Thiourea Modified Doxorubicin: A Perspective pH-Sensitive Prodrug

Article abstract of DOI:10.1021/acs.bioconjchem.8b00885

A novel approach to the synthesis of pH-sensitive prodrugs has been proposed: thiourea drug modification. Resulting prodrugs can release the cytotoxic agent and the biologically active 2-thiohydantoin in the acidic environment of tumor cells. The concept of acid-catalyzed cyclization of thioureas to 2-thiohydantoins has been proven using a FRET model. Dual prodrugs of model azidothymidine, cytotoxic doxorubicin, and 2-thiohydantoin albutoin were obtained, which release the corresponding drugs in the acidic environment. The resulting doxorubicin prodrug was tested on prostate cancer cells and showed that the thiourea-modified prodrug is less cytotoxic (average IC50 ranging from 0.5584 to 0.9885 μM) than doxorubicin (IC50 ranging from 0.01258 to 0.02559 μM) in neutral pH 7.6 and has similar toxicity (average IC50 ranging from 0.4970 to 0.7994 μM) to doxorubicin (IC50 ranging from 0.2303 to 0.8110 μM) under mildly acidic conditions of cancer cells. Cellular and nuclear accumulation in PC3 tumor cells of Dox prodrug is much higher than accumulation of free doxorubicin.

Full text of DOI:10.1021/acs.bioconjchem.8b00885

Subscriber access provided by EKU Libraries
Article
THIOUREA MODIFIED DOXORUBICIN: A NOVEL
PERSPECTIVE PH-SENSITIVE PRODRUGS
Olga Krasnovskaia, Vladislav Malinnikov, Natalia Dashkova, Vasily M Gerasimov,
Irina V Grishina, igor I. Kireev, Svetlana V. Lavrushkina, Pavel A. Panchenko, Marina
Zakharko, Pavel Ignatov, Olga A Fedorova, Gediminas Jonusauskas, Dmitry Skvortcov,
Sergey Kovalev, Elena Beloglazkina, Nikolai Vasil'evich Zyk, and Alexander G. Majouga
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00885 • Publication Date (Web): 06 Feb 2019
Downloaded from http://pubs.acs.org on February 7, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 12
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 12
1
2
3
4
5
6
7
8
9
THIOUREA MODIFIED DOXORUBICINE: A NOVEL
PERSPECTIVE PH-SENSITIVE PRODRUGS
*
*
*
§
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
⊥
Olga O. Krasnovskaya , Vladislav M. Malinnikov , Natalia S. Dashkova , Vasily M. Gerasimov , Irina
,
†
†
⊥
*
∥
∥
V. Grishina , Igor I. Kireev , Svetlana V. Lavrushkina , Pavel A. Panchenko , Marina A. Zakharko ,
,
†
⊥
⊥
Pavel A. Ignatov , Olga A. Fedorova , Jonusauskas Gediminas‡, Dmitry A. Skvortsov*, Sergey S. Ko-
*
⊥
*
¤
*
*
valev , Elena K. Beloglazkina , Nikolay V. Zyk , Alexander G. Majouga .
*Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1/3, 119991 Moscow, Russian Federation
∥Department of Biology, Lomonosov Moscow State University, Leninskie Gory, 1/12, 119234 Moscow, Russian Federation
§ Institute of Biochemistry and Genetic Russian Academy of Science (IBG RAS), Ufa Scientific Centre, Oktyabra Prospect
71, 450054, Ufa, Russian Federation
†A. N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, 119991, Vavilova str. 28,
Moscow, Russia
⊥D. Mendeleev University of Chemical Technology of Russia, 125047, Miusskaya sqr. 9, Moscow, Russia
‡Laboratoire Ondes et Matière d’Aquitaine (LOMA), UMR CNRS 5798, Bordeaux University, 33405, 351 Cours de la
Libération, Talence, France
¤Laboratory of Oncoproteomics, N.N. Blokhin Cancer Research Centre, Moscow, Russia
Acidic extracellular pHe is a major feature of tumor tissue; extra-
ABSTRACT: A novel approach to the synthesis of pH-sensitive
cellular acidification is considered to occur in the presence of 6,7
-
prodrugs has been proposed: thiourea drug modification. Resulting
prodrugs can release the cytotoxic agent and the biologically active
2-thiohydantoin in the acidic environment of tumor cells. The con-
cept of acid-catalyzed cyclization of thioureas to 2-thiohydantoins
has been proven using a FRET model. Dual prodrugs of model az-
idothymidine, cytotoxic doxorubicin, and 2-thiohydantoin albutoin
were obtained, which release the corresponding drugs in the acidic
environment. The resulting doxorubicin prodrug was tested on
prostate cancer cells, and showed that the thiourea-modified pro-
drug is less cytotoxic (average IC50 ranging from 0.5584 to 0.9885
μM) than Doxorubicin (IC50 ranging from 0.01258 to 0.02559μM)
in neutral pH 7.6, and similar toxicity (average IC50 ranging from
0.4970 to 0.7994 μM) to Doxorubicin (IC50 ranging from 0.2303
to 0.8110μM) under mildly acidic conditions of cancer cells. Cel-
lular accumulation in PC3 tumor cells of Dox prodrug is much
higher than accumulation of free Doxorubicin.
lactic acid, which accumulates due to anaerobic glycolysis in hy-
poxic cancer cells; excess of CO2, which accumulates due to pen-
tose phosphate pathway, and then oxidized by the enzyme carbonic
anhydrase (CA), which leads to the increase of the proton concen-
tration.8,9 Acidic pHe not only leads to activation of some lysosomal
enzymes, but also increases resistance to certain types of chemo-
therapy10 through the unforeseen metabolism of therapeutic mole-
cules resulting in high outflow of drugs from tumor cells. In partic-
ular, acidic pHe reduces cytotoxicity of antitumor drugs with weak
base pKa: Doxorubicin, Mitoxantrone and Daunorubicin.11 In the
early stages of breast cancer, high CAIX enzyme level is a predic-
tive marker of doxorubicin resistance.12 Acidic pHe also plays role
in the drug resistance of tumor cells due to the increased p-glyco-
protein expression, that increases drug efflux.13,14
There are several strategies that can be used to develop pH-sensi-
tive prodrugs capable of cytotoxic drug release in the acidic envi-
ronment of tumor cells and are described in lots of newest scientific
publications. pH-dependent release of Dox from its micellous con-
jugate with vitamin E, via pH-sensitive hydrazone bond,15 adaman-
tane-modified Dox via pH-sensitive hynrazone linker, capable to
release free Dox at pH 4.5;16 DOX-conjugated smart poly-
meric self-assambled micelles, prepared via an imine linkage,
which exhibited the pH-triggered charge-conversion property and
accelerated drug release at tumor pH;17 polymeric micelles delivery
system based on block copolymers of poly(L-lactic acid)-β-
poly(ethylene glycol) β-poly (L- histidine)-TAT (transactivator of
transcription) and poly(L-histidine)-β-poly(ethylene glycol). Such
micelles have been proven to increase the cytotoxicity of doxoru-
bicin in several multidrug-resistant tumor cell lines through the life-
time increase of the active molecule.18 Also, conjugation of do-
cosahexaenoic acid (DHA) to Doxorubicin (Dox) with a pH-sensi-
tive hydrazone linker at 13 position formed a lipophilic prodrug,
INTRODUCTION
Doxorubicin - antitumor antibiotic - is an effective and commonly
used chemotherapeutic agent and is able to embed into the double
helical structure of cell DNA, inhibiting RNA and DNA synthesis,
and thereby leading to apoptosis of cancer cells.1 Doxorubicin and
its modified form Doxil are widely used in cancer therapy.2 Despite
the effectiveness of doxorubicin, there is a high chemotherapeutic
failure rate due to its low specificity towards tumor tissues, dose-
dependent resistance to therapy, and strong cardiotoxicity. 3
Development of doxorubicin prodrugs, modified with target-recog-
nizing fragment4, as well as pH-sensitive prodrugs capable of re-
leasing a cytotoxic agent in a weakly acidic tumor tissues5, is a rel-
evant task for medical chemistry.
