Synthesis and Antitumor Activity of Conjugates Based on the Phe-D-Trp-Lys-Thr Peptide Fragment of Somatostatin

Article abstract of DOI:10.1134/S1068162019040034

Abstract: New somatostatin analogs containing the fragments of adamantane, coumarin, tetrahydrocarbazole, and palmitic acid of the general formula R-Phe-D-Trp-Lys(Boc)-Thr-OMe have been synthesized. The structure of the conjugates combines a peptide fragm

Full text of DOI:10.1134/S1068162019040034

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2019, Vol. 45, No. 4, pp. 248–252. © Pleiades Publishing, Ltd., 2019.
Russian Text © The Author(s), 2019, published in Bioorganicheskaya Khimiya, 2019, Vol. 45, No. 4, pp. 374–379.
Synthesis and Antitumor Activity of Conjugates Based
on the Phe-D-Trp-Lys-Thr Peptide Fragment of Somatostatin
D. V. Avdeeva, b, 1, M. V. Sidorovaa, M. V. Ovchinnikova, N. I. Moiseevac,
V. N. Osipovc, d, A. N. Balaevd, and D. S. Khachatryane
aResearch Center of Cardiology, Moscow, 121552 Russia
bHigher College of Chemistry, Russian Academy of Sciences, Moscow, 125047 Russia
cBlokhin Russian Oncological Research Center, Russian Academy of Medical Sciences, Moscow, 115478 Russia
dAO Farm-Synthesis, Moscow, 121357 Russia
eResearch Center Kurchatov Institute—IREA, Moscow, 107076 Russia
Received November 19, 2018; revised January 14, 2019; accepted February 12, 2019
Abstract—New somatostatin analogs containing the fragments of adamantane, coumarin, tetrahydrocarba-
zole, and palmitic acid of the general formula R-Phe-D-Trp-Lys(Boc)-Thr-OMe have been synthesized. The
structure of the conjugates combines a peptide fragment, which has affinity for somatostatin receptors (sstr),
and a nonpeptide fragment, which potentially possesses antitumor activity. Presumably, the compounds syn-
thesized are the agonists of sstr. The amide bond between the peptide and nonpeptide fragments has been
formed using the carbodiimide method and the method of activated esters. The structures of the conjugates
have been confirmed by mass spectrometry (ESI+) and 1H NMR spectroscopy. The antitumor activity of the
conjugates has been examined by the MTT test on human lung adenocarcinoma (A549), human prostate ade-
nocarcinoma (PC3), human colon cancer (HCT-116), human breast cancer (MCF7), and acute human
T-cell leukemia (Jurkat) cell lines. Compounds selectively inhibiting the growth of A549 cells have been iden-
tified.
Keywords: somatostatin, analogs, peptides, synthesis, chimeric molecules, antitumor activity
DOI: 10.1134/S1068162019040034
as doxorubicin, methotrexate, camptothecin, and cis-
platin [7]. One modern-day approach to this problem
is the design of chimeric molecules combining peptide
and nonpeptide fragments. A peptide fragment pro-
vides a high selectivity of action of the conjugate by
binding to receptors, and a nonpeptide fragment
enhances antitumor activity. In addition, the presence
of the peptide fragment in the molecule diminishes the
toxicity of the preparation [8]. The present study is a
continuation of investigations on the synthesis and
examination of the cytostatic activity of somatostatin
analogs containing adamantane and naphthalene
fragments some of which have shown a high cytostatic
effect on MSF-7, PC3, and HCT-116 tumor cell lines [9,
10]. The tetrapeptide sequence -Phe-D-TRP-Lys-Thr-,
which is a pharmacophore in somatostatin [11], was cho-
sen as a peptide component in these investigations.
At present, several drugs based on the fragments of
somatostatin have been used; this hormone inhibits
many physiological functions in the hypothalamus,
anterior part of the hypophysis, the alimentary tract,
and the pancreas [1–6]. Among these drugs are okt-
reotid (trade name Sandostatin), lanreotid (Somatu-
line), bapreotid (Sanvar), and pasireotid (Signifor).
