Effect of Derivatives of Hydroxamic Acids on Vasculogenic Mimicry

Article abstract of DOI:10.1134/S106816202002017X

Abstract—: Vasculogenic mimicry, the formation of vascular channels lined with tumor cells of a highly malignant phenotype, is currently considered as an additional system of blood supply of the tumor. Experimental studies in vivo have repeatedly demonstrated that vascular channels form in the areas of a tumor with a low density of blood vessels. It is supposed that the formation of a network of these channels inside the tumor maintains homeostasis and prevents early necrosis within it. In this work, bifunctional compounds based on a combination of quinazoline and hydroxamic acid in one molecule were examined for the ability to inhibit the migration of tumor cells and vasculogenic mimicry.

Full text of DOI:10.1134/S106816202002017X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2020, Vol. 46, No. 2, pp. 252–263. © Pleiades Publishing, Ltd., 2020.
Russian Text © The Author(s), 2020, published in Bioorganicheskaya Khimiya, 2020, Vol. 46, No. 2, pp. 207–219.
Effect of Derivatives of Hydroxamic Acids on Vasculogenic Mimicry
A. A. Vartaniana, D. A. Khochenkova, Yu. A. Khochenkovaa, Yu. S. Machkovaa,
D. S. Khachatryanb, A. V. Kolotaevb, A. N. Balaevc, K. A. Ohmanovichc, and V. N. Osipova, 1
aBlokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation, Moscow, 115478 Russia
bInstitute of Chemical Reagents and High Purity Chemical Substances, National Research Center Kurchatov Institute,
Moscow, 123098 Russia
c JSC Farm-Sintez, Moscow, 121357 Russia
Received September 25, 2019; revised November 8, 2019; accepted November 14, 2019
Abstract—Vasculogenic mimicry, the formation of vascular channels lined with tumor cells of a highly malignant phe-
notype, is currently considered as an additional system of blood supply of the tumor. Experimental studies in vivo have
repeatedly demonstrated that vascular channels form in the areas of a tumor with a low density of blood vessels. It is
supposed that the formation of a network of these channels inside the tumor maintains homeostasis and prevents early
necrosis within it. In this work, bifunctional compounds based on a combination of quinazoline and hydroxamic acid
in one molecule were examined for the ability to inhibit the migration of tumor cells and vasculogenic mimicry.
Keywords: hydroxamic acids, quinazoline derivatives, melanoma, breast cancer, kidney cancer, migration,
vasculogenic mimicry
DOI: 10.1134/S106816202002017X
INTRODUCTION
present, there is not a single physiological process in
adult and children that is an analog of VM. The only
example of VM in humans is the formation of vascular
channels by cytotrophoblasts in the placenta during
embryogenesis; therefore, VM can be considered as a
tumor-specific process. This fact opens up new ways
for blocking the tumor growth with the minimal
impact on normal physiological processes.
The concept that VEGF-induced angiogenesis is a
factor that limits tumor growth is currently generally
accepted. According to the data of some clinical stud-
ies, antiangiogenic therapy significantly increases the
total survival of oncological patients [1]. At the same
time, evidence has accumulated indicating that most
tumors barely respond to anti-VEGF therapy [2]. One
reason for the survival of tumor cells during antiangio-
genic therapy may be the heterogeneity of blood ves-
sels. The formation of vessels in a tumor occurs against
the background of uncontrolled mitogenic stimulation
and the altered extracellular matrix. This leads to the
replacement of vascular endothelium by tumor cells;
sometimes, there can be no endothelial cells in vessels
at all. The term vasculogenic mimicry (VM), the for-
mation by tumor cells of vascular channels covered
with the basal membrane was introduced late in 1999
[3]. The generation of vascular channels lined with
tumor cells is a unique capacity of cells with a highly
malignant phenotype. It was found that there is a high
statistical correlation between the origination of VM in
a tumor and the incidence of metastasizing [4]. A
detailed examination of the role of some antiangio-
genic drugs used in the clinic in the modulation of VM
in vitro showed that these agents do not affect the for-
mation of a vascular network by tumor cells [5]. At
A promising strategy for creating new pharmaceu-
tical agents is currently the design and synthesis of
hybrid compounds that consist of two or more differ-
ent bioactive fragments and act through the activa-
tion/blocking of several targets. A combination of two
active groups in one molecule can lead to a more pro-
nounced therapeutic effect. Multipurpose hybrids also
offer several advantages over a combined therapeutic
approach. They are distinguished for better bioacces-
sibility, low toxicity, predictable pharmacokinetic and
pharmacodynamic profiles, a simpler regime of appli-
cation by a patient, a higher efficacy of treatment, and
a lower cost of the therapy (see for the review [6]).
Hydroxamic acids, which are capable of forming com-
plexes with copper, zinc, magnesium, and calcium
ions, block the activity of many metal-containing pro-
teins. In addition, hydroxamic acid derivatives exhibit
hypotensive [7], antimalarial [8], antituberculosis [9],
and fungicidal activities [10]. On the other hand, the
quinazoline cycle is present in the molecules of more
than a hundred medicines. At present, various
quinazoline and dihydroquinazoline derivatives are
being used in clinical practice [11].
Abbreviations: HOBt, N-hydroxybenztriazole; NMM, N-meth-
ylmorpholine;
TBTU,
2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethylaminium tetrafluoroborate; VEGF, vascular epithe-
lial growth factor; FCS, fetal calf serum; VM, vasculogenic
mimicry; CLS, capillary-like structure.
The modern methods of chemical synthesis make it
possible to introduce a hydroxamic group into various
natural and synthetic compounds, including the known
1 Corresponding author: phone: +7 (916) 412-61-43; e-mail:
ovn65@yandex.ru.
252

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
253
pharmaceuticals [12], increasing their therapeutic sizing rate, and, as a consequence, short-term survival
effect. We proposed that a combination of a quinazoline of patients [3]. Interestingly, vascular channels lined
fragment and hydroxamic acid in one molecule would with tumor cells are found in tumor areas with a low den-
enable one to create promising new antitumor drugs. sity of blood vessels. The results we have obtained in
The goal of the present study was the screening of com- recent years enabled us to formulate the conception that
pounds combining the quinazoline cycle and a antiangiogenic therapy activates the formation of vascu-
hydroxamic group in one molecule for the capacity to lar channels and transfers the tumor into a phase of more
affect the migration of tumor cells and VM.
aggressive growth [15]. Taking into account that vascular
channels are formed by tumor cells with a highly malig-
nant phenotype (today we are unable to kill these cells),
the search for a low-molecular-weight inhibitor of VM is
becoming an increasingly urgent problem. The goal of
our study was the synthesis of bifunctional compounds
and their screening for the capacity to block VM.
RESULTS AND DISCUSSION
Neoangiogenesis, the formation of microvessels on
the basis of a network of preexisting vessels in the tis-
sue, is a necessary condition for tumor growth [13]. A
great number of microvessels in a tumor promote its
rapid proliferation owing to the constant supply of
nutrients and oxygen and the excretion of toxic prod-
ucts of metabolism. In recent years, more than
Synthesis
We proposed that bifunctional molecules that con-
40 antiangiogenic agents have passed phase II and sist of two different bioactive fragments and act
phase III clinical trials, and some of them in combina- through the activation/blocking of several targets can
tion with chemotherapy have proved effective [1]. effectively inhibit VM. We synthesized several groups
However, most tumors do not respond to antiangio- of compounds containing a quinazoline cycle and a
genic therapy [2]. The key factor may be the heteroge- hydroxamic function. Compounds (IV) and (VII) were
neity of tumor vessels: classical angiogenesis in a synthesized by a general method (Scheme 1). At the first
tumor goes parallel with the formation of mosaic ves- stage, ethers were obtained from amino acids after which,
sels; in addition, the vessel cooption takes place in by the reaction with isatoic anhydride, derivatives of
which the tumor grows along vessels already existing in anthranilic acid amides were synthesized. Then, the
the tissue [14]. Numerous recent investigations have methyl esters of acids were converted into hydroxamates
shown that the presence of VM components in the by the reaction with hydroxylamine. At the final stage,
tumor materials of patients correlates with the rapid hydroxamic acids were condensed with 4-chloro-
progression of the tumor, an increase in the metasta- quinazoline to form target compounds (IV) and (VIII).