ACS Paragon Plus Environment

Page 3 of 12
Bioconjugate Chemistry
and demonstrated higher anticancer activity in vivo than free doxo-
rubicin, authors suggested a pH-sensitive Dox release and higher
cellular accumulation than free Dox, but have not confirmed it ex-
perimentally. 19
1
2
3
We have proposed another approach to the synthesis of pH-sensi-
tive prodrugs: Dox-thiourea drug modification, that would be able
to simultaneously release two different therapeutic agents in the
acidic pH (pH ≥ 6.5/7.5)20. Thioureas are perspective prodrugs be-
cause in the acidic environment of tumor tissues they are cyclized
to corresponding thiohydantoins, releasing free Doxorubicin. At
the same time the resulting thiohydantoin, depending on the struc-
ture of the initial thiourea, may have different pharmacological
properties. 2-thiohydantoins exhibit a wide variety of biological
properties: antitumor, 21 antiviral, 22anti-inflammatory,23 anticon-
vulsant.24, 25 One of the most convenient methods of 2- thiohydan-
toins synthesis is the acid-catalyzed cyclization of the correspond-
ing thioureas.26
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this study, we have chosen thiohydantoin Albutoin as a simple
structure model drug - an anticonvulsant 27,28 – that was evaluated
by the United States Food and Drug Administration, but not ap-
proved . 29Albutoin, in contrast to the structural similar anticonvul-
sant diphenylhydantoin, didn’t not have toxicity-induced side ef-
fects. 30
Doxorubicin is highly hydrophilic, has short half-life, and its use is
associated with severe side effects at high doses. 31 Doxorubicin
implements its cytotoxicity by inhibiting Topoisomerase II en-
zyme.32 The amino group in the 3'-position is not essential for
topoisomerase II-targeting activities, because it can be replaced by
a hydroxyl group without reduction of activity (Doxorubicin analog
Annamicyn). However, conjugation of 3’-amino group through an
amide bond reduced the anticancer activity against the leukemia,
breast, ovarian, and colon cancer cell lines, suggesting that the pres-
ence of a free amino group is required for anticancer activity of
doxorubicin.33 Thus, we have suggested that the modification of
amino group of Doxorubicin with thiourea will reduce the general
toxicity of the corresponding prodrug.
Figure 1. Model pH-sensitive thiourea as naphthalenimide flu-
orescein FRET-pair
Thus the study of FRET-pair fluorescence, where resonant energy
transfer is observed, can show whether fluorophores are part of the
same molecule or not. In case the acid catalyzed cyclization occurs,
the resonance transfer of energy will cease, clearly demonstrating
the acid-catalyzed cyclization of thiourea with the release of the
amide fragment and the formation of the 2-thiohydantoin.
The spatial structure of FRET-model optimized by molecular me-
chanics in the gas phase is shown in Fig 2. The calculated distance
between the elements of the FRET pair is about 12.8 Å, and there
is also a significant overlap of the emission spectrum of the donor
fragment and the absorption spectrum of acceptor moiety (see sec-
tion 2.1.2.1), which indicates the possibility of resonant transport
in this system.
To prove the ability of sterically hindered thioureas to cyclize in a
weakly acidic medium, we have synthesized a model of pH-sensi-
tive thiourea: FRET- pair based on naphthalenimide and fluores-
cein. The present paper reports our initial studies on the synthesis
of the novel thiourea derivative of Doxorubicin - the acid-hydro-
lyzable twin prodrug by the modification of the N- 3’ position of
Dox through amide bond formation and its anticancer activity. The
cytotoxic effects and cellular accumulation of the synthesized com-
pound were evaluated against androgen receptor negative PC3
prostate cancer cell lines.
Synthesis of the pH-sensitive thiourea as a FRET-model molecule
The synthesis of the model FRET-pair (thiourea 10) was carried out
according to the Scheme 1.
FRET-pair was synthesized through the ten-stage synthesis, the key
stages in which were copper-catalyzed azide-alkyne cycloaddition
reaction of propargyl ester 2 with azide 8 in mild conditions and
the reduction of the resulting triazole 9 with the formation of thio-
urea 10. Target compounds were isolated by high-performance liq-
uid chromatography.
RESULTS AND DISCUSSION
The studies of pH-sensitive thioureas-based prodrugs using a
FRET-pair model
The detailed description of the experimental procedures is given in
the Supporting Information.
To assess the cyclization of thiourea derivatives in a weakly acidic
medium, we have proposed to synthesize a sterically hindered flu-
orescent derivative of 2-thiohydantoin acid containing the donor
naphthalimide and acceptor fluorescein fragments (Scheme 1). In
the case of this FRET-pair, the Förster resonance energy transfer
must be disrupted by acid-catalyzed cyclization, leading to frag-
mentation of the initial molecule to naphthalenimide and fluores-
cein components, which will lead to a significant change in the flu-
orescence spectrum and can be easily monitored by spectral meth-
ods (Fig. 1).
Study of FRET-model pH- sensitivity
Theoretical study of resonant energy transfer in FRET- model
Effective resonant energy transfer between fluorophores is possible
if the distance between them is less than 10 nm, and the emission
spectrum of the donor is overlapping with the acceptor absorption
spectrum.34 The superposition of experimental fluorescence spectra
of the donor naphthalimide (NI) and acceptor fluorescein isothio-
cyanate (FITC) absorption are given in Fig. 3.
2
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 12
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Geometry of naphtalineimide-fluorescein FRET-
model optimized by MM2 – method
Results of the calculations based on the experimentally measured
spectral characteristics of fluorophores are given in Table 2.
Table 2. Theoretical calculation of the resonance energy
transfer process characteristics according to the Förster
model in the NI-FIC compound 10 and in the protonated
form of NI-FITC
퐽(휆)
푘FRET/푐−1
푙 ∙ 푛푚4
Compound
푅0/Å 푟/Å
ΦFRET
Scheme 1. Synthesis of pH-sensitive thiourea 10 as a FRET-
model molecule.
/ 푚표푙 ∙ с푚
FRET-
model
4.35·1014
2.08·1014
44.7
39.5
12.8 2.0·1011
12.8 9.6·1010
0.9994
Table 1. Spectral characteristics of individual chromo-
phores NI, FITC and protonated FITC form in methanol-
water mixture (v / v = 1: 1).
FRET-
0.9988
model +H+
휺
∙ ퟏퟎ−ퟑ
/ М−ퟏ
∙ см−ퟏ
퐚퐛퐬
퐟퐥
흉퐒
/ нс
흀
퐦퐚퐱
흀
퐦퐚퐱
Also, we have studied the resonant energy transfer in FRET-model
in comparison of absorption and fluorescence spectra of model thi-
ourea 10 and equimolar amount of the free corresponding donor
(NI) and acceptor (FITC). The data obtained indicate the high effi-
ciency of resonant transport in the FRET-pair 10, consistent with
the theoretical calculation. For experimental details, see Supporting
Information (Figure S3).