The action of these drugs is determined by their bind-
ing to somatostatin receptors whose hyperexpression
occurs in various tumor cells [2–4]. Many somatosta-
tin analogs have been created, and studies concerned
with the search for new compounds are in progress [5,
6]. Recent investigations along this line are devoted to
the development of analogs with improved properties:
a higher metabolic stability, selectivity of action, and
enhanced antitumor effect, which is achieved, e.g., by
incorporating the known cytostatic preparations, such
This work is devoted to the synthesis and study of
the antitumor activity of new somatostatin analogs
based on the sequence -Phe-D-TRP-Lys-Thr-. As
distinct from previous investigations in which the con-
jugation was accomplished through the carboxyl group
of threonine, the nonpeptide fragment in the present
study was conjugated with the peptide through the for-
Abbreviations: Boc, tert-butyloxycarbonyl; DCC, N,N'-dicyclo-
hexylcarbodiimide; DMEM, Dulbecco’s Modified Eagle’s
Medium; ESI, electrospray chemical ionization under atmo-
spheric pressure; HOBt, 1-hydroxybenzotriazole; TFA, trifluo-
roacetic acid; NMM, N-methylmorpholine; HEF, human
embryonic fibroblasts.
1 Corresponding author: phone: +7 (915) 230-37-53, e-mail:
mityaavdeev93@mail.ru.
248

SYNTHESIS AND ANTITUMOR ACTIVITY
249
mation of the amide bond with its terminal α-amino duction of protective groups into the -Phe-D-Trp-
group.
Lys-Thr- sequence and the substitution of the residue
L-Trp by D-Trp should increase the metabolic stabil-
ity of the tetrapeptide. Organic residues that poten-
tially possess antitumor activity and hold promise in
the development of antitumor preparations: a carbazole
RESULTS AND DISCUSSION
At the stage of synthesis, chimeric molecules were
obtained by the condensation of the protected tetra-
peptide H-Phe-D-Trp-Lys(Boc)-Thr-OMe and the
nonpeptide component at the α-amino group of phe-
derivative
(3-(1,2,3,4-tetrahydro-9H-carbazol-9-yl)
propanoic acid) [12], an adamantane derivative, and
nylalanine (Scheme 1). It is supposed that the intro- coumarin analogs were chosen as nonpeptide fragments.
DCC, HOBt
H-Phe-D-Trp-Lys(Boc)-Thr-OMe + RCOOH
RCO-Phe-D-Trp-Lys(Boc)-Thr-OMe
DMF, 0°C
(I)−(IV)
N
R:
O
O
O
,
,
,
O
O
O
(I)
(II)
(III)
(IV)
Scheme 1. General scheme of the synthesis of conjugates.
It has been shown earlier that the conjugates of somatostatin receptors (Table 1). Transformed HEFs
adamantane with the dipeptide Boc-Tyr-D-Trp-OH were used as control pseudonormal cells.
protected at the amine group exhibit a high antitumor
It should be noted that the initial peptide H-Phe-
activity in HCT-116 line cells [13]; the elimination of
D-TRP-Lys(Boc)-Thr-OMe showed no antitumor
the Boc protection led to a decrease in the activity of
activity on these cell lines; it exhibited only insignificant
the resulting compounds [13]. Some compounds
among coumarin derivatives are known to inhibit the
cytotoxicity on MCF-7 breast cancer cells (IC50 75 μM).
The study of the antitumor activity of compounds
(I)–(V) showed that conjugate (V) containing palmitic
acid produces a rather weak cytostatic action as compared
with the other analogs. Compound (I) showed a high anti-
tumor effect almost on all cell lines except for Jurkat cells;
however, its selectivity (IC50(HEF)/IC50(tumor cell)) was
close to 1, indicating a potential toxicity of this conju-
gate. Compounds (II)–(IV) showed the best results,
particularly in the inhibition of the growth of human
lung adenocarcinoma (A549) cells. In addition, the effi-
ciency of action of compounds (III) and (IV) on tumor
cells (A549) was almost by one order of magnitude higher
than on control cells (IC50(HEF)/IC50(A549) > 9).