O
O
O
NHOH
N
H
NH2
N
H
NH2
i
ii
iii
5
5
H2N(CH2)nCOOH
H2N(CH2)nCOOCH3
n = 5 (Ia), 6 (Ib)
O
O
(II)
(III)
iv
O
NHOH
5
N
H
NH
O
(IV)
N
N
NH2
NH2
O
O
i
ii
iii
N
H
NH2
N
H
NH2
O
NHOH
O
O
(V)
(VI)
(VII)
OH
O
O
O
iv
O
N
H
NH
NHOH
O
N
(VIII)
N
Scheme 1. Reagents and conditions: (i) SOCl2/MeOH; (ii) isatoic anhydride/Na2CO3/DMF/H2O;
(iii) HCl ⋅ H2NOH/MeONa/MeOH; (iv) 4-chloroquinazoline/DMF/Δ.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020

254
VARTANIAN et al.
For the synthesis of compounds (XIIa) and (XIIb), aminoethers (Ia) and (Ib) were added; at the final
methyl-4-aminobenzoate was first condensed with 4- stage, hydroxamic acids were obtained (Scheme 2).
chloroquinazoline, the ether was hydrolyzed, and
O
O
O
O
O
HN
N
HN
OH
N
N
i
ii
N
(IX)
(X)
NH2
iii
O
O
HN
N
HN
NH(CH2)nCOOMe
NH(CH2)nCONHOH
N
N
iv
(XIIa, b)
n = 5 (XIa), 6 (XIb)
N
Scheme 2. Reagents and conditions: (i) 4-chloroquinazoline/DMF/Δ; (ii) LiOH/MeOH/H2O;
(iii) (Ia) or (Ib)/TBTU/HOBt/NMM/DMF; (iv) HCl ⋅ H2NOH/MeONa/MeOH.
Compounds (XIIIa)–(XIIIc) were obtained by the boiling methanol or ethanol in the presence of cata-
condensation of (III) with different carbonyl com- lytic amounts of p-toluenesulfonic acid (PTSA).
pounds (Scheme 3). The reaction was carried out in
O
H
N
CHO
CHO
OH
N
O
O
O
Cl
H
N
N
H
(XIIIa)
O
N
OH
F
Cl
O
(III)
O
N
H
O
(XIIIc)
O
CHO
H
N
O
OH
N
O
O
N
H
F
(XIIIb)
Scheme 3. Reagents and conditions: MeOH or EtOH/PTSA.
Under these conditions, the reaction of 4,5-dime- ducted in two stages using compound (II) as an initial
thoxy-2-formylbenzoic acid with compound (III) leads reagent; in this case, condensed tetracycle (XIIId) similar
to a complex mixture of products. The synthesis was con- to those described in [16] formed (Scheme 4).
O
O
CHO
O
O
COOH
O
O
O
O
N
N
i
N
ii
N
(IIa)
O
O
O
O
OH
O
N
H
(XIV)
(XIIId)
Scheme 4. Reagents and conditions: (i) PhCl/PTSA/Δ; (ii) HCl ⋅ H2NOH/MeONa/MeOH.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 2
2020

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
255
Compounds (XVa)–(XVc) were obtained by the tives of cyclic hydroxamic acid were obtained (Scheme 5).
condensation of anthranilic acid hydroxyamide with With the use of 2-formylbenzoic acid derivatives, con-
different carbonyl compounds; as a result, the deriva- densed tetracycles also formed.
O
OH
N
CHO
N
H
O
O
O
F
O
F
O
CHO
O
(XVa)
OH
NHOH
COOH
N
NH2
N
(XVb)
(XVc)
O
CHO
O
N
O
COOH
OH
N
O
O
O
O
Scheme 5. Reagents and conditions: MeOH or PhCl/PTSA/Δ.
Determination of Cytotoxic Activity
Inhibition of Migration Capacity
In recent decades, the efforts of many oncologists
were directed toward studying the mechanisms of ini-
tiation and progression of malignant tumors. With the
passage of time, it became clear that the main threat of
cancer is its spread throughout the body. Patients with
malignant tumors die not from the original tumor but
from metastases, which disorganize the functioning of
tissues they damage. Metastasizing, a process of
migration of tumor cells from the primary focus with
subsequent formation of secondary tumor foci (metas-
tases) is one of the major problems in the therapy of
oncological diseases. The migration of tumor cells is
currently considered as a key step of metastasizing. A
search for preparations inhibiting the movement of
tumor cells from the primary focus to adjacent or
remote organs is of fundamental importance and can
make possible the creation of new antitumor drugs
increasing the efficacy of therapy.
The migration of tumor cells was assessed by the
“wound-healing” method with some modifications. A
cell monolayer was broken by scraping, and the width
of the migration area was analyzed 24 h after the addi-
tion of compounds (IV) and (XIIIa)–(XIIId). A
monolayer of cells incubated in a culture medium
containing 10% FCS served as a positive control. Cells
growing in a serum-free medium served as a negative
control, which determines the width of a “wound,” a
cell-free space on the surface of a well. It was shown
VM is differently represented in different tumor
types. Thus, the blood supply through VM channels is
more than 60% in the case of skin melanoma, 50% in
the case of triple negative breast cancer, about 40% in
the case of kidney cancer, more than 35% in the case
of soft tissue sarcoma, more than 15% in the case of
ovary cancer, and about 10% in the case of colon can-
cer [15]. Against the background of extremely imper-
fect blood circulation in a tumor, 10–15% of VM
channels would hardly significantly change anything;
however, in the amount of 40–60%, they clearly may
be very important. Based on the above arguments, the
cytotoxic activity of compounds was tested on mela-
noma, breast cancer, and kidney cancer cells.
Compounds (IV) and (XIIIa)–(XIIId) with the
IС50 value less than 10 μM (Table 1) were chosen for
further examination of functional activity.
Other tests (migration and formation of CLS) were
performed at a noncytotoxic concentration of com-
pounds, IC10 (90% live cells after 24 h). The use of this
principle makes it possible to avoid false-positive
results, when, e.g., the blocking of migration by 60%
relative to the control is associated with the death of
cells within 24 h.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020

256
VARTANIAN et al.
Table 1. Cytotoxic activity of compounds toward SN-12C kidney cancer, Mel Z metastatic human melanoma, and MCF-7
breast cancer cells in vitro
SN-12С
Mel Z
MCF-7
Number
of compound
IC50, μM
IC10, μM
IC50, μM
IC10, μM
IC50, μM
IC10, μM
(IV)
(VIII)
4.7 1.5
>50
2.1 0.5
>50
4.1 0.8
>50
0.9 0.1
>50
9.5 1.58
>50
2.8 1.1
>50
(XIIa)
(XIIb)
(XIIIa)
(XIIIb)
(XIIIc)
(XIIId)
(XVa)
>50
25.0 3.2
6.2 0.6
1.7 0.2
3.1 0.9
0.7 0.2
1.6 0.3
>50
>50
23.0 4.8
6.6 2.7
1.2 0.3
>50
0.9 0.3
5.5 1.2
>50
>50
>50
28.6 2.7
>50
41.7 11.1
3.8 1.2
4.8 0.8
1.3 0.6
5.7 0.2
>50
28.7 11.2
8.1 0.3
8.9 1.3
1.9 0.3
5.7 1.4
>50
7.6 2.8
7.0 0.1
1.8 0.5
24.4 4.6
>50
0.7 0.2
0.7 0.3
0.7 0.2
4.7 1.8
>50
(XVb)
>50
>50
>50
>50
>50
>50
(XVc)
>50
>50
>50
>50
>50
>50
Sunitinib
2.4 0.3
1.0 0.5
2.7 0.4
1.2 0.3
2.9 0.3
1.3 0.2
that compounds (IV), (XIIIa), (XIIIb), and (XIIId) at
nontoxic concentrations (IC10) block the migration of
cells of all three types of tumor cells into the region of
the wound by 35 to 65% compared with the control.
