous imaging and targeted non-invasive treatment of cancer have become of
increasing interest over the last decade [10].
Figure 1. Structure of a proposed conjugate.
A number of synthetic or semi-synthetic chlorins (NPe6, Foscan, Fotolon, Photoditasine, and
Tookad – a palladium bacteriopheophorbide PS, related to chlorins) are already used as
photosensitizers in clinics [11-15]. Chlorin-e derivatives (Figure 2), belonging to the natural
6

ACCEPTED MANUSCRIPT
metabolites of chlorophyll a, are prone to simple chemical functionalization, exhibit strong
fluorescence in the red part of the visible spectrum and display good quantum yield of singlet
oxygen and ROS production [16]. Owing to the presence of three carboxyl groups, these
compounds are relatively hydrophilic [17]. At the same time, chlorin-e derivatives manifest
6
amphiphilic properties, since the carboxyl groups are placed on the same side of the
heterocyclic core. Due to their pronounced photocytotoxicity, chlorin-e (Photolon) or its
6
derivatives (Radachlorin or Photoditasine) are used for the treatment of sarcoma and brain
cancers [18]. A less hydrophilic benzochlorin Verteporfin (trade name Visudyne) is used in
UK for the treatment of ophthalmic, pancreatic and skin cancer [18b]. The attachment of L-
aspartic acid moiety to the chlorin core increases the hydrophilicity of the molecule giving
compound known as Talaporfin (or NPe6) – mono-L-aspartyl chlorin-e . It is less
6
photocytotoxic than the parent compound, and has been approved in USA for cure of liver,
colon and brain cancers [18b]. Completely synthetic and highly hydrophilic Temoporfin
(Foscan) is also used for the treatment of these types of cancer.
The incorporation of a non-transition metal into the chlorin core can influence the balance
between the quantum yields of singlet oxygen and the compound fluorescence. So, the
complexation of chlorin-e with Sn(IV) increases the singlet oxygen yield. This effect was
6
used in the development of SnEt /Purlytin, the drug used for the treatment of the skin and
2
breast cancers [18]. The complexation with zinc leads to a drop of the singlet oxygen yield,
with a simultaneous increase in the fluorescence quantum yield. The incorporation of
3
paramagnetic metals completely inhibit O production [17].
2
The PS aggregation can profoundly influence the efficiency of a compound for PDT therapy.
Chlorin-e itself is monomeric in the aqueous solutions. However, π–π stacking and
6
hydrophobic interactions between the molecules of chlorin-e derivatives might cause their
6
self-aggregation and thus a harsh drop of the ROS generation via self-quenching [19].
Extensive aggregation along with poor accumulation of chlorins in the tumor tissues, and

ACCEPTED MANUSCRIPT
long-lasting phototoxicity for healthy tissues are the major problems preventing broader use
of chlorin-e PSs [20].
6
Fig.2. Chlorin-type photosensitizers and epidermal growth factor receptor
(EGFR)/vascular endothelial growth factor receptor (VEGFR) ligands.
To improve the selectivity of PS accumulation in the tumor tissue, we performed the
conjugation of the chlorin core with 4-arylaminoquinazolines (Figure 2) belonging to a family
of high affinity ligands of epidermal growth factor and vascular endothelial growth factor
receptors ((EGFR and VEGFR). Such receptors are often highly expressed by rapidly growing
tumor tissues, due to the necessity to develop their own vasculature [21]. Substituted 4-
arylaminoquinazolines such as Vandetanib (Figure 2, approved in 2011 by FDA for the
treatment of late-stage medullary thyroid cancer) bind to these types of receptors, inhibiting
their tyrosine kinase activity [22].
In order to improve water solubility of the target conjugates [23], we attached hydrophilic
tetraalkylammonium moieties to the chlorin core of the molecule.

ACCEPTED MANUSCRIPT
2
. Results and discussion
Chemistry
The substituted 4-arylaminoquinazoline 1 was synthetized from vanillic acid according to the
known procedure [24], and was converted into azido-ether 2 via two-step sequence (Scheme
1
).
Scheme 1. Synthesis of azido-functionalized derivative 2.
The photoactive parts of the conjugates were prepared from naturally occurring
methylpheophorbide-a (3) (Scheme 2) [25]. At the first step, one of the two ester groups in 3
was selectively cleaved under the acidic conditions to afford acid 4 in 93% yield (Scheme 2).
Its amidation with propargylamine according to the Steglich protocol [26] gave terminal
alkyne 6, isolated in 81% yield. It was subjected to a nucleophilic ring-opening [27] by the
treatment with an excess of amine 7. Zinc insertion in the next step afforded compound 8 in
6
8% yield.
Scheme 2. Synthesis of alkyne-functionalized chlorin-e derivative.
6
At the next step, alkyne 8 was subjected to the [3+2]-cycloaddition conjugation with azide 2
in the presence of CuSO /AscNa/TBTA catalytic system in t-BuOH/CHCl /H O mixture [28]
4
3
2

ACCEPTED MANUSCRIPT
that afforded conjugate 9 in 72% yield (Scheme 3). To increase its water solubility, chlorin 9
was aminomethylated by bis(dimethylamino)methane 10 via the in situ formation of the
Eschenmoser salt, that proceeded at the peripheral vinyl fragment of the chlorin core [29].
The target conjugate 11, bearing three tertiary amino groups was isolated in 63% yield.
Finally, the quaternization of chlorin 11 with methyl iodide afforded quantitatively water-
soluble and stable conjugate 12 (Scheme 3).
Scheme 3. Synthesis of conjugate 12.
In order to investigate the contribution of the quinazoline fragment to the antitumor activity
and photophysical properties of conjugate 12, a water-soluble chlorin 15 without
EGFR/VEGFR ligand part was prepared in four steps starting from methylpheophorbide-a
(Scheme 4).

ACCEPTED MANUSCRIPT
Scheme 4. Synthesis of water-soluble chlorin-e derivative without EGFR/VEGFR ligand.
6
The nucleophilic ring opening of the five-membered exocycle in methylpheophorbide-a 3
using amine 7 followed by the zinc insertion gave chlorin 13 in 72% yield. Its further
aminomethylation with bis(dimethylamino)methane 10 afforded triamine 14 in 76% yield,
which after quaternization gave water-soluble salt 15.
The tetraalkylammonium salts 12 and 15 demonstrated desired solubility in water (~ 0.01
mol/L).
Photophysical properties
Compounds 12 and 15 possess absorption spectra, characteristical for chlorins, with intense
Soret and Q-bands at ~410 nm and ~630 nm, respectively (Table 1, Figure 3). These bands
exhibit high molar absorptivities (log ε higher than 4.3), with higher values for the short-wave
than for the long-wave region, also typical for chlorin dyes [30].
Table 1.Photophysical characterization of compounds 12 and 15 in water.
a
b
Compound λabs(nm) (log ε)
λ (nm) Φ (%)
em F
4
6
12 (4.80)
32 (4.39)
1
1
2
5
638
7.3
4
6
08 (4.96)
28 (4.44)
b
636
9.5
a
Exited at 410 nm. Relative to Rhodamine B in water.