Compounds (III) and (IV) were comparable in IC50 val-
ues with the known antitumor drug 5-fluorouracil
(11.13 μM) in A549 cells [16]. It is known that 5-fluo-
rouracil has low cytotoxicity due to a low selectivity of
action, which was not noticed in the case of com-
pounds (III) and (IV). Compound (III) also showed a
high cytotoxic effect toward the primary glioblastoma
(IC50 13.5 μM), one of the most aggressive tumors.
growth of HCT-116, A549, and MSF-7 tumor line
cells [14–16]; in addition, there is evidence indicating
that coumarin amides possess the antitumor activity
comparable with that of the known antitumor drugs
5-fluorouracil [16] and entinostat [17]. We have also
attempted to increase the antineoplastic activity of
H-Phe-D-TRP-Lys(Boc)-Thr-OMe by conjugating it
with palmitic acid [18].
In the present work, the peptide H-Phe-D-TRP-
Lys(Boc)-Thr-OMe was synthesized as described in
[19]. In the synthesis of compounds (I)–(IV), the
amide bond was formed by the carbodiimide method.
Compound (V) was synthesized using the palmitic
acid N-hydroxysuccinimide ester obtained by means
of the transetherification reagent N-(trifluoroace-
toxy)succinimide, since the carbodiimide method
often turned out to be unsuitable for producing highly
lipophilic derivatives of peptides with fatty acids in
organic solvents [20]. All compounds were obtained
with high yields. Their structures were confirmed by
mass spectrometry (ESI+) and 1H NMR spectroscopy
(see the Experimental section).
As a result of the study, new somatostatin analogs
The antitumor activity of compounds (I)–(V) was containing the fragments of adamant, coumarin, tet-
determined by the MTT test on human lung (A549) rahydroxy carbazole, and palmitic acid were obtained.
and prostate adenocarcinoma (PC3), colorectal can- Compounds possessing in vitro cytotoxic activity and
cer (HCT-116), breast cancer (MCF7), and acute pronounced selectivity of action toward human lung
T-cell leukemia (Jurkat) cell lines expressing the adenocarcinoma (A549) were identified.
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AVDEEV et al.
γCH2, δCH2, Lys), 1.35 (s, 9H, -Boc), 1.4–1.67 (m,
EXPERIMENTAL
2H, βCH2, Lys), 1.68–1.87 (m, 4H, (CH2)2, Сrb),
2.24–2.46 (m, 2H, –NCH2CH2, Prp), 2.51–3.28 (m,
10H, βCH2, D-Trp; (CH2)2, Сrb; εCH2, Lys; βCH2,
Phe), 3.60 (s, 3H, –OCH3, Thr(OMe)), 4.01–4.13
(m, 3H, –NCH2CH2(O), Prp; αCH, Phe), 4.25 (dd,
1H, J 8.2, 3.4, βCH, Thr), 4.37 (q, 1H, J 7.5, αCH,
Lys), 4.57 (dt, 1H, J 13.1, 6.6, αCH, D-Trp), 4.95 (d,
1H, J 5.7, –OH, Thr), 6.66–6.81 (m, 2H, HAr, Phe;
εNH, Lys), 6.85–7.12 (m, 8H, HAr, Phe; HAr, D-Trp),
7.16 (d, 1H, J 2.3, HAr, Crb), 7.20–7.38 (m, 3H,
HArCrb), 7.69 (d, 1H, J 7.7, HAr, D-Trp), 7.97 (t, 2H,
J 8.1, αNH, Lys; αNH, D-Trp), 8.21 (d, 1H, J 8.1,
αNH, Phe), 8.38 (d, 1H, J 8.2, αNH, Thr), 10.77 (s,
1H, NH, D-Trp).
The derivatives of L/D-amino acids DMF, DCC,
NMM, and HOBt, as well as difluoromethane and
2
1
methanol (Fluka, Switzerland) were used. H NMR
spectra were recorded on an AVANCE III NanoBay
spectrometer in DMSO-d6 at 300 MHz and 300 K
with DMSO-d6 (δ 2.500) as an internal standard; the
concentration of the compounds was 2–3 mg/mL.