Сompound
흋퐟퐥*
/нм
/нм
449
518
514
Naphthalimide 371
NI (Donor)
8.0
1.0
9.0
FITC (Accep- 492
tor)
31.4
0.23 3.6
0.11 2.4
Study of acid-catalyzed cyclization of FRET-model 10 using sta-
tionary and time-resolved fluorescence spectroscopy
FITC+HCl
(10-4)
453;480 7.8;7.2
To investigate the cyclization process of FRET-model 10 a series
of solutions at acidic pH was prepared (5*10-6M of 10) in a water:
methanol (1 : 1) mixture with HCl in 0 M, 1*10-5 M, and 1*10-4 M
concentrations. The absorption spectra of these mixtures are pre-
sented in Fig. 4.
The most important spectral characteristics of fluorophores were
determined by the Förster model to calculate the characteristics of
resonant transport: the absorption maximum wavelength, the max-
imum fluorescence wavelength, the extinction coefficient at the ab-
sorption maximum, the quantum yield fluorescence, lifetime of the
excited state.35 The absorption and fluorescence spectra of individ-
ual naphthalimide and FITC are shown in Fig S1 and S2 in Sup-
porting Information. Spectral characteristics of individual chromo-
phores NI, FITC and protonated FITC form in methanol-water mix-
ture (v / v = 1: 1), are presented in Table1.
The results of stationary fluorescence and fluorescence kinetics
analysis are shown in Fig. 5, Fig. S4. Fluorescence kinetics (Fig.
S4) analysis allowed to calculate the contribution of an exponent
with a characteristic time of 6.0 ns corresponding to free naph-
thalimide–which was 52% (calculated from the known values36 of
the pre-exponential factors = 619 /(619 + 561) = 0.52),
i.e. there is an increase in contribution of this component if com-
pared to neutral pH, where it was 37%. In thestationary spectra a
significant decrease in the fluorescence intensity of the acceptor (λ
= 518 nm), and the growth of donor fluorescence in-
3
ACS Paragon Plus Environment

Page 5 of 12
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
Figure 5. Fluorescence spectra of the FRET-model 10 in meth-
anol-water mixture (v/v = 1: 1) in the presence and absence of
hydrochloric acid.
Figure 3. Superposition of the experimentally observed fluores-
cence spectra of the donor (NI, 1) and acceptor (FITC, 2).
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pH sensitive thioureas prodrugs based on 3'-amino-3'- deoxy-
thymidine
tensity (λ = 449 nm) are also observed, confirming the proposed
hypothesis about compound 5 decomposition in a weakly acid me-
dium with the interruption of resonance energy transfer and a
change in spectrum of fluorescence.
To develop synthetic approaches to the acid-sensitive thiourea de-
rivatives with sugar moieties we synthesized a model conjugate of
the Albutoin precursor with 3'- amino-3'-deoxythymidine.
Synthesis of 3'-amino-3'-deoxythymidine analogs of pH-
sensitive thioureas
After multiple optimizations, we have proposed to introduce the
drug fragment at the last stage of the synthesis, through the ester
formation of the corresponding thiourea in the reaction with N-hy-
droxysuccinimide as an approach to twin 3'-amino-3'-deoxythymi-
dine-based prodrug (Scheme 2):
-6
Figure 4. Absorption spectra of Fret-pair 10 (5×10 M) in a
methanol-water mixture (v / v = 1: 1) in the presence and ab-
sence of hydrochloric acid.
Thereby, in studied FRET-model of pH-sensitive thiourea an effec-
tive resonance energy transfer was observed, but decomposition of
thiourea 10 took place in acidic medium (pH = 5) which was proved
by a change in the fluorescence spectrum.
Scheme 2. Synthesis of pH-sensitive 3'-amino-3'-deoxy-
thymidine-Albutoin prodrug 13.
Thus, the hypothesis about the ability of sterically hindered deriv-
atives of 2-thiohydantoin acid to cyclize in a weakly acidic medium
was confirmed. Taking this into account it can be assumed the prin-
ciple possibility of working out of pH-sensitive prodrugs of cyto-
static molecules based on the same derivatives, capable of releasing
the biologically active substance in the acidic medium of tumor tis-
sue.
Product 13 was isolated by preparative chromatography on silica
1
gel and characterized by H NMR spectroscopy and high-resolu-
tion mass spectrometry. For a detailed description of the synthesis
see Supporting Information.
Evaluation of pH- sensitivity of twin 3'-amino-3'- deoxythymidine -
based prodrug 13 by LC-MS.
The thiourea-based prodrugs should release of biologically active
substance and thiohydantoine in a weakly acidic medium. The
model drug release from the conjugate of 3'-amino-3'- deoxythymi-
dine with Albutoin precursor 13 was studied in vitro under physio-
logical conditions at different pH.
4
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 12
1
2
3
4
5
6
7
8
9
Scheme 3. The proposed way of acid-catalyzed decomposi-
tion of the model conjugate 13 in a weakly acid medium.
Compound 13 was dissolved in formate buffer solutions with a pH
of 5.5; 6.5; 7.4. The resulting mixtures were incubated at 37° C for
24 hours. Samples for analysis were taken at different time inter-
vals: 0, 20, 40, 60, 90, 120, 240, 480, 1440 minutes, and analyzed
by the LC-MS method. Relative rates of hydrolysis of the com-
pound 13 over a 48h period were calculated using the percent of
prodrug 13 hydrolysis in acidic condition; the relative rate of free
aminothymidine accumulation was also measured. Conjugate 13
flow rate is shown in Fig. 6, the accumulation rate of the cyclization
product 3'- amino-3'-deoxythymidine - in Fig. 7. We have sug-
gested the following scheme for the release of 3'-amino-3'- deoxy-
thymidine and Albutoin, which proceeds in a slightly acidic solu-
tion pH ~ 6.5 (Scheme 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. The dependence of 3'-amino-3'-deoxythymidine con-
centration from the time of incubation in medium with differ-
ent pH.
to the tumor tissue. Next step is the synthesis of the anti-cancer drug
(Doxorubicin)-based thioureas.
pH-sensitive thiourea prodrug based on Doxorubicin
We have proposed a synthesis of twin Doxorubicin-Albutoin pro-
drug that can possess both antitumor and anticonvulsant properties.
It was suggested that the introduction of an Albutoin precursor
fragment would reduce the toxicity in the neutral pH due to a sig-
nificant change in the structure of the initial cytotoxic agent, but
such molecule will be able to release the initial drug in a weakly
acidic pH of the tumor tissue medium, also, higher lipophilicity of
the thiourea-doxorubicin prodrug should improve cell penetration
compared to unmodified Doxorubicin, thereby lowering the effec-
tive drug dose reducing toxicity.
We also have determined the kinetic parameters of this process.
The reaction order for both the product and the reagent is zero. The
ratio of the rate constants of the process at pH 7.4 (k1), pH 6.5 (k2),
pH 5.5 (k3) was k1: k2: k3 = 1: 6.6: 8.5. This data indicates the
almost 10-fold increase in the rate of aminothymidine release at pH
= 5.5 compared to neutral medium (pH = 7.4).
It should be noted that during the LC-MS analysis of compound 13
the signal of Albutoin (m/z = 211.0911) was observed in all cases,
which confirms that the cyclization reaction proceeds according
with the Scheme 3.
Synthesis of Doxorubicin derivative of pH-sensitive thiourea
To obtain a twin Doxorubicin-based prodrug 14 we have used a
previously described three-steps approach with intermediate prep-
aration of the N-hydroxysuccinimide ester 12. The synthesis was
carried out according to the Scheme 4:
Scheme 4. Synthesis of pH-sensitive Doxorubicin-Albutoin
prodrug 14.