The target inhibitor VEGFR2 sunitinib (Selleckchem)
at a nontoxic concentration of 1 μM, which inhibits
migration activity of Mel Z cells by 22.3%, of MCF-7
cells by 28.9%, and of SN-12C cells by 38.5%, served
as a reference preparation. Compound (IV) exhibited
the highest inhibitory activity toward Mel Z mela-
noma and SN-12C kidney cancer cells: the migration
activity was inhibited by 63.5 and 65.4%, respectively
(Fig. 1). Compounds (XIIIa) and (XIIId) inhibited the
migration of MCF-7 breast cancer cells by 64.9 and
69.3%, respectively. Compound (XIIIc) produced an
insignificant inhibitory effect on the migration of mel-
anoma and breast cancer cells (28–30%). With the use
of compound (XIIIc), the width of the migration area
in the case of kidney cancer cells was comparable with
that of the control sample (2.3%). The data on the
effect of compounds (IV) and (XIIIa)–(XIIId) on the
migration of tumor cells are presented in Table 2. The
blocking of migration by compound (IV) was maximal
in the case of SN-12C kidney cancer cells (65.4%) and
Mel Z melanoma cells (63.5%). Compound (XIIId)
appeared to be an efficient inhibitor of the migration
of MCF-7 breast cancer cells (64.9%). Presumably,
compounds (IV) and (XIIId) act on one of regulatory
pathways underlying the rearrangement of the actin
cytoskeleton of the cell. These results led us to the con-
clusion that it is advisable to create new analogs based
on compound (IV) and, probably, compound (XIIId)
Inhibition of Formation of CLS
The generation of vascular channels by tumor cells
involves a number of successive events: migration of
cells, recognition of homotypic cells, formation of
cell–cell contacts, cell sprouting, and formation of
homotypic structures similar to honey-like combs.
The formation of CLS by tumor cells in a 3D culture
serves as a test for VM in vitro [17]. In our studies,
Matrigel, a lyophilized natural extracellular gel was
used as a gel matrix. As noted above, compounds (IV)
and (XIIId) produced the most pronounced inhibitory
effect on cell migration. These compounds should also
block CLS to one degree or another, since migration is
the basic stage of VM. However, the other three com-
pounds could also participate in the multistage VM
process. Therefore, we examined the effect of all five
compounds on the formation of CLS in the 3D cul-
ture. In the control on Matrigel, three types of selected
tumor cells formed CLS. In melanoma cells growing
in the presence of compounds (IV) and (XIIIa), the
formation of CLS was inhibited by 54.4 and 47.4%,
respectively. Sunitinib inhibited the formation of a
network of Mel Z cells; the total length of CLS was
64.3% of the control. On Matrigel, along with the for-
mation of small clusters of cells, islets of the network
characteristic of VM were seen. Compounds (XIIIb)–
(XIIId) very little affected the ability of melanoma
cells to participate in VM. Compound (IV) produced
an unexpectedly strong effect on the formation of CLS
by SN-12C kidney cancer cells; it inhibited the forma-
tion of CLS by 72.7% (Fig. 2). Compounds (XIIIa),
(XIIIb), and (XIIId) inhibited the formation of VLS
by the same cells from 56.4 to 5.3%. Sunitinib induced
the blocking of CLS from SN-12C cells by 47.2% of
as potential inhibitors of the migration of tumor cells. the control (Table 3). It should be noted that none of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 2
2020

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
(а) (b) (c) (d)
257
Mel Z
SN-12C
MCF-7
Fig. 1. Inhibition of the migration of melanoma Mel Z, kidney cancer SN-12C, and breast cancer MCF-7 cells (staining with
trypan blue). (a) Negative control: wound width; (b) positive control: an increment in the cell monolayer growth in complete
medium after 24 h; (c) an increment in the cell monolayer growth in complete medium in the presence of compound (IV) at IC10
after 24 h; (d) an increment in the cell monolayer growth in complete medium in the presence of sunitinib at IC10 after 24 h.
the five compounds affected the formation of CLS by tion of CLS by breast cancer cells. The formation of
VM is a complex biological process, which, along with
the migration and homotypic recognition of cells,
involves several signaling pathways. Presumably, com-
pound (IV) is involved, in some or another way, in
driver signaling pathways that control VM.
breast cancer cells (data not shown).
Thus, new hydroxamic acid derivatives coupled
with the quinazoline cycle were synthesized. Their
effect on the migration of tumor cells and formation of
CLS was examined. We showed using the wound-
healing method that compound (IV) markedly blocks
the migration of melanoma, breast cancer, and kidney
cancer cells. In addition, in the presence of compound
(IV) the capacity of kidney cancer cells for communi-
EXPERIMENTAL
All reagents and solvents were commercially avail-
able products and were used without additional purifi-
cation, which is necessary for the formation of CLS, cation. NMR spectra were recorded on a Bruker
AVANCE III NanoBay Fourier NMR spectrometer
significantly decreased. In metastatic melanoma cells,
this effect was somewhat less pronounced. None of the
1
13
(300 MHz for H NMR and 76 MHz for C NMR).
five compounds had significant effect on the forma- Spectra were recorded in the deuterium stabilization
Table 2. Inhibition of migration activity of tumor cells by the action of hydroxamic acid derivatives at a concentration of IC10
in Mel Z metastatic human melanoma, SN-12C kidney cancer, and MCF-7 breast cancer cells
Inhibition of migration of tumor cells (% of cells without compounds)
Compound
OVFV
SN-12С
Mel Z
MCF-7
(IV)
65.4
58.6
14.1
2.3
45.7
38.5
63.5
48.5
35.5
34.1
62.0
22.3
55.8
64.9
54.1
28.0
69.4
28.9
(XIIIa)
(XIIIb)
(XIIIc)
(XIIId)
Sunitinib
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020

258
VARTANIAN et al.
(b)
(а)
(c)
SN-12C
Mel Z
Fig. 2. Effect of compound (IV) on the formation of CLS on Matrigel by SN-12C kidney cancer cells and Mel Z melanoma cells.
(a) Cells in complete medium RPMI-1640; (b) cells in complete medium RPMI-1640 in the presence of compound (IV) at IC10
(c) cells in complete medium RPMI-1640 in the presence of sunitinib at IC10
,
.
mode; thermal stabilization at 25°С, with tetramethyl- acid (tranexamic acid) as white crystals. Yield 93.4%.
1H NMR: 8.14 (br s, 3H), 3.57 (s, 2H), 2.60 (d, 2H, J
silane in DMSO-d6 as an internal standard. Chemical
shifts are given in ppm (δ), and the spin-spin coupling 6.9), 2.24 (tt, 1H, J 12.2, 3.5), 1.97–1.75 (m, 4H),
constants are in Hz. Electrospray ionization mass 1.64–1.47 (m, 1H), 1.28 (qd, 2H, J 12.9, 3.2), 0.97
(qd, 2H, J 12.9, 3.3). ESI-MS, m/z 172.1 [М + H]+.
spectra (ESI-MS) were recorded on an Agilent
LC/MS 1200 chromatographic system equipped with
an Agilent Ion Trap 6310 mass detector. High-resolu-
tion mass spectra were measured in the electrospray
ionization mode using an HPLC-MS analytical sta-
tion Agilent Infinity 1260/Thermo Scientific Orbitrap
Fusion Lumos. Melting points were determined in an
open capillary on a Mettler Toledo MP 90 melting
point analyzer.