The values of chemical shifts (δ, ppm) and spin–spin
coupling constants (J, Hz) are presented. Mass spectra
were recorded on an AmazonBruker device by electro-
spray ionization (ESI) in the mode of positive and
negative ions (capillary voltage 3500 V). The range of
mass scan m/z was 70–2200. Samples dissolved in eth-
anol or acetonitrile were injected by a syringe. The
sprayer gas was nitrogen, and the interface tempera-
ture was 100°С. Analytical HPLC was carried out on a
Shimadzu chromatograph (Japan) using Reprosil–
PurBasic C18 columns (5 μm), 4.6 × 240 mm. The
mobile phase: buffer A 0.1% TFA in water; buffer B
0.1% TFA in acetonitrile. The elution was in a gradient
of the concentration of buffer B in buffer A from 5 to
100% in 30 min, the flow rate was 1 mL/min, and
detection was at 220 nm.
Nα-[2-((3S,5S,7S)-Adamantan-1-yl)acetyl]-Phe-
D-Trp-Lys(Boc)-Thr-OMe (II) was synthesized from
2-((3S,5S,7S)-adamantan-1-yl) acetic acid (0.20 g,
1.03 mmol) to obtain 0.70 g (0.92 mmol, 89.5%) of a
white powder. ESI-MS, m/z (Irel, %): 893.7(100)
[M + Na]+, 909.62(45) [M + K]+. Purity (HPLC):
1
89.84%. H NMR: 1.07 (d, 3H, J 6.4, γCH3, Thr),
1.13–1.27 (m, 4H, γCH2, δCH2, Lys), 1.35 (s, 9H,
-Boc), 1.40–1.86 (m, 17H, βCH2, Lys; CH15, Adm),
1.86–2.08 (m, 2H, CH2, AcAdm), 2.65–2.74 (m, 1H,
β'-CH2, Phe), 2.75–3.00 (m, 4H, βCH2, D-Trp;
A General Method for Obtaining Compounds (I)–(IV)
The peptide H-Phe-D-Trp-Lys(Boc)-Thr-OMe εCH2, Lys), 3.12 (dd, 1H, J 14.6, 5.0, β''-CH2, Phe),
(0.69 g, 1 mmol), a corresponding organic acid
(1.03 mmol) (see scheme), and HOBt (0.14 g, 1.033
mmol) were successively dissolved in DMF (10 mL).
A solution of DCC (0.21 g, 1 mmol) in DMF (1.67 mL)
was added dropwise to the reaction mixture cooled to
0°С, and the mixture was stirred for 2 h at 0°С and
then for an additional 12 h at room temperature. A
precipitate of dicyclohexyl urea was filtered and
washed with DMF (2 mL). A 5% solution of citric acid
(100 mL) was added to the combined filtrate. The pre-
cipitate was filtered, washed with water (2 × 20 mL), a
5% potassium bicarbonate solution (10 mL), and
water (2 × 20 mL) and reprecipitated from a
C2H5OH–H2O mixture 0.5 : 1 (1 × 10 mL). The pre-
cipitate was washed with C2H5OH–H2O 1 : 1 (1 × 10
mL) and air-dried to a constant weight.
3.61 (s, 3H, –OCH3, Thr(OMe)), 4.10 (d, 1H, J 7.0,
αCH, Thr), 4.25–4.36 (m, 2H, αCH, D-Trp; αCH,
Lys), 4.46–4.62 (m, 2H, αCH, Phe; βCH, Thr), 5.17
(s, 1H, –OH, Thr), 6.72 (t, 1H, J 5.7, εNH, Lys),
7.21–6.91 (m, 8H, HAr, Phe; HAr, D-Trp), 7.29 (d, 1H,
J 7.9, HAr, D-Trp), 7.63 (d, 1H, J 7.8, HAr, D-Trp),
7.79 (d, 1H, J 8.3, αNH, Lys), 8.09 (d, 1H, J 8.3,
αNH, D-Trp), 8.27 (d, 1H, J 8.3, αNH, Phe ), 8.39
(d, 1H, J 8.4, αNH, Thr), 10.80 (s, 1H, –NH,
D-Trp).