ACCEPTED MANUSCRIPT
Fig. 3. Absorption and fluorescence spectra of 12 and 15 (both at 5 µM) in water.
Fluorescence was excited at 410 nm.
Both compounds manifested fluorescence in water with the maxima in red region (~640 nm)
with the relative quantum yields (Rhodamine 6G) of 7.3% and 9.5% for 12 and 15,
respectively. Conjugate 12 demonstrated lower fluorescence than free photosensitizer 15,
what can be attributed to the partial quenching by the arylquinazoline part of the molecule.
Biology
Cellular uptake
The ability of the synthesized compounds 12 and 15 to penetrate and accumulate in living
cells was studied on cell lines with various expression levels of epidermal growth factor
receptor (EGFR): EGFR-negative CHO (chinese hamster ovary cells) [31], HeLa (human
cervical carcinoma) [32] and A431 (human epidermoid carcinoma) [33]. All the chosen cell
lines have minimal or negligible expression of VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1)
[
34].
To quantify the relative level of EGFR expression, the cells were stained by anti-EGFR
antibodies labeled with phycoerythrin (PE) and analyzed using flow cytometry (Figure 4).

ACCEPTED MANUSCRIPT
HeLa cells demonstrated low level of EGFR-expression, with moderate staining by anti-
EGFR antibodies (ca. 7-fold increase in the fluorescence signal to CHO-cells). On the
contrary, A431 cells reveal high level of EGFR expression (fluorescence signal increased by
two orders of magnitude after the labeling).
Fig. 4. Flow cytometry analysis of EGFR expression by cells of CHO (a), HeLa (b) and A431
(c) lines. Cells were stained with PE-labeled anti-EGFR antibodies (red) or isotype control
(black) and their fluorescence (λex 488 nm, λem 564-606) was measured by FACS. The
distribution of 30 000 cells depending on PE fluorescence intensity is shown. (d) Relative
level of EGFR expression was calculated from flow cytometry data as the ratio of geometric
mean fluorescence intensities in EGFR-labeled and isotype control cells.
When conjugate 12 was incubated with CHO, HeLa and A431 cells during 4 hours, an
increased accumulation of this compound in A431 cells was observed (Figure 5). The cellular
uptake of 12 in CHO and HeLa cells was significantly lower. Thus, the conjugate 12,
containing arylaminoquinazoline vector to EGFR, preferentially accumulates in the cells with
high expression of such receptors. We presume, however, that both non-specific and EGFR-

ACCEPTED MANUSCRIPT
facilitated mechanisms contribute comparably to the conjugate 12 internalization in HeLa
cells. Interestingly, compound 15 accumulates in the cells of all three lines (CHO, HeLa and
A431) at a significantly lower level.
Fig. 5. (a) Confocal images on CHO, HeLa and A431 cells after incubation with 5 µM for 4h,
conjugate 12 (bottom line), compound 15 (top line). λ 405 nm, λ 600–740 nm. Images size
ex
em
1
43 µm × 143 µm. (b) Comparison of the corresponding intracellular fluorescence intensity
for 12 and 15. At least ten cells in two-three fields of view were analyzed; mean ± SEM are
presented.
Dark and photoinduced cytotoxicity
A standard MTT-assay was used to determine the concentration of 12 and 15 required to
inhibit cell viability by 50 % after the incubation in the culture medium for 24 h. Along with
the dark cytotoxicity, we determined the light cytotoxicity of these compounds under
2
2
irradiation with a dose of 20 J/cm (λ = 615-635 nm, power 20 mW/cm ) (Figure 6). The IC
5
0
values are summarized in Table 2. Despite the fact that A431 cells accumulate 12 in higher
amounts than HeLa or CHO, their dark sensitivity to this compound seems to be lower. The
differences in the cell lines sensitivity to the toxic agents are associated with their different
metabolic activity and genes expression, and especially mutations in oncogenes that are
specific for certain cancer cell lines. Also, while the accumulation of 15 is 5-10 folds lower in
the investigated cell lines, its IC50dark values are 1.5-3 folds higher. Thus, this compound is
several times more toxic for cells than 12.

ACCEPTED MANUSCRIPT
Conjugate 12 demonstrates photoinduced cytotoxicity in low-micromolar concentrations (2.0
–
3.0 µM), while its dark toxicity was 11-18 times lower. We observed slightly higher
photoactivity of 12 against A431 (about two-fold increase in IC50dark/IC50light). Compound 15,
lacking the molecular fragment responsible for the EGFR affinity was found to be less active,
both in the dark and light conditions, with similar ratio between IC50dark and IC50light.
Fig. 6. Relative viability of CHO, HeLa and A431 cells after treatment with 12 (a) or 15 (b).
Cells were incubated with the compound for 4 h, then the medium was exchanged with full
2
fresh growth medium, and the cells were irradiated in dose 20 J/cm (λ = 615-635 nm, power
2
4
0 mW/cm ) or stayed in dark. After the additional incubation for 24 h, cell viability was
measured by MTT-assay and expressed as the percentage to untreated cells. Mean ± SEM are
presented; the experimental data are fitted using four parameters model for lognormal
distribution.
Table 2. In vitro light and dark cytotoxic activity of 12 and 15.
1
2
15
Cells
IC , µM
IC , µM
50
5
0
IC50dark/IC
IC50dark/IC50light
light
50
[
95% confidence
[95% confidence

ACCEPTED MANUSCRIPT
intervals]
light dark
.7 32
2.4;3.0] [28;36]
.2 25
2.0;2.3] [21;29]
.6 48
2.5;2.7] [43;55]
intervals]
light
dark
2
6.1
[5.8;6.4]
6.5
CHO
HeLa
A431
12
11
18
>100
72
-
[
[
[
2
11
13
[5.7;7.3] [64;80]
5.5 72
[4.9;6.2] [63;81]
2
Ratio of IC50dark/IC50light for compound 12 in A431 cells is the highest of all the combinations
tested. It correlates well with the increased uptake of 12 by A431 cells, expressing more
EGFR than the other cell types (Fig. 5). We assume that the conjugate photoactivity is
resulted from the same photochemical processes in all the cells, and its primary intracellular
targets are also the same. In this case, the enhancement of A431 response to compound 12 can
be a consequence of EGFR blockade.
Although IC50dark / IC50light is only 1.5-1.7 fold higher for this combination than for the others
(excluding compound 15/CHO cells, where the cells seem to be resistant to the dark toxicity
of 15), this demonstrate the efficiency of the agents for combined photodynamic treatment
and the inhibition of growth factors receptors. We attribute the moderate increase to the
comparably high non-specific binding of both compounds in the cells.
Animal study
After intravenous administration into the tumor-bearing Balb/c mice, compound 12 quickly
disappears from the normal tissues, but shows relatively prolonged retention in the tumor
(Figure 7). Its tumor-to-tissue ratio reaches 4.2 after 24 h. The behavior of compound 12 is
similar to well-studied chlorin photosensitizer mTHPC, characterized by the tumor-to-skin
ratio of ca. 4 [35], and a rather long period between the administration and the optimal PDT
treatment time [36].