Figure 6. The dependence of prodrug 13 concentration from
the time of incubation at in medium with different pH.
The product 14 was isolated by preparative chromatography on sil-
ica gel and characterized by 1H NMR spectroscopy and high-reso-
lution mass spectrometry. For a detailed description see Supporting
Information.
Thus, the resulting conjugate 13 is capable to release of the starting
drug and Albutoin in a weakly acidic medium, but practically is not
disintegrating under neutral conditions. This suggests that such
compounds in in-vivo models would be selectively released in tu-
mor (рН~6,5),37 while in healthy tissues release of the drug should
not occur.
Evaluation of pH-sensitivity of twin Doxorubicin-Albutoin prodrug
14 of by LC-MS.
Relative rates of compound 14 hydrolysis over a 48 h period were
evaluated using the experimentally determined percentage of the
initial Doxorubicin-Albutoin prodrug 14 under the hydrolyzes in
acidic conditions at different pH.
Based on our result, we conclude that the cyclization of thioureas
prodrug can occur under conditions corresponding
Prodrug 14 were incubated in aqueous formate buffer at pH 5.5,
6.5, and 7.4 at 37°C over a 20 h. LC-MS technique was employed
to analyze how much of compound 14 was hydrolyzed. We have
demonstrated the ability of the derivative 14 to enter the acid-cata-
lyzed cyclization reaction with the formation of Albutoin and
5
ACS Paragon Plus Environment

Page 7 of 12
Bioconjugate Chemistry
Doxorubicin in a weakly acid solution. Sampling for LC-MS anal-
hydrolysis of amides, easily occurs in weakly acid media. The next
1
2
3
4
ysis was carried out at 0, 70, 1210 minutes. The content of the ini-
tial conjugate was evaluated; the resulting concentration/time de-
pendence in semilogarithmic coordinates is shown in Figure 8.
step was to study the effect of thiourea modification on cytotoxi-
city.
In vitro studies of compound 14 cytotoxicity against PC3 cell line
under pre-established acidic cancer cell culture medium (pH <
7.0).
5
After the studying the pH-sensitive dual prodrug 14 ability to re-
lease Doxorubicin and Albutoin in acidic environment, we studied
the effect of compound 14 on tumor cells of prostate cancer PC3.
The cells treated with same concentration of free Doxorubicin were
used as negative control.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We have prepared two different cell cultures for comparison: one
was incubated in standard nutrient medium with pH = 7.6, and the
other one was adapted to conditions of tumor tissue with pH = 6.6.
Previously it has been shown that a pH decrease of the nutrient me-
dium from 7.4 to 6.7 does not affect the growth rate and the popu-
lation of cells.39 This means that the cytotoxicity can be attributed
exclusively to the effect of the test drug, but not to the change in
external conditions.
Doxorubicin prodrug 14 and free Doxorubicin were added to the
aforementioned cultures. The MTT method40 was used to study cell
survival after incubation for 48 hours.
Figure 8. Semi-logarithmic dependence concentration of com-
pound 14 on time incubation at various pH conditions
We have suggested the following scheme for the release of Doxo-
rubicin and Albutoin, which proceeds in a slightly acidic solution
pH ~ 6.5 (Scheme 5):
We have detected a decrease in the toxicity of the conjugate 14 un-
der study in a weakly alkaline medium corresponding to healthy
tissues, compared with its toxicity in more acidic medium. IC50
values for Doxorubicin prodrug 14 and Doxorubicin in various me-
dia also were calculated, and were found to be dramatically differ-
ent (Table 3). The cytotoxicity curve of 14 and Dox at different
concentrations are given in Supporting information (Fig. S5).
Table 3. IC50 values of Doxorubicin prodrug 14 and free
Doxorubicin against PC3 cells.
IC50,мМ pH = 6.6
pH = 7.6
Scheme 5. The proposed way of acid-catalyzed decomposi-
tion of the twin Doxorubicin-Albutoin prodrug 14 in
aweakly acid medium.
Average Doxorubicin
14
Doxorubicin
14
0,4322
95% Confidence Intervals
0.2303 to
0,6303
0,01794
0,7430
The release of free Doxorubicin reaches a value of 94% at pH =
5.5; 93% at pH = 6.5; 89% at pH = 7.4. Analysis of the data in semi-
logarithmic coordinates allows to assume the first order of the re-
action for the conjugate 14. On this base, the half-transformation
times at different pH values were calculated, which are 300 min at
pH = 5.5; 320 min at pH = 6.5; 380 min at pH = 7.4.
0.4970 to
0,7994
0,01258 to
0,02559
0,5584 to
0,9885
0,8110
Such a different IC50 values for Doxorubucin in different pH can
be explained by low cellular penetration, which occurs due to pro-
tonation. In addition to lipophilicity, the degree of molecular ioni-
zation, which is dependent on pH and pKa, determines the transport
action. Doxorubicin act as weak base (pKa 8.34).41 Therefore, a de-
creasing extracellular pH leads to an increasing ionization of drug
molecules and, hence, drug transport into cells is rendered more
difficult.42
Thus, the hypothesis of faster release of conjugate 14 in an acid
medium was confirmed.
Previously, the fatty acid derivatives of Doxorubicin have been
synthesized .38 These prodrugs are amides, and they possess gener-
ally lower toxicity compared to Doxorubicin, which was explained
by the high stability of amides to non-enzymatic hydrolysis under
physiological conditions. Also, recently, adamantane-modified
doxorubicin via amide and ester linker was developed, and, also,
this conjugates were found to be non-toxic and didn’t show the abil-
ity to release free Dox in weakly acid medium.16
Thus, due to pKa value, Doxorubicin shows increased toxicity to
pH-neitral healthy tissues compared with pH-acidic tumor tissues,
therefore, more hypophilic and penetrating prodrugs are promising
analogues. Comparison of the cellular penetration of prodrug 14
with free doxorubicin is given in 2.3.4.
Thereby, previous attempts to obtain pH-sensitive prodrugs of
Doxorubicin, by modifying the 3’-NH2-position with an amide
bond, did not give satisfactory results on drug release and toxicity.
The proposed concept of thiourea modification shows a proton-sen-
sitive release even at pH 6.5. Due to the special structure of the
introduced thiourea fragment, resulting prodrug does not undergo
hydrolysis according to the classical mechanism of amide hydroly-
sis, instead initiating the formation of a 2-thiohydantoin derivative
with the release of the amide fragment. This reaction, unlike the
At pH = 7.6 high-lipophilic conjugate 14 penetrates into the cells,
but Dox release does not take place sufficiently. However, a rather
high toxicity even in a neutral medium compared to the low toxicity
of prodrugs incapable of release16 suggests Dox releasing inside the
cell in a weakly acidic environment of the endosomes. (pH~6.0).
This explains the similarity of the cytotoxicity values of 14 at dif-
ferent acidity of the medium - in both experiments, one substance
acts by one mechanism - most likely, Topoisomerase II inhibition.
6
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 12
Thus, the resulting conjugate 14 has toxicity similar to Doxorubicin
expected, 14 penetrates the cell much better than doxorubicin due
1
2
3
4
5
6
7
8
in a tumor tissue environment and is 30 times less toxic in a healthy
tissue environment.
to increased lipophilicity.
After penetrating the cell membrane and releasing from the endo-
some, most likely we observe a mixed signal of Dox, which have
released from 14, and the conjugate 14. (Figure 9, C3).