Nα-2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)-
butanoyl-Phe-D-Trp-Lys(Boc)-Thr-OMe (III) was
synthesized from 2-((4-methyl-2-oxo-2H-chromen-
7-yl)oxy) butanoic acid (0.271 g, 1.03 mmol) to obtain
0.85 g (0.92 mmol, 91.8%) of a white powder. ESI-
MS, m/z (Irel, %): 939.5(100) [M]+, 839.5(3)
Nα-[3-(1,2,3,4-Tetrahydro-9H-carbazol-9-yl)pro-
panoyl]-Phe-D-Trp-Lys(Boc)-Thr-OMe (I) was syn-
thesized from 3-(1,2,3,4-tetrahydro-9H-carbazol-9-
yl) propanoic acid (0.25 g, 1.03 mmol) to obtain 0.9 g
(0.98 mmol, 97.8%) of a white powder. ESI-MS, m/z
(Irel, %): 920.6(100) [M]+, 820.5(5) [M-Boc]+, 410.8 (2)
[M-Boc]+. Purity (HPLC): 92.83%. 1H NMR
(DMSO-d6): 0.77 (q, 3H, J 7.4, –CH3СH2(Chr)),
1.03 (d, 3H, J 6.4, γCH3, Thr), 1.07–1.31 (m,
4H,γCH2, δCH2, Lys), 1.35 (s, 9H, -Boc), 1.38–1.77
(m, 4H, βCH2, Lys; –CH3СH2(Chr)), 2.37 (d, 3H, J
8.6, ‒CH3(Chr)), 2.71 (d, 1H, J 11.6, β'-CH2, Phe),
2.77–2.98 (m, 4H, βCH2, D-Trp; εCH2, Lys), 3.07 (d,
1H, J 14.7, β''-CH2, Phe), 3.60 (s, 3H, –OCH3,
Thr(OMe)), 3.98–4.11 (m, 1H, αCH, Thr), 4.18–
4.27 (m, 1H, βCH, Thr), 4.31–4.41 (m, 1H, αCH,
Lys), 4.45–4.76 (m, 2H, αCH, D-Trp; αCH, Phe),
1
[(M-Boc)/2]+. Purity (HPLC) 92.47%. H NMR:
1.06 (d, 3H, J 6.3, γCH3, Thr), 1.10–1.32 (m, 4H,
2 Spectra were described using the following abbreviations for the
designation of residues in compounds: Crb, 3-(1,2,3,4-tetrahy-
dro-9H-carbazol-9-yl; Prp, propanoyl; Adm, adamantly; Chr,
(4-methyl-2-oxo-2H-chromen-7-yl)oxy); FuroChr, (9-ethyl-4-
methyl-2-oxo-2H-furo[2,3-h]chromen); Palm, palmitoyl.
3
4.98 (s, 1H, –OH, Thr), 6.21 (d, 1H, J 8.9, H,
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SYNTHESIS AND ANTITUMOR ACTIVITY
251
Table 1. Antitumor activity (IC50, μM) of conjugates of the general formula RCO-Phe-D-Trp-Lys(Boc)-Thr-OМe
Cancer cells
MCF-7
IC50 (HEF)/
IC50 (A549)
Compound
R
HEF
HCT-116
12
PC3
11
A549
10
Jurkat
47
(I)
11
13
1.18
N
(II)
10
16
8.4
17
65
25
8.2
5
8.4
16
17
2.07
9.2
(III)
46
O
O
O
(IV)
(V)
75
50
72
>100
>100
11
71
87
>100
90
9.09
0.9
O
O
O
CH3(CH2)14
>100
>100
HEF, transformed human embryonic fibroblasts.