ACCEPTED MANUSCRIPT
Fig. 7. Biodistribution of conjugate 12 in vivo.
a) Dynamics of fluorescence intensity in tumor (red) and normal tissue (black) of the animals
injected with conjugate 12. Mean ± SEM are presented (n=5). λ = 615 nm, λ =660–740 nm.
(
ex
em
(b) Fluorescent image of a mouse, injected with 12 (24 h post-injection). Tumor is indicated
by arrow. (c) Tumor-to-normal tissue fluorescence ratio at different time points, indicating
selective accumulation of 12 in tumor.
These preliminary results demonstrate the ability of 12 to selectively accumulate in tumor
cells and support the further development of this type of compounds as possible leads for
tumor treatment.
3
. Conclusions
In conclusion, we have synthesized a new water-soluble conjugate consisting of a chlorin-
based photosensitizer and an EGFR/VEGFR ligand. The newly prepared hybrid molecules are
of interest as a new class of theranostic agents. An increased accumulation of this compound
in A431 cells with high EGFR expression was observed, in comparison with the uptake by the
CHO and HeLa cells with low EGFR expression. The prepared conjugate exhibits dark and
photoinduced cytotoxicity at low micromolar concentrations with pronounced IC50dark/IC50light
ratio of 11-18. After intravenous administration into tumor-bearing mice, the conjugate
preferentially accumulates in the tumor tissue as it was detected by fluorescence

ACCEPTED MANUSCRIPT
measurements. The replacement of EGFR/VEGFR-binding part by another high affinity
receptor ligand might be a simple way to change the selectivity of the conjugate action.
4
. Experimental
4
.1. Materials.
1
13
H NMR and C NMR spectra were recorded on Agilent DD2 400 MHz spectrometer.
Chemical shifts (δ) are reported in ppm for the solution of compound in CDCl , DMSO-d or
3
6
CD OD, with the residual peak of solvent as an internal reference, J values in Hertz. Mass
3
spectra were recorded using the MALDI method on a time-of-flight Bruker Microflex LT
mass-spectrometer. TLC analyses were carried out on Merck TLC Silica gel 60 F . Column
2
54
chromatography separation was performed using Macherey-Nagel Kieselgel 60 (70-230
mesh). Commercially available reagents (Aldrich, Alfa Aesar) were used without additional
purification. Solvents were purified according to the standard procedures. Petroleum ether
used was of bp 40-70 °C.
4
.2. Preparation of compounds.
Synthesis of 4-(amino-4'-bromo-2'-fluorophenyl)-6-methoxy-7-(3"-azidopropyloxy)-
quinazoline 2 [C H BrFN O ]. An argon-filled Schlenk flask was charged with quinazolinol
18
16
6
2
1
(0.250 g, 0.68 mmol), 1-bromo-3-chloropropane (0.125 g, 0.81 mmol) and K CO (0.283 g,
2 3
2
.05 mmol). Anhydrous DMF (6 mL) was added, and the mixture was stirred for 24 h at 60
°
С, cooled and concentrated under reduced pressure. The residue was dissolved in CHCl3
(
100 mL), washed with H O (3 × 50 mL), dried (Na SO ), and concentrated in a Schlenk
2
2
4
flask. NaN (0.165 g, 2.54 mmol), and anhydrous DMF (3 mL) were added, and the mixture
3
was stirred for another 24 h at 50 °С. The solvent was removed under reduced pressure and
the residue was dissolved in CHCl (100 mL), washed with H O (3 × 50 mL), dried (Na SO ),
3
2
2
4
and concentrated. The product was purified by column chromatography (EtOAc/petroleum

ACCEPTED MANUSCRIPT
1
ether 90:10) to obtain a white solid. Yield 0.215 g (70%); mp 142 °C. H NMR: (400 MHz,
DMSO-d ) δ 9.54(s, 1H, NH), 8.36 (s, 1H, CH), 7.80 (s, 1H, CH), 7.66 (d, J = 10 Hz, 1H,
6
CH), 7.58-7.43 (m, 2H, 2CH), 7.21 (s, 1H, CH), 4.22 (t, J = 6.1 Hz, 2H, CH ), 3.95 (s, 3H,
2
1
3
CH O), 3.55 (t, J = 6.7 Hz, 2H, CH ), 2.12-2.00 (m, 2H, CH ). C NMR: (101 MHz, DMSO-
3
2
2
d6) δ 157.90, 156.88, 155.40, 153.44, 152.93, 149.01, 146.88, 129.55, 127.47, 126.44,
1
2
26.32, 119.43, 119.20, 117.99, 117.90, 108.72, 107.82, 102.01, 65.55, 56.15, 47.64,
+
79
+ 81
7.91.MS (MALDI): m/z 447.1 [M+H] ( Br), 449.1 [M+H] ( Br).
Synthesis of pheophorbide-a 4 [C H N O ]. Conc. HCl (1 mL) was added dropwise to the
3
5
36
4
5
solution of methylpheophorbide-a (0.300 g, 0.49 mmol) in acetone (15 mL). The mixture was
stirred for 48 h at rt, diluted with CHCl (200 mL), washed with H O (3 × 100 mL), dried
3
2
(Na SO ), and concentrated. The product was purified by column chromatography
2 4
(
CHCl /MeOH 95:5) to obtain a deep-green solid. Yield 0.272 g (93%). The physical and
3
spectroscopic data obtained for the compound were in agreement with the published data [37].
Synthesis of chlorin 6 [C H N O ]. A Schlenk flask was filled with argon and charged with
3
8
39
5
4
EDC·HCl (0.138 g, 0.72 mmol) and pheophorbide-a (4, 0.213 g, 0.36 mmol). Anhydrous
CHCl (4 mL) was added, and the mixture was stirred for 30 min at 0 °С. Another flask was
3
charged with propargyl amine (5, 0.04 g, 0.73 mmol), DMAP (0.022 g, 0.18 mmol) and
anhydrous CHCl (4 mL). The mixture from the second flask was transferred by a syringe into
3
the first flask. The reaction mixture was stirred at 0 °С for 1 h, at rt for 18 h, and concentrated
under reduced pressure. The residue was dissolved in CHCl (100 mL), washed with H O (3 ×
3
2
1
00 mL), dried (Na SO ), and concentrated. The product was purified by column
2 4
chromatography (CHCl /MeOH 98:2) to obtain a deep-green solid. Yield 0.188 g (81%); mp
3
1
1
39 °C. H NMR: (400 MHz, CDCl ) δ 9.49 (br.s, 2H), 8.63 (s, 1H), 7.98 (dd, J = 17.5, 11.5
3
Hz, 1H), 6.30 (d, J = 18.2 Hz, 1H), 6.27 (s, 1H), 6.20 (d, J = 19.9 Hz), 5.36 (br.s, 1H), 4.53
(br.s, 1H), 4.27 (br.s, 1H), 3.87 (s, 3H), 3.78-3.55 (m, 7H), 3.41 (s, 3H), 3.25 (s, 3H), 2.70
(br.s, 1H), 2.46 (br.s, 1H), 2.33-2.15 (m, 2H), 1.94 (s, 1H), 1.83 (d, J = 6.6 Hz, 3H), 1.68 (t, J