Cellular accumulation studies of compound 14 in PC3 cell line
To study the cell penetration ability of 14, and comparison of pen-
etration with doxorubicin, we have investigated intracellular ac-
cumulation in PC3 cells after 2 hours of incubation with 14 and
Dox. The results are shown in Figure 9 (PC3 cells), 10 (PC3 cells
labeled with DAPI)
Also, high nucleous accumulation was observed. ((Figure 10, C3).
It is well-known, that Dox is capable of nuclear accumulation, re-
sulting in Topoisomerase II inhibition.43 Prodrug 14, as well as
higher cellular penetration, shows higher nuclear penetration in
comparison with free Doxorubicin (Figure 10, C3), which indicates
that the prodrug 14, which did not undergo hydrolysis in the pro-
teasome, penetrates into the cell nucleus as easily as into the cell,
due to its lipophilicity. Also, cell nuclei accumulation was quanti-
fied, average fluorescence signals for prodrug 14 and Doxorubicin
are presented in Supporting Information (Fig. S6, Table S1).
It should be noted that Doxorubicin is not protonated in neural pH
7.4, and posess a strong cytotoxic effect (Table 3), but its penetra-
tion into the cell and nuclei is still extremely low (Figure 9, 10, C2).
In the acidic pH of the tumor cells in vivo, Doxorubicin will be
protonated, and show even lower cellular accumulation. This
would leads to a low effective dose of the drug, and high toxicity.
Bioconjugation – is an attach a bioactive molecule to another mol-
ecule via a covalent bond, leads to the formation of a novel chemi-
cal structure with may have enhanced properties compared to those
of the original molecule. At the same time, as can be seen from the
literature data, the bioconjugation of various molecules to Doxoru-
bicin often leads to a rapid decline in cytotoxic activity, and biodis-
tribution failure. Conjugating the dexamethasone molecule to Dox
results in a more lipophilic conjugate, which also, like prodrug 14,
more easily penetrates into the cell compared to the original Doxo-
rubicin. However, the conjugate with dexomethasone showed no
ability to accumulate in the cell nuclei, and also, as in the examples
described above, shows toxicity to MCF-7 cells more than 20 times
less compared to Dox44. Prodrug 14, due to the pH-sensitive thio-
urea fragment, which increases the lipophilicity of the molecule,
distribution rate, but which at the same time can be easily removed
by cyclization, easily penetrates both into the cell and into cell nu-
clei.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 9. Cellular accumulation of 14 in PC3cells (A) PC3 cells
treated with water (A1), Doxorubicin (A2), 14 (A3); (B) visual-
ization of drugs in the red fluorescent channel (561nm) (C)
merge of A and B.
Conjugate 14 is less toxic than doxorubicin in neutral pH (Table 3),
however, it exhibits better cellular accumulation even at this pH
value, at pH 6.5 14 shows toxicity similar with doxorubicin (Table
3), due to a combination of high cellular penetration and pH-sensi-
tive release Doxorubicin and Albutoin.
CONCLUSION
Summarizing the results, we have developed a methodology for the
preparation of a naphthalimide-fluorescein FRET pair, which con-
tains in the structure the fragment of thiohydantoin acid and is ca-
pable of an acid-catalyzed cyclization. The presence of resonant
energy transfer in this system was predicted theoretically and con-
firmed experimentally, and the hypothesis about the FRET pair cy-
clization in a weakly acidic medium was proved.
The method for the synthesis of new pH-sensitive twin Doxo-rubi-
cin-thiourea prodrug was proposed. The ability of the resulting pro-
drug 14 to release cytotoxic and thiohydantoin components in a
weakly acidic medium was confirmed. Using in vitro cytotoxicity
studies, the selectivity of the pH-sensitive twin Doxorubicin-Albut-
oin prodrug towards PC3 prostate cancer cell line was compared to
free Doxorubicin. It was shown that the obtaining pH-sensitive
twin Doxorubicin-Albutoin prodrug has more than 30-fold selec-
tivity increase towards healthy tissues compared to the free Doxo-
rubicin. Despite the fact that in neutral pH the toxicity of Dox and
prodrug 14 is dramatically different, in acidic pH it is almost equal.
Figure 10. Cell nucleous accumulation of 14 in DAPI-labeled
PC3 cells (A) DAPI-labeled PC3 cells treated with water (A1),
Doxorubicin (A2), 14 (A3), vizualization of DAPI in blue fluo-
rescent channel (450 nm); (B) visualization of drugs in the red
fluorescent channel (561nm) (C) merge of A and B.
Transport of anthracyclines is essentially influenced by the param-
eters pKa and polarity of the molecule. The intracellular drug con-
centration at steady state increases with increasing lipophilicity in
the order doxorubicin, epirubicin, and aclacinomycin.42 As we
7
ACS Paragon Plus Environment

Page 9 of 12
Bioconjugate Chemistry
Thiourea-modified Doxorubicin 14 have showed better cellular and
Geometry estimation of naphtalineimide-fluorescein FRET-
1
2
3
4
5
6
7
8
nuclei penetration in comparison with free Dox. Thus, the proposed
approach to the modification of Doxorubicin with pH-sensitive thi-
oureas opens up possibilities for obtaining drugs of the same phar-
macological efficacy on tumor tissues having a lower effective dose
due to higher cellular penetration and the possibility of adding a
second pharmacological action depending on the structure of the
released thiohydantoin.
model 10 optimized by PM6 semiempirical method
The three dimentional structure of 10 was built with MOPAC 2016
program package using PM6 semiempirical method.45 The calcula-
tions were performed at optimized geometries, which reached gra-
dient variations less than 0.01 kcal/mol. The solvent effect was in-
cluded in geometry optimizations following the «Conductorlike
Screening Model» (COSMO) implemented in MOPAC. A dielec-
tric constant of ε = 60 and a refraction index of solvent (n) such that
n2 = 2 were used
MATERIALS AND METHODS
Experimental study of resonant energy transfer in FRET-
modelTo confirm the presence of resonant energy transfer in
FRET-model, the absorption and fluorescence spectra of the
equimolar mixture naphthalimide + fluorescein isothiocyanate (NI
5*10-6M + FITC 5*10-6M) and the solution of FRET-model 5 * 10-
6M were compared. (excitation wavelength 370 nm). (Figure S3).
Due to the instability of FRET-model, there was a partial decay
during transportation to the place of investigation according to Fig
S3. This resulted in the destruction of the FRET pair with the re-
lease of the compound containing the naphthalimide moiety. This
process causes the appearance of an additional peak of fluorescence
at λ = 449 nm. The time-resolved fluorescence spectra of FRET-
model and naphthalimide were also recorded. The results are pre-
sented in the form of spectral-temporal maps and a plot of fluores-
cence intensity at different wavelengths versus time (Fig S3). It was
found that the lifetime of the excited impurity state is 5.6 ns, and
that of the test compound is 3.0 ns. The contribution of the expo-
nential with the characteristic time 5.6 ns corresponding to the
naphthalimide-containing impurity is 37% (calculated from the
known values of the pre-exponential factors as 976 / (976 + 1612)
= 0.37). Taking into account the difference between the quantum
yields of fluorescence FRET-model and naphthalimide, it was
found that the impurity content is approximately 15%. Fluores-
cence of the acceptor-fluorescein appears almost immediately after
photoexcitation; fast relaxation of naphthalimide with a simultane-
ous rapid increase in the fluorescence intensity of the acceptor is
not observed. The obtained data indicate the high efficiency of res-
onant transport in the system under investigation, consistent with
the theoretical calculation. Spectral-temporal maps of fluorescence
FRET-model and naphthalimide, fluorescence kinetics of FRET-
model is show in Supporting information. (Figure S3).