Methyl (6-aminohexanoate) hydrochloride (Ia).
Thionyl chloride (6.0 g, 50 mmol) was added dropwise
at –5°С to methanol (50 mL). Then, 6-aminohexa-
noic acid (6.6 g, 25 mmol) was added, and the mixture
was stirred for 3 h at room temperature. The solvent
was removed in vacuum, methanol (20 mL) was
added, and the solvent was removed once again. The
product was obtained as white crystals. Yield 6.9 g
(95.0%). 1H NMR: 8.17 (s, 3H), 3.57 (s, 3H), 2.71 (t,
J 7.4, 2H), 2.28 (t, J 7.4, 2H), 1.65–1.42 (m, 4H),
1.36–1.20 (m, 2H). ESI-MS, m/z: 146.1 [М + H]+.
Methyl (7-aminoheptanoate) hydrochloride (Ib) was
obtained from 7-aminoheptanoic acid in a similar
way. Yield 54.2%; white crystals. 1H NMR: 8.11 (br s,
3H), 3.57 (s, 3H), 2.77–2.65 (m, 2H), 2.29 (t, 2H,
J 7.4), 1.61–1.44 (m, 4H), 1.36–1.18 (m, 4H). ESI-
MS, m/z 160.1 [М + H]+.
Methyl trans-4-aminomethylcyclohexan-1-carbox-
ylate hydrochloride (V) was obtained in a similar way
from trans-4-aminomethylcyclohexane carboxylic
Methyl 6-(2-aminobenzamido)hexanoate (II).
Methyl ether of 6-aminohexanoic acid (15 g, 83 mmol)
was dissolved in water (20 mL), and the pH of the
solution was brought to 7–8 with a 10% sodium hydro-
carbonate solution. The resulting solution was poured
into a suspension of isatoic anhydride (10 g, 61 mmol) in
dimethylformamide (DMF) (100 mL) in a flat-bot-
tom flask of volume 250 mL, and the resulting mixture
was stirred for 2 h at room temperature. The mixture
was poured into water (200 mL), and the pH of the
solution was brought to 3–4 with a 10% citric acid solu-
tion. After extraction with sulfuric ether (2 × 100 mL),
the organic layer was washed with water (50 mL). The
solvent was distilled off, and the residue was crystal-
Table 3. Formation of CLS by the action of hydroxamic
acid derivatives
Length of CLS within the network
Compound
(% of control)
OVFV
SN-12С
Mel Z
(IV)
27.3
43.6
71.8
94.7
47.2
45.6
52.6
77.2
80.3
64.3
(XIIIa)
(XIIIb)
(XIIIc)
Sunitinib
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 2
2020

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
259
1H NMR: 12.60 (s, 1H, NH–OH), 10.33 (s, 1H,
lized from hexane (50 mL). Yield 15.0 g (92%); white
1
crystals; mp 53–55°С. H NMR: 8.16 (t, 1H, J 5.7), NH–OH), 8.98 (d, 1H, NH, J 1.2), 8.90 (t, 1H,
7.46 (dd, 1H, J 7.9, 1.5), 7.15–7.08 (m, 1H), 6.68 (dd, CH2NH, J 5.6), 8.72 (s, 1H, Ar), 8.66 (s, 1H, Ar), 8.17
1H, J 8.3, 1.2), 6.36 (br s, 2H, NH2), 3.58 (s, 3H), 3.26–
(d, 1H, Ar, J 8.2), 7.95–7.81 (m, 3H, Ar), 7.75 (t, 1H,
3.13 (m, 2H), 2.30 (t, 2H, J 7.4), 1.64–1.42 (m, 4H), Ar, J 7.5), 7.60 (t, 1H, Ar, J 7.4), 7.19 (td, 1H, Ar, J 7.6,
1.39–1.22 (m, 2H). ESI-MS, m/z 265.1 [М + H]+.
1.2), 3.18 (td, 2H, J 6.9, 4.7), 1.94 (t, 2H, COCH2,
J 7.3), 1.62–1.45 (m, 4H, CH2), 1.38–1.21 (m, 2H,
Methyl 4-((2-aminobenzamido)methyl)cyclohexan-
1-carboxylate (VI) was obtained in a similar way from
the methyl ester of tranexamic acid. Yield 14.3 g
(89%); white crystals; mp 100–103°С. 1H NMR: 8.17
(t, 1H, J 5.8), 7.47 (dd, 1H, J 8.0, 1.5), 7.15–7.08 (m,
1H), 6.67 (dd, 1H, J 8.2, 1.2), 6.54–6.46 (m, 1H),
6.36 (br s, 2H, NH2), 3.58 (s, 3H), 3.06 (t, 2H, J 6.3),
2.33–2.17 (m, 1H), 1.90 (dd, 2H, J 13.4, 3.5), 1.77
(dd, 2H, J 13.2, 3.4), 1.59–1.40 (m, 1H), 1.28 (qd, 2H,
J 13.0, 3.3), 0.96 (qd, 2H, J 12.9, 3.4). ESI-MS, m/z
292.2 [М + H]+.
CH2). 13C NMR: 169.58, 169.24, 169.17, 157.23,
154.73, 149.78, 140.26, 140.11, 133.73, 132.38, 128.72,
128.55, 127.82, 122.73, 121.89, 121.34, 115.97, 32.65,
28.99, 26.54, 25.32. HRMS: m/z 394.1874 [M]+. Cal-
culated for (C21H23N5O3)+: 394.1877.
N-((4-(Hydroxycarbamoyl)cyclohexyl)methyl)-2-
(quinazolin-4-ylamino)-benzamide (VIII) was obtained
from 4-chloroquinazoline (164 mg, 1 mmol) and
compound (VII) (291 mg, 1 mmol) in a similar way as
compound (IV). Yield 97.4%; white crystals; mp
1
200.6–201.8°С. H NMR: 12.48 (s, 1Н, NH–OH),
2-Amino-N-(6-(hydroxyamino)-6-oxohexyl)ben-
zamide (III). A solution of hydroxylamine in methanol
(30 mL) obtained from sodium (1.15 g, 0.05 mol) and
hydroxylamine hydrochloride (2.10 g, 0.03 mol) was
added to a solution of compound (II) (2.64 g,
0.01 mol) in methanol (30 mL). The reaction mixture
was stirred for 6 h at room temperature. Methanol was
distilled off in vacuum, water (20 mL) was added, and
the solution was acidified to the pH 4 with 5% citric
acid. The precipitate was filtered and washed with
water (10 mL). Yield 2.20 g (83.3%); white crystals;
10.32 (s, 1Н, NH–OH), 8.94 (d, 1Н, NH, J 1.2), 8.87
(t, 2Н, CH2NH, J 5.0), 8.71 (s, 1H, Ar), 8.62 (s, 1H,
Ar), 8.16 (d, 1Н, Ar, J 7.8), 7.95–7.80 (m, 3H, Ar),
7.75 (t, 1Н, Ar, J 7.2), 7.60 (t, 1Н, Ar, J 7.8), 7.20 (t,
1Н, Ar, J 7.6), 3.18 (t, 2H, CH2, J 6.3), 2.01–1.85 (m,
1H, CH), 1.79 (d, 2H, CH2, J 12.7), 1.64 (d, 2H, CH2,
J 11.8), 1.34 (q, 2H, CH2, J 12.6), 0.95 (q, 2H, CH2, J
12.4). 13C NMR: 172.23, 172.15, 168.89, 156.87,
156.78, 154.36, 149.54, 139.72, 139.56, 133.22, 131.90,
128.33, 128.26, 127.29, 122.31, 121.38, 115.52, 45.33,
45.21, 41.18, 36.77, 29.67, 28.62. HRMS: m/z
420.2030 [M]+. Calculated for (C23H25N5O3)+:
420.2034.