ACCEPTED MANUSCRIPT
1
3
=
7.6 Hz), -1.66 (br.s, 2H). C NMR: (101 MHz, CDCl ) δ 189.36, 172.27, 171.76, 169.56,
3
1
1
37.93, 128.86, 104.64, 104.26, 97.59, 79.16, 64.77, 52.97, 51.52, 50.25, 29.69, 28.83, 23.31,
+
9.55, 17.19, 12.16, 11.26. MS (MALDI): m/z 630.1 [M] .
Synthesis of chlorin 8 [C H N O Zn]. Amine 7 (0.640 g, 7.34 mmol) was added to a
42
49
7
4
solution of chlorin 6 (0.180 g, 0.29 mmol) in CHCl (7 mL), and the mixture was stirred at rt,
3
until 6 was completely consumed (TLC). Solution of Zn(OAc) (0.360 g, 1.97 mmol) in
2
MeOH (3 mL) was added, the mixture was stirred for 2 h, then diluted with CHCl (100 mL)
3
and washed with H O (3 × 50 mL). The organic phase was dried (Na SO ) and concentrated
2
2
4
under reduced pressure. The product was purified by column chromatography (CHCl /MeOH
3
1
9
5:5 to 85:15) to obtain a deep-green solid. Yield 0.205 g (92%); mp 145 °C. H NMR: (400
MHz, DMSO-d ) δ 9.51 (s, 2H), 8.96 (br.s, 1H), 8.66 (s, 1H), 8.34 (t, J = 5.4 Hz, 1H), 8.22
6
(dd, J = 17.8, 11.6 Hz, 1H), 6.22 (d, J = 17.9 Hz, 1H), 6.00 (d, J = 11.6 Hz, 1H), 5.41 (d, J =
1
3
2
3
1
1
4
9.1 Hz, 1H), 5.07 (d, J = 18.9 Hz, 1H), 4.42 (q, J = 7.0 Hz, 1H), 4.20 (d, J = 9.9 Hz, 1H),
.91- 3.75 ( m, 6H), 3.69 (br.s, 4H), 3.37 (s, 3H), 3.35 (s, 3H), 3.26-3.16 (m, 3H), 3.05 (t, J =
.3 Hz, 1H), 2.73 (br.s, 6H), 2.47-2.38 (m, 1H), 2.07 (d, J = 8.8 Hz, 2H), 1.67 (t, J = 7.5 Hz,
13
H), 1.61 (d, J = 7.0 Hz). C NMR: (101 MHz, DMSO-d ) δ 173.24, 171.72, 170.35, 165.14,
6
62.77, 151.66, 147.96, 146.14, 144.07, 143.10, 141.20, 140.74, 138.58, 137.23, 133.04,
32.29, 130.66, 119.20, 101.79, 101.75, 99.87, 93.16, 81.19, 72.81, 52.24, 51.65, 46.26,
3.88, 37.46, 32.02, 29.99, 27.78, 22.80, 18.87, 17.89, 12.30, 11.76, 10.93. MS (MALDI):
m/z 779.0 [M]+.
Synthesis of conjugate 9 [C H BrFN O Zn]. Chlorin 8 (0.060 g, 0.08 mmol) and
6
0
65
13
6
compound 2 (0.045, 0.09 mmol) were placed in a flask with mixture of t-BuOH/CHCl /H O
3
2
(
2:1:2) (9 mL). In another flask, CuSO ·5H O (0.005 g, 0.02 mmol), TBTA (0.008 g, 0.01
4 2
mmol) and sodium ascorbate (0.006 g, 0.03 mmol) were dissolved in 6 mL H O, and this
2
solution was immediately added into the first flask. The mixture was stirred for 1.5 h at 50 °С,
cooled, diluted with CHCl (100 mL), washed with H O (3 × 50 mL), dried (Na SO ), and
3
2
2
4

ACCEPTED MANUSCRIPT
concentrated. The product was purified by column chromatography (CHCl /MeOH 95:5 to
3
1
8
0:20) to obtain a deep-green solid. Yield 0.068 g (72%); mp 175 °C. H NMR: (400 MHz,
DMSO-d ) δ 10.81 (s, 1H), 9.50 (s, 1H), 9,47 (s, 1H), 9.16 (br.s, 1H), 8.57 (s, 1H), 8.51 (br.s,
6
1H), 8.20 (dd, J = 17.8, 11.7 Hz, 1H), 8.04 (s, 1H), 7.80 (br.s, 1H), 7.61 (d, J = 9.9 Hz, 7.55-
7.26 (m, 3H), 6.20 (d, J = 18.2 Hz, 1H), 5.99 (d, J = 11.8 Hz, 1H), 5.23 (br.s, 2H), 4.53 (br.s,
2H), 4.44-4.33 (m, 2H), 4.26 (d, J = 5.1 Hz, 1H), 4.18-4.09 (m, 1H), 4.07-4.01 (m, 1H), 4.00-
3.88 (m, 3H), 3.86 (s, 3H), 3.83-3.73 (m, 2H), 3.67 (s, 3H), 3.56- 3.48 (m, 2H), 3.25-3.16 (m,
4H), 2.98 (s, 6H), 2.44-2.26 (m, 4H), 2.07 (d, J = 8.2 Hz, 2H), 1.65 (t, J = 7.4 Hz, 3H), 1.45
1
3
(
d, J = 6.0 Hz, 3H). C NMR: (101 MHz, DMSO-d ) δ 173.52, 171.94, 170.28, 165.09,
6
1
1
1
1
4
62.46, 157.82, 156.65, 155.32, 153.18, 152.15, 151.63, 148.70, 148.03, 146.21, 145.13,
44.15, 143.13, 141.18, 140.84, 139.00, 137.20, 133.37, 133.03, 132.39, 130.59, 129.59,
27.46, 125.97, 125.85, 122.73, 119.39, 119.22, 119.16, 117.89, 117.81, 113.79, 108.03,
02.31, 101.99, 101.96, 99.95, 93.19, 65.21, 57.70, 56.14, 52.80, 52.17, 51.62, 46.27, 46.10,
3.90, 37.19, 34.06, 32.08, 29.56, 22.71, 18.86, 17.85, 12.28, 11.82, 10.92. MS (MALDI):
+
m/z 1226.6 [M+H] .
Synthesis of conjugate 11 [C H BrFN O Zn]. Bis(dimethylamino)methane (10, 0.524 g,
6
6
79
15
6
5
.14 mmol) was added to a solution of conjugate 9 (0.070 g, 0.06 mmol) in AcOH/THF (1:1,
8
mL), and the mixture was stirred for 1 h at rt. It was diluted with CHCl (100 mL), washed
3
with 2% NaOH (3 × 100 mL) and H O (1 × 100 mL), dried (Na SO ), and concentrated. The
2
2
4
product was purified by column chromatography (CHCl /MeOH-Et N 97:2:1 to 95:5:1) to
3
3
obtain a deep-green solid. Yield 0.048 g (63%); mp 174 °C. 1H NMR: (400 MHz, DMSO-d6)
δ 9.61 (s, 1H), 9.46 (s, 1H), 9.43 (br.s, 1H), 8.73 (s, 1H), 8.57 (br.s, 1H), 8.51 (s, 1H), 8.10 (s,
1H), 7.61-7.50 (m, 3H), 7.43-7.24 (m, 2H), 7.23-7.10 (m, 1H), 5.26 (br.s, 2H), 4.59-4.51 (m,
2H), 4.51-4.41 (m, 2H), 4.34 (d, J = 6.8 Hz, 1H), 4.27-4.19 (m, 1H), 4.16-4.11 (m, 2H), 4.05-
3.89 (m, 3H), 3.82 (br.s, 8H), 3.64 (br.s, 5H), 3.61-3.49 (m, 3H), 3.26-3.13 (m, 4H), 2.71-2.66
(m, 3H), 2.35 (br.s, 10H), 2.22-2.13 (m, 10H), 2.11-1.99 (m, 3H), 1.66 (t, J = 7.4 Hz, 3H),