(Chr)), 6.57–7.19 (m, 11H, HAr, Phe; HAr, D-Trp; HAr(FuroChr)), 7.98 (d, 1H, J 8.1, αNH, Lys), 8.13 (d,
εNH, Lys; HAr, Chr), 7.29 (d, 1H, J 8.2, HAr, D-Trp), 1H, J 8.1, αNH, D-Trp), 8.26 (d, 1H, J 8.0, αNH,
Phe), 8.56 (d, 1H, J 8.1, αNH, Thr), 10.80 (s, 1H,
7.55 (dd, 1H, HAr(Chr)), 7.68 (d, 1H, J 7.7, HAr, D-Trp),
8.00 (d, 1H, J 8.4, αNH, Lys), 8.19–8.40 (m, 2H,
αNH, Phe; αNH, Thr), 10.78 (s, 1H, NH, D-Trp).
‒NH, D-Trp).
Nα-Palmitoyl-Phe-D-Trp-Lys(Boc)-Thr-OМe (V).
N-Hydroxysuccinimide (2.0 g, 17.4 mmol) was dis-
solved in a round-bottomed flask with a volume of
150 mL in trifluoroacetic anhydride (5 mL, 26 mmol).
After 15 min, the resulting solution was evaporated to
dryness and washed with hexane after which the sol-
vent was decanted. After drying the residue in vacuo,
3.6 g (17 mmol, 98%) of colorless crystals of N-triflu-
oroacetoxysuccinimide (TPS) was obtained to which a
solution of palmitic acid (4 g, 15 mmol) in methylene
chloride (15 mL) and pyridine (2 mL) were added.
The reaction mass was stirred for 40 min at room tem-
perature. The solvent was removed in vacuo (40°С,
15 mmHg), and water (100 mL) was added to the res-
idue. The precipitate was filtered, washed with water
Nα-(9-Ethyl-4-methyl-2-oxo-2H-furo[2,3-h]-
chromen-8-carbonyl)-Phe-D-Trp-Lys(Boc)-Thr-OMe
(IV) was synthesized from (9-ethyl-4-methyl-2-oxo-
2H-furo[2,3-h]chromen-8-carboxylic acid (0.05 g,
0.18 mmol) to obtain 0.11 g (0.11 mmol, 62.5%) of a
white powder. ESI-MS, m/z (Irel, %): 949.9(100)
[M]+, 242.6(3) [M/4 + 5H]+, 397.5 (2.2) [M/3 +
1
DMSO + 2H]+. Purity (HPLC): 90.12%. H NMR
(DMSO-d6): 0.98–1.23 (m, 8H, γCH3, Thr;
‒CH3CH2(FuroChr), γCH2, Lys), 1.26–1.8 (m, 12H,
δCH2, Lys; -Boc; βCH2, Lys), 2.7–3.02 (m, 8H,
βCH2, Phe; βCH2, D-Trp; εCH2, Lys;
‒CH3CH2(FuroChr), 3.09–3.23 (m, 1H, αCH,
Phe), 3.58 (s, 3H, –OCH3, Thr(OMe)), 4.01–4.15 (2 × 10 mL), a 5% sodium bicarbonate solution
(10 mL), and a C2H5OH–H2O mixture 1 : 1 (1 × 10 mL),
(m, 1H, αCH, Thr), 4.23 (dd, 1H, J 8.0, 3.5, βCH,
Thr), 4.31–4.44 (m, 1H, αCH, Lys), 4.58–4.86 (m, and air-dried to a constant weight. The product (5 g,
1H, αCH, D-Trp), 4.94 (d, 1H, J 5.7, –OH, Thr), 14.14 mmol, 94.2%) was obtained as a white powder of
3
N-hydroxysuccinimide ester of palmitic acid (Palm-
6.41 (s, 1H, H(FuroChr)), 6.70 (s, 1H, εNH, Lys),
6.87–7.23 (m, 8H, HAr, Phe; HAr, D-Trp), 7.3 (d, 1H,
J 7.9, HAr, D-Trp), 7.59 (d, 1H, J 7.7, HAr(FuroChr)),
7.71 (d, 1H, J 7.7, HAr, D-Trp), 7.82 (d, 1H, J 8.8,
ONSu).