9
Chemicals Unless otherwise noted, all preparations were carried
out in reagent grade solvents. All chemicals used in the synthesis
were obtained from Acros or Sigma-Aldrich and were used without
further purification. Solvents were deoxygenated/distilled/purified
by bubbling through a stream of argon or by conventional methods
and dried over molecular sieves. Column chromatography was per-
formed using silica gel 60-120 mesh, 100-200 mesh.
1H NMR spectra were recorded on a Brucker-Avance instrument
(operating at 400 MHz for 1H). As the solvent, deuterochloroform
(CDCl3) and dimethylsulfoxide-d6 (DMSO-d6) were used. Chemi-
cal shifts are given in parts per million on a scale δ with respect to
hexamethyldisiloxane as an internal standard.
High resolution mass spectra (HRMS) were recorded on an Or-
bitrap Elite (Thermo Scientific) mass spectrometer with an IRET.
To inject solutions with a concentration of 0.1 to 9 μg/ml (in 1%
formic acid in acetonitrile), direct injection into the ion source us-
ing a syringe pump (5 μl / min) was used. The spray voltage is ±
3.5 kV, the temperature of the capillary is 275ºC. Mass spectra were
recorded using an Orbitrap analyzer with a resolution of 480,000 (1
microscan). The maximum input time is 900 ms, averaging over 9
spectra, the mass range is 90-2000 Da, in some cases 200-4000 Da.
For internal calibration, DMSO and diisooctylphthalate signals (m
/ z 157.03515 and 413.26623) were used in the positive mode and
the dodecyl sulfate signal (m / z 265.14790) in the negative mode.
LCMS For purification and analysis of samples we used Shimadzu
Prominence LC-20 system with column oven and fraction collector
coupled to single quadrupole mass-spectrometer Shimadzu LCMS-
2020 with dual DUIS-ESI-APCI ionization source. Analytical and
preparative column was Phenomenex Luna 3u C18 100A (150 x
4.6 mm). Mobile phases: A - 0.1% formic acid in water, B - 10 mM
ammonium formate in water, D -acetonitrile.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Adaptation of PC3 culture cells to pH = 6.6. The PC3 line cells
were incubated in the medium at t = 37 ° C until spontaneous pH =
6.7. The cells were then transferred twice to RPMI-1640 medium
with pH = 6.7 to adapt to a weakly acidic medium. After adaptation
for 24 hours, the pH of the medium was adjusted to 6.6 using
KH2PO4. Cells were incubated in acid medium for 48 hours.
Cytotoxicity study of 14. PC3 line cells incubated at pH 6.6 were
suspended and transferred to 2 plates per 96 wells. Similarly, the
culture of PC3 cells incubated at pH = 7.6 was reported. Subse-
quently, solutions of 14 and Doxorubicin in media with a corre-
sponding acid growth medium in concentrations of 1*10-4 g/L,
1*10-3 g/L, 1*10-2 g/L, 1*10-1 g/L and 1 g/L. Cells were incubated
for 48 hours. Then, using the MTT method (using 3- (4,5-dimethyl-
thiazol-2-yl) -2,5-diphenyltetrazolium bromide as a colorant, the
percentage of surviving cells was determined by the formula ,
where x is the proportion of surviving cells, - the optical density of
the solution of the test sample, - the optical density of the control
sample at λ = 700 nm. As a control sample, cells not treated with
cytotoxic drugs were used.
LCMS parameters for analyses were: gradient flow of 1 ml/min (0-
0.5 min - 5% D, 0.5 -10.5 min - 5% to 100% D, 10.5-12 min - 100%
D, 12-14.5 min - 100% to 5% D), column oven temperature 40 C,
optional UV detection of some compounds.
MS parameters: drying gas 15.0 L/min, nebulizing gas 1.5 L/min,
DL temperature 250 C, heat block temperature 400 C, interface
voltage -3.5 kV, corona needle voltage -3.5 kV. Positive (mass
range 250-2000 Da, in some cases 155-2000 Da) and negative ions
(mass range 215-2000 Da) were registered. For hydrolysis kinetics
study SIM mode was used with registration of the molecular ions
and adducts [M+H]+, [M+Na]+, [M+K]+, [M-H]-, [M+HCOO]-.
HPLC For purification of Fret-pair 10 we used identical LC pa-
rameters except gradient which was tailored for this compound (0-
0.5 min - 35% D, 0.5-5.5 min - 35% to 55% D, 5.5-9.5 min - 55%
to 100% D, 9.5-10.5 min - 100% D, 10.5-14 min - 100% to 35%
D). Fractionation was based on UV detection only (absorbance on
485 nm), fractions were collected based on UV signal level and
slope.
PC3 cellular/nucleus accumulation study PC3 cells (105
cells/ml) were seated on Petri dishes with a glass bottom with a
thickness of 0.17 mm. The experiment was carried out next day (for
the complete spreading of the cells and the acquisition of
Analytical thin-layer chromatography TLC was performed on
Merck silica gel aluminium plates with F-254 indicator. Com-
pounds were visualized by irradiation with UV light or iodine stain-
ing.
8
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 12
characteristic morphology by them). There were 3 samples: cells
3. Albert, Z. L, Biswajit, C., Mohammed, Al-O., Hwee, T., Da-
1
2
3
4
5
6
7
8
incubation with DOX (C ~ 0.14 uM), cells incubation with 14 (C ~
1.192 uM), and incubation with a solvent (H2O) for 2 hours. Dur-
ing incubation, cells were in phosphate-buffered saline (PBS) with
the addition of 10 mM HEPES (to reduce autofluorescence due to
components of the complete nutrient medium). Living cells were
kept in a chamber with a maintained level of CO2, temperature and
humidity. DIC and DOX/14 fluorescence images (in the red fluo-
rescent channel (561 nm) were obtained using a motorized inverted
fluorescent microscope Eclipse Ti-E (Nikon) equipped with an
iXon cooled EM-CCD camera (Andor), PerfectFocus (Nikon) au-
tofocus system and Plan Apo 40x lens (NA = 0.95). For DAPI vi-
zualization, the cell preparations were fixed in 3.7% formaldehyde
(Sigma-Aldrich, USA) prepared on PBS. Washing from the re-
tainer - 3 times for 5 minutes. Next, 0.2 ug/ml DAPI (Cayman
Chemical Company, USA) was added. Washing - 3 times for 5
minutes (PBS). Further shooting in 2 channels - 450 nm for DAPI
and 561 for DOX and 14. Image processing was performed in the
NIS-elements imaging software and in ImageJ.
vid, A. H., Subodh, V. (2018) Role of Endothelium in Doxo-
rubicin-Induced Cardiomyopathy. JACC: Basic to translation
science. 3,
6 , 861-870. https://doi.org/10.1016/j.jac-
bts.2018.06.005
4. Guo-Bin, D., Junqing, S., Peng, Y., Binchun, L., Ying, G.,
Zhuoyu, L. (2018) A Novel Doxorubicin Prodrug with
GRP78 Recognition and Nucleus-Targeting Ability for Safe
and Effective Cancer Therapy. Mol. Pharmaceutics. 15,
1, 238–246. DOI: 10.1021/acs.molpharmaceut.7b00830
5. González-Méndez, I., Solano, J. D., Porcu, P. (2019) Opti-
mized synthesis, characterization and in vitro systematic eval-
uation of adamantane-doxorubicin prodrugs sensitive to pH in
breast cancer cells. Journal of Molecular Structure. 1177, 5
143-151. https://doi.org/10.1016/j.molstruc.2018.09.044
6. Kato, Y., Ozawa, S., Miyamoto, C., Maehata, Y., Suzuki, A.,
Maeda, T., Baba, Y. (2013) Acidic extracellular microenvi-
ronment and cancer. Cancer Cell International. 13, 1, 89-97.
https://doi.org/10.1186/1475-2867-13-89
7. Griffiths, J. R. (1991) Are cancer cells acidic? British Journal
of Cancer. 64, 3, 425-427.