ACCEPTED MANUSCRIPT
1
3
1
1
1
1
1
3
.39 (d, J = 6.8 Hz, 3H). C NMR: (101 MHz, CD OD) δ 179.04, 176.43, 175.30, 174.12,
3
66.97, 163.45, 159.21, 158.25, 156.70, 155.14, 153.61, 152.90, 150.88, 149.13, 148.65,
46.57, 145.76, 145.58, 145.30, 142.98, 142.78, 140.96, 139.51, 136.20, 134.63, 133.32,
30.09, 128.29, 126.38, 124.64, 120.41, 120.17, 119.65, 109.34, 103.96, 103.01, 102.16,
02.02, 94.19, 66.10, 58.99, 57.43, 56.70, 53.87, 52.69, 48.16, 47.67, 45.35, 44.24, 39.18,
8.71, 35.35, 33.49, 30.68, 23.43, 23.32, 20.29, 18.15, 14.20, 13.20, 12.13, 11.37, 11.21. MS
+
(
MALDI): m/z 1343.9 [M+H] .
Synthesis of conjugate 12 [(C H BrFN O Zn)I ]. CH I (0.228 g, 1.60 mmol) was added to
6
9
88
15
6
3
3
a solution of conjugate 11 (0.025 g, 0.02 mmol) in THF (5 mL), and the mixture was stirred at
rt until a precipitate formed. It was collected on a filter, washed with a small amount of THF
and dried under reduced pressure to obtain a deep-green solid. Yield 0.032 g (quantitative);
1
mp 168 °C. H NMR: (400 MHz, DMSO-d ) δ 9.59 (d, J = 10.6 Hz, 1H), 9.50 (br.s, 2H), 9.19
6
(
s, 1H), 8.57 (br.s, 2H), 8.07 (s, 1H), 7.95 (s, 1H), 7.68-7.51 (m, 2H), 7.45-7.32 (m, 2H), 7.23-
.05 (m, 1H), 5.23 (br.s, 2H), 4.55 (br.s, 2H), 4.48-4.34 (m, 2H), 4.32-4.19 (m, 2H), 4.12
br.s, 2H), 4.04-3.89 (m, 3H), 3.86-3.78 (m, 11H), 3.69 (s, 5H), 3.10 (br.s, 12H), 2.89 (s, 9H),
.73 (s, 9H), 2.45-2.29 (m, 3H), 2.18 (d, J = 6.5 Hz, 3H), 2.13-1.94 (m, 3H), 1.65 (t, J = 6.8
7
(
2
13
Hz, 3H), 1.43 (d, J = 5.8 Hz, 3H). C NMR: (101 MHz, DMSO-d ) δ 173.42, 171.93, 169.85,
6
1
1
9
3
62.71, 157.86, 156.92, 156.70, 155.36, 153.21, 148.76, 147.87, 146.10, 145.09, 143.33,
41.15, 140.98, 133.18, 132.82, 129.59, 127.50, 122.78, 119.41, 119.19, 117.85, 102.06,
9.55, 93.27, 65.24, 63.65, 56.16, 54.91, 52.78, 52.38, 51.77, 46.28, 37.30, 34.04, 32.10,
1.28, 30.39, 29.51, 29.00, 27.88, 22.70, 21.05, 18.85, 17.88, 13.96, 11.91, 11.06.
Synthesis of chlorin 13 [C H N O Zn]. Compound 7 (0.807 g, 9.17 mmol) was added to a
40
48
6
5
solution of methylpheophorbide-a (3, 0.200 g, 0.33 mmol) in CHCl (8 mL), and the mixture
3
was stirred at rt until 3 was completely consumed (TLC). Solution of Zn(OAc) (0.300 g, 1.64
2
mmol) in MeOH (3 mL) was added, the mixture was stirred for 2 h, diluted with CHCl (100
3
mL) and washed with H O (3 × 50 mL). The organic phase was dried (Na SO ) and
2
2
4

ACCEPTED MANUSCRIPT
concentrated under reduced pressure. The product was purified by column chromatography
(
CHCl /MeOH 98:2 to 90:10) to obtain a deep-green solid. Yield: 0.180 g (72%); mp 138 °C.
3
1
H NMR: (400 MHz, DMSO-d ) δ 9.53 (s, 1H), 9.51 (s, 1H), 8.71 (t, J = 5.3 Hz, 1H), 8.66 (s,
6
1
H), 8.23 (dd, J = 17.8, 11.6 Hz, 1H), 6.22 (d, J = 17.9 Hz, 1H), 6.01 (d, J = 11.6 Hz, 1H),
.43 (d, J = 19.1 Hz, 1H), 5.13 (d, J = 19.1 Hz, 1H), 4.43 (q, J = 6.9 Hz, 1H), 4.28 (d, J = 9.2
5
Hz, 1H), 3.82 (q, J = 7.5 Hz, 2H), 3.67 (s, 3H), 3.54 (s, 3H), 3.37 (s, 3H), 3.27-3.16 (m, 1H),
2
7
1
1
5
1
.67 (t, J = 6.9 Hz, 2H), 2.61-2.54 (m, 1H), 2.33 (s, 6H), 2.12 (d, J = 10.1 Hz, 2H), 1.68 (t, J =
1
3
.5 Hz, 3H), 1.60 (d, J = 7.0 Hz, 3H). C NMR: (101 MHz, DMSO-d ) δ 173.34, 173.28,
6
69.91, 164.97, 162.11, 151.62, 148.15, 146.06, 144.00, 143.15, 141.40, 140.73, 138.56,
37.23, 134.06, 133.05, 132.24, 130.69, 119.20, 101.99, 101.75, 99.95, 93.04, 58.11, 51.98,
1.59, 51.26, 46.11, 45.36, 37.68, 37.35, 30.01, 29.19, 22.84, 18.90, 17.92, 12.31, 11.68,
+
0.95. MS (MALDI): m/z 755.9 [M] .
Synthesis of chlorin 14 [C H N O Zn]. Bis(dimethylamino)methane (10, 0.749 g, 7.34
4
6
62
8
5
mmol) was added to a solution of chlorin 13 (0.150 g, 0.20 mmol) in mixture of AcOH/THF
1:1) (10 mL), and the mixture was stirred for 1 h at rt. It was diluted with CHCl (100 mL),
(
3
washed with 2% NaOH (3 × 100 mL) and H O (100 mL), dried (Na SO ), and concentrated.
2
2
4
The product was purified by column chromatography (CHCl /MeOH/Et N from 98:1:1 to
3
3
1
9
6:3:1) to obtain a deep-green solid. Yield 0.132 g (76%); mp 155 °C. H NMR: (400 MHz,
DMSO-d ) δ 9.63 (s, 1H), 9.52 (s, 1H), 8.71 (br.s, 1H), 8.62 (s, 1H), 7.21-7.12 (m, 1H), 5.44
6
(d, J = 18.6 Hz, 1H), 5.13 (d, J = 19.1 Hz, 1H), 4.43 (d, J = 7.0 Hz, 1H), 4.28 (d, J = 9.3 Hz,
1
H), 3.82 (d, J = 7.3 Hz, 2H), 3.67 (br.s, 5H), 3.55 (br.s, 5H), 2.76-2.70 (m, 1H), 2.66 (t, J =
.9 Hz, 2H), 2.62-2.53 (m, 1H), 2.32 (br.s, 9H), 2.16 (br.s, 11H), 1.68 (t, J = 7.3 Hz, 3H), 1.59
6
1
3
(
br.s, 3H). C NMR: (101 MHz, DMSO-d ) δ 173.33, 173.27, 170.02, 164.97, 162.09,
6
1
1
51.14, 148.12, 145.70, 143.17, 142.88, 142.82, 141.37, 140.71, 138.59, 138.49, 134.10,
33.55, 133.39, 132.81, 102.01, 101.76, 100.50, 92.97, 69.55, 58.14, 55.50, 51.98, 51.59,