To a solution of H-Phe-D-Trp-Lys(Boc)-Thr-
OMe (0.50 g, 0.72 mmol) in DMF (5.00 mL), NMM
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 45 No. 4 2019

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AVDEEV et al.
Conflict of Interest
(120 μL) to pH 8 and then Palm-ONSu (0.26 g,
0.69 mmol) were added, and the mixture was stirred at
room temperature for 18 h. A 2% citric acid solution
(70 mL) was added to the reaction mixture. The pre-
cipitate was filtered, washed with water (2 × 20 mL)
and a 5% sodium bicarbonate solution (10 mL), and
reprecipitated from a C2H5OH–H2O mixture 0.5 : 1
(1 × 10 mL) after which the precipitate was air-dried
to a constant weight. The product (0.40 g, 0.43 mmol,
59.5%) was obtained as a white powder; Rf 0.6 (chlo-
roform–methanol–acetic acid 9 : 1 : 0.5); ESI-MS,
m/z (Irel, %): 955.87(100) [M + Na]+, 971.79 (14)
The authors declare that they have no conflict of interest.
REFERENCES
1. Guillemin, R. and Gerich, J.E., Annu. Rev. Med., 1976,
vol. 27, pp. 379–388.
2. Patel, Y.C., Front. Neuroendocrinol., 1999, vol. 20,
pp. 157–198.
3. Lamberts, S.W., van der Lely, A.J., and Hofland, L.J.,
Eur. J. Endocrinol., 2002, vol. 146, pp. 701–705.
1
[M + K]+; H NMR (DMSO-d6): 0.85 (t, 3H, J 6.5,
4. Barnett, P., Endocrine, 2003, vol. 20, pp. 255–264.
5. Janecka, A., Zubrzycka, M., and Janecki, T., J. Peptide
‒CH3(Palm)), 0.95–1.54 (m, 42H, γCH3, Thr; γCH2,
δCH2, Lys; -Boc; –CH2(Palm)), 1.54–1.71 (m, 2H,
βCH2, Lys), 1.88–2.05 (m, 2H, –CH2C(O)(Palm)),
2.68 (d, 1H, J 10.5, β'-CH2, Phe), 2.76–2.99 (m, 4H,
βCH2, D-Trp; εCH2, Lys), 3.12 (dd, 1H, J 14.6, 5.0,
β''-CH2, Phe), 3.61(s, 3H, OCH3, Thr(OMe)), 4.02–
4.17 (m, 1H, αCH, Thr), 4.26 (dd, 1H, J 8.3, 3.5,
βCH, Thr), 4.3–4.4 (m, 1H, αCH, Lys), 4.44–4.66
(m, 2H, αCH, D-Trp; αCH, Phe), 5.0 (d, 1H, J 5.4,
‒OH, Thr), 6.70 (t, 1H, J 5.7, εNH, Lys), 6.9–7.19
(m, 8H, HAr, Phe; HAr, D-Trp), 7.29 (d, 1H, J 7.9, HAr,
D-Trp), 7.66 (d, 1 H, J 7.7, HAr, D-Trp), 7.83 (d, 1H,
J 8.2, αNH, Lys), 7.99 (d, 1H, J 8.2, αNH, D-Trp),
8.18 (d, 1H, J 8.0, αNH, Phe), 8.33 (d, 1H, J 8.2,
αNH, Thr), 10.78 (s, 1H, NH, D-Trp).
The antitumor activity of the new somatostatin ana-
logs was studied by the MTT test on human lung ade-
nocarcinoma (А549), human prostate adenocarci-
noma (PC3), human colon cancer (НСТ-116), human
breast adenocarcinoma (MCF7), a primary glioblastoma
culture kindly presented by E.Yu. Rybalkina, and acute
human T-cell leukemia (Jurkat) cell lines. Trans-
formed HEFs were used as control cells. А549, MCF-7,
primary glioblastoma cells, and HEFs were cultured in
medium DMEM (PanEko, Russia); НСТ-116, PC3,
and Jurkat cells were grown in medium RPMI 1640
(PanEko, Russia) supplemented with 10% fetal bovine
serum (HyClone, United States) and gentamycin
(40 μg/mL).