1
mp 125–126°С. H NMR: 10.28 (s, 1H, NH–OH),
8.60 (s, 1H, NH–OH) 8.11 (t, 1H, CH2NH, J 5.6),
7.44 (dd, 1H, Ar, J 8.0, 1.5), 7.14–7.08 (m, 1H, Ar),
6.67 (dd, 1H, Ar, J 8.2, 1.2), 6.55–6.45 (m, 1H, Ar),
6.30 (br s, 2H, NH2), 3.19 (td, 2H, NCH2, J 7.5, 5.6),
1.95 (t, 2H, COCH2, J 7.5), 1.51 (m, 4H, 2CH2), 1.28
4-(Quinazolin-4-ylamino)benzoic acid (X). A solu-
tion of methyl 4-aminobenzoate (300 mg, 2 mmol) in
DMF (5 mL) was added to a solution of 4-chloro-
quinazoline (230 mg, 2 mmol) in DMF (5 mL). The
reaction mixture was stirred for 30 min at 40–50°С
and cooled to room temperature. The precipitate was
filtered and washed with DMF (5 mL) and sulfuric
ether (2 × 10 mL). To the resulting compound (IX), a
solution of NaOH (120 mg, 3 mmol) in a mixture of
water (5 mL) and methanol (10 mL) was added. The
mixture was refluxed for 2 h, water (10 mL) was added,
and the solution was acidified with 5% citric acid to
the pH 4. The sediment was filtered and washed with
water (10 mL). Yield 416 mg (78.5%); white crystals;
(p, 2H, CH2, J 7.5). ESI-MS, m/z 266.2 [М + H]+.
4-((2-Aminobenzamido)methyl)cyclohexan-1-
hydroxyaminocarbamide (VII) was obtained from com-
pound (VI) (164 mg, 1 mmol) in a similar way as com-
1
pound (III). Yield 97.4%; white crystals. H NMR:
10.33 (s, 1H, NH–OH), 8.62 (s, 1H, NH–OH), 8.16
(t, 1H, J 5.5), 7.46 (dd, 1H, Ar, J 7.9, 1.1), 7.12 (td, 1H,
Ar, J 8.2, 1.4), 6.67 (dd, 1H, Ar, J 8.1, 1.3), 6.50 (td,
1H, Ar, J 8.0, 1.1), 6.34 (br s, 2H, NH2), 3.05 (t, 2H,
CH2, J 5.5), 2.31–2.14 (m, 1H), 1.88 (dd, 2H, CH2, J
13.2, 3.5), 1.75 (dd, 2H, CH2, J 13.2, 3.5), 1.57–1.41
(m, 1H), 1.27 (qd, 2H, CH2, J 13.0, 3.3), 0.95 (qd, 2H,
1
mp 296.1–299.2°С (with decomposition). H NMR:
10.67 (s, 1H, OH), 8.85 (s, 1H, NH), 8.68 (d, 1H, Ar,
J 8.3), 8.06–7.97 (m, 5H, Ar), 7.87 (d, 1H, Ar, J 7.2),
7.79 (t, 1H, Ar, J 7.7).
CH2, J 12.9, 3.4). ESI-MS, m/z 290.3 [М + H]+.
N-(6-(Hydroxyamino)-6-oxohexyl)-2-(quinazolin-
4-ylamino)benzamide (IV). A solution of compound
Methyl 6-((4-(quinazolin-4-ylamino)benzoyl)amino)-
(III) (265 mg, 1 mmol) in DMF (5 mL) was added to hexanoate (XIa). Compound (X) (530 mg, 2 mol) and
a solution of 4-chloroquinazoline (164 mg, 1 mmol) in thereafter methyl 6-amino hexanoate hydrochloride
DMF (5 mL). The reaction mixture was stirred for (362 mg, 2 mmol) were added to a solution of TBTU
30 min at 40–50°С and cooled to room temperature. (642 mg, 2 mmol), hydroxybenztriazole hydrate
The sediment was filtered and washed with DMF (304 mg, 2 mmol), and N-methylmorpholine (606 mg,
(5 mL) and sulfuric ether (2 × 10 mL). Yield 384 mg 6 mmol) in DMF (20 mL). The mixture was stirred for
(97.6%); light beige crystals; mp 180.8–187.5°С. 5 h at room temperature, water (30 mL) was added,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020

260
VARTANIAN et al.
and the extraction with ethyl acetate (2 × 25 mL) was vacuum. The resulting solid residue was triturated with
carried out. The organic layer was washed with 5% cit-
ric acid (20 mL), water (10 mL), a 5% sodium bicar-
bonate solution (10 mL), and water (10 mL). The sol-
vent was distilled off in vacuum. Yield 576 g (73.5%);
beige crystals. ESI-MS, m/z 393.2 [М + H]+. The
product was used in further syntheses without addi-
tional purification.
Methyl 7-((4-(quinazolin-4-ylamino)benzoyl)amino)-
heptanoate (XIb) was obtained in a similar way as
compound (XIa) using methyl 7-aminoheptanoate
hydrochloride (390 mg, 2 mmol). Yield 610 mg
(75.1%). ESI-MS, m/z 407.3 [М + H]+. The product
was used in further syntheses without additional puri-
fication.
diethyl ether (5 mL) and air-dried. Yield 289 mg
1
(74.8%); mp 111.4–112.3°С. H NMR: 10.31 (s, 1Н,
NH–OH), 7.63 (d, 1Н, Ar, J 7.4), 7.45–7.29 (m, 4Н,
Ar), 7.19 (t, 1Н, Ar, J 7.6), 6.70–6.57 (m, 2Н, Ar),
5.86 (s, 1Н, N–CH–N), 3.96–3.79 (m, 1H, NCH2),
2.79–2.64 (m, 1H, NCH2), 1.91 (t, 2Н, СОСН2, J
7.3), 1.61–1.37 (m, 4H, 2CH2), 1.33–1.16 (m, 2H,
CH2CH2–CO). HRMS: m/z 388.1422 [M]+. Calcu-
lated for (C20H22ClN3O3)+: 388.1426.