8. Han, J., Burgess, K. (2010) Fluorescent Indicators for Intra-
cellular pH. Chemical Reviews. 110, 5, 2709-2728,
https://doi.org/10.1021/cr900249z
9. Helmlinger, G., Sckell, A., Dellian, M., Forbes, NS., Jain, RK.
(2002) Acid production in glycolysis-impaired tumors pro-
vides new insights into tumor metabolism. Clin Cancer Res.
8, 4, 1284–1291.
10. Poomthavorn, P., Wong, S.H., Higgins, S., Werther, G.A.,
Russo, V.C. (2009) Activation of a prometastatic gene expres-
sion program in hypoxic neuroblastoma cells. Endocr Relat
Cancer. 16, 991- 1004. https://doi.org/10.1677/ERC-08-0340
11. Vukovic, V., Tannock, H. (1997) Influence of low pH on cy-
totoxicity of paclitaxel, mitoxantrone and topotecan. British
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information
Synthesis details, structural data, materials and methods.
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*Phone: +7 495 9394020.
Fax: +7 495 9328846.
E-mail: krasnovskayao@gmail.com.
Author Contributions
Journal
of
Cancer.
75,
8,
1167-1172.
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script.
https://doi.org/10.1038/bjc.1997.201
12. Betof, A.S., Rabbani, Z.N., Hardee, M.E., Kim, S.J.,
Broadwa, G., Bentley, R.C., Snyder, S.A., Vujaskovic, Z.,
Oosterwijk, E., Harris, L.N. (2012) Carbonic anhydrase IX
is a predictive marker of doxorubicin resistance in early-
stage breast cancer independent of HER2 and TOP2A am-
plification. British Journal of Cancer. 106, 5, 916–922.
https://doi: 10.1038/bjc.2012.32
13. Lotz, C., Kelleher, D. K., Gassner, B., Gekle, M., Vaupel, P.,
Thews, O. (2007) Role of the tumor microenvironment in the
activity and expression of the p-glycoprotein in human colon
carcinoma cells. Oncol Reports. 17, 1, 239–244.
https://doi.org/10.1677/ERC-08-0340
14. Thews, O., Dillenburg, W., Rosch, F., Fellner, M. (2013)
PET imaging of the impact of extracellular pH and MAP ki-
nases on the p-glycoprotein (Pgp) activity. Adv Exp Med
Biol. 765, 279–286. https://doi.org/10.1007/978-1-4614-
4989-8_39
15. Xiong, S., Wang, Z., Liu, J., Deng, X., Lei, Y. C. X., Tang,
G. (2019) A pH-sensitive prodrug strategy to co-deliver DOX
and TOS in TPGS nanomicelles for tumor therapy. Colloids
and Surfaces B: Biointerfaces. 173, 1, 346-355.
https://doi.org/10.1016/j.colsurfb.2018.10.012.
ABBREVIATIONS
Dox, Doxorubicin; FRET, Förster resonance energy transfer.
FITC, fluorescein isothiocyanate.
ACKNOWLEDGMENT
The study was supported by Russian Foundation for Basic Re-
search [grant number 18-33-01038], Russian Science Foundation
[grant number 17-74-30012].
Synthesis of naphthalimide dye, steady-state spectroscopic studies
of FRET dye, calculations of resonance energy transfer process
characteristics was supported by Russian Foundation for Basic
Research [grant number № 18-33-20111]
Cellular accumulation study was supported by Russian Science
Foundation [grant number 17-15-01290].
REFERENCES
1. Shu, Y., Xie, B., Liang, Z., Chen, J. (2018) Quercetin reverses
the doxorubicin resistance of prostate cancer cells by down-
regulating the expression of c-met. Oncology Letters. 15,
2252-2258. https://doi.org/10.3892/ol.2017.7561
2. Yongle, D., Long, X., Ami, J., Richey, M. D., Philippe., Mar-
ion, F. E., David, G. I. K.. (2018) Synthesis and Evaluation of
Doxorubicin-Loaded Gold Nanoparticles for Tumor-Targeted
Drug Delivery. Bioconjugate Chemistry. 29 (2), 420-430.
DOI: 10.1021/acs.bioconjchem.7b00756JP
16. González-Méndez, I., D.Solano, J., Porcu, P., Ruiu, A.,
Yareli, R., Rivera, E. (2019) Optimized synthesis, characteri-
zation and in vitro systematic evaluation of adamantane dox-
orubicin prodrugs sensitive to pH in breast cancer cells. Jour-
nal
of
Molecular
Structure.
1177,
143-151.
https://doi.org/10.1016/j.molstruc.2018.09.044
17. Ma, B., Zhuang, W., Wang, Y., Luo, R., Wang, Y. (2018)
9
ACS Paragon Plus Environment

Page 11 of 12
Bioconjugate Chemistry
modifications at the 3' and 4' positions of doxorubicin on the
drug ability to trap topoisomerase II and to overcome multi-
drug resistance. Mol Pharmacol. 45, 5, 908-915.
pH-sensitive doxorubicin-conjugated prodrug micelles with
charge-conversion for cancer therapy. Acta Biomaterialia. 70,
1, 186-196. https://doi.org/10.1016/j.actbio.2018.02.008
1
2
3
4
5
6
7
8
9
33. Gallois, L., Fiallo, M., Garnier-Suillerot, A. (1998) Compari-
son of the interaction of doxorubicin, daunorubicin, idarubicin
and idarubicinol with large unilamellar vesicles: Circular di-
chroism study. Biochimica et Biophysica Acta (BBA) -
18. Lee, E.S., Gao, Z., Kim, D., Park, K., Kwon, I.C., Bae, Y.H.
(2008) Super pH-sensitive multifunctional polymeric mi-
celle for tumor pHe specific TAT exposure and multidrug
resistance.
J
Control Release. 129, 3, 228–236.
Biomembranes
1370.
1,
31-40.
https://doi.org/10.1016/j.jconrel.2008.04.024
https://doi.org/10.1016/S0005-2736(97)00241-1
19. Wang, Y., Li, L., Jiang, W., Yang, Z., Zhang, Z. (2006) Syn-
thesis and preliminary antitumor activity evaluation of a
DHA and doxorubicin conjugate. Bioorg Med Chem Lett.
16, 2974-2977. https://doi.org/10.1016/j.bmcl.2006.02.066
20. Swietach, P., Vaughan-Jones, R.D., Harris, A.L., Hulikova,
A. (2014) The chemistry, physiology and pathology of pH in
cancer. Philosophical Transactions of the Royal Society B:
Biological Sciences. 369, 1638, 2013009-2013018.
https://doi.org/10.1098/rstb.2013.009
34. Nikiforov, T.T., Beechem, J.M. (2006) Development of ho-
mogeneous binding assays based on fluorescence resonance
energy transfer between quantum dots and Alexa Fluor fluor-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ophores.
Anal
Biochem.