ACCEPTED MANUSCRIPT
5
1.27, 46.11, 45.40, 45.17, 37.73, 37.34, 30.09, 29.16, 22.82, 18.90, 17.91, 11.67, 11.29,
+
1
0.89. MS (MALDI): m/z 872.9 [M] .
Synthesis of chlorin 15 [(C H N O Zn)I ]. CH I (0.228 g, 1.60 mmol) was added to a
4
9
71
8
5
3
3
solution of chlorin 14 (0.03 g, 0.03 mmol) in THF (5 mL), and the mixture was stirred at rt
until a precipitate formed. It was collected on a filter, washed with a small amount of THF
and dried under reduced pressure to give a deep-green solid. Yield 0.032 g (quantitative); mp
1
1
1
4
3
51 °C. H NMR: (400 MHz, DMSO-d ) δ 9.62 (d, J = 15.2 Hz, 1H), 9.56 (s, 1H), 9.19 (br.s,
6
H), 8.66 (s, 1H), 7.23-7.10 (m, 1H), 5.37 (d, J = 19.0 Hz, 1H), 5.13 (d, J = 19.1 Hz, 1H),
.45 (d, J = 6.9 Hz, 1H), 4.32 (d, J = 8.2 Hz, 1H), 4.03-3.77 (m, 6H), 3.72 (s, 3H), 3.54 (s,
H), 3.16 (br.s, 9H), 2.86 (br.s, 1H), 2.65-2.55 (m, 1H), 2.22 (d, J = 14.7 Hz, 2H), 2.17-2.08
1
3
(
m, 2H), 1.71-1.65 (m, 3H), 1.60 (br.s, 3H). C NMR: (101 MHz, DMSO-d ) δ 173.30,
6
1
1
6
1
4
73.23, 170.71, 165.32, 162.21, 151.07, 147.77, 146.11, 143.33, 142.61, 142.47, 141.14,
41.02, 138.87, 137.15, 134.05, 133.24, 132.82, 102.08, 101.89, 97.17, 93.18, 75.19, 68.76,
8.28, 65.71, 63.64, 62.72, 52.80, 52.42, 51.84, 51.30, 37.53, 34.00, 29.94, 29.14, 22.76,
8.86, 17.92, 11.88, 11.45, 11.07.
.3. Photophysical measurements
Photophysical properties of 12 and 15 were measured in deionized water at 5 µM solutions.
Absorption and fluorescence spectra were registered using a Synergy MX spectrophotometer-
spectrofluorometer (BioTek, USA). Fluorescence was excited at 410 nm.
The molar extinction coefficient ε was determined using the following equation:
ꢁ
ꢀ
= ꢄ
ꢂꢃ
,
where D – optical density; l – path length; c – concentration.
The fluorescence quantum yield φ was calculated using the equation:
1
ꢇ ꢉ ꢋ
ꢊ ꢈ
ꢈ
ꢅ =
ꢆ
,
ꢉ ꢋ
ꢈ
ꢊ
where F and D – integral fluorescence intensity and optical density of 12 (or 15),
1
1
respectively;

ACCEPTED MANUSCRIPT
φ – quantum yield of Rhodamine B (Sigma, USA) in water (0.31); F and D – integral
2
2
2
fluorescence intensity and optical density of rhodamine B, respectively.
The fluorescence was excited at 410 nm, the optical density was measured at the same
wavelength. The fluorescence signal was detected at 550–850 nm.
4
.4 Cell lines and culturing conditions
Cell lines of human epidermoid carcinoma A431, human cervival carcinoma HeLa, chinese
hamster ovary CHO were obtained from the Russian cell culture collection of vertebrates. The
cells were cultured in Eagle MEM medium (PanEco, Russia) with 10% (v/v) fetal calf serum
(
HyClone, USA) and 2 mM L-glutamine in 5% CO at 37 °C. At each passaging stage the
2
cells were treated with Versene solution (PanEco, Russia).
Cells of mouse colon adenocarcinoma CT-26 (ATCCnumber CRL-2638) were cultured in
RPMI medium (HyClone, USA) with the same supplements and treated with 0.25% tripsin-
EDTA solution (PanEco, Russia) at passaging stages.
4
.5. EGFR analysis
6
Cells (0.3×10 ) were detached from culturing flask and resuspended in 500 µl 3% (w/v)
solution of bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Anti-EGFR
antibodies conjugated with phycoerythrin (Abcam, UK) were added (0.1 µg/m, 3 µl), the cells
were incubated for 30 min under rotation at 15 rpm, and then washed twice with 1% BSA in
PBS.
Fluorescence of the stained cells was immediately analyzed using FACSCalibur flow
cytometer (BD Biosciences, USA) using 488 nm laser and registration of phycoerythrin (PE)
emission at 585/42 nm. 30 000 cells were analyzed for every cell line.
To assess nonspecific antibodies adherence on cell surface, the cells were stained by
antibodies of the same isotype conjugated with phycoerythrin (Abcam, UK) having no
specific target on cell surfaces and undergo the same flow cytometry procedure (isotypic
control).