N-Hydroxy-6-(2-(3-methoxyphenyl)-4-oxo-1,2-
dihydroquinazolin-3(4H)-yl)hexamide (XIIIb). A solu-
tion of compound (III) (265 mg, 1 mmol) and 4-chlo-
robenzaldehyde (147 mg, 1.05 mmol) in ethanol
(5 mL) was stirred for 6 h under boiling in an atmo-
sphere of argon, and the solvent was distilled off in
vacuum. The resulting solid residue was triturated with
diethyl ether (5 mL) and air-dried. Yield 299 mg
N-(6-(Hydroxyamino)-6-oxohexyl)-4-(quinazolin-
4-ylamino)benzamide (XIIa). Hydroxylamine hydro-
chloride (208 mg, 3.00 mmol) was added to a solution
of sodium methylate obtained by dissolving sodium
(92 mg, 4.00 mmol) in methanol (10 mL) at 0°C, the
mixture was stirred for 20 min at 0°C, and compound
(XIa) (392 mg, 1 mmol) was added. The reaction mix-
ture was stirred for 5 h at room temperature (TLC con-
trol), water (10 mL) was added, the mixture was acid-
ified with 5% hydrochloric acid to the pH 4–5, and
methanol was distilled off. The precipitate was fil-
tered, washed with water, and air-dried. Yield 320 mg
(81.2%); white crystals; mp 212.9°С. 1H NMR: 10.34
(s, 1Н, NH–OH), 9.94 (s, 1Н, NH–OH), 8.67 (br s,
2H, NH + Ar), 8.59 (d, 1H, Ar, J 8.3), 8.37 (t, 1H,
CH2NH, J 5.5), 8.01 (d, 2H, Ar, J 8.5), 7.93–7.79 (m,
4H, Ar), 7.67 (t, 1H, Ar, J 7.5), 3.30–3.19 (m, 1H,
CH2NH), 1.96 (t, 2H, COCH2, J 7.3), 1.61–1.45 (m,
4H, CH2), 1.38–1.20 (m, 2H, CH2). HRMS: m/z
1
(78.1%); mp 119.0–120.2°С. H NMR: 10.31 (s, 1Н,
NH–OH), 7.66 (dd, 1Н, Ar, J 7.9, 1.6), 7.28 (td, 1H,
Ar, J 8.2, 1.8), 7.18 (td, 1H, Ar, J 7.6, 1.4), 7.13 (d, 1H,
Ar, J 7.6), 7.05 (d, 1H, Ar, J 8.2), 6.86 (t 1H, Ar, J 7.5),
6.70–6.60 (m, 2H, Ar), 6.14 (s, 1H, N–CH–N),
3.96–3.82 (m, 1H, NCH2), 3.70 (s, 3H, OCH3),
2.80–2.63 (m, 1H, NCH2), 1.91 (t, 2Н, СО–СН2, J
7.3), 1.63–1.40 (m, 4H, 2CH2), 1.37–1.17 (m, 2H,
CH2–CH2–CO). 13C NMR: 169.09, 162.22, 159.31,
146.18, 142.77, 133.13, 129.64, 127.34, 118.14, 117.15,
115.03, 114.22, 113.25, 112.36, 69.81, 55.05, 44.29,
32.17, 27.22, 25.94, 24.85. HRMS: m/z 384.1918 [M]+.
Calculated for (C21H25N3O4)+: 384.1922.
N-Hydroxy-6-(2-(4-methoxy)-3-(2-fluorophenoxy)-
methylphenyl)-4-oxo-1,2-dihydroquinazolin-3(4H)-yl)-
hexamide (XIIIc). A solution of compound (III)
(159 mg, 0.60 mmol) and 4-methoxy-3-(2-fluoro-
phenoxy)methylbenzaldehyde (164 mg, 0.63 mmol)
in methanol (5 mL) was stirred for 6 h under boiling in
an atmosphere of argon, and the solvent was distilled
off in vacuum. The resulting solid residue was tritu-
rated with diethyl ether (5 mL) and air-dried. Yield
301 mg (98.8%); mp 102.8–104.7°С. 1H NMR: 10.30
(s, 1Н, NH–OH), 7.61 (dd, 1H, Ar, J 7.8, 1.6), 7.42
(d, 1H, Ar, J 2.3), 7.32–7.06 (m, 6H, Ar), 7.03 (d, 1H,
Ar, J 8.6), 6.98–6.88 (m, 1H, Ar), 6.67–6.58 (m, 2H,
Ar), 5.79 (s, 1H, N–CH–N), 5.06 (s, 2H, OCH2),
3.88–3.80 (m, 2H, NCH2), 3.79 (s, 3H, OCH3),
2.74–2.58 (m, 1H, NCH2), 1.90 (t, 2Н, СО–СН2, J
7.3), 1.60–1.33 (m, 4H, 2CH2), 1.25–1.11 (m, 2H,
394.1800 [M]+. Calculated for (C21H23N5O3)+:
394.1877.
N-(7-(Hydroxyamino)-7-oxoheptyl)-4-(quinazolin-
4-ylamino)benzamide (XIIb) was obtained from com-
pound (XIb) (406 mg, 1 mmol) in a similar way as
compound (XIIa). Yield 310 mg (76.1%); white crys-
1
tals; mp 237.8–238.3°С. H NMR: 10.34 (s, 1H,
NHOH), 9.95 (s, 1H, NHOH), 8.66 (br s, 2H, NH +
Ar), 8.60 (d, 1H, Ar, J 8.4), 8.37 (t, 1H, CH2NH, J
5.6), 8.01 (d, 2H, Ar, J 8.4), 7.93–7.79 (m, 4H, Ar),
7.67 (t, 1H, Ar), 3.30–3.18 (m, 2H, CH2), 1.95 (m,
2H, CH2, J 7.3), 1.60–1.41 (m, 4H, 2CH2), 1.39–1.24
(m, 4H, 2CH2). 13C NMR: 169.19, 165.62, 157.49,
154.33, 149.74, 141.72, 133.21, 129.35, 127.88, 127.63,
126.47, 123.08, 121.00, 115.22, 32.25, 29.13, 28.39,
26.28, 25.12. HRMS: m/z 408.2030 [M]+. Calculated
for (C22H25N5O3)+: 408.2034.
2CH2). 13C NMR: 169.11, 162.21, 157.04, 153.46,
150.24, 146.28, 146.23, 146.15, 133.06, 132.96, 127.41,
127.35, 124.78, 124.74, 124.23, 121.31, 121.22, 117.08,
116.15, 115.92, 115.42, 114.98, 114.19, 110.94, 69.64,
65.66, 55.68, 44.04, 32.19, 27.12, 25.97, 24.83.
HRMS: m/z 508.2242 [M]+. Calculated for
(C28H30FN3O5)+: 508.2246.
N-Hydroxy-6-(2-(4-chlorophenyl)-4-oxo-1,2-
dihydroquinazolin-3(4H)-yl)hexamide (XIIIa). A solu-
tion of compound (III) (265 mg, 1 mmol) and 4-chlo-
robenzaldehyde (147 mg, 1.05 mmol) in ethanol
(5 mL) was stirred for 6 h under boiling in an atmo-
sphere of argon, and the solvent was distilled off in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 2
2020

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
261
Methyl 6-(9,10-dimethoxy-5,11-dioxoindolo[2,1- (d, 1H, NH, J 2.3), 7.41 (dd, 1H, Ar, J 8.5, 2.3), 7.36
a]quinazolin-6(5H,6aH,11H)-yl)hexanoate (XIV). A (d, 1H, J 1.8), 7.28–7.15 (m, Ar, 4H), 7.15–7.03 (m,
solution of compound (IIa) (264 mg, 1.0 mmol) and Ar, 2H), 7.01–6.88 (m, Ar, 1H), 6.76–6.63 (m, Ar,
2,3-dimethoxy-6-formylbenzoic acid (242 mg, 2H), 5.87 (d, 1H, J 1.7), 5.07 (s, 2H, CH2), 3.81 (s,
3H). 13C NMR: 162.94, 157.35, 153.41, 150.18, 146.48,
146.35, 146.22, 134.59, 133.35, 131.74, 131.70, 129.50,
128.52, 128.39, 127.28, 127.19, 127.10, 124.85, 124.80,
121.31, 121.23, 121.15, 117.34, 114.06, 110.75, 74.60,
65.80, 55.74. HRMS: m/z 395.1402 [M]+. Calculated
for (C22H19FN2O4)+: 395.1405.
1.15 mmol) in chlorobenzene (5 mL) in the presence
of a catalytic amount of p-toluenesulfonic acid mono-
hydrate was refluxed for 4 h and cooled to room tem-
perature after which diethyl ether (10 mL) was added.