357,
1,
68-76.
https://doi.org/10.1016/j.ab.2006.06.006
35. Hanulia, T., Inami, W., Ono, A., Kawata, Y. (2018) Fluores-
cence life-time measurement excited with ultraviolet surface
plasmon resonance. Optics Communications. 427, 266- 270,
doi.org/10.1016/j.optcom.2018.06.069.
36. Singharoy, D., Chowdhury, S., Mati, S.S., Ghosh, S. , Chatt,
P. (2017) Electron Transfer Switching Mechanism of a Naph-
thalimide Derivative with its Solvatochromic Behaviour An
Experimental and Theoretical Study with In Cell Investiga-
tions. Chem. Eur. J. 23, 16516–16524.
37. Wei, Y., Liao, R., Mahmood, A. A., Xu, H., Zhou, Q. (2017)
pH-responsive pHLIP (pH low insertion peptide) nanoclusters
of super-paramagnetic iron oxide nanoparticles as a tumor-se-
lective MRI con-trast agent. Acta Biomaterialia. 55, 194-
203. https://doi: 10.1016/j.actbio.2017.03.046.
38. Chhikara, B. S., St. Jean, N., Mandal, D., Kumar, A., & Pa-
rang, K. (2011) Fatty acyl amide derivatives of doxorubicin:
Synthesis and in vitro anticancer activities. European Journal
21. Majumdar, P., Bathula, C., Basu, S.M., Das, S.K., Agarwal,
R., Hati, S., Singh, A., Sen, S., Das, B.B. (2015) Design, syn-
thesis and evaluation of thiohydantoin derivatives as potent
topoisomerase I (Top1) inhibitors with anticancer activity. Eur
J
Med
Chem,
102.
540-551.
https://doi.org/10.1016/j.ejmech.2015.08.032
22. Chang, W.J., Kulkarni, M. V., Sun C.M. (2006) Traceless and
Stere- oselective Synthesis of Tetrahydro-β-carbolinethiohy-
dantoins by Microwave Irradiation. J. Comb. Chem. 8, 141-
144. https://doi.org/10.1021/cc050098j
23. Roué, N., Bergman, J. (1999) Synthesis of the marine alkaloid
leucettamine
B.
Tetrahedron.
55,
14729-14738.
https://doi.org/10.1016/S0040-4020(99)00918-7.
24. Bader, J., Salameh, A.B., Angeles, P. M., Francisco, de Cór-
doba J.F., Gasch C. (2006) Stereocontrolled synthesis of thio-
hydantoin spironucleosides from sugar spiroacetals. Tetrahe-
dron. 62, 97-111. https://doi.org/10.1016/j.tet.2005.09.128
25. Beloglazkina, E. K., Majouga, A. G., Romashkina, R. B., Zyk,
N. V. (2006) A novel catalyst for alkene epoxidation: a poly-
mer-supported CoIILCl2 {L = 2-(alkylthio)-3-phenyl-5-(pyr-
of
Medicinal
Chemistry.
46,
6,
2037-2042.
https://doi.org/10.1016/j.ejmech.2011.02.056
39. Legigan, T., Clarhaut, J., Tranoy‐Opalinski, I., Monvoisin,
A., Renoux, B., Thomas, M., Le Pape, A., Lerondel, S., Papot,
S. (2012) The First Generation of β‐Galactosidase‐Responsive
Prodrugs Designed for the Selective Treatment of Solid Tu-
mors in Prodrug Monotherapy. Angew. Chem. Int. Ed. 51,
11606-11610. https://doi: 10.1002/anie.201204935
40. Hassan, F., El-Hiti, G. A., Abd-Allateef, M., & Yousif, E.
(2017) Cytotoxicity anticancer activities of anastrozole
against breast, liver hepatocellular, and prostate cancer cells.
idine-2-ylmethylene)-
complex. Tetrahedron.
https://doi.org/10.1016/j.tetlet.2006.02.098
3,5-dihydro-4H-imidazole-4-one}
47, 2957-2959.
26. Kuznetsova, O., Antipin, R. L., Udina, A. V., Krasnovskaya,
O. O., Beloglazkina, E. K., Terenin, V. I., Koteliansky, V. E.,
Zyk, N. V., Majouga, A. G. (2016) An Improved Protocol for
Synthesis of 3‐Substituted 5‐Arylidene‐2‐thiohydantoins:
Two‐step Procedure Alternative to Classical Methods. J. Het-
Saudi
Medical
Journal.
38,
4,
359–365.
https://doi: 10.15537/smj.2017.4.17061
41. Hematyar, M., Soleimani, M., Es-haghi, A., Rezaei Mokar-
ram, A. (2018) Synergistic co-delivery of doxorubicin and
melittin using functionalized magnetic nanoparticles for can-
cer treatment: loading and in vitro release study by LC–
MS/MS. Artificial Cells, Nanomedicine, and Biotechnology.
19, 1-10. https://doi: 10.1080/21691401.2018.1536063.
42. Kleeberger., Rottinger M. E. (1993) Effect of pH and moder-
ate hyperthermia on doxorubicin, epirubicin and aclacinomy-
cin A cytotoxicity for Chinese hamster ovary cells. Cancer
Chemotherapy and Pharmacology. 33, 2, 144–148.
erocyclic
Chem.
53,
1570-1577,
https://doi.org/10.1002/jhet.2464
27. Tripathi, L. (2011) Design & synthesis of N'-[substituted] pyr-
idine-4- carbohydrazides as potential anticonvulsant agents.
Eur.
J.
Med.
Chem.
46,
2,
509-518.
https://doi.org/10.1016/j.ejmech.2010.11.030
28. Finnerup, N.B. (2015) Pharmacotherapy for neuropathic pain
in adults: a systematic review and meta-analysis. The Lancet.
14, 162-173. https://doi.org/10.1016/S1474-4422(14)70251-0
29. Shorvon, S. D. (2009). Drug treatment of epilepsy in the cen-
tury of the ILAE: The second 50 years, 1959-2009. Epilep-
sia. 5093,130. https://doi:10.1111/j.15281167.2009.02042.x
30. John, R. Green, L., Miller, H., Patsy, D., Burnett, A., Ward,
A. (1969) Clinical evaluation of albutoin. Neurology. 19,
12, 1207-1215. DOI: 10.1212/WNL.19.12.1207
31. Bridewell, D.J., Finlay, G.J., Baguley, B.C. (1997) Differen-
tial actions of aclarubicin and doxorubicin: the role of topoi-
somerase I. Oncol Res. 9, 535-42.
32. Capranico, G., Supino, R., Binaschi, M., Capolongo, L.,
Grandi, M. S, Zunino, F. (1994) Influence of structural
43. Nitiss, JL. (2009) Targeting DNA topoisomerase II in cancer
chemotherapy. Nat Rev Cancer. 9, 5, 338-350.
44. Chaikomon, K., Chattong, S., Chaiya, T., Tiwawech,
D., Sritana-Anant, Y., Sereemaspun, A., Manotham, K.
(2018) Doxorubicin-conjugated dexamethasone induced
MCF-7 apoptosis without entering the nucleus and able to
overcome MDR-1-induced resistance. Drug Des Devel Ther.
12, 2361–2369. https://doi: 10.2147/DDDT.S168588
45. Stewart, J.P. (2007) Optimization of parameters for
10
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 12
semiempirical methods V: Modification of NDDO approxi-
1
2
mations and application to 70 elements. J. Mol. Model. 13,
1173–1213.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
ACS Paragon Plus Environment

Page 13 of 12
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
ACS Paragon Plus Environment