ACCEPTED MANUSCRIPT
Ratio of geometric mean fluorescence intensities in EGFR-labeled and isotype control cells
was calculated to express the relative level of EGFR expression.
4
.6. Study of cellular uptake
3
Cells were seeded in glass bottom 96-well plates (Falcon, Germany) at the density of 4×10
cells per well and allowed to attach overnight. The medium was then exchanged with fresh
serum-free growth medium containing 5 µM of tested compound. Cells were incubated for 4
hours, washed thrice with PBS, fixed with 4% formaldehyde for 30 min and washed again.
Cells were imaged using laser scanning confocal microscope Axio Observer Z1 LSM 710
NLO/Duo (Carl Zeiss, Germany) equipped with C-Apochromat 63×water immersion
objective lens with numerical aperture 1.2. Fluorescence was excited at 405 nm and registered
in the range of 600-740 nm.
The fluorescence intensity of the cytoplasmic region of the cells was measured using ZEN
2
012 program; at least 10 cells in two-three fields of view were analyzed.
4
.7. Cytotoxicity study
The effect of tested compounds on cell viability was estimated using the microculture
tetrazoline test (MTT) [38].
3
Cells were seeded in 96-well plates at the density of 4×10 cells per well and allowed to
attach overnight. The medium was then exchanged with fresh serum-free growth medium
containing tested compound in different concentration. After 4 h of incubation, the medium
was exchanged with full fresh growth medium.
To estimate the photoinduced toxicity of the tested compound, the cells were exposed to light
2
2
irradiation (615–635 nm, 20 mW/cm , 20 J/cm ) using LED light source providing a
homogeneous light distribution in 96-well plates [39]. Irradiated cells were then incubated for
2
4 h before cell viability was measured. To this aim, the cells were incubated with serum-free
medium containing 0.5 mg/mL MTT-reagent (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazole bromide, Alfa Aesar, USA) for 4 hours. The formazan formed from the reduction of

ACCEPTED MANUSCRIPT
MTT by cells’ dehydrogenases was dissolved in DMSO, and the absorbance was measured at
5
70 nm with a Synergy MX plate reader (BioTeck, USA).
A similar procedure was used for the estimation of the dark toxicity. The cells, grown
overnight, were incubated in the medium with the compound being tested, washed with full
fresh growth medium, then incubated for 24 h and undergo MTT assay.
Cell viability was expressed as the ratio of the optical density of treated and untreated cells (in
percentage). Three independent experiments (all in triplicate) were performed. Experimental
data were fitted using four parameters model for lognormal distribution to obtain dose-effect
relationship and calculate inhibition concentration IC :
5
0
ꢏꢐꢑꢒꢓꢏꢐꢔꢕ
Y = Yꢌꢍꢎ
+
,
ꢆꢖꢆꢗ(ꢘꢙ ꢚ( ꢛꢜꢝꢞ)ꢓꢟ)∙ꢠꢡ
where Ymax and Ymin – maximal and minimal Y values, respectively; SF –slope factor.
Data analysis was performed using the GraphPad Prism 6 software.
4
.8. Animal study
Balb/c female mice (age 8 weeks; 17 g, n=5) were inoculated subcutaneously in the left leg
6
with 10 CT-26 cells in 0.2 mL PBS. Nine days after the inoculation, when tumors reached
3
~
100 mm , the mice were injected with conjugate 12 solved in PBS, in dose 25 µg/kg
intravenously into the tail vein.
Whole-body in vivo fluorescence imaging was performed using a fluorescence imaging
system (Institute of Applied Physics RAS, Russia). Fluorescence was excited using narrow-
band LED with central wavelength 615 nm and collected with cooled CCD-camera (ORCA II
BT-512G, Hamamatsu Photonics K.K., Japan) in the range 660-740 nm. Images were
obtained before and at different time points post-injection.
All experimental procedures were approved by the local Committee on Bioethics (protocol
No. 7 of 06 July 2017).
The mean signal was calculated using ImageJ software in two regions of interest (ROI): tumor
area and a region of the same area and position on the other leg (‘normal tissue’).

ACCEPTED MANUSCRIPT
ACKNOWLEDGEMENTS
We thank the Russian Foundation of Basic Research (grant number 16-03-00464). O.I.
Koifman thank the Ministry of Education and Science of the Russian Federation (government
assignment number 4 1929.2017-4.6)
Supplementary data
The following is the supplementary data related to this article:
1
13
H, C NMR, IR and MS spectra.
Corresponding Authors
*
*
(I.V.B.) Tel/Fax: +7 831-462-32-13. E-mail: irin-b@mail.ru
(A.Yu.F.) Fax: +7 831-462-30-85. E-mail: afnn@rambler.ru
Notes
The authors declare no competing financial interest.
REFERENCES
[
1] (a) D. Van Straten, V. Mashayekhi, H.S. de Bruijn, S. Oliveira, D.J. Robinson, Oncologic
photodynamic therapy: basic principles, current clinical status and future direction, Cancers, 9
2017) 19; (b) E.J. Dennis, G.J. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for
(
cancer, Nat. Rev. Cancer 3 (2003) 380–387; (c) P. Agostinis, K. Berg, K.A. Cengel, T.H.
Foster, A.W. Girotti, S.O. Gollnick, S.M. Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M.
Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic
therapy of cancer: an update, CA Cancer J. Clin. 61 (2011) 250–281; (d) C.A. Robertson, D.
Hawkins Evans, H. Abrahamse, Photodynamic therapy (PDT): a short review on cellular
mechanisms and cancer research applications for PDT, J. Photochem. Photobiol. B 96 (2009)
1
–8.
2] (a) M.T. Wan, J.Y. Lin, Current evidence and applications of photodynamic therapy in
dermatology, Clin. Cosmet. Investig. Dermatol. 7 (2014) 145–163; (b) P. Babilas, S. Schreml,
[

ACCEPTED MANUSCRIPT
M. Landthaler, R.-M. Szeimies, Photodynamic therapy in dermatology: state-of-the-art,
Photodermatol. Photoimmunol. Photomed. 26 (2010) 118–132; (c) S. Choudhary, K. Nouri,
M.L. Elsaie, Photodynamic therapy in dermatology: a review, Lasers Med. Sci. 24 (2009)
9
71–980; (d) R. Darlenski, J.W. Fluhr, Photodynamic therapy in dermatology: past, present,
and future, J. Biomed. Opt 18 (2012) 061208.
3] (a) J. Marotti, A.C.C. Aranha, C.D.P. Eduardo, M.S. Ribeiro, Photodynamic therapy can
be effective as a treatment for herpes simplex labialis, Photomed. Laser Surg. 27 (2009) 357–
63; (b) M.E. Lima, Y.-l. Leeb, Y. Zhanga, J. Jang Hann Chu, Photodynamic inactivation of
viruses using upconversion nanoparticles, Biomaterials 33 (2012) 1912–1920.
4] (a) R.F. Donnelly, P.A. McCarron, M.M. Tunney, Antifungal photodynamic therapy,
[
3
[
Microbiol. Res. 163 (2008) 1-12; (b) J.P. Lyon, C. de M.P. e S. Azevedo, L.M. Moreira, C.
Jose´ de Lima, M.A. de Resende, Photodynamic Antifungal Therapy Against
Chromoblastomycosis, Mycopathologia 172 (2011) 293–297.
[
5] (a) A.A. Takasaki, A. Aoki, K. Mizutani, F. Schwarz, A. Sculean, C.-Y. Wang, G. Koshy,
G. Romanos, I. Ishikawa, Y. Izumi, Application of antimicrobial photodynamic therapy in
periodontal and peri-implant diseases, Periodontol. 2000 51 (2009) 109–140; (b) A. Tavares,
S.R.S. Dias, C.M.B. Carvalho, M.A. Faustino, M.G.P.M.S. Neves, J.P.C. Tomé, A.C. Tomé,
J.A.S. Cavaleiro, Â. Cunha, N.C.M. Gomes, E. Alves, A. Almeida, Mechanisms of
photodynamic inactivation of a Gram-negative recombinant bioluminescent bacterium by
cationic porphyrins, Mar. Drugs 8 (2010) 91–105.
[
6] A.T. Dharmaraja, Role of reactive oxygen species (ROS) in therapeutics and drug
resistance in cancer and bacteria, J. Med. Chem. 60 (2017) 3221–3240.
7] P. Mroz, A. Yaroslavsky, G.B. Kharkwal, M.R. Hamblin, Cell death pathways in
photodynamic therapy of cancer, Cancers 3 (2011) 2516–2539.
8] (a) R.J.Y. Ho, J. Chien, Trends in translational medicine and drug targeting and delivery:
new insights on an old concept–targeted drug delivery with antibody–drug conjugates for
[
[