The precipitate was filtered and air-dried. Yield
392 mg (89%); white crystals; mp 123.9–124.4°C. 1H
NMR: 8.04–7.90 (m, 2H, Ar), 7.75–7.64 (m, 1H,
Ar), 7.60 (d, 1H, Ar, J 8.3), 7.46 (d, 1H, Ar, J 8.3), 7.36
(t, 1H, Ar, J 7.6), 6.40 (s, 1H, NCHN), 3.92 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 3.71–3.63 (m, 2H,
NCH2), 3.56 (s, 3H, CO2CH3), 2.22 (t, 2H, CH2, J
7.3), 1.56–1.35 (m, 4H, 2CH2), 1.34–1.07 (m, 2H,
6-Hydroxy-6,6a-dihydroisoindolo[2,1-a]quinazolin-
5,11-dione (XVb). A solution of anthranilic acid
hydroxyamide (0.45 g, 3.30 mmol) and 2-formylben-
zoic acid (0.50 g, 3.30 mmol) in the presence of a cat-
alytic amount of p-toluenesulfonic acid monohydrate
in chlorobenzene (13 mL) was refluxed for 7 h, and the
reaction mixture was evaporated to dryness and tritu-
rated with diethyl ether (10 mL). The precipitate was
filtered and air-dried to a constant weight. Yield 0.77 g
(96%); white crystals; mp 198–199ºC. 1H NMR: 9.41
(s, 1H, NOH), 8.08 (dd, 1H, Ar, J 8.2, 0.6), 7.97 (dd,
1H, Ar, J 7.8, 1.6), 7.89 (br d, 2H, Ar, J 8.1), 7.79 (td,
1H, Ar, J 7.4, 1.3), 7.74–7.64 (m, 2H, Ar), 7.35 (td,
1H, Ar, J 7.6, 1.1), 6.52 (s, 1H, NCHN). 13C NMR:
165.26, 164.88, 139.55, 136.41, 133.68, 133.22, 131.27,
130.58, 128.22, 126.72, 125.24, 123.85, 120.00, 119.25,
72.72. HRMS: m/z 267.0764 [M]+. Calculated for
(C15H10N2O3)+: 267.0769.
CH2). ESI-MS, m/z 439.5 [М + H]+.
6-(9,10-Dimethoxy-5,11-dioxoisoindolo[2,1-a]-
quinazolin-6(5H,6aH,11H)-yl)-N-hydroxyhexanamide
(XIIId). Hydroxylamine hydrochloride (139 mg,
2 mmol) was added to a solution of sodium methylate
obtained by dissolving sodium (69 mg, 3 mmol) in
methanol (7 mL) at 0°С. The mixture was stirred at
0°C for 20 min after which compound (XIV) (378 mg,
0.86 mmol was added. The reaction mixture was
stirred for 3 h at room temperature (TLC control),
water was added (5 ml), the solution was acidified with
5% hydrochloric acid to a pH value of 4–5, and meth-
anol was evaporated. The precipitate was filtered,
washed with water, and air-dried. Yield 200 mg
(52.9%); white crystals; mp 136.1–141.8°C. 1H NMR:
10.31 (s, 1H, NHOH), 8.65 (s, 1H, Ar), 7.97 (d, 1H,
Ar, J 6.9), 7.94 (d, 1H, Ar, J 7.3), 7.68 (t, 1H, Ar, J
7.9), 7.59 (d, 1H, Ar, J 8.3), 7.47 (d, 1H, Ar, J 8.3),
7.36 (t, 1H, Ar, J 7.6), 6.40 (s, 1H, NCHN), 3.92 (s,
3H, OCH3), 3.91 (s, 3H, OCH3), 3.79–3.54 (m, 2H,
NCH2), 1.88 (t, 2H, COCH2, J 7.3), 1.54–1.36 (m,
6-Hydroxy-9,10-dimethoxy-6,6a-dihydroisoindolo-
[2,1-a]quinazolin-5,11-dione (XVc). A solution of anthra-
nilic acid amide (0.45 g, 3.30 mmol) and 6-formyl-2,3-
dimethoxybenzoic acid (0.80 g, 3.80 mmol) in the pres-
ence of a catalytic amount of p-toluenesulfonic acid
monohydrate in chlorobenzene (15 mL) was refluxed
for 7 h, and the reaction mixture was evaporated to
dryness and triturated with diethyl ether (10 mL). The
precipitate was filtered and air-dried to a constant
weight. Yield 0.94 g (92%); white crystals; mp 240°C
(with decomposition). 1H NMR: 9.28 (s, 1H, NOH),
8.04 (d, 1H, Ar, J 7.8), 7.95 (dd, 1H, Ar, J 7.8, 1.6),
7.68 (td, 1H, Ar, J 7.8, 1.6), 7.52 (d, 1H, Ar, J 8.3), 7.44
(d, 1H, Ar, J 8.3), 7.33 (td, 1H, Ar, J 7.6, 1.1), 6.36 (s,
1H, NCHN), 3.91 (s, 3H, OCH3), 3.88 (s, 3H,
4H, 2CH2), 1.34–1.10 (m, 2H, CH2). 13C NMR:
169.05, 162.71, 162.58, 153.83, 147.09, 136.51, 133.07,
130.75, 128.32, 124.99, 123.82, 121.64, 120.46, 120.24,
117.53, 68.85, 61.84, 56.44, 41.97, 32.13, 27.66, 25.80,
24.80. HRMS: m/z 440.1816 [M]+. Calculated for
(C23H25N3O6)+: 440.1819.
OCH3). 13C NMR: 164.76, 163.40, 153.81, 146.70,
136.47, 133.51, 131.79, 128.13, 125.16, 123.26, 122.27,
120.25, 119.39, 117.80, 71.65, 61.76, 56.50. HRMS:
m/z 327.0903 [M]+. Calculated for (C17H14N2O5)+:
327.0979.
3-Hydroxy-2-(3-(4-methoxy-(2-fluorophenoxy)-
methyl)-phenyl)-2,3-dihydroquinazolin-4(1H)-one (XVa).
A mixture of 2-aminobenzhydroxamic acid (0.30 g,
2 mmol) and 3-((2-fluorophenoxy)methyl)-4-methoxy-
benzaldehyde (0.52 g, 2 mmol) in methanol (7 mL) in
the presence of a catalytic amount of p-toluenesul-
fonic acid monohydrate was refluxed for 4 h in an
atmosphere of argon, cooled to –15°C, and allowed to
stand for 8 h. The precipitate was filtered, the filtrate
Cell Сulture
Mel Z melanoma cells were used, which were
was evaporated in vacuum, and the resulting residue derived as a cell line from metastatic nodes of a patient
was triturated with diethyl ether (10 mL) and air-dried with skin melanoma, who underwent treatment at the
to yield 0.62 g (79%) of the product as flesh-colored Blokhin National Medical Research Center of Oncol-
crystals; mp 209.7°C (with decomposition). 1H NMR:
ogy, Ministry of Health of the Russian Federation
9.64 (s, 1H, NOH), 7.65 (dd, 1H, Ar, J 8.0, 1.6), 7.54 [18]. MCF-7 breast cancer cells (ATCC® HTB-22™)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020

262
VARTANIAN et al.
and SN-12C kidney cancer cells [19] were obtained tomycin (PanEko, Russia) and incubated until the for-
from the bank of cell cultures at the Blokhin National mation of a monolayer. Then, the monolayer was bro-
Medical Research Center of Oncology, Ministry of ken by scraping a part of cells (by a 200 μL tip) in a
Health of the Russian Federation.
straight line through the center. Cells were incubated
for 24 h in complete growth medium RPMI-1640 or
DMEM containing 10% FCS (HyClone), 2 mM glu-
tamine, and each of the test compounds at noncyto-
toxic concentrations (IC10). As a negative control,
which determines the width of a “wound,” cells cul-
tured in serum-free medium RPMI-1640 or DMEM
were used. Cells growing in complete medium RPMI-
1640 or DMEM containing 10% FCS without test
compounds served as a positive control, which deter-
mines a maximum growth of the monolayer in the
wound area. Sunitinib at a noncytotoxic concentration
of 1 μМ (Selleckchem) served as a reference com-
pound. After the termination of incubation, the cell
monolayer on plates was washed twice with PBS and
stained with trypan blue (PanEkon, Russia). The
degree of the inhibition of migration activity was
defined as the ratio (in percent) of the increment in the
cell monolayer area by the action of a test compound
to the increment in the growth of cells in the positive
control. The width of the wound in the negative con-
trol was used as the basic value for the calculation of
the wound width.