Res., 2001, vol. 58, pp. 91–107.
6. Weckbecker, G., Lewis, I., Albert, R., Schmid, H.A.,
Hoyer, D., and Bruns, C., Nat. Rev. Drug Discov., 2003,
vol. 2, pp. 999–1017.
7. Zamoria, A.M., Gandioso, A., Brabec, V., Ruiz, J.,
and Marchan, V., Chem. Commun., 2017, vol. 53,
pp. 5523–5526.
8. Srinivasarao, M., Galliford, C.V., and Low, P.S., Nat.
Rev. Drug Discov., 2015, vol. 14, pp. 203–219.
9. Balaev, A.N., Osipov, V.N., Okhmanovich, K.A., Ruc-
hko, E.A., Baryshnikova, M.A., and Khachatryan, D.S.,
Izv. Akad. Nauk, Ser. Khim., 2016, vol. 11, pp. 2766–
2769.
10. Balaev, A.N., Osipov, V.N., Okhmanovich, K.A., Ruc-
hko, E.A., Baryshnikova, M.A., and Khachatryan, D.S.,
Izv. Akad. Nauk, Ser. Khim., 2016, vol. 12, pp. 2948–2951.
11. Modlin, I.M., Pavel, M., Kidd, M., and Gustafsson, B.I.,
Aliment. Pharmacol. Ther., 2010, vol. 31, pp. 169–188.
12. Sambasivarao, K., Saifuddin, M., and Aswar, V.R.,
Org. Biomol. Chem., 2016, vol. 14, pp. 9868–9873.
13. Kuriyama, I., Miyazaki, A., and Tsuda, Y., Anticancer
Res., 2010, vol. 30, pp. 4841–4849.
14. Saidu, N.E., Valente, S., Bana, E., Kirsch, G., Bagrel, D.,
and Montenarh, M., Bioorg. Med. Chem., 2012, vol. 20,
pp. 1584–1593.
15. El-Samahy, F.A., Abd El Salam, H.A., El-Sayed, N.F.,
Shalaby, E.M., and Dondeti, M.F., Z. Naturforsch., A:
Phys. Sci., 2017, vol. 72, pp. 705–716.
Adhesive tumor cell cultures were seeded on a
96-well plate in an amount of 5 × 103 per well, and Jur-
kat cells were plated in an amount of 15 × 103. On the
next day, test substances at final concentrations from
100 to 5 μM were added to cells. After 72 h of incuba-
tion, the MTT reagent at a concentration of 5 mg/mL
was added into wells, 20 μL per well. After incubation
16. Soni, R., Umar, S., Shah, N.N., Balkrishnan, S., and
Somana, S.S., J. Heterocycl. Chem., 2017, vol. 49,
pp. 3232–2510.
17. Abdizadeh, T., Kalani, M.R., Abnous, K., Tayarani-
Najaran, Z., Khashyarmanesh, B.Z., Abdizadeh, R.,
Ghodsi, R., and Hadizadeh, F., Eur. J. Med. Chem.,
2017, vol. 132, pp. 42–62.
for three to four hours, the resulting formazan crystals 18. Mäde, V., Els-Heindl, S., and Beck-Sickinger, A.G.,
Beilstein J. Org. Chem., 2014, vol. 10, pp. 1197–1212.
were dissolved in DMSО (60 μL), and the optical den-
sity of the solution was determined on a MultiSkanFC
spectrophotometer (ThermoScientific, United States)
at 540 nm.
19. Balaev, A.N., Osipov, V.N., Okhmanovich, K.A., Fe-
dorov, V.E., and Reshetnikov, E.V., Ross. Bioterapevt.
Zh., 2011, vol. 4, pp. 43–46.
20. Andreev, S.M., Sidorova, M.V., Rakova, O.A., Tsvet-
kov, D.E., and Fonina, L.A., Bioorg. Khim., 1987,
vol. 13, pp. 696–700.
COMPLIANCE WITH ETHICAL STANDARDS
This article does not contain any studies involving animals or
human participants performed by any of the authors.
Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 45
No. 4
2019