ACCEPTED MANUSCRIPT
cancers, J. J. Pharm. Sci. 103 (2014) 71–77; (b) L. Dai, J. Liu, Z. Luo, M. Li, K. Cai, Tumor
therapy: targeted drug delivery systems, J. Mater. Chem. B 4 (2016) 6758–6772; (c) T.
Luhmann, L. Meinel, Nanotransporters for drug delivery, Curr. Opin. Biotechnol 39 (2016)
3
5–40; (d) H.-J. Hsu, J. Bugno, S. Lee, S. Hong. Dendrimer-based nanocarriers: a versatile
platform for drug delivery, WIREs Nanomed. Nanobiotechnol 9 (2016) 1409; (e) S.Z. Vahed,
R. Salehi, S. Davaran, S. Sharifi, Liposome-based drug co-delivery systems in cancer cells,
Mater. Sci. Eng. C 71 (2017) 1327–1341; (f) M.A. Grin, A.F. Mironov, A.A. Shtil, Anti-
cancer agents in medicinal chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer
Agents) 8 (2008) 683–697.
[
9] (a) P.R. Hamann, L.M. Hinman, I. Hollander, C.F. Beyer, D. Lindh, R. Holcomb, W.
Hallett, H.-R. Tsou, J. Upeslacis, D. Shochat, A. Mountain, D.A. Flowers, I. Bernstein,
Gemtuzumab Ozogamicin, A potent and selective anti-CD33 antibody−calicheamicin
conjugate for treatment of acute myeloid leukemia, Bioconjugate Chem. 13 (2002) 47–58; (b)
A. Gabizon, H. Shmeeda, Y. Barenholz, Pharmacokinetics of pegylated liposomal
doxorubicin, Clin. Pharmacokinet 42 (2003) 419–436; (c) J.D. Bagnato, A.L. Eilers, R.A.
Horton, C.B. Grissom, Synthesis and characterization of a cobalamin−colchicine conjugate as
a novel tumor-targeted cytotoxin, J. Org. Chem. 69 (2004) 8987–8996. (d) A. Homma, H.
Sato, A. Okamachi, T. Emura, T. Ishizawa, T. Kato, T. Matsuura, S. Sato, T. Tamura, Y.
Higuchi, T. Watanabe, H. Kitamura, K. Asanuma, T. Yamazaki, M. Ikemi, H. Kitagawa, T.
Morikawa, H. Ikeya, K. Maeda, K. Takahashi, K. Nohmi, N. Izutani, M. Kanda, R. Suzuki,
Novel hyaluronic acid–methotrexate conjugates for osteoarthritis treatment, Biorg. Med.
Chem. 17 (2009) 4647–4656; (e) B. Moulari, A. Béduneau, Y. Pellequer, A. Lamprecht,
Lectin-decorated nanoparticles enhance binding to the inflamed tissue in experimental colitis,
J. Control. Release 188 (2014) 9–17; (f) P. Ringhieri, C. Diaferia, S. Galdiero, R. Palumbo, G.
Morelli, A. Accardo, Liposomal doxorubicin doubly functionalized with CCK8 and R8
peptide sequences for selective intracellular drug delivery, J. Pept. Sci. 21 (2015) 415–425;

ACCEPTED MANUSCRIPT
(
g) L.Q. Chen, W. Huang, Z.G. Gao, W.S. Fang, M. Jin,Lx2-32c–loaded polymeric micelles
with small size for intravenous drug delivery and their inhibitory effect on tumor growth and
metastasis in clinically associated 4T1 murine breast cancer, J. Int. J. Nanomedicine 11
(2016) 5457–5472; (h) H.J. Lee, H.H. Park, Y. Sohn, J. Ryu, J.H. Park, W.J. Rhee, T.H.
Park, α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for
effective treatment of Fabry disease, Appl. Microbiol. Biotechnol 100 (2016) 10395–10402;
(
i) S. Jaferian, M. Soleymaninejad, B. Negahdari, A. Eatemadi, Stem cell, biomaterials and
growth factors therapy for hepatocellular carcinoma, Biomed. Pharmacother. 88 (2017) 1046–
053.(j) M. Zhang, P. Yuan, N. Zhou, Y. Su, M. Shao, C. Chi, pH-Sensitive N-doped carbon
1
dots–heparin and doxorubicin drug delivery system: preparation and anticancer research, RSC
Adv. 7 (2017) 9347–9356.
[
10] J.F. Lovell, T.W. B. Liu, J. Chen, G. Zheng, Activatable photosensitizers for imaging
and therapy, Chem. Rev. 110 (2010) 2839–2857.
11] J. Usuda, S. Ichinose, T. Ishizumi, H. Hayashi, K. Ohtani, S. Maehara, S. Ono, H.
[
Honda, N. Kajiwara, O. Uchida, H. Tsutsui, T. Ohira, H. Kato, N. Ikeda, Outcome of
photodynamic therapy using NPe6 for bronchogenic carcinomas in central airways >1.0 cm in
diameter, Cancer Therapy: Clinical16 (2010) 2198–2204.
[
12] G. Kniebühlera, T. Pongratza, C. Betzb, B. Gökec, R. Srokaa, H. Steppa, J. Schirra,
Photodynamic therapy for cholangiocarcinoma using low dose mTHPC (Foscan®),
Photodiagn. Photodyn. 10 (2013) 220–228.
[
13] Y. Istomina, T. Lapzevicha, V. Chalaua, S. Shliakhtsin, T. Trukhachova, Photodynamic
therapy of cervical intraepithelial neoplasia grades II and III with Photolon®, Photodiagn.
Photodyn. 7 (2010) 144–151.
[
14] N.V. Koudinova, J.H. Pinthus, A. Brandis, O. Brenner, P. Bendel, J. Ramon, Z. Eshhar,
A. Scherz, Y. Salomon, Photodynamic therapy with Pd-bacteriopheophorbide (TOOKAD):