Mel Z and SN-12C tumor cells were cultured on
complete growth medium RPMI-1640 (Gibco) con-
taining 10% FCS (HyClone), 2 mM glutamine, and
50 mg/mL of penicillin/streptomycin (PanEko, Rus-
sia) in Eppendorf cell culture flasks at 37°С in an
atmosphere of 5% CO2. MCF-7 cells were cultured on
complete growth medium DMEM (Gibco) supple-
mented with 10% FCS (HyClone), 2 mM glutamine,
and 50 mg/mL of penicillin/streptomycin (PanEko,
Russia) in Eppendorf cell culture flasks at 37°С in an
atmosphere of 5% CO2. Cells were maintained in the
logarithmic growth phase by the constant reseeding of
the culture every two to three days.
MTT Test
The cytotoxic activity of compounds was estimated
by the standard MTT test using the MTT reagent
(3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
bromide); the test is based on the ability of dehydroge-
nases of live metabolically active cells to reduce the MTT
reagent to blue insoluble formazan crystals. For each com-
pound, a graph of the dose-effect dependence was plotted,
and the IС50 and IС10 values were determined using the
program GraphPad Prizm 5.0 (GraphPad). The experi-
mental error was no more than 5%.
MCF-7, Mel Z, and SN-12C tumor cells (5 ×
104 cell/mL) were placed into the wells of a 96-well
plate (Nunc) in complete growth medium RPMI-
1640 or DMEM (for MCF-7) supplemented with 10%
FCS (HyClone), 2 mM glutamine, and 50 mg/mL of
penicillin/streptomycin (PanEko, Russia). After 24 h,
compounds at concentrations of 10–4–10–10 were
added, and cells were incubated for 48 h after which
the MTT solution (Sigma, Chemical Co, United
States) at a final concentration of 0.5 mg/mL was
added, 10 μL to each well. Cells were incubated for an
additional 4 h and precipitated by the centrifugation of
plates for 3 min at 1000 rpm on a Hettich 460R centri-
fuge (Hettich Lab Technology, Germany). Medium
was taken, and DMSO (200 μL) was added to cells.
Cells were resuspended and incubated for 10 min at
37°С after which the optical density of the formazan
solution was immediately determined on a Multiskan
FC microplate photometer (Thermo Scientific) at
570 nm using DMSO as a zero control.
Capillary-Like Structure Formation in 3D Culture
Matrigel (Corning) was defrosted at 4°С, applied
onto the wells of a 24-well plate (Nunc) (100 μL onto
each) on ice, and allowed to stand under sterile condi-
tions for matrix polymerization for 1 h at room tempera-
ture and for 30 min at 37°С in a СО2 incubator. Mel Z
and SN-12C cells at a concentration of 4 × 105 cell/mL
in complete growth medium RPMI-1640 (Gibco)
supplemented with 10% FCS were applied onto gel
and incubated at 37°С for 12 h in the presence of test
compounds at noncytotoxic concentrations (IС10).
Cells incubated in medium RPMI-1640 containing
10% FCS (Gibco) without test compounds served as a
positive control. After the termination of incubation,
the resulting network of CLS was photographed using
a digital camera (Canon), and the length of CLS from
tumor cells within the closed network was estimated
using the program Image J v.1.73 (NIH, United States,
freeware).
Statistical Analysis
All experiments were performed three times inde-
pendently of one another. The data are given as the
mean standard deviation. Differences were consid-
Estimation of the Blocking of Migration Capacity
of Cells by the Wound-Healing Method
Tumor cells (3 × 105 cell/mL) were placed into the ered statistically significant if P values were less than
wells of a 24-well plate (Nunc) in medium RPMI- 0.05. All statistical analyses were carried out using the
1640 or DMEM containing 10% FCS (HyClone), software GraphPad Prism (GraphPad Software, La
2 mM glutamine, and 50 mg/mL of penicillin/strep- Jolla, San-Diego, California, United States).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 2
2020

EFFECT OF DERIVATIVES OF HYDROXAMIC ACIDS ON VASCULOGENIC MIMICRY
263
FUNDING
6. Berube, G., Expert Opin. Drug Discov., 2016, vol. 11,
no. 3, pp. 281–305.
The study was supported by the Ministry of Science and
Higher Education of the Russian Federation (Agreement
no. 075-11-2018-172 of 03.12.18). The unique identifier of
the project is RFMEFI62418X0051.
7. Yoon, S. and Eom, G.H., Chonnam Med. J., 2016,
vol. 52, no. 1, pp. 1–11.
8. Giannini, G., Battistuzzi, G., and Vignola, D., Bioorg.
Med. Chem. Lett., 2015, vol. 25, no. 3, pp. 459–461.
9. Rao, M., Valentini, D., Zumla, A., and Maeurer, M.,
COMPLIANCE WITH ETHICAL STANDARDS
Int. J. Infect. Dis., 2018, vol. 69, pp. 78–84.
10. Muri, E.M., Nieto, M.J., Sindelar, R.D., and William-
son, J.S., Curr. Med. Chem., 2002, vol. 9, pp. 1631–
1653.
This article does not contain any studies involving ani-
mals or human participants performed by any of the
authors.
11. Ahmad, S. and Ahmad, I., Med. Chem. Commun., 2017,
vol. 8, pp. 871–885.
Conflict of Interests
12. Asif, M., Int. J. Med. Chem., 2014, article ID 395637.
The authors declare that there is no conflict of interest.
13. Folkman, J., Curr. Mol. Med., 2003, vol. 3, pp. 643–
651.
14. Hillen, F. and Griffioen, A.W., Cancer Metastasis Rev.,
REFERENCES
2007, vol. 26, pp. 489–502.
1. Persy, I.S., Wick, J.Y., and Kaufman, B., Nat. Rev.
15. Vartanian, A., Golubeva, I., and Shprakh, Z., Horizons
Clin. Oncol., 2009, vol. 6, pp. 569–577.
Cancer Res., 2017, vol. 65, pp. 13–34.
2. Azam, F., Mehta, S., and Harris, A.L., Eur. J. Cancer,
16. Sashidhara, K.V., Palnati, G.R., Dodda, R.P., Avula, S.R.,
2010, vol. 46, pp. 1323–1332.
and Swami, P., Synlett, 2013, vol. 24, pp. 105–113.
3. Maniotis, A., Folberg, R., Hess, A., Seftor, E.A.,
Gardner, L.M., Pe’er, J., Trent, J.M., Meltzer, P.S.,
and Hendrix, M.J., Am. J. Pathol., 1999, vol. 55,
pp. 739–752.
17. Vailhe, B., Vittet, D., and Feige, J., Lab. Invest., 2001,
vol. 81, pp. 439–452.
18. Mikhailova, I.N., Lukashina, M.I., Baryshnikov, A.Yu.,
et al., Vestn. Ross. Akad. Med. Nauk., 2005, vol. 7,
pp. 37–40.
4. Zhang, S., Zhang, D., and Sun, B., Cancer Lett., 2007,
vol. 254, pp. 157–164.
19. Naito, S., von Eschenbach, A.C., Giavazzi, R., and
5. Van der Schaft, W., Seftor, R.E., Seftor, E.A., Hess, A.R.,
Gruman, L.M., Kirschmann, D.A., Yokoyama, Y.,
Griffoen, A.W., and Hedrix, M.J., J. Natl. Cancer Inst.,
2004, vol. 96, no. 19, pp. 1473–1477.
Fidler, I.J., Cancer Res., 1986, vol. 46, pp. 4109–4115.
Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 2